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Introduction
The typical crop improvement cycle takes 10-15 years to complete and includes germplasm manipulations,
genotype selection and stabilization, variety testing, variety increase, proprietary protection and crop
production stages. Plant tissue culture and genetic engineering procedures that form the basis of plant
biotechnology can contribute to most of these crop improvement stages.

This review provides an overview of the opportunities presented by the integration of plant biotechnology
into plant improvement efforts and raises some of the societal issues that need to be considered in their
application.

Humans began to modify the characteristics of plants used for food and fibre approximately ten to twenty
thousand years ago. Even primitive seeding, cultivating, harvesting and storing practices would have exerted
selection pressures on those plant species which became domesticated that were different from the pressures
their progenitors encountered in the wild. Over time, but particularly in the last 150 years, plant breeding has
developed into a complex discipline that now incorporates information from many branches of science and
mathematics.

The most recent development is the utilization of biotechnology for plant improvement (Ratner, 1989). Plant
biotechnology can be defined as the application of tissue culture and molecular genetics to develop or
produce a commodity from plants. Tissue culture refers to themaintenance and propagation of plant parts (as
small as a single cell) in biologically pure (axenic) and controlled environments (Fig. 1; Evans et al., 1983;
Vasil, 1984).

Moleculargenetics includes techniques for isolating, characterizing, recombining and multiplying and
transferring discrete fragments of DNA that contain genes coding for specific traits (Fig. 2; Maniatis et al.,
1982; Gelvin et al., 1988; Watson et al., 1987). The fact that a whole plant can be regenerated from a single
cell makes tissue culture a valuable procedure for proliferating genetically identical material and selecting
interesting variants.

Totipotency also allows a genetic change, made at the cellular level, to become an established trait of a
whole plant. The newly introduced or selected trait can, subsequently, be passed on to future generations of
the species by conventional crossing methods. To be effective, plant biotechnology must be well integrated
into established plant breeding and crop production practices. For many field crop species the average
amount of time that is required to develop, test and release a new variety is 10-15 years.

The procedure has many stages including: germplasm manipulation, parent selection, genotype selection,
genetic stabilization, testing, variety increase, proprietary protection, crop production and crop quality
control. Fig. 3 illustrates that biotechnology can contribute to most stages of crop development and
production.

Given the range of expertise and level of integration that is required in a modem plant breeding program it is
important that mutually beneficial partnerships among industry, government and university institutions are
developed in the application of plant biotechnology to plant improvement. Not only is this partnership
approach likely to be necessary to collect a sufficient critical mass around a particular topic, but it is
probably the only way in which the societal issues related to the application of biotechnology will be
properly addressed (Wrubel et al., 1992; Buttel, 1986).
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Figure 1. Plant tissue culture. Plant cells can be induced to proliferate in a variety of forms
including nondifferentiated callus and suspension cultures in vitro. Single ceils like protoplasts
express totipotency, i.e. the ability of a single cell to regenerate into a whole plant.
Regeneration may occur via various processes including somatic embryogenesis. In this process
structures that resemble zygotic embryos are formed in the tissue culture.

Plant biotechnology, a major component of agricultural biotechnology, deals with various aspects of plant
tissue culture, genetic transformation, and molecular biology techniques. Tissue culture methods offer a rich
scope for creation, conservation, and utilization of genetic variability for the improvement of field, fruit,
vegetable, and forest crops, and medicinal/aromatic plants. Micropropagation technology ensures true to
type, rapid and mass multiplication of plants that possesses special significance in vegetatively propagated
plant species. This technology has witnessed a huge expansion globally, with an estimated global market of
15 billion US$/annum for tissue-culture products. Some basic techniques of tissue culture, such as
anther/microspore culture, somaclonal variation, embryo culture, and somatic hybridization, are being
exploited to generate useful genetic variability for obtaining incremental improvement in commercial
cultivars.
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Production of secondary metabolites, such as food flavors, food colors, dyes, perfumes, drugs, and scented
oils used in aromatherapy, through cell cultures and hairy root cultures, are leading examples of molecular
farming. Cryopreservation of germplasm at the cell/tissue/organ levels, in liquid nitrogen at −196◦C, is
highly rewarding for establishing germplasm banks, especially for vegetatively propagated crops and rare,
endangered plant species. During the past 15 years, remarkable achievements have been made in the
production, characterization, field evaluation, and release of transgenic varieties/hybrids in several crops.

Transgenic varieties/hybrids of maize, cotton, soybean, potato, tomato, and papaya are now being
commercially grown on about 134 million hectares spread across 25 countries. Research in genomics allows
high-resolution genetic analysis for physical mapping and positional gene cloning of useful genes for crop
improvement. Molecular (DNA) markers help in precise characterization of germplasm, construction of
saturated linkage maps, and DNA fingerprinting of crop varieties. Molecular markers are now increasingly
being used for marker-assisted gene pyramiding and alien gene introgression. Current research, involving
large-scale DNA sequencing, microarrays, and robotics, is heading towards gene revolution and
nanobiotechnology.

Biotechnology in a broad sense has been practiced for centuries for curd making, food preservation, pickle
making, and fermentation. However, biotechnology received a boost during the 1970s with the discovery of
restriction enzymes, which led to the development of a variety of gene technologies and is, thus, considered
the greatest scientific revolution of the 20th century. Biotechnology, in a true sense, deals with changing and
improving, more efficiently than traditional technologies can, the characteristics of an organism at cellular
and molecular levels for the benefit of mankind.

Depending upon the biological system (organism) involved, the field of biotechnology may be divided into
plant biotechnology, microbial biotechnology, animal biotechnology, and human biotechnology. Plant
biotechnology deals with cell and tissue culture, genetic transformation, gene cloning, DNA markers, and
other molecular approaches. Unlike conventional plant breeding, the biotechnological techniques for genetic
modifications largely operate at organ, tissue, cell, protoplast, and molecular levels. These innovative
techniques are considered an adjunct to the conventional methods for efficient and precision plant breeding
(Kang et al. 2007).

Plant tissue culture broadly refers to the in vitro cultivation of plants, seeds, and plant parts (tissues, organs,
embryos, single cells, protoplasts) on nutrient media under closely controlled and aseptic conditions. It
includes several specialized areas, such as induction of callus and plant regeneration, micropropagation,
somatic embryogenesis, somaclonal variation, meristem culture, anther culture, embryo culture, protoplast
culture, cryopreservation, and production of secondary metabolites. Tissue-culture methods hold
Biotechnology & Crop Improvement 155 significant promise for creation, conservation, and utilization of
genetic variability for improvement of a wide variety of crop plants. Among these, micropropagation,
somaclonal variation, and embryo, anther and protoplast culture have direct applications in crop
improvement.
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Figure 2. Plant molecular genetics. A foreign gene, coupled to a selectable marker gene, is
cloned into a disarmed Ti plasmid of Agrobacterium tumefaciens. The foreign gend and the
selectable marker gene axe transferred to the plant by cocultivating the bacteria with the explant
(leaf disk) and regenerating plants from the cells that express resistance to the selective agent
in tissue culture. The foreign gene becomes stably integrated into the DNA of the plant and
is inherited by future generations of plants derived from the original transgenic plant.

Writing about biotechnology for crop improvement in the next millennium does not appear to be an easy
task owing to the rapid progress in this field. Within the last 100 years the world has seen the rise of genetics
as a scientific discipline (1900s), the finding of DNA as the hereditary Ortiz, R. 153 material (1944), the
elucidation of the double helix structure of the DNA molecule (1953), the cracking of the genetic code
(1966), the ability to isolate genes (1973), and the application of DNA recombinant techniques (from 1980
onwards).

Methods of crop improvement have also changed dramatically throughout this century. Mass and pure line
selection in landraces, consisting of genotype mixtures, were the popular breeding techniques until the 1930s
for most crops. In the 1930s maize breeders started the commercial development of double cross hybrids
that was followed by the extensive utilization of single crop hybrids since the 1960s (Troyer 1996).
Pedigree-, bulk-, backcrossand other selection methods were also developed especially for self-pollinating
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crop species. Such scientific advances in plant breeding led to the so-called ‘Green Revolution’, one of the
greatest achievements to feed the world in the years of the Cold War (Perkins 1997). Owing to this
agricultural betterment, cereal production, which accounts for more than 50% of the total energy intake of
the world’s poor, kept in pace with the high average population growth rate of 1.8% since 1950 (Daily et al.
1998). Today, 370 kg of cereals per person are harvested as compared to only 275 kg in the 1950s; i.e., in
excess of 33% per capita gain. Similar progress in other food crops resulted in 20% per capita gains since
the early 1960s, according to FAO (1995). There are 150 million fewer hungry people in the world today
than 40 years ago, though there are twice as many human beings. Despite this splendid progress in crop
productivity, even greater progress must be made in order to feed an additional two billion people by the
early part of the 21st century (Anderson 1996a). Around 800 million people are hungry today and another
185 million pre-school children are still malnourished owing to lack of food and water, or disease (Herdt
1998). Hence as suggested by the Nobel Peace Laureate, Norman Bourlag (1997), new biotechniques, in
addition to conventional plant breeding, are needed to boost yields of the crops that feed the world.

Careful choice of such biotechniques as well as a realistic assessment of their potential in crop improvement
are needed to avoid not only the criticism of the anti-science lobbyists but also the permanent distrust of
pragmatic traditional breeders (Simmonds 1997). For example, a World Bank panel recently released for
discussion a well based report concerning bioengineering of crops (Kendall et al. 1997). In this working
paper, the panel members recommend “to give priority to all aspects of increasing agricultural productivity
in the developing world while encouraging the necessary transition to sustainable methods”. Indeed, plant
biotechnology has been regarded as a priority area for technology transfer (Altman and Watanabe 1995),
because genetically modified food, feed, and fibre are of vital concern to the developing world (Ives and
Bedford 1998). Therefore, the rich industrialized world should share their biotechniques and avoid policies
that do not allow the progress of agriculture in poor, nonindustrialized parts of the world (Erbisch and
Maredia 1998), where this economic activity still provides 60 to 80% employment and 50% of national
income (Anderson 1996a). Such support will assist the developing world towards food self-reliance (Herdt
1998), which will be very important to avoid hunger and keep peace in many regions of the tropics, where
the agricultural sector remains the most important basis for economic growth. Furthermore, a wealthy
society provides high living standards to its citizens.

Tissue culture was developed in the 1950s and became popular in the 1960s. Today, micropropagation and
in vitro conservation are standard techniques in most important crops, especially those with vegetative
propagation. At the beginning of the 1980s genetic engineering of plants remained a promise of the future,
although gene transfer had already been achieved earlier in a bacterium. The first transgenic plant, a tobacco
accession resistant to an antibiotic, was reported in 1983. Transgenic crops with herbicide, virus or insect
resistance, delayed fruit ripening, male sterility, and new chemical composition have been released to the
market in this decade (NCGR 1998; USDAAPHIS 1997).

In 1996, there were about 3 million ha of transgenic crops grown in the world (mainly in North America)
whereas in excess of 34 million ha (a 12-fold addition) of transgenic crops will be harvested this year in
North America, Argentina, China, and South Africa among other countries. Argentina is the leading
developing country with an excess of 4 million ha of transgenic herbicideresistant soybean. There are 4.4
million ha of transgenic corn (14% of total acreage), 5 million ha of transgenic soybean (20%), and 1.6
million ha of transgenic canola (42%) grown only in North America (Moore 1998). It hasbeen calculated
that in 1998 US farmers are growing over 50% of their cotton fields with transgenic seeds, the largest
percentage for any crop ever. Trees are the next target in the agenda of genetic engineering.
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Allozymes were available as the first biochemical genetic markers in the 1960s. Population geneticists took
advantage of such marker system for their early research. In the 1970s, restriction fragment length
polymorphisms (RFLP) and Southern blotting were added to the tool box of the geneticists. Taq polymerase
was found in the 1980s, and the polymerase chain reaction (PCR) developed shortly afterwards. Since then,
marker-aided analysis based on PCR have become routine in plant genetic research and marker systems
have shown their potential in plant breeding (Paterson 1996). Furthermore, new single nucleotide
polymorphic markers based on high density DNA arrays, a technique known as ‘gene chips’ (Chee et al.
1996), have recently been developed. With ‘gene chips’, DNA belonging to thousand of genes can be
arranged in small matrices (or chips) and probed with labeled Cdna from a tissue of choice. DNA chip
technology uses microscopic arrays (or micro-arrays) of molecules immobilized on solid surfaces for
biochemical analysis (Lemieux et al. 1998; Marshall and Hodgson 1998; Ramsay 1998). An electronic
device connected to a computer may read this information, which will facilitate marker-assisted

selection in crop breeding. In summary, since Mendel’s work on peas, there have been five eras in genetic
marker evolution (Liu 1997): morphology and cytology in early genetics (until late 1950s), protein and
allozyme electrophoresis in the pre-recombinant DNA time (1960 - mid1970s), RFLP and minisatellites in
the pre-PCR age (mid 1970s - 1985), random amplified polymorphic DNA, microsatellites, expressed
sequence tags, sequence tagged sites, and amplified fragment length polymorphism in the oligoscene period
(1986 - 1995), and complete DNA sequences with known or unknown function as well as complete protein
catalogs in the current computer robotic cyber genetics generation (1996 onwards) The driving force for
such a development has been the scientific interest of human beings to understand and manipulate the
inheritance of their own characters.

Responses to biotechnology in crop improvement

The advances in plant transgenics and genomics described above have not been isolated from society (Busch
et al. 1991). Some of these achievements have been acclaimed by end-users whereas other accomplishments,
e.g. release of genetically modified organisms (GMO), are being attacked, not only in words but also in
deeds, by political activists. Some of these educated middle-class campaigners are expressing in this way
their rampant ‘eco-paranoia’, while others hide their real agenda to manipulate the fashionable ecological
movement. This controversy has attracted the attention of non-scientific partizans to each side. There have
been negative comments about transgenic plants by a crown prince and contrasting positive comments by a
former president, both of whom may not have the required technical knowledge to assess the potential of
biotechnology for crop improvement. Irrespective of this ideological dispute and ensuing democratic
disagreements, biotechnology products will be accepted by people who support scientific-based progress, in
a similar way that new cultivars or innovative crop husbandry techniques have previously become integral
parts of farming systems elsewhere. However, without end-user’s consent, the impact of a new technology
in the society will be small or nil.

Scientific honesty seems to the best policy to convince people about the advantages of biotechnology for
crop improvement (Frewer et al. 1998). What to do? Scientists, farmers, consumers, and policy-makers
should objectively assess the potential hazards of crop biotechnology in farming and food systems regarding
the current situation and the likelihood that such hazards may occur. For example scientists should explain
to the people that gene recombination (or reassortment) already occurs in nature. However, the ecological
success of viable recombinants after gene reassortment is unpredictable owing to the high fitness of current
isolates. For this reason, more scientific research will be needed to identify unpredictable risks and the
chances of their occurrence.
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The need for profit, as in any other business, has attracted the interest of the private sector to defend their
investments in crop biotechnology with patents, intellectual property rights, and new protection methods,
e.g. ‘terminator’ technology that inhibits germination of self-pollinated seeds. This technology protection
system prevents farmers from saving seeds from their harvest for further utilization as next season planting
propagules. Three genes, each with a specific promoter, are inserted into the ‘terminator’ plant (D.E. Culley,
Washington State Univ. in RAFI 1998). One of the genes (e.g. CRE/LOX system from bacteriophages)
produces a recombinase that removes a spacer between the gene producing, for example, a ribosomal
inhibitor protein and its promoter such as late embryonic abundance, which only becomes active during the
late stages of embryo development. This spacer with specific recognition sites blocks the gene (for the
ribosomal inhibitor protein) from being activated. Another gene (e.g. tetracycline repressor system)
produces a repressor that keeps off the recombinase gene until an outside stimulus is applied to the
‘terminator’ plant, e.g. a chemical such as the tetracycline, or temperature and osmotic shocks. The United
States

Department of Agriculture (USDA) and a cotton seed enterprise jointly acquired a patent for this concept
(U.S. patent 5,723,765). Two months after this patent was announced, one of the leading agro-chemical
transnationals bought the cotton seed company, although one of its officers said that it may take many years
before this ‘terminator gene’ idea becomes a proven technology in the seed industry. Strategic alliances,
joint ventures, research partnerships, new investments, company mergers, cross-ownerships, and take-overs
in the seed and agro-chemical business have also been in the news in recent months. Likewise, some leading
scientists are leaving their academic appointments to join the new private enterprises in plant biotechnology.
These events are happening because the private sector wants to use biotechnology to accelerate its growth in
agri-business in the short-term. Nonetheless, funds to support basic and strategic research by public
researchers are needed for a long-term sustainable transfer of public goods (both knowledge and technology)
to the private sector or other users.

Bioinformatics

Another important factor in the successes of the genetic improvement of crops was the development of fast
and more reliable computers, which allowed easier management and analysis of data as well as publication
of scientific reports. The impact of the informatic revolution in crop improvement can be partially assessed
by counting the number of publications indexed in Plant Breeding Abstracts (CAB International,
Wallingford, Oxon, UK). There was ca. 22-fold increase of publications in the 1930-1997 period (Fig. 1). It
was in the 1970s that indexed publications in plant breeding exceeded 10,000 per year. More publications
and easy means for retrieving this information accounted Ortiz, R. 155 for such growth of knowledge
dissemination in plant genetics and breeding. Today, rapid information exchange has been facilitated with
electronic mail and access to the internet to read electronic publications such as this journal.

Nowadays, information technology and DNA science are beginning to fuse into a single operation.
Computers are deciphering, and organizing the huge genetic information that may become “the raw resource
of the emerging biotech economy” in the next century (Rifkin 1998). Scientists working in the new field of
“bioinformatics” are developing biological data banks to download the genetic information accumulated
during millions of years of life evolution, and perhaps reconstruct some of the living organisms of the
natural world.
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Figure 3. Number of publications indexed in Plant Breeding Abstracts (CAB International,

Wallingford, Oxon, UK) since its publication in early 1930s.publication in early 1930s

Plant genomics

This new term, defined by the development of biotechnology, refers to the investigations of whole genomes
by integrating genetics with informatics and automated systems. Genomic research aims to elucidate the
structure, function and evolution of past and present genomes (Liu 1997).

Some of the most dynamic fields concerning agriculture are the sequencing of plant genomes, comparative
mapping across species with genetic markers, and objective assisted breeding after identifying candidate
genes or chromosome regions for further manipulations. As a result of genomics, the concept of gene pools
has been enlarged to include transgenes and native exotic gene pools that are becoming available through
comparative analysis of plant biological repertoires (Lee 1998). Understanding the biological traits of one
species may enhance the ability to achieve high productivity or better product quality in another organism.
DNA markers and gene sequencing provides quantitative means to determine the extent of genetic diversity
and to establish objective phylogenetic relationships among organisms. ‘Gene chips’ and transposon tagging
will provide new dimensions for investigating gene expression.

Molecular biologists will study not only individual genes but how circuits of interacting genes in different
pathways control the spectrum of genetic diversity in any crop species. For example, more information will
be available on why plant resistance genes are clustered together, or what candidate genes should be
considered when manipulating quantitative trait loci (QTL) for crop improvement (Paterson 1997).
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Farming in environmentally friendly systems

The aims of applied plant science research for agriculture are to enhance crop yields, improve food quality,
and preserve the environment where human beings and other organisms live. The best way for conservation
of plant biodiversity and its environment, would be to achieve highcrop productivity per unit area. In this
regard, Briggs (1998) reported that as yields treble, soil erosion per ton of food decreases by two-thirds.
There has been a significant yield improvement owing to enhanced crop husbandry, but in the next years
progress will be achieved by changing plants that could be more suitable to sustainable and environmentally
friendly farming systems. Agro-chemical corporations are developing pest and disease resistant transgenic
crops to avoid pollution with pesticides in the farming system. Furthermore, food quality will become more
important than crop productivity in a wealthy society. Consumers will prefer transgenic crops if they have
the desired characteristics.

In the next decades meiotic-based breeding will still generate cultivars for farmers. Genetic improvement
through biotechnology needs conventional breeding because (1) the elite cultivars will be the parents of the
next generation of improved genotypes, (2) field testing across locations or cropping systems and over years
will be needed to determine the best selections due to the genotype-byenvironment interaction (Kang and
Gauch 1996). As stated by Briggs (1998), “transgenes must be viewed as improvements rather than
replacements for elite germplasm”. Indeed, genetic engineering may provide a means to add value by
introducing synthetic or natural genes that enhance crop quality and yield, as well as protect the plant against
pest and diseases. Farmers will pay more for transgenic crop propagules if they obtain extra-income after
adopting biotech-derived products. For example, seeds of insect resistant transgenic crops will be more
expensive than those of available cultivars but the farmer will not need to apply pesticides in their transgenic
fields. Of course, patents make transgenic seeds more expensive but also farmer’s benefits may be higher.

Gene banks, DNA banking and virtual plant breeding

The sequencing of crop genomes opened new frontiers in conservation of plant biodiversity and its genetic
enhancement. The advances in gene isolation and sequencing in many plant species allows to envisage that
Critical role of plant biotechnology for the genetic improvement of food crops: perspectives for the next
millennium 156 within a few years, gene-bank curators may replace their large cold stores of seeds with
crop DNA sequences that will be electronically stored. The characterization of plant genomes will ultimately
create a true gene bank, which should possess a large and accessible gene inventory of today’s non-
characterized crop gene pools. Of course, seed banks of comprehensively investigated stocks should remain
because geneticists and plant breeders, the main users of gene banks, will need this germplasm for their
work. Genomics may accelerate the utilization of candidate genes available at these gene banks through
transformation without barriers across plant species or other living kingdoms. Nonetheless, genetic
engineering should be seen as one of the methods of plant breeding that permits the direct alteration and re-
building of a crop population. “Shutting-off” genes coding for undesired characteristics may be another
application of transgenics in crop improvement.

Plant breeders will change their modus operandi with the development of objective marker-assisted
introgression and selection methods. Backcross breeding will be shortened by eliminating undesired
chromosome segments (also known as linkage drags) of the donor parent or selecting for more chromosome
regions of the recurrent parent. Parents of elite crosses may be chosen based on a combination of DNA
markers and phenotypic assessment in a selection index, such as best linear unbiased predictors (Bernardo
1998). To achieve success in these endeavours, cheap, easy, decentralized, and rapid diagnostic marker
procedures are required.
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There are many areas of basic and strategic research in plant breeding and genetics that are being facilitated
by marker-aided analysis (Paterson 1996). With molecular markers, plant biologists are reviewing crop
evolution and gathering new knowledge. Such information should be incorporated into genetic enhancement
programmes, especially those with an evolutionary breeding scheme. Likewise, plant ideotypes for each
crop should drive the work of plant breeders. Specific plant morphotypes have been defined in rice and
wheat based on accumulated knowledge of crop physiology and crop protection. The needed characteristics
required to develop improved plant prototypes ensuing from such a ‘virtual breeding’ approach may be
available in gene banks of the crop or in those of other species. Otherwise, breeders may obtain novel
transgenes to develop the required ideotype.

Nowadays, the finding of new genes that add value to agricultural products seems to be very important in
the private agri-business. Unique gene databases are being assembled by the industry with the massive
amount of data generated by genomics research. A new term ‘biosource’ was coined recently to refer to a
fast and effective licensed technology of pinpointing genes. With this method, a ‘benign’ virus infects a
plant with a specific gene that allows researchers to observe directly its phenotype.

Biosource replaces the standard time-consuming approach of first mapping a gene to subsequently
determine its exact function. Gene identification in DNA libraries coupled with biosource technology and an
enhanced ability to put genes into plants will be routine for improving crops in the next decade.

Genomics may provide a means for the elucidation of important functions that are essential for crop
adaptedness (Wallace and Yan 1998). Regions of the world should be mapped by combining data of
geographical information systems, crop performance, and genome characterization in each environment. In
this way, plant breeders can develop new cultivars with the appropriate genes that improve fitness of the
promising selections. Fine-tuning plant responses to distinct environments may enhance crop productivity.
Development of cultivars with a wide range of adaptation will allow farming in marginal lands. Likewise,
research advances in gene regulation, especially those processses concerning plant development patterns,
will help breeders to fit genotypes in specific environments. Photoperiod insensitivity, flowering initiation,
vernalization, cold acclimation, heat tolerance, host response to parasites and predators, are some of the
characteristics in which advanced knowledge may be acquired by combining molecular biology, plant
physiology and anatomy, crop protection, and genomics. Multidisciplinary co-operation among researchers
will provide the required holistic approach to facilitate research progress in these subjects.

Pharming and Farmer-ceuticals

Growth of cities in the developed world has already replaced farmland with shopping malls, parking lots,
and housing developments. Peri-urban agriculture and home gardening are also becoming very important for
national food security in the developing world as a result of rapid urban expansion. Hence, new cultivars
will be needed to fit into intensive production systems, which may provide the food required to satisfy urban
world demands of the next century. Specific plant architecture, tolerance to urban pollution, efficient
nutrient uptake, and crop acclimatization to new substrates for growing are, among others, the plant
characteristics required for this kind of agriculture. Genes controlling these characteristics may be available
in gene banks for further cross breeding, which can be assisted by genomics. Peri-urban and home garden
“farmers” will have to adapt to new demands from emerging urban populations with higher income. These
consumers may request a more varied diet. For example, food crops with low fats, and high in specific
amino acids may be needed to satisfy people who wish to change their eating habits. If genes controlling
these characteristics do not exist in a specific crop pool they may be incorporated into the breeding pool
using transgenics. Some publications anticipated that in the next millennium food will not need to be
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harvested from farmer’s fields (Anderson 1996b). Tissue culture of certain parts of the plant may provide a
means to achieve success in this endeavour. For example, edible portions of fruit crops could be grown in
vitro. A steady and cheap supply of these edible plant parts will be required in this new agri-business. It will
take some time before such a process can be scaledup for commercial output. Nonetheless, a patent was
submitted in 1991 by a Californian biotech company for producing a vanilla extract through cell culture. Of
course, this technique will not replace farming as we know it today.This biotechnique, as well as other new
farming methods, offers a means for new ways of producing food, feed or fibre.

Often plants provide the raw materials for agro-industry, and not only for food or fibre processing. Active
ingredients of plants have been transformed into commercial products such as medicines, solvents, dyes, and
non-cooking oils for many years. Hence, it would not be surprising to see, in few years from now, entire
farms without food crops but growing transgenic plants to produce new products, e.g. edible plastic from
peas or plant oils to manufacture hydraulic fluids and nylon (Grace 1997). This new rural activity may result
in important changes in the national economic sector. ‘Pharming’ has been added to the dictionary to
indicate a new kind of system to obtain medicines (Anderson 1996b). For example, oral vaccines appear to
be a convenient delivery system for vaccination throughout the world. Biotechnology has been used to
engineer plants that contain a gene derived from a human pathogen (Tacker et al. 1998).

An antigenic protein encoded by this foreign DNA can accumulate in the resultant plant tissues. Results
from preclinical trials showed that antigenic proteins harvested from transgenic plants were able to keep the
immunogenic properties if purified. These antigenic proteins caused the production of specific antibodies in
injected mice. Mice, which ate these transgenic plant tissues, also showed also a mucosal immune response.
Arakawa et al. (1998) recently demonstrated the ability of transgenic food crops to induce protective
immunity in mice against a bacterial enterotoxin such as cholera toxin B subunit pentamer with affinity for
GMI-ganglioside. Also, potato tubers have been used successfully as a biofactory for high-level output of a
recombinant single chain antibody (Artsaenko et al. 1998).

Within the next 10 or 20 years, five research areas may become very important for crop improvement: (i)
apomixes to fix hybrid vigour, (ii) male sterility systems with transgenics for hybrid seed in self-pollinating
crops, (iii) parthenocarpy for seedless vegetables and fruit trees, (iv) short-cycling for rapid improvement of
forest and fruit trees, and (v) converting annual into perennial crops for sustainable agricultural systems. The
development of perennial crops will be especially important to protect the soil from erosion. Plant
biotechnology will play, of course, an important role in achieving research and development success in these
areas. Banning transgenic crops in the farming system will be foolish because the potential benefits are so
great.

Environmentalists should recall or re-read ‘Silent Spring’ by Rachel Carlson (1962). Whatever scientists do
to develop crops that eliminate or reduce the utilization of polluting agro-chemicals in the farming systems
must be welcome by farmers and consumers. For example, one interesting approach for developing resistant
transgenic crops may be through the improvement of the plant’s own defence system. Inducible and tissue
specific promoters could assist in this endeavour.

The general public should see biotechnology as a safe tool for scientific crop improvement, because it helps
in the fight against hunger and poverty. Therefore, research funding should be allocated accordingly to long-
term plant breeding programmes, which include biotechnology as one of its tools. In this way, we may
effectively face the serious challenge of feeding the rapidly growing world population in the next
millennium.
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PLANT TISSUE CULTURE

Introduction and Definition

Agriculture today is on the verge of a technological revolution, in a manner it has never been seen before.
As we enter the new millennium, one key development that comes to our mind is the emergence of
biotechnology, which offers some of the best opportunities and solutions to some of the uncontrollable
problems faced by us. Biotechnology is a group of technologies that share two things in common; they
manipulate living cells and their molecules and have a wide range of uses that can improve our lives. The
major techniques of biotechnology are genetic engineering, cell culture, tissue culture, bioprocessing,
protein engineering etc. Plant Tissue Culture, Cell Culture or Micropropagation is the technique of
producing selected plants of known desirable agriculture qualities, in large numbers of plants from small
pieces of plant in relatively short periods of time. It is a method of rapid propagation under controlled
disease free conditions. Entire crop population with premium qualities can be created from a single elite
specimen plant. Depending on the species in question, the original tissue piece may be taken from shoot tip,
leaf, lateral bud, stem or root tissue of the mother plant. Ex-plants from selected mother plant are established
and multiplied under 'In-Vitro' conditions, providing the optimum pre-requisite for plant growth. These ex-
plants go through the initiation, multiplication and rooting methods for producing a cell into a full-fledged
plant. These ready plantlets are then hardened in climatically controlled green houses or poly houses.
Depending on the species, the plants become ready for plantation in the field. This technique of plant
propagation greatly reduces the labour and spacerequirement, for producing new varieties and can also
markedly enhance propagation rates. Mettiods of plant propagation and establishment are of particular
interest to our country and work on a wide range of vegetables, fruit crops and trees are in progress. Several
scientists have been experimenting to extend the application of tissue culture to make plant species
commercially important. For example experiments on crops like coconut, date palm. Cashew, Mango,
Orange etc. are being made in the different research laboratories. With the advent of plant tissue technology,
it is now possible to propagate fine varieties of flowers, forest, and fruit trees by tiny plantlets.
Commercialization of these crops has already taken place. In Ornamental crops, Orchids, Carnation,
Gladiolus, Gerbera, Anthurium etc. has been commercially grown. In Fruit crops, Banana, Sugarcane etc.
has been commercially grown. In Forest trees. Teak, Eucalyptus etc. has been commercially grown.
Medicinal plants are also being experimented by tissue culture method and soon would be ready for
commercial plantation. Biotechnology is an area with a tremendous potential in solving basic problems of
food, fiber, fuel and medicine particularly in developing Asian countries.

Advantages of Plant Tissue Culture are as follows

• Mass multiplication of elite clones: Micropropagation allows the production of large numbers of plants
from small pieces of the mother plant. The production requires relatively short periods of time to grow
plants. Depending on the species under production, a single ex-plant can be multiplied into several thousand
plants in less than one year.

• Elimination of diseases in planting material: Another purpose for which plant tissue culture is uniquely
suited is in the obtaining, maintaining, and mass propagating of specific disease-free plants. The concept
behind indexing plants free of pests is closely allied to the concept of using tissue culture as a selection
system. Plant tissues known to be free of the disease under consideration (viral, bacterial or fungal) are
physically selected as the explants for tissue culture. Tissue culture could be a useful way of circumventing
or eliminating disease, which can accrue in stock plants.
 Page 13 of 94

• Plant improvement through tissue culture: Creation of superior varieties of agricultural crops is possible
through tissue culture method, which otherwise is not possible through conventional plant breeding
methods.

• True to Type production: Large number of true to the type plants could be propagated within a short time
and space and that too throughout the year. For example, it may be possible to propagate Two to Four lakhs
of tissue cultured plants from a single bush or rose against 10 to 15 plants by conventional means. Also, it
may take about Two to Four months to produce a healthy planting material by tissue culture means, whereas
a minimum of Six to Eight months is required for most species by the latest method of plant propagation.

• Higher Yields: Tissue Culture Plants may have increased branching and flowering, greater vigour and
higher yield, mainly due to possibility of elimination of diseases.

• Beneficial when conventional propagation is difficult: The method may succeed to propagate plants
where seeds or conventional propagation is not possible or difficuk or undesirable.

• Efficient method in saving space and energy: The method saves space and energy of the farmer. For
example, in a conventional method the plants are grown in the open farm requiring an area of about 25,000m
same number of plants would require only 10 m^ space, if they are grown in the tissue culture laboratory.

• Flexible method: The flexibility of nurseries can be improved. As the capital investment on mother plant
is reduced to almost zero, it may be easier to adapt to changing conditions. Additionally, a better programme
of production is possible, because of the greater plant uniformity and the availability in the mass at any time.

• Innovation of new varieties: Tissue culture can be utilized for breeding new varieties.

Objective of Tissue Culture Project

The primary objective of tissue culture project could be propagation of large quantity of good quality
planting material from elite mother plants within short time, space with minimum cost per plant.

Conceptual Foundation of Plant Tissue Culture Technology

Plant tissue culture refers to the cultivation "In-Vitro" (Vitro-Glass) of all plant parts, whether a single cell, a
tissue or organ, under disease-free conditions on nutrient medium. In the life cycle of any organism, two
gamates of opposite sex fuse to form a single cell-zygote. From this single-celled zygote originates the entire
multicellular and multiorganed body of a higher organism. In a flowering plant, for example, structures as
functionally diverse as underground roots, green leaves and flowers all arise from the single-celled zygote
through millions of divisions of cells.

Theoretically, therefore, all the cells in a plant body, whether residing in the flowers, conducting tissues or
root tips, should have received the same genetical material as originally present in the zygote. There must be
some other factors superimposed on the genetic characteristics of cells, which bring about this vast variation
expressed by the genetically identical cells. The process of variations is called differentiation. This
differentiation is actually preceded by certain cellular and sub cellular changes. A question that arises at this
stage is whether the cellular changes underlying differentiation of various types of cells are permanent and
irreversible or whether there is merely an adaptive change to suit the functional need of organism in general.
During the normal life cycle of a plant, it is believed, that the events leading to differentiations are of
permanent nature. However, the experiments of Vochting on polarity in cuttings (1878) suggested otherwise.
He had observed that all cells along the length are capable of forming roots as well as shoots but their
density is decided by their relative positioning of the cutting. The best way to answer this question and
 Page 14 of 94

understand more about the interrelationship between different cells of an organ and different organs of an
organism would however be to remove them from the influence of their neighbouring cells and tissue and
grow them in isolation on nutrient media. This has led to the foundation of a new branch of biology as 'Cell
and Tissue Culture'. It is applicable to both plant and animal cells. Plant tissue culture has acquired many
practical applications in agriculture, horticulture and forestry. It is increasingly becoming popular as a part
of recent field of Biotechnology

History of Plant Tissue Culture

The German Botanist Guttlieb Haberlandt first proposed the importance of plant tissue and cell culture in
isolation, in 1902. He is regarded as the father of plant tissue culture. He used tissue of Lamium Puroureum
and Eichhornia crassipes, the epidemis of Ornithooalium and epidermal hairs of Pulmonaria Mollissima. He
grew them on a particular salt solution with sucrose and observed obvious growth in the cells. The cells
remained alive for up to 1 month. They grew in size, changed shape; thickening of cell walls occurred and
starch appeared in the chloroplasts, which initially lacked it. However, none of the cells divided. The failure
was that he was handling highly differentiated cells and the present day growth hormones, necessary for
inducing division in mature cells, were not available to him. Hanning (1940) had initiated a new line of
investigation, which later developed into an important applied area of in-vitro techniques. Hanning excised
nearly mature embryos of some plants like Raphanus Sativus and successfully grew them to maturity on
mineral salts and sugar solution. Van Overbeck (1941) and co-workers demonstrated for the first time the
stimulatory effect of coconut milk, which was similar to embryo sac fluid, on embryo development and
callus formation in Datura. This proved a turning point in the field of embryo culture, for it enabled the
culture of young embryos which failed to grow on a mixture of mineral salts, vitamins, amino acids and
sugar. Subsequent detailed work by Raghavan and Torrey (1963), Norstog (1965) and others led to the
development of Synthetic media for the culture of younger embryos. Laibach (1925, 1929) demonstrated the
practical applicafion of embryo culture in the field of plant breeding. He isolated embryos from nonviable
seeds of a particular plant and reared them to maturity on a nutrient medium. In 1922, working
independently Robbins (USA) and kotte (Germany) reported some success with growing isolated root tips.
White made the first successful report of continuously growing tomato root tips in 1934. During 1939 - 1950
extensive work on root culture was undertaken by Street to understand the role of vitamins implant growth
and shoot-root relationship. Gautheret (1934), White (1939) and Nobecourt successfully cultured cells of
Salix, Nicotiana-Hybrid and carrot on synthetic media. They, for the first time, demonstrated that growth
regulators and vitamins if added to media enhanced the growth forming mess of cells called callus. Skoog
(1944), Tsui (1951), and Miller (1955) demonstrated the induction of divisions in isolated, mature and
differentiated cells by using synthetic as well as natural compounds. Muir (1953) developed a technique of
growing single cells into liquid medium in case of Tenetes Erecta and Nicotiana Tabacum. Vasil and
Hildeprandt (1965) raised whole plants starting from single cells of tobacco. Skoog and Miller (1957)
showed that changing the relative concentrations of the two substances in the medium could regulate the
organ differentiation. The first reports of some embryo formation from Carrot tissue appeared in 1958-59 by
Reinert (Germany) and Steward (USA). Ball (1946) successfully raised whole plants of Lupinus and
Tropaeolum by culturing shoot tips. Morel and Martin (1952), for the first time, recovered virus-free Dahlia
plants from infected individuals by culturing their shoots.Murashige (USA) used this technique to muUiply
plants in large number for several species ranging from ferns to foliage, flower and fruit plants. Guha and
Maheshwari (1966) demonstrated the possibility of raising large numbers of plantlets from pollen grains of
Dhatura. In 1972 Carlson and others produced the first somatic hybrid between two plants by fiising their
protoplasts.
 Page 15 of 94

Applications of Plant Tissue Culture

In the early fifties it was observed that plant cells are amenable to chemical manipulations in the medium
whereby they can be induced to form organized structures and complete plants. This discovery is considered
to be very important for the application of cell and tissue culture methods to overcome several problems
connected with agriculture, horticulture and plant breeding.

1) The technique provides a way for rapid multiplication of desirable and rare plants. 20,000 plants/year/bud
in turmeric, 1,00,000 plants/year/bud in Eucalyptus were found.

2) As the experiments reveal, virus infected plants also contain some healthy stocks as such they can be
obtained by separating shoot tips for their in-vitro propagation. This has given successfiil results in
Strawberries and Sugarcane.

3) The development of haploids through the technique of anther culture has a potential significance in basic
and applied genetics and plant breeding. During the past 20 years the technique has been successfully
extended to about 20 plant species including some economic plants.

4) The embryo culture has been useful in overcoming seed dormancy. It is also utilized for producing viable
plants from crosses, which normally fail due to the death of immature embryos. Experiments were
successftil in case of Jute and Rice.

5) The embryo tissue culture is also applied for the propagation of rare plants.

In some experiments, coconuts developed soft, solid and fatty tissue in place of the liquid endosperm
(Mohan Ram, 1976). These are rare and very expensive, served only at special banquets in Philippines.
Under normal conditions the coconut seeds fail to germinate. Using the technique of in-vitro culture of
excised embryos De Guzman (1969) succeeded in making plantlets from makapuno nuts.

6) Another important use of embryo culture is found in obtaining some rare hybrids. It is possible to raise
complete hybrid plants through embryo culture. This method has been profitably used for many interspecific
crosses of crops like Tomato, Papaya and Cotton.

7) It is possible to isolate and culture single cells of plants. This helps in mutantselection in relation to crop
improvement, as done in Tobacco, Datura etc. The technique is also useful in the production of some
chemical substances in the industry. In some cases cell cultures contained twenty times more chemical
content than the roots.

8) Recently tissue culture is used in protoplast culture of different varieties of plants and these protoplasts
are used for somatic hybridization.

9) A few high performance crop varieties have been widely adopted, resulting in the disappearance of a
large number of older varieties. The forests, which house the wild races of most of the crops, are being cut
on large scale. Henc tissue culture can be used to preserve germplasm i.e. tissue conservation of these plants
can be done identically.
Page 16 of 94
 Page 17 of 94

Important Steps in Plant Tissue Culture

1. Preparation of medium and sterilization in Autoclave: In

the commercial laboratory, the medium is prepared for initiation,
subculture and rooting purposes. The medium comprises of the
nutrients such as Micro Nutrients, Macro Nutrients, Vitamins,
Irons and growth regulators required for the growth of the plant.
The medium is sterilized in the electrically operated Autoclave at
Fifteen pounds temperature. This sterilization is important to
avoid the bacterial contamination. This medium is then poured
into the culture bottles and these bottles are again sterilized in the
autoclave. Then the medium is cooled and is ready for
inoculation.

2. Selection of a mother plant and sterilization: The commercial laboratory decides the plant species,
which are to be multiplied in the laboratory. Accordingly, the mother plants are selected from the virus free
areas. Healthy, disease and virus free plants are selected as mother plants. The actual plant part, which is
called as ex-plant is selected for inoculation. This plant part is washed with water, liquid soap and antiseptic
solution. This ex-plant is washed again with the chemical solution to avoid any fungal contamination
coming from the field environment. To remove the remnants of the chemicals it is thoroughly washed by
distilled water.

3. Inoculation of explants on sterilized medium of known composition:

Inside the laminar flow station, the ex-plant is

treated again with a disinfectant- Chemical called
Sodium Hypochloride. After this treatment the ex-
plant is washed thrice with double distilled water to
remove the chemical remnants. The explants are
inoculated on the sterilized medium. The
inoculation takes place in the Inoculation room, on
the Laminar Air Flow station, which maintains a
continuous air current, which keeps this bench
without the risk of contamination. The expert
technicians in the laboratory do the inoculation.

4. Shifting of cultures to the growth room:

Inoculated cultures are then shifted to the growth room where a typical temperature of Fifteen to Twenty
degree centigrade with the help of air conditioners and Class 1000 clean air is maintained. The artificial
lighting arrangement is also made for the growth of the plant. (Figure 3)

5. Shifting of cultures for Subculture: After three to six weeks from inoculation, the inoculated ex-plant
shows growth in multiple shoots. These shoots are transplanted on the subculture or multiplication medium
for ftirther growth of the plants. For subculture, separate growth medium is prepared. Several subculture
cycles are done in the laboratory for mass production from the ex-plant. (Figure 2)

6. Separation of in-vitro shoots and rooting: The shoots are separated in the laminar airflow stations and
these fully grown shoots are transferred to the rooting medium for root generation. Depending upon the
species, rooting requires One to Three weeks. (Figure 2)
 Page 18 of 94

7. Transfer to green house for hardening: Rooted shoots are removed from the laboratory and are
transferred to the green house for hardening. In the hardening procedure the plants are first kept in the
humidity chambers for acclimatization and then are transferred to the green house. (Figure 4).

Commercialization of Plant Tissue Culture technique

In Western Europe, commercial micro-propagation started

as late as early 1980's and there were total two hundred
and forty commercial plant tissue culture laboratories
producing millions of plants per year by the end of 1988.
Even Holland started commercial micro-propagation in
1980's and by the end of 1988 Holland was producing
sixty two million plants with the help of sixtyseven tissue
culture laboratories. Even Israel had five commercial
tissue culture laboratories producing five million plants
per year by the end of 1988.4 Also in other countries
many commercial tissue culture laboratories were set up
and produced millions of plants for their country. These
countries included, Poland, Yugoslavia, and Soviet Union
etc. In America commercial micro-propagation started in
1965 and there were about hundred commercial tissue
culture laboratories then.

Indian Scenario

In 1980's while all these countries were producing millions of plants, India had only four commercial tissue
culture laboratories. Eventually the laboratories
increased, but they were unable to produce the quantity
that agriculture and horticulture market needed. Many
commercial plant tissue culture laboratories
commenced operations in 1990's. Currently the plant
tissue culture is well studies, experimented and
Biotechnologies for Agriculture and Aqua Culture.
Chapter 27-Present capabilities in Commercial Tissue
Culture and the Potential for Growth. Dr. Jitendra
Prakash 195-199 accepted in India. India has achieved a
milestone in this technique by conducting research and
development with well-equipped research laboratories
like; Indian Council for Agriculture Research, Delhi;
Indian Institute of Horticulture Research, Bangalore
and National Chemical Laboratory, Pune. Now the need
is to make this technique strong in the commercial area
of production. Despite of the support of the national level Research Institutes, Agricultural Universities and
Government Agriculture Department, commercial tissue culture is still facing multifarious problems.
 Page 19 of 94

Assistance for Plant Tissue Culture business from different sources

It was found that different type of financial and technical assistance was available for commercial plant
tissue culture laboratories. The commercial laboratories did not identify these sources. Following are the
different assistance schemes available for commercial tissue culture laboratories.

A. Assistance from Government Agencies:

Various Central and State Government agencies have been trying hard to boost agriculture business and
agriculture processing sector in the State. The lacking factor is, a dialogue between the farmers, agriculture
industry and the Government development agencies. To fill up this gap. Government departments have
come up with different schemes. Most of the commercial laboratories were not aware about these schemes.
Commercial laboratories should study these schemes and make use of them in making their business cost-
effective.

Schemes implemented by Central and State Government for promotion of Agri Business and Agro
Processing Sector:

1. Name of the scheme: To establish Tissue Culture Laboratory in private sector.

Scheme implementing agency: Central Government

Nature of Assistance: Assistance is available for one unit per year for private sector for establishing tissue
culture laboratory. Subsidy available is Rupees Ten Lakhs.

2. Name of the scheme: Assistance for establishing Green Houses.

Scheme implementing agency: Central Government

Nature of Assistance: Under this scheme for establishing Green Houses for high tech agriculture following
assistance is available.

a) G.H.I: Green house frame and U.V. Film would be subsidized upto Fifty percent with a limit of rupees
Thirty-one thousand two hundred and fifty only for one green house.

b) G.H.2: For partially controlled green houses using Fan and Pad, subsidy at the rate of forty percent with
the limit of rupees One lakh is available. Both the schemes of green house are provided with the assistance
only for five hundred sq.mtr. of area for each beneficiary.

3. Name of the scheme: Assistance for Drip Irrigation for High Value Crops.

Scheme implementing agency: Agriculture Department, Government of Maharashtra

Nature of Assistance: Under Centrally sponsored scheme assistance is provided for drip irrigation system
for fruits, flowers and vegetables. The rate of subsidy is ninety percent for B.C., S.T., small and marginal
farmers and women. For other farmers the rate of subsidy is seventy percent. Subsidy is available for an area
upto limits prescribed under Agricultural Land Ceiling Act. The rate of subsidy is rupees twenty five
thousand per hector as the maximum limit.

4. Name of the scheme: Property tax on Green House

Scheme implementing agency: Government of Maharashtra

 Page 20 of 94

Nature of Assistance: Green houses and Poly houses build for high tech cultivators of Vegetables, Flowers
and Nursery plants will not be charged property tax by village panchayats.

5. Name of the scheme: To provide electricity at a concessional rate for high tech. agriculture.

Scheme implementing agency: Maharashtra State Electricity Board.

Nature of Assistance: Electricity rate for high tech agriculture like tissue culture, green houses in Private
and Public sector will have concessional rate as follows:

a) Rupees two and twenty-five paise per unit for high tech agriculture, which would include tissue culture
and mushroom cultivation. To avail the benefit from Maharashtra State Electricity Board for a concession in
electricity charges, the laboratory should be located out side the Industrial area.

B. Assistance from Micro propagation technology parks:

In order to promote tissue culture activities, the Department of Biotechnology, Ministry of Science and
Technology, Government of India has developed Micropropagation Technology Parks. At present there are
two Micropropagation Technology Parks in existence. One is located in Maharashtra at the National
Chemical Laboratory, Pune and the other is at Tata

Energy Research Institute, New Delhi. The technology parks are developed to provide an effective platform
for transfer of proven technologies to the entrepreneurs in the field of commercial plant tissue culture. The
technology parks also act as an interface between the Research Institutes and the tissue culture business for
accelerating commercialization of the tissue culture technology.

Micropropagation technology parks offer following services to the tissue culture business:

1. Technology transfer: Transfer of proven technologies to the users, training, implementation and
adoption of the technology at the client's site.

2. Contract research: Development of process for the newer crops, refinement of existing protocol.

3. Technical assistance for production of plants: An indigenously designed highly sophisticated

laboratory and green house exists to produce plants on large scale. A strong group of scientists and highly
experienced staff are available with the unit and give technical assistance for production of plants for
plantation programs or field trials.

4. Training of the personnel: Training programs are organized on general training for plant tissue culture
and specialized training on specific plant of interest.

5. Setting up and commissioning of tissue culture laboratory and green

house: Designing of the laboratory and green house, equipping the laboratory and the green house.

6. Advisory consultancy for overall running of the laboratory:

Consultancy provided for running a tissue culture unit, which includes all the aspects, right from washing of
glassware till production and hardening of plants in the green house.

7. Turn-key project based on the client's need: All the above services can be taken singly or in
combinations as per the needs. Turn-key project is also offered which includes all the above services
together and any other related aspects or problems.
 Page 21 of 94

C. Assistance from Financial Institutes:

Financial assistance is available to tissue culture business houses from the State Bank of India, Bank of
Baroda, Central Bank of India, Dena Bank and NABARD. The terms and conditions of assistance differ
from institution to institution.

D. Assistance for Marketing:

National Horticulture Board, Ministry of Agriculture, Government of India was set up in 1984 for integrated
development of horticulture in the country. National horticulture board encourages and promotes the
development of horticulture industry in the country. The board helps in increasing production and marketing
of horticulture produce.

National Horticulture Board's developmental program in marketing:

Development of marketing of horticulture produces through participation of Soft Loan.

Objective: To increase flow fresh and processed horticultural produce to targeted domestic and external
markets.

Commodities: Fruits, vegetables of commercial importance

Assistance: The loan support will be made upto forty percent of the loan portion upto a limit of rupees
hundred lakhs per project @ four percent surcharge per annum, repayable in five installments after a
moratorium of three to five years.

D.Assistance from Biotech Consortium India Limited:

Biotech Consortium India Limited was set up with the objective of providing the linkages to facilitate
accelerated commercialization of biotechnology. This organization was incorporated as a public limited
company in 1990 under the Indian Companies Act 1956. It is promoted by the Department of
Biotechnology, Government of India. Biotech Consortium India Limited has been engaged in technology
development, technology transfer, project consultancy, fund syndication, information dissemination,
manpower training and placement related to biotechnology. It has assisted over One hundred and twenty
clients including scientists, technologists, research institutions, universities, first generation entrepreneurs,
the corporate sector, government, banks and financial institutions.

Importance of tissue culture

In a relatively short time and space a large number of plantlets can be produced starting from the single
explant.

Taking an explant does not usually destroy the mother plant, so rare and endangered plants can be cloned
safely.

It is easy to select desirable traits directly from the culture setup (in vitro) thereby decreasing the amount
of space required, for field trials.

Once established, a plant tissue culture line can give a continuous supply of young plants throughout the
year.
 Page 22 of 94

The time required is much shortened, no need to wait for the whole life cycle of seed development. For
species that have long generation time, low level of seed production, or seeds that readily do not germinate,
rapid propagation is possible.

In vitro growing plants usually free, from the bacterial and fungal diseases. Virus eradication and
maintenance of plants in virus free state. This facilitates movement of plant across international boundaries.

Plant tissue banks can be frozen and then regenerated through tissue culture. It preserves the pollen and
cell collections from which plants may be propagated.

Advantages

In a relatively short time and space a large number of plantlets can be produced starting from the single
explants.

In the living plant the behavior of each part of tissue is strongly influenced by correlative controls
imposed by the rest of the plant by isolating it in vitro, the nature of some of these correlative controls can
be determined.

The production of exact copies of plants that produce particularly good flowers, fruits, or have other
desirable traits.

To quickly produce mature plants.

The production of multiples of plants in the absence of seeds or necessary pollinators to produce seeds.

The regeneration of whole plants from plant cells that have been genetically modified.

The production of plants in sterile containers that allows them to be moved with greatly reduced chances
of transmitting diseases, pests, and pathogens.

The production of plants from seeds that otherwise have very low chances of germinating and growing,
i.e.: orchids and nepenthes.

Applications

Micro propagation is widely used in forestry and in floriculture. Micro propagation can also be used to
conserve rare or endangered plant species.

A plant breeder may use tissue culture to screen cells rather than plants for advantageous characters, e.g.
herbicide resistance/tolerance.

Large-scale growth of plant cells in liquid culture inside bioreactors as a source of secondary products,
like recombinant proteins used as biopharmaceuticals.

To cross distantly related species by protoplast fusion and regeneration of the novel hybrid.

To cross-pollinate distantly related species and then tissue culture the resulting embryo this would
otherwise normally die (Embryo Rescue). For production of doubled monoploid plants from haploid cultures
to achieve homozygous lines more rapidly in breeding programs, usually by treatment with colchicines
which causes doubling of the chromosome number.
 Page 23 of 94

Conclusion

Propagation by tissue culture offers good commercial prospects in ornamental plants, vegetables and fruit
plants, where value of the product is high. In India the tissue culture technique has reportedly been
successful in more than a hundred species of plants. It has been estimated that in India, more than Three
hundred and fifty million tissue cultured plants are being produced annually through tissue culture method.
Plant Tissue Culture has come to stay as a tool in plant biology. Plant Tissue Culture has the potential to
resolve the problems of experimental biology, which otherwise through conventional methods is difficult to
tackle. In the near future this technique will play a very prominent role in genetic engineering, breeding and
afforestation programs. Tissue culture technique is a boon for the agriculture and the horticulture industry
because of its numerous advantages. Tissue culture can produce several number of healthy, virus free and
true-totype plants. The advantage of this technique is such that these plants can be planted anytime during
the year, which solves farmers' seasonal plantation problems. Disease-free plants, multiplied through tissue
culture, produce higher yields than infected ones. This biotechnology application can be used for both,
traditional and new varieties. Carefully monitored production would ensure clean plantlets for distribution.
Tissue culture activity is taking a shape of an industry as many farmers are planting tissue culture grown
plantlets; agro-traders are buying and selling tissue culture grown plantlets while some are exporting either
the plantlets or the produces of plants grown by tissue culture, especially varieties of flowers like roses,
anthuriums and gerberas are enjoying high profits. Tissue cukure is the greatest advancement in plant
breeding. Agriculture and horticulture industry should take full advantage of this technique in reaching the
greatest heights at national and international levels. In India, tissue culture is rapidly becoming a commercial
method for propagating new and rare species and difficult-to-propagate plants. From a few research
laboratories several years ago, a whole new industry is emerging. Currently, the demand for micro
propagated plants is greater than the supply with some plants. Some growers specialize in only the micro
propagation of plantlets, leaving the growing-on i.e. hardening activity to others. Many growers are
integrating a tissue culture laboratory into their overall operation. While many plant tissue culture
laboratories are coming up, some of the laboratories are being closed down due to various reasons and many
of the existing laboratories are found to be complaining about the problems they are facing. Commercial use
of plant tissue culture technique has vast business potential if tissue culture laboratories are freed of their
constraints, particularly financial and marketing. They are the businesses that would enable India retain its
self-sufficiency on agricultural production front. Finance being the heart of any business, finances will have
to be controlled and regulated systemically in tissue culture business as well. A thorough study of the
financial viability and commercial prospects of the tissue culture venture should be carried out by the new
entrant. It is the need of the time with the backdrop of an era of globalization and the emergence of World
Trade Organization.

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 Page 24 of 94

MICROPROPAGATION

Micropropagation of Plants

Micropropagation is one of the most popular techniques of tissue culture. It is the practice of rapidly
multiplying stock plant material to produce a large number of progeny plants, using modern plant tissue
culture methods. Micro propagation is used to multiply novel plants, such as those that have been genetically
modified or breed through conventional plant breeding methods. It is also used to provide a sufficient
number of plantlets for planting from a stock plant which does not produce seeds, or does not respond well
to vegetative reproduction. Generally interest in the use of this technique for clonal propagation of crop
plants originated from the success in this area with orchids, the credit for which goes to French botanist G.
Morel (1960). During last three decades progress in this field has been such that multiplication of many
ornamental and fruit cultivars is being practiced on commercial feasible method of clonal propagation.
Micropropagation can be defined as a technique in which any vegetative (meristmatic) part of plant such as
shoot tip, shoot bud etc is excised aseptically and cultured on sterile media under controlled conditions to
give rise to plantlet which is exact copy of its donor plant. In Simple words, it can be defined as clonal
propagation in vitro. The word ‘clone’ was first used by Webber for cultivated plants that were propagated
vegetatively. The word derived from Greek (clone= twig, spray or a slip ,like those broken off as propagules
for multiplication).It signifies that plants grown from such vegetative parts are not individuals in the
ordinary sense, but are simply transplanted parts of the same individual and such plants are identical. Thus,
Clonal propagation is the multiplication of genetically identical individuals by asexual reproduction. Plant
regeneration can be achieved by culturing tissue sections either lacking a preformed meristem (adventitious
origin) e.g. Axillary Bud Proliferation approach or from Callus and Cell cultures (De Novo Origin). It is the
stimulation of Axillary buds, which are usually present in the axil of each leaf to develop into a shoot. This
technique exploits the normal ontogenic route for branch development by lateral (Axillary) meristem. In
nature these buds remain dormant for various periods depending upon the growth pattern of plant. In some
species, removal of terminal bud is essential to break the apical dominance and stimulate the Axillary bud to
grow into shoot. Due to continuous application of cytokine in cultured medium the shoot formed by the bud,
which is present on explants (nodal segment/shoot tip cutting) develops Axillary buds. The shoot is then
separated and rooted to produce plants or shoots are used as propagules for propagation. The merit of using
Axillary bud proliferation from meristem, shoot tip or bud culture as a mean of regeneration is that the
incipient shoot has already differentiated in vivo. Thus, only elongation and root differentiation are require
to establish a complete plant. Another, major advantage of this technique is that, it preserves the precise
arrangement of all layers necessary if a chimeral plant genetic section is to be maintained. In a typical
chimera, the surface layers of developed meristem are of differing genetic background and it is their
contribution in particular arrangement to the plant organ that produces the desisted characteristics. As long
as the integrity of the meristem remains intact and development is normal in vitro, then the chimeral pattern
will be preserved. If however, callus tissue were allowed to form and shoot proliferation subsequently was
from adventitious origin, then there would be a risk that the chimeral layers of original explants may not all
be represented in the specially require from in the adventitious shoots.

Micropropagation Methods

Among the various applications of plant tissue culture, micropropagation of plant species has attained the
status of large plant based study. The development in the study of various aspects of plant growth and
differentiation were rapid during 1960s and 70s. The technique of culturing plants becomes a wide subject
embracing morphology, physiology, biochemistry, molecular biology and genetic engineering.
 Page 25 of 94

1. Somatic Embryogenesis

2. Axillary Bud

3. Adventitious Budding

General Technique of Micropropagation

The process of plant micropropagation aims to produce clones (true copies of a plant in large numbers). The
process is usually divided into the following stages:

Stage 0: Pre-propagation Stage

The pre-propagation stage requires proper maintenance of the mother plants in the greenhouse under disease
and insect free conditions with minimal dust. Clean enclosed areas, glasshouses, plastic tunnels and net
covered tunnels, provide high quality explant source plants with minimal infection. Collection of explants
for clonal propagation should be done after appropriate pre-treatment of the mother plants with fungicides
and pesticides to minimize contamination in the in vitro cultures. This improves growth and multiplication
rates of in vitro cultures. The control of contamination begins with the pretreatment of the donor plants.The
choice of explant depends on the methods of shoot multiplication to be followed. All plant organs viz. nodal
segment, inter-nodal segments, shoot tip, root tip. For axillary bud induction, callus culture, somatic
embryogenesis explants nodal segments, internodes and leaves are collected.

Stage 1: Initiation of Aseptic Culture:

In this stage sterilization of explants and establishment of explants were done. The plant organ used to
initiate a culture is called explant. The choice of explant depends on the method of shoot multiplication to be
followed.

For micropropagation work the explant of choice is nodes

For callus culture work the explant of choice is internodes and leaves.

For somatic embryogenesis the explant is internodes and leaves.

Stage 2: Multiplication of Culture:

This is the most important stage and the rate of multiplication determines the largely success of
micropropagation system this can be achieved by-Enhanced axillary branching

Adventitious bud formation

Through callusing

Enhanced axillary branching:

The axillary bud present in the axil of each leaf either develops into a single shoot or form a cluster of shoots
in the presence of cytokinins (BAP 1.0mg/l) in the medium.

Adventitious Bud Formation:

Buds arising from any part other than the leaf axils or shoot apex are called adventitious buds. It is a
standard horticulture practice.
 Page 26 of 94

Through Callusing:

Plant cells are totipotent. In tissue culture, the mass of differentiated cells commonly known as callus. This
either gives rise to shoot bud or bipolar structure resembling embryo (somatic embryo). This method is used
when aim is to induce variability especially in self-pollinating species with narrow genetic base.

Stage 3: In Vitro Rooting of Shoots

In-vitro grown shoots lack root system. For induction of roots they were transferred to rooting medium. For
rooting half strength MS medium supplemented with 1.0mg/l auxin was used.

Stage 4: Hardening and Acclimatization of Tissue Culture Plantlets

This is the final stage and requires careful handling of plants. The transplantation from completely
controlled conditions should be gradual. This process of gradually preparing the plants to survive in the field

conditions is called acclimatization. The plants produced in tissue culture, although green in color; do not
prepare sufficient food for their own survival. Also inside the culture vessels humidity is very high and thus
the natural protective covering of cuticle is not fully developed. Therefore immediately after transfer plants
were maintained under high humidity. Optimum conditions were provided to plants in green house.

Advantages of Micropropagation

Micropropagation has a number of advantages over traditional plant propagation techniques:

The main advantage of micropropagation is the production of many plants that are clones of each other.

Micropropagation can be used to produce disease-free plants.

Micropropagation produces rooted plantlets ready for growth, saving time for the grower when seeds or
cuttings are slow to establish or grow.

It can have an extraordinarily high frequency rate, producing thousands of propagules while conventional
techniques might only produce a fraction of this a number.

It is the only viable method of regenerating genetically modified cells or cells after protoplast fusion.

It is useful in multiplying plants which produce seeds in uneconomical amounts, or when plants are sterile
and do not produce viable seeds or when seed can't be stored.

Micropropagation often produces more robust plants, leading to accelerated growth compared to similar
plants produced by conventional methods - like seeds or cuttings.

Some plants with very small seeds, including most orchids, are most reliably grown from seed in sterile
culture.

A greater number of plants can be produced per square meter and the propagules can be stored longer and
in a smaller area.

Disadvantages of Micropropagation

Micropropagation is not always the perfect means of multiplying plants, conditions that limits its use
include:
 Page 27 of 94

It is very expensive, and can have a labour cost of more than 70%.

A monoculture is produced after micropropagation, leading to a lack of overall disease resilience, as all
progeny plants may be vulnerable to the same infections.

An infected plant sample can produce infected progeny. This is uncommon if the stock plants are
carefully screened and vetted to prevent culturing plants infected with virus or fungus. Not all plants can be
successfully tissue cultured, often because the proper medium for growth is not known or the plants produce
secondary metabolic chemicals that stunt or kill the explant.

Different tasks

Sometimes plants or cultivars do not come true to type after being tissue cultured; this is often dependent on
the type of explant material utilized during the initiation phase or the result of the age of the cell or
propagule line.

MICROPROPAGATION OF BAMBOO

Introduction to Explant Material

The bamboos are a group of woody perennial evergreen (except for certain temperate species) plants in the
true grass family Poaceae, subfamily Bambusoideae, tribe Bambuseae. Some are giant bamboos, the largest
members of the grass family. Bamboos are the fastest growing woody plants in the world. They are of
economic and high cultural significance in East Asia and South East Asia where they are used extensively in
gardens, as a building material, and as a food source.

Diversity: Around 92 genera and 5,000 species.

Mass flowering

Although some bamboos flower every year, most species flower infrequently. In fact, many bamboos only
flower at intervals as long as 60 or 120 years. These taxa exhibit mass flowering (or gregarious flowering),
with all plants in the population flowering simultaneously. The longest mass flowering interval known is
130 years, and is found for all the species Phyllostachys bambusoides (Sieb. & Zucc.). In this species, all
plants of the same stock flower at the same time, regardless of differences in geographic locations or
climatic conditions, then the bamboo dies.

Medicine

Bamboo is used in Chinese medicine for treating infections and healing. It is a low-calorie source of
potassium. It is known for its sweet taste and as a good source of nutrients and protein. In Ayurveda, the
Indian system of traditional medicine, the silicious concentration found in the culms of the bamboo stem is
called banslochan. It is known as tabashir or tawashir in Unani-Tibb the Indo-Persian system of medicine.
In English it is called "bamboo manna". This concretion is said to be a tonic for the respiratory diseases. It
was earlier obtained from Melocanna bambusoides and is very hard to get; it has been largely replaced by
synthetic silicic acid. In most Indian literature, Bambusa arundinacea is described as the source of bamboo
manna. The fiber of bamboo has been used to make paper in China since early times.

A high quality hand-made paper is still produced in small quantities. Coarse bamboo paper is still used to
make spirit money in many Chinese communities.
 Page 28 of 94

Classification

Dendrocalamus strictus

Vernacular Names- Bengal - Karali, Gujarat - Nakur bans; Kiri bidiru;

Maharashtra - Male bamboo, narvel; Orissa - Salia; Tamilnadu - Kalmungil;

Andhra - Sadanapa Veduru; Tripura - Lathi bans; Kerala – Kallumula

Classification of Bamboo

Kingdom: Plantae

Phylum: Magnoliophyta

Class: Liliopsida

Order: Poales

Genus: Dendrocalamus

Family: Poaceae(alt. Gramineae)

Subfamily: Bambusoideae

Tribes: Bambuseae.

Soort Dendrocalamus strictus (Roxb.) Nees

Common names:

Bambú-grande ,Bans ,Calcutta bamboo ,Male bamboo ,Solid bamboo

Economic importance:

Human food: Cereal; Vegetable

Materials: Cane; Fiber (used for rafters, baskets, sticks, etc.)

Clonal propagation of bamboo (Dendrocalamus strictus)

Bamboo grows naturally in many types of forests. About 50% of the annual production of bamboo in our
country is used by various industries like pulp, paper, rayon, mat boards, besides agricultural implements. It
is also used for making baskets, bridges, coffins, beds, toys and weapons. A grove of bamboo at ground zero
in the area that destroyed Hiroshima in 1945 sprouted new shoots within 1 month. The distribution of
bamboo in India is largely governed by rainfall, temperature, and altitude and soil types. In recent years
bamboos are in great demand; but no availability of sufficient quantity of saplings and seeds are the major
problem. Commonly called as solid or lathy bamboo. The species is widely distributed in dry deciduous
forests and grows rapidly in all climatic conditions. It grows better in the drier parts and on sandstone,
granite and coarse grained soils with low moisture-retaining capacity and soils with pH 5.5–7.6. It grows
more than 8 feet in 6 months. The pulp is used for making quality paper and the clumps, being strong and
elastic, are used for lathies,shafts, axe handles, walking sticks, agricultural, and industrial implements and
 Page 29 of 94

therefore it is rightly called as ‘poorman’s timber'. of D. strictus is the erratic flowering and nonavailability
of seeds on regular basis, besides low viability of seeds. Moreover the seeds have to be stored in 3 to 5°C
after reducing the moisture (8%) or stored in a desiccator with anhydrous Calcium chloride.

MATERIAL AND METHODS

Lab requirements

The present investigation was carried out at Arid Forest Research Institute, Jodhpur (Rajasthan). The
experimental material for the present investigation on Micropropagation of Dendrocalamus stritus

APPARATUS

1. pH Meter

2. Microwave oven

3. Electronic Weighing Balance

4. Refrigerator

5. Defreeze

6. Oven

7. Autoclave

8. Laminar Air Flow

9. Incubator

CHEMICALS

1. Ammonium Nitrate

2. Potassium Nitrate

3. Boric Acid

4. Potassium Di Hydrogen ortho Phosphate

5. Potassium Iodide

6. Sodium Molybdate di hydrate

7. Cobaltous Chloride

8. Calcium Chloride

9. Magnesium Sulphate

10.Manganese Sulphate

11.Zink Sulphate

12.Cupric Sulphate
 Page 30 of 94

13.Sodium Ethylene di amine tetra acetic acid

14.Ferrous Sulphate

15.Thiamine HCl

16.Nicotinic Acid

17.Pyridoxine

18.Glycine

19.Myoinositole

20.Sodium Hydroxide

21.Hydro Chloric Acid

22.Sucrose

23.Agar-Agar

24.Citric Acid

25.Glutamic Acid

26.Adenine Sulphate Di hydrate

27.Asparagine

28.Arginine

29.Mercuric Chloride

30.Ascorbic Acid

31.Sodium Hypochlorite

32.Banzyl Amino Purine(BAP)

33.Indole Acetic Acid(IAA)

34.α-Naphthalene Acetic Acid(NAA)

35.2,4-Di chlorophenoxyacetic acid(2,4-D)

Tissue culture media

Generally all culture media are made up of:

Macronutrients

Micronutrients

Vitamins

Growth regulations
 Page 31 of 94

Carbohydrates (Sucrose)

Formulation designed by Murashige and Skoog (1962), revised by Linsmair and Skoog (1965) can be
regarded as standard. Special plant groups like conifers have nutritional requirements, which appear, not to
meet by standard media, and then some additional nutrients are required in media.

MEDIA CONSTITUENTS

Inorganic Nutrients:-

Mineral elements are very important in the life of a plant.

Mg is a part of chlorophyll molecules.

Ca is a component of cell wall.

N is an essential part of amino acids, vitamins, proteins and nucleic acid.

Fe, Zn, and Mo are part of certain enzymes.

Besides C, H, and O there are 12 elements, known to be essential for plant growth viz. N, P, S, K, Ca,
Mg, Fe, Mn, Cu, Zn, B and Mo.

Macro elements

C- Carbon forms the backbone of many plants Bio-molecules, including starches and cellulose. It is fixed
through photosynthesis from the carbon synthesis in the air and is a part of the carbohydrates that store
energy in the plant.

H- Hydrogen also is necessary for building the plant and it is obtained almost entirely from water.

O- Oxygen is necessary for cellular respiration. Cellular respiration is the process of generating energy
rich adenosine tri phosphate (ATP) via the consumption of sugars made in photosynthesis. Plants produce
oxygen gas during photosynthesis to produce glucose but then require oxygen to undergo aerobic cellular
respiration and break down this glucose and produce ATP.

N- Nitrogen is an essential component of all proteins. Nitrogen deficiency most often results in stunted
growth.

P- Phosphorus is important in plant bioenergetics as a component of ATP. It is needed for the conversion
of light energy to chemical energy (ATP) during photosynthesis. Phosphorus can also be used to modify the
activity of various enzymes by phosphorylation and can be used for cell signaling. Since ATP can be used
for the biosynthesis of many plant bio molecules, it is important for plant growth and flower/seed formation.

K- Potassium regulates the opening and closing of the stomata by a potassium ion pump. Since stomata
are important in water regulation, potassium reduces water loss from the leaves and increases drought
tolerances. Potassium deficiency may cause necrosis or interveinal chlorosis.

Ca- Calcium regulates transport of other nutrients into the plant. It is also involved in the activation of
certain plant enzymes. Calcium deficiency results in stunting.

Mg- Magnesium is an important part of chlorophyll, a critical plant pigment important in photosynthesis.
It is important in the production of ATP through its role as an enzyme cofactor. There are many other
 Page 32 of 94

biological roles for magnesium in biological system for more information. Magnesium deficiency can result
in intervenial chlorosis.

S- Sulphur is a structural component of some amino acids and vitamins. It is essential in the
manufacturing of chloroplasts.

Microelements: These are essential as catalysts for many biochemical reactions; microelement deficiency
symptoms include Leaf chlorosis (Fe, Zn, and Mn) Shoot tip necrosis (B, Co, Ni) inhibits ethylene synthesis.

Fe- Iron is necessary for photosynthesis and is present as an enzyme cofactor in plants. Iron deficiency
can result in interveinal chlorosis and necrosis.

Zn- Zinc is required in a large number of enzymes and plays an essential role in DNA transcription. A
typical symptom of zinc deficiency is the stunted growth of leaves, commonly known as “little leaf” and is

caused by the oxidative degradation of the growth hormone auxin.

Mn- Manganese is necessary for building the chloroplasts. Manganese deficiency may result in coloration
abnormalities, such as discolored spots on the foliage.

B- Boron is important for binding of pectin in the RG II region of primary cell wall; secondary roles may
be in sugar transport, cell division and synthesizing certain enzymes. Boron deficiency causes necrosis in
young leaves and stunting.

Co- Cobalt has proved to be beneficial to at least some plants, but is essential in others, such as legumes
where it is required for nitrogen fixation.

Ni- In higher plants, Nickel is essential for activation of ureases, an enzyme involved with nitrogen
metabolism that is required to process urea. Without Nickel, toxic leaves of urea accumulate, leading to the
formation of necrotic lesions. In lower plants, Nickel activates several enzymes involved in a variety of
processes and can substitute for Zinc and iron as a cofactor in some enzymes.

Si- Silicon deposited in the cell walls and contributes to its mechanical properties including rigidity and
elasticity.

Na- Sodium involved in the regeneration of phosphoenolpyruvate in CAM and C4 plants. It is also
substitute for potassium in some circumstances.

V- Vanadium may be required by some plants, but at very low concentrations. It may also substitute for
Molybdenum.

Se- Selenium and Sodium may also be beneficial. Sodium can replace potassium’s regulation of stomatal
opening and closing.

Organic Nutrients :-

Vitamins: Plants can produce their requirements of vitamins. However, plant cell cultures need to be
supplemented with certain vitamins like Thiamine (vit B1), Niacin (vit B3), Pyridoxine (vit B6), and Myo-
inositol (Member of the vit. B complex).

Thiamine: Involved in the direct biosynthesis of certain amino acids and essential co-factor of
carbohydrates metabolism.
 Page 33 of 94

Vit E – Antioxidants.

Vit C- To prevent blacking during explant isolation.

Vit D- Growth regulatory effect

Amino Acids – Glycine- has little benefit in the growth of plant. They may be directly utilized by plant own
be provided as N2 source.

Carbon Sources- Sucrose (is most commonly used carbon source) at a concentration of 3%, glucose and
fructose also known to support plant growth. Sucrose in the medium is necessary for various metabolic
activities.

Growth Regulators :

Auxin – Auxin are involved in cell division and elongation and in cell wall synthesis. IAA, IBA, NAA, 2, 4-
D are the most frequently used auxin in plant tissue culture. The principal naturally occurring auxin, the IAA
is not often used in the tissue culture, because it is unstable. IBA is slightly more potent than IAA and is not
easily broken down. Hormones of this group are involved with elongation of stems and inter nodes, tropism,
apical dominance abscission, rooting etc.

Cytokinin –These hormones, are concerned with cell division, modification of apical dominance, shoot
differentiation etc. Most commonly used cytokinins are BAP, BA, Kinetin, 2 ip and Zeatin. They usually
promote cell division if added together with an auxin. Of these, BAP is the most effective cytokinins for
stimulating axillary shoot proliferation.

Gibberellins – There are over 20 known gibberellins. Of these, generally, GA3 is used. They are rarely used
and reported to stimulate normal development of plantlets from in vitro formed adventives embryos.

Others – Abscisic acid is most often required for normal growth and development of somatic embryos and
only in its presence they resemble zygotic embryos.

Gelling Agent :-

In static cultures if liquid medium is used the tissue would get submerged and die due to lack of oxygen. A
gelling agent is generally used to circumvent this problem. The most desirable property of a gelling agent is
that it should with stand sterilization by autoclaving and the medium should be liquid when hot but form a
semisolid gel when cool. Some important gelling agents are – Agar, Agarose ,Gelrite

Agar – This is obtained from red algae, especially Gelidium amansii. Complex mixture of related
polysaccharides built up from the sugar, galactose. These include the natural polymer fractions, agarose,
which gives strength to the gel and the highly charged anionic polysaccharides agaropectins which give agar
its viscosity. Agar is used at varying concentration from 0.8 to 1%.

Agarose- Is commonly preferred over agar for protoplast culture.

Gelrite- Is a good alternative to agar not only because of its lower cost per liter of medium (0.1-0.2% is
sufficient) but also for the many advantage it offers.
 Page 34 of 94

Preparation of the stocks solutions:-

It is difficult to weigh and mix all the constituents just before preparation of medium. It is time consuming
and a tedious job. Again if 100 ml medium is to be prepared, then it is very difficult to weigh some
constituents that are used in very small quantity for 1 liter medium. So it is convenient to prepare
concentrated stock solution of macro salts, micro salts, Vitamins, amino acids, hormones etc. and all stocks
solution should be stored in a refrigerator and should be checked visually for contamination with
microorganism or precipitation of ingredients.

Major types of media

1. White’s medium - is one of the earliest plant tissue culture media

2. MS medium - The most extensively used nutrient medium is MS medium

(developed by Murashige and Skoog in 1962)

3. B5 medium - developed by Gamborg for cell suspension and callus

culture and at present its modified form used for protoplast culture.

4. N6 medium - formulated by Chu and used for cereal anther culture.

5. Nitsch’s medium- developed by Nitsch and used for anther culture.

 Page 35 of 94

Sterilization

It is very important to maintain aseptic environment during the in vitro culture of plant cells and tissues.
Following are some of the methods adopted for sterilization:

(a) Sterilization of Glassware- The glassware can be

sterilized in a hot air oven at 160-1800C for 2-4 hours.
(b) Sterilization of instruments- The metallic instruments
are incinerated by dipping them in 75% ethanol follow
-ed by flaming and cooling.
(c) Sterilization of nutrient media- The culture media are
transferred into glass container, plugged with cotton or
sealed with plastic closures and sterilized by autoclaving
at 15 psi for 30 min. The autoclaving denatures the
vitamins, plant extracts, amino acids and hormones
therefore the solution of these compounds are sterilized by
using Millipore filter paper with pore size of 0.2 micrometer diameter. Surface sterilization of
juvenile material is generally not difficult. However, if older trees are used, as it is the initial and
basic material for tree breeders when selection is done contamination of explants is sometimes a
serious problem, unless the tree produce juvenile sprouts. In some cases spores are deposited on field
grown trees by insects. This contamination can be reduced by spraying these trees with insecticides
and fungicides and by subsequently protecting expending shoot against insects by enclosing them in
bags made of transparent film before collecting the explants. Surface of plant part carries a wide
range of microbial contaminants. To avoid this source of infection the tissue must be thoroughly
surface sterilized before planting it on the nutrient medium. To disinfect plant tissues various
sterilizing agents have been used. Hypochlorite solutions have proved too effective in most cases.
Ethyl and isopropyl alcohol have also been used to surface sterilize of plant tissues. Explants were
washed in distilled water to remove dust particles, then they were washed in detergent solution and
surface sterilized in 0.1% solution of HgCl2, NaCl for 5 minutes. To remove the sterilant nodal
segments were again washed with sterile distilled water.
 Page 36 of 94

(d) Sterilization method of Explants:-

Table: - Some of the agents used for surface sterilization of explants

 Page 37 of 94

Tissue culture media

To prepare the medium, many researchers mix the stock solutions which were made previously since the
medium compositions are generally complicated. For example, MS medium is prepared as follows:

The present studies pertained to the Micropropagation of Dendrocalamus strictus.This aspect deals with
following studies for the Micropropagation of the bamboos:

1. Selection of explants.

2. Standardization of sterilization technique.

3. Establishment of explant.

4. To get maximum rate of shoot proliferation.

SELECTION OF EXPLANT

Micropropagation is preferred because of genetic stability. For this experiment axillary bud and nodal
explants were collected from 2-3 years old clumps of Bamboo plant. Explants were collected from soft;
rapidly growing shoots provided the box material for initiation of culture.

STERILIZATION OF EXPLANT

It was observed that explants were very susceptible to HgCl2 and majority of them turned black and died.
Surface sterilization of nodal explants with 0.1% HgCl2 for 12-14 minutes gave up to 80% response, which
was to be best among all the treatments for surface of D. Strictus.It was observed that explants very
susceptible to time of HgCl2 if time increased more than or less than 12 min. then the survival of explants
effects.

AXILLARY BUD BREAK (SHOOT INDUCTION)

Bud break was achieved in 20-25 days in different media compositions. Maximum percentage of bud break
was achieved on MS medium supplemented with BAP (3 mg/l) in case of Dendrocalamus strictus. Multiple
shoots were separated and regular sub culturing was done on preestablished cultures on MS medium
supplemented 3.0 mg/l BAP.

Table: Effect of plant growth regulators (BAP) on number of shoot buds per explants formed in
Dendrocalamus strictus after one week.

------------------------------
 Page 38 of 94

Somaclonal variation
Introduction

Plant tissue culture techniques proffer a substitute method of vegetative propagation of horticultural crops
(Krishna et al. 2005; Alizadeh et al. 2010). Clonal propagation through tissue culture (popularly known as
micropropagation) can be
realized relatively rapidly
within a small space (Krishna
et al. 2008; Eftekhari et al.
2012). The uniformity of
individual plants within a clone
population is a major advantage
of clonal cultivars in
commercial production
(Krishna and Singh 2013).
However, genetic variations do
occur in undifferentiated cells,
isolated protoplasts, calli,
tissues and morphological traits
of in vitro raised plants (Bairu
et al. 2011; Currais et al. 2013).
In 1981, Larkin and Scowkraft
coined a general term
‘‘somaclonal variation’’ for
plant variants derived from any form of cell or tissue cultures.

At present, micropropagated plants, in various crops, such as strawberry, papaya, banana, grapes, pineapple,
citrus, tomato, cucumber, watermelon, rhododendron, orchids, etc., are preferred over plants propagated
through conventional means. However, ever since the first formal report of morphological variants in
sugarcane plants produced in vitro in 1971 (Heinze and Mee 1971), several instances of somaclonal
variations have been reported indifferent horticultural crops.

The notable example could be banana in which occurrence of off-types from tissue cultured plantlets ranged
from 6 to 38 % in Cavendish cultivars (Sahijram et al. 2003); however, it could be as high as 90 % (Smith
1988). From the point of commercial micropropagation, variation of any kind, in particular, genetic
variations may be considered obstructive and worthless; since, such variations may lead to loss of genetic
fidelity. However, plant cell and tissue cultures render increased genetic variability comparatively faster and
without applying a sophisticated technology.

This technology holds ample scope in crop improvement of horticultural crops, which are largely propagated
vegetatively, partly, due to reasons like longer juvenile phase as in perennial fruit crops, occasional
inbreeding depression, self and cross incompatibility, narrow genetic base especially in ornamentals, etc.
Further, somaclonal variations require less space and time for screening of desirable traits in vitro unlike
cross seedlings of perennial crops, which require a great deal of land area and time.

Somaclones may itself have numerous applications in plant breeding and genetic improvements and
recovery of such novel variants can be enhanced by applying suitable in vitro selection pressure (Jain 2001;
Lestari 2006). Sources of variations detected in plant tissue culture Tissue culture is an efficient method of
 Page 39 of 94

clonal propagation; however, the resulting regenerants often has a number of somaclonal variations (Larkin
and Scowcroft 1981). These somaclonal variations are mainly caused by newly generated mutations arising
from tissue culture process (Sato et al. 2011b).

The triggers of mutations in tissue culture had been attributed to numerous stress factors, including
wounding, exposure to sterilants during sterilization, tissue being incomplete (protoplasts as an extreme
example), imbalances of media components such as high concentration of plant growth regulators (auxin
and cytokinins), sugar from the nutrient medium as a replacement of photosynthesis in the leaves, lighting
conditions, the disturbed relationship between high humidity and transpiration (Joyce et al. 2003; Sato et al.
2011b; Smulders and de Klerk 2011).

Much of the variability expressed in micropropagated plants may be the result of, or related to, oxidative
stress damage inflicted upon plant tissues during in vitro culture (Cassells and Curry 2001; Tanurdzic et al.
2008; Nivas and DSouza 2014).

Oxidative stress results in elevated levels of pro-oxidants or reactive oxygen species (ROS) such as
superoxide, hydrogen peroxide, hydroxyl, peroxyl and alkoxyl radicals. These ROS may involve in altered
hyperand hypo-methylation of DNA (Wacksman 1997); changes in chromosome number from polyploidy to
aneuploidy, chromosome strand breakage, chromosome rearrangements, and DNA base deletions and
substitutions (Czene and Harms-Ringdahl 1995), which in turn may lead to mutations in plant cells in vitro
(Fig. 1).

Somaclonal variation shows a similar spectrum of genetic variation to induced mutation as both of them
result in qualitatively analogous gamut of DNA changes (Cassells et al. 1998). Different factors affect the
frequency of development of somaclones under in vitro conditions.

Explant/explant source

Differences in both the frequency and nature of somaclonal variation may occur when regeneration is
achieved from different tissue sources (Sahijram et al. 2003). Highly differentiated tissues such as roots,
leaves, and stems generally produce more variations than explants with preexisting meristems, such as
axillary buds and shoot tips (Duncan 1997).

In general, the older and/or the more specialized the tissue is used for regeneration, the greater the chances
that variation will be recovered in the regenerated plants (Table 1) as under such conditions, adventitious
shoot regeneration (shoot organogenesis) takes place from atypical points of origin directly or indirectly
through a callus stage (e.g., from leaves, petioles, shoot internodes, root segments, anthers, hypocotyls,
cotyledons, etc.; Pijut et al. 2012). Somaclonal variation can also arise from somatic mutations already
present in the donor plant, i.e., presence of chimera in explants (Karp 1994).

Mode of regeneration

Both culture initiation and subsequent subculture expose explants to oxidative stress (Krishna et al. 2008),
which may result in mutations (Cassells and Curry 2001). It seems evident that ‘extreme’ procedures such as
protoplast culture and also callus formation impose stress (Smulders and de Klerk 2011). Magnitude of this
stress depends on the tissue culture technique.
 Page 40 of 94

Fig. 1 Mechanism of somaclonal variation in micropropagated

plants as a result of oxidative burst upon in vitro culture.

Therefore, the production of plants via axillary branching does not normally result in the production of
variants, while cultures that go through a callus phase are the ones that theoretically promote a higher
mutation rate (Zayova et al. 2010). Investigations indicate more chromosome variability in the callus phase
than in adventitious shoots (Saravanan et al. 2011), indicating a loss of competence in the more seriously
disturbed genomes.

This could be explained by the different grade of disturbance with which the cells are confronted. In the first
case, cells follow a pattern of division which is the normal one in the developing plant.

On the other hand, callus formation implies a dedifferentiation phase followed by uncontrolled cell
divisions (Va´zquez 2001). Some types of tissue culture mimic, in some aspects, other stressful situations as,
for example, protoplast preparation in which cell wall degradation resembles the infective process of some
pathogens.

Therefore, the type and magnitude of the stress imposed on cultured cells varies according to the technique
used. In contrast to popular belief that the growth of unorganized callus is necessary for induction of genetic
variation, variability could be noticed in plants regenerated from explants adventitiously (Farahani et al.
2011; Bhojwani and Dantu 2013).

Sometimes for regeneration under in vitro conditions, somatic embryogenesis is the preferred pathway for
generating propagules.
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It has been suggested that regeneration via embryogenesis has better chance of obtaining genetically uniform
plants than through organogenic differentiation (Va´zquez 2001). This is so, because DNA in the initial
stages of development in somatic embryogenesis contains lower levels of methylation than in the later stages
(Sahijram et al. 2003). Variation in in vitro cultures raised through somatic embryogenesis has been reported
in severalhorticultural crops like hazel nut (Diaz-Sala et al. 1995), Citrus paradisi (Hao et al. 2004), oil palm
(Jaligot et al. 2004), rose (Xu et al. 2004), potato (Sharma et al. 2007), grapevine (Schellenbaum et al.
2008), coffee (Mene´ndez-Yuffa´ et al. 2010), olive (Leva et al. 2012), tamarillo (Currais et al. 2013) and
brinjal (Naseer and Mahmood 2014).

Effect of length of culture period and number of subculture cycles

The longer a culture is maintained in vitro, the greater the somaclonal variation is (Kuznetsova et al. 2006;
Gao et al. 2010; Farahani et al. 2011; Jevremovic´ et al. 2012; Sun et al. 2013).

Variant karyotypes are found to amass with increasing age of callus and as a result the chances of variant
plants produced during successive subculture also increases, in general (Zayova et al. 2010).

Furthermore, the rapid multiplication of a tissue, during micropropagation, may affect its genetic stability.
Khan et al. (2011) reported that after the eighth subculture, the number of somaclonal variants increased
with a simultaneous decrease in the multiplication rate of propagules in banana.

Similarly, Clarindo et al. (2012) suggested a limit of less than 4 months storage of coffee cell aggregate
suspensions for true-to-type mass propagation as ploidy instability was noticed in long-term in vitro culture.

Similarly when Farahani et al. (2011) raised olive cultivars, under in vitro conditions, through internode
cuttings, significant difference was observed in morphological characters among the regenerated plants after
seventh subculture, which was later confirmed by RAPD analysis.

However, C-value analysis showed that no significant change has occurred during subculturing in both olive
genotypes. This indicates that the genetic changes accompanied by somaclonal variation could be due to the
changes in the nucleotide content of the genome, probably, owing to mutations (insertions/deletions) and not
due to quantitative changes.

Not only the number of subculture but their duration also contributes to enhancing the rate of somaclonal
variations, especially cell suspension and callus cultures (Bairu et al. 2006; Sun et al. 2013).

Studies have shown that somaclonal variation is more apparent in plants regenerated from long-term
cultures (Etienne and Bertrand 2003). Rival et al. (2013) noticed that in vitro proliferation induces DNA
hypermethylation in a time-dependent fashion and changes in DNA methylation is involved in modulating
the expression of embryogenic capacity of oil palm during tissue culture.
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Culture environment

External factors like growth regulators, temperature, light, osmolarity and agitation rate of the culture
medium are known to influence the cell cycle in vivo in plants, considerably, which indicates that
inadequate control of cell cycle in vitro is one of the causes of somaclonal variation (Karp 1994; Nwauzoma
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and Jaja 2013). Normal cell cycle controls, which prevent cell division before the completion of DNA
replication, are presumed to be disrupted by tissue culture, resulting in chromosomal breakage (Phillips et al.
1994). Chromosome breakage and its consequences (deletions, duplications, inversions, and translocations)
cause aberrations in vitro (Duncan 1997). Plant growth regulators can affect the rate of somaclonal variation
both directly and indirectly by increasing the multiplication rate and inducing adventitious shoots (Gao et al.
2010). According to D’Amato (1985), it cannot be excluded that some plant growth regulators (PGRs) at
certain concentrations or in combination with other growth regulators and/or particular constituents of a
culture medium, may act as mutagens. Several growth regulators, such as 2,4-dichlorophenoxy acetic acid
(2,4-D), naphthalene acetic acid (NAA) and BAP (6-benzylaminopurine), synthetic phenylurea derivatives
(4-CPPU, PBU and 2,3-MDPU) have been most frequently considered to be responsible for genetic
variability (Siragusa et al. 2007; Sun et al. 2013; Sales and Butardo 2014). Prolonged cultivation in medium
containing 2,4-D influences higher DNA ploidy levels in callus cells (da Silva and Carvalho 2014). In their
experiment with banana, Sales and Butardo (2014) observed that addition of synthetic auxin 2,4-D in culture
medium led to high level of methylation events, particularly, cytosine methylation either at the internal or
external cytosine end, which largely resulted in variations in tissue cultured plants. Alteration in genomic
DNA methylation rate is being attributed for the development of ‘mantled’ somaclonal variant in oil palm
(Eeuwens et al. 2002; Jaligot et al. 2011). Similarly, Arnhold- Schmitt (1993) observed that indole-3-acetic
acid (IAA) and inositol in the growth medium induced DNA rearrangements and methylation changes in
carrot (Daucus carota) callus cultures. Matsuda et al. (2014) observed thatpercentage of somaclonal
variations dramatically increased when PGRs (0.5 ppm BA and 0.1 ppm NAA) were added to the medium
inoculated with leaf/leaf segments explants of African violet. Kinetin has been shown to cause extensive
hypomethylation of DNA in proliferating cultures of carrot root explants within 2 weeks (Arnhold-Schmitt
1993), and auxins, including NAA, have the opposite effect and cause hypermethylation (LoSchiavo et al.
1989). Moreover, there is evidence that differential expression in chromatin remodeling genes and histone
methylation genes happens during tissue culture, which leads to disruption in the methylation pathway in a
non-specific manner and hypo/ hypermethylation patterns of DNA induced in tissue culture. This can be
stabilized and transmitted to plants regenerated from these cultures (Shearman et al. 2013). Not only the
concentration, but also the ratio of different growth regulators affects the occurrence of variations in vitro.
Eeuwens et al. (2002) observed that, in general, a relatively high auxin/cytokinin ratio resulted in the lowest
incidence of variant ‘mantled’ flowering in oil palm, while using media supplemented with relatively high
cytokinins/auxin ratio resulted in a high incidence of mantled flowering. The role of cytokinin was further
confirmed by Ooi et al. (2013), who noticed that the mantled inflorescences of oil palm contained higher
levels of cytokinins like isopentenyladenine 9-glucoside and lower levels of trans-zeatin 9-glucoside,
dihydrozeatin riboside, and dihydrozeatin riboside 50- monophosphate compared with normal
inflorescences.

Genotype and ploidy

Though, the in vitro morphogenesis seems to be highly dependent on plant growth regulators and media
used for culture, it is again genotype specific (Alizadeh et al. 2010; Eftekhari et al. 2012). Among factors
affecting somaclonal variation, plant genotype is probably the most important determinant of variation (Shen
et al. 2007; Tican et al. 2008; Nwauzoma and Jaja 2013). Earlier, Eeuwens et al. (2002) characterized oil
palm clones as low/moderate risk and high risk with regard to ‘mantle’ flowering (wherein anther primordia
in both male and female flowers turn into fleshy supplementary carpels), on the basis of terminal
inflorescence data generated under in vitro conditions. Clones classified as high risk at the outset gave a
significantly higher incidence of mantled flowering in the field than low/medium risk clones, confirming
that data on terminal inflorescences produced in vitro allows effective screening of material with regard to
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the risk of mantled flowering. It is likely that this result from a combination of differences in genotype and
differences in epigenetically inherited changes are induced during the pre-embryogenic stages of the culture
process, i.e., callus initiation and maintenance.

Identification of variation in tissue culture

Both genetic and epigenetic alterations are associated with in vitro propagation, which may have phenotypic
consequences, and are collectively called somaclonal variation (Larkin and Scowcroft 1981; Guo et al.
2007). As a result, somaclonal variation is characterized by the intricacy of the changes, which are exhibited
at various levels, including phenotypic, cytological, biochemical and genetic/epigenetic (Kaeppler et al.
2000). Therefore, the strategy for the detection of somaclones should be based on such manifestations. A
wide variety of tools are available for the detection and characterization of somaclonal variants which are
primarily based on the differences in morphological traits (Pe´rez et al. 2009, 2011; Nhut et al. 2013),
cytogenetical analysis for the determination of numerical and structural variation in the chromosomes
(Clarindo et al. 2012; Currais et al. 2013; Abreu et al. 2014), biochemical (Vujovic et al. 2010; Kar et al.
2014), molecular DNA markers (Krishna and Singh 2007; Pathak and Dhawan 2012; Hossain et al. 2013;
Bello-Bello et al. 2014) or their combinations (Hora´cˇek et al. 2013; Dey et al. 2015; Stanisˇic´ et al. 2015).
The best test for assessing somaclonal variation is to fruit out the plants and conduct an extensive
horticultural evaluation, which is unfortunately a long-term endeavor with woody fruit crops, particularly
(Grosser et al. 1996). Every tool has its own advantages and limitations in assessment of the variations
(Table 2), which govern their use for restricted or large-scale application. The choice of technique for any
given application depends upon the material used and the nature of the question being addressed (Karp
2000).

Molecular basis of somaclonal variation

How a single plant genotype can result in a variety of phenotypic outcomes under the same in vitro culture
conditions is still far from being completely understood.

Several bases for somaclonal variation have been proposed, which include changes in chromosome number
(Mujib et al. 2007; Leva et al. 2012), point mutations (D’Amato 1985; Ngezahayo et al. 2007), somatic
crossing over and sister chromatid exchange (Duncan 1997; Bairu et al. 2011), chromosome breakage and
rearrangement (Czene and Harms-Ringdahl 1995; Alvarez et al. 2010), somatic gene rearrangement, DNA
amplification (Karp 1995; Tiwari et al. 2013), changes in organelle DNA (Cassells and Curry 2001;
Bartoszewski et al. 2007), DNA methylation (Guo et al. 2007; Linacero et al. 2011), epigenetic variation
(Kaeppler et al. 2000; Guo et al. 2006; Smulders and de Klerk 2011), histone modifications and RNA
interference (Miguel and Marum 2011), segregation of preexisting chimeral tissue (Brar and Jain 1998;
Va´zquez 2001; Ravindra et al. 2012; Nwauzoma and Jaja 2013) and insertion or excision of transposable
elements (Gupta 1998; Sato et al. 2011b).

In particular, transposable elements are one of the causes of genetic rearrangements in in vitro culture
(Hirochika et al. 1996; Sato et al. 2011a). Tissue culture is reported to activate silent transposable elements,
resulting in somaclonal variations. Insertions of transposable elements and retrotransposons can function as
insertional mutagens of plant genomes, whereas widespread activation may result in a wide gamut of
chromosomal rearrangements (Tanurdzic et al. 2008). In turn, these rearrangements can lead to
misregulation of genes, aneuploidy and new transposon insertions (Smulders and de Klerk 2011). However,
many aspects of the mechanisms, which result in somaclonal variations, remain undefined. It is therefore,
inevitable to explore the genome-wide change through sequencing of whole-genome of the concerned crop.
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Nextgeneration sequencing technology has enabled the wholegenome sequencing of individual plants
(Miyao et al. 2012). A new generation of sequencing technologies,

fromIllumina/Solexa, ABI/SOLiD, 454/Roche, and Helicos, has provided unprecedented opportunities for
high-throughput functional genomic research (Morozova and Marra 2008; Metzker 2010). Somaclonal
variations vis-a`-vis crop improvement Genetic variation is an essential component of any conventional crop
breeding program.

The typical crop improvement cycle takes 10–15 years to complete and includes germplasm manipulations,
genotype selection and stabilization, variety testing, variety increase, proprietary protection and crop
production stages. Plant tissue culture is an enabling technology from which many novel tools have been
developed to assist plant breeders (Karp 1992; Mathur 2013). Tissue culture- induced somaclonal variation
is akin to variations inducedwith chemical and physicalmutagens (Jain 2001) and offers an opportunity to
uncover natural variability for their potential exploitation in crop improvement. Like any other technology,
in vitro induced somaclonal variation has its own merits and demerits, like the two sides of the same coin.

Advantages

The advantages comprise:

(1) it is cheaper than other methods of genetic manipulation and does not require ‘containment’ procedures.
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(2) Tissue culture systems are available for more plant species than can be manipulated by somatic
hybridization and transformation at the present time.

(3) It is not necessary to have identified the genetic basis of the trait, or indeed, in the case of transformation,
to have isolated and cloned it.

(4) Novel variants have been reported among somaclones, and evidences indicate that both the frequency
and distribution of genetic recombination events can be altered by passage though tissue culture. This
implies that variation may be generated from different locations of the genome than those, which are
accessible to conventional and mutation breeding (Karp 1992).

(5) There Table 2 Strengths and weaknesses of different marker systems for the assessment of clonal
fidelity.

Disadvantages

One of the serious limitations of somaclonal variation which makes it comparatively difficult to use is that,
despite the identification of factors affecting the variation response of a given plant species, it is still not
possible to predict the outcome of a somaclonal program (Karp 1992) as it is random and lacks
reproducibility.

Further, as a large number of genetic changes are based on point mutations or chromosome rearrangements,
most R1 segregate. Therefore for quantitative traits such as yield, it is virtually impossible to select
individuals with improvements in the R1 generation. Though techniques for selection of somaclones
resistant to various biotic and abiotic stresses had been worked out in many horticultural crops,
unfortunately, no in vitro selection methods exist for complicated traits such as yield, soluble solids,
sweetness, texture or shelf life (Evans 1989). Somaclonal variation can become a part of plant breeding
provided they are heritable and genetically stable. Only a limited numbers of promising varieties so far had
been released using somaclonal variations.

This is perhaps due to the lack of interaction between plant breeders and tissue culture scientists, and non-
predictability of somaclones (Jain 2001). Further, though the new varieties have been produced by
somaclonal variation, in a large number of cases improved variants have not been selected due to

(1) the variations were all negative;

(2) positive changes were also altered in negative ways;

(3) the changes were not novel, or

(4) the changes were not stable after selfing or crossing (Karp 1992).

Recovery of somaclonal variants

The recovery of variants can be improved by promoting the factors which are responsible for the
development of somaclonal variations such as protoplast culture (Kothari et al. 2010) and employing callus
and cell suspension culture for several cycles and regeneration of large number of plants from long-term
cultures (Barakat and El-Sammak 2011).
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Indirect organogenesis is an important means of retrieving genetic variation through somaclones with useful
traits of agronomic or industrial use. Besides, plant genotype is a major factor, which determines the type
andfrequency of somaclonal variation.

For instances, Solanaceous plants like potato (Sharma et al. 2007) and tomato (Bhatia et al. 2005) produce a
gamut of somaclonal variation than many other commercial horticultural crops. However, to be of practical
value, the frequency of somaclonal variation should be sufficient enough to select desirable traits, and the
selected lines should perform well under multiple environments (Duncan 1997).

The efficiency of recovering variants in vitro can further be enhanced by applying selection pressure through
screening of desirable traits, e.g., in vitro selection for tolerance against abiotic and biotic stresses (Barakat
and El-Sammak 2011). This attains more significance in view of the fact that the selection of desirable traits
takes several years and many generations under field conditions.

In vitro selection can shorten considerably the time for the selection of desirable traits under in vitro
selection pressure with minimal environmental interaction, and can complement field selection (Jain 2001).
The recovery of somaclones can be increased by combining micropropagation with induced mutagenesis in
vitro (Afrasiab and Iqbal 2010). Kuksova et al. (1997) noted that somaclonal variation and mutagens can be
combined to increase the frequency of induced mutation.

Likewise, irradiation followed by adventitious bud regeneration has been reported to have allowed the
recovery of mutants with useful agronomic traits in Gypsophila paniculata L. (Barakat and El-Sammak
2011). Yang and Schmidt (1994) treated in vitro leaves of the cherry rootstock ‘209/1’ (Prunus cerasus 9 P.
canescens) with X-rays with LD50 close to 20 Gy.

Among plants regenerated from leaves with 20 Gy, one was phenotypically different, and was subsequently
isolated and cloned. This somaclone was extremely dwarfed and was stable in both greenhouse and field
tests. Employing more than one mutagen results in further improvement in recovery of somaclones in vitro.
Murti et al. (2013) exposed the strawberry ‘DNKW001’ to the doses of 0, 30, 80, 130, 180, 230, 280, 300
and 325 Gy and similar doses of gamma rays ? EMS 7 lM treatments.

Their results showed that Gamma ray irradiation ? EMS was more effective to generate more type and
magnitude of variants. Purwati and Sudarsono (2007) regenerated four variant lines in abaca banana from
(1) embryogenic calli; (2) ethyl methyl sulphonate (EMS)-treated embryogenic calli; (3) EMS-treated
embryogenic calli, followed by in vitro selection on Foc (Fusarium oxysporum f.sp. cubense) culture filtrate
(EMS ? CF line) and (4) EMS-treated embryogenic calli, followed by in vitro selection on fusaric acid.

The Foc resistance abaca variants were successfully identified from four tested abaca variant lines, although
with different frequencies. However, more Foc resistance abaca plants were identified from EMS ? CF line
than the others. Earlier, Bidabadi et al. (2012) suggested that the subjecting of shoot tips cultures of banana
to EMS (200 mM) treatments could provide an alternative strategy for inducing variants. Recently, Iuliana
and Cerasela (2014) suggested irradiation of in vitro raised plants with ultraviolet radiations (UV-C) for
induction of somaclones in potato.
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Application of somaclonal variations

It is well accepted that somaclonal variations arising out of unique tissue culture environment are very often
noticed phenomenon in clonally propagated plants, which can advantageously be utilized as a source of new
variation in horticultural crops (Karp 1995).

However, suitable tools for detection, evaluation, identification and improvement of resistant clones should
be designed in order to realize the benefits of such variations (Sahijram et al. 2003). Crop improvement
through somaclonal variation enables breeders to obtain plants tolerant to the biotic or abiotic stress, such as
 Page 51 of 94

drought, high salinity, high or low soil pH and disease tolerance (Yusnita et al. 2005). A number of cultivars
have been developed through somaclonal variation in different horticultural crops for a range of useful traits,
which are presented in Table 3.

Conclusions

Several strategies have been followed to ascertain the genetic fidelity of the in vitro produced progenies in
view of the fact that the commercial viability of micropropagation technology is reliant upon maintenance of
genetic fidelity in the regenerated plants. Therefore, a thorough assessment of micropropagated plants
becomes very critical, especially, for perennial crops such as fruit species, which have a long pre-bearing
growth period.

The efficiency and sensitivity of new molecular tools has enabled us to detect somaclonal variation at an
early stage. These tools have become very useful for the rapid detection and accurate identification of
variants.

Nevertheless, the morphological and cytological assays should continue to remain as the primary and
essential assay for the sustained success of fidelity tests associated with production of clonal plants. Though,
on one hand, tissue culture-induced variations pose a major threat to the genomic integrity of regenerated
plants, they provide tools for improvement to plant breeders, particularly for crops with a narrow genetic
base, i.e., self pollinated and vegetatively propagated.

Irrespective of our goal either for production of true-to-the type planting material or creation of variability, a
multidisciplinary approach (involving concerned sciences of horticulture, genetics and plant breeding,
physiology, cytology and molecular biology) with all our previous knowledge and experience should be
followed to achieve the desideratum.

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Somatic Hybridization
INTRODUCTION

Plant breeding throughout the 21th century has been successful because of the incorporation of resistance
from exotic germplasm into modern cultivars. Somatic hybridization broadens the base of accessible
germplasm and offers additional opportunities for introgression of desirable traits into cultivars. Even
though measurable success of somatic hybridization in terms of cultivar release may still be limited, the
potential for their use in plant breeding remains great. In general, somatic hybridization provides excellent
opportunities for research on plant improvement, first by exploring genetic variations among the existing
crops and then by attempting to transfer the available genetic information from one species to another
through fusion of protoplasts isolated from somatic tissues of these crops.

Somatic hybridization, also called somatic cell fusion or protoplast fusion, refers to the fusion of plant
protoplasts from somatic cells of different species and the subsequent regeneration of hybrid plants from the
fused protoplasts. Somatic hybridization of plants by protoplast fusion is a technique that has captivated the
imagination of plant breeders for three decades (Hoffmann et al., 1995).

It offers the possibility of accessing sexually incompatible germplasm between crop species and distant
relatives, merging genomes of sexually dysfunctional cultivars or breeding lines, and substituting one
cytoplasm for another with little effect on the nuclear genome. Since Carlison et al., (1972) first reported
successes with parasexual hybridization of tobacco (Nicotiana tabacum L.), hundreds of reports have been
published to extend the procedures to additional plant genera and to evaluate the potential of somatic hybrids
in many crops.

Somatic hybridization has even been conducted under microgravity as part of a space laboratory experiment
(Hoffmann et al., 1995). Somatic hybridization is one of the most important uses of protoplast culture. This
is particularly significant for hybridization between species or genera, which can not be made to cross by
conventional method of sexual hybridization.

Although somatic hybridization was successfully achieved first in animals and only later in plants, its
significance has been realized fully in plants because the hybrid cells can be induced to regenerate into
whole plants (Evans & Bravo, 1983). Therefore, somatic hybridization provides excellent opportunities for
research on plant improvement, first by exploring genetic variations among the existing crops and then by
attempting to transfer the available genetic information from one species to another through fusion of
protoplasts isolated from somatic tissues of these crops.

SOMATIC HYBRIDIZATION AS A PROTOPLAST FUSION

Protoplast fusion has often been suggested as a means of developing unique hybrid plants which cannot be
produced by conventional sexual hybridization. Protoplasts can be produced from many plants, including
most crop species (Feher and Dudits 1994). However, while any two plant protoplasts can be fused by
chemical or physical means, production of unique somatic hybrid plants is limited by the ability to
regenerate the fused product and sterility in the interspecific hybrids (Gleddie et al., 1986) rather than the
production of protoplasts.

Perhaps the best example of the use of protoplasts to improve crop production is that of Nicofiana, where the
somatic hybrid products of a chemical fusion of protoplasts have been used to modify the alkaloid and
diseaseresistant traits of commercial tobacco cultivars (Pandeya and White, 1994). Somatic hybrids were
produced by fusing protoplasts, using a calcium-polyethylene glycol treatment, from a cell suspension of
 Page 53 of 94

chlorophylldeficient N. rusfica with an albino mutant of N. tabacum (Douglas et al., 1981a,). The wild N.
rusficaparent possessed the desirable traits of high alkaloid levels and resistance to black root rot. Fusion
products were selected as bright green cell colonies, the colour being due to the genetic complemention for
chlorophyll synthesis the hybrid cells.

Plants recovered by shoot organogenesis showed a wide range of leaf alkaloid content but had a high level
of sterility. However, after three backcross generations to the cultivated N. tabacum parent, plant fertility
was restored in the hybrid lines, although their alkaloid content and resistance to blue mould and black root
rot were highly variable. Interestingly, neither parent was known to possess significant resistance to blue
mould.

Where mutant cell lines of donor plants are not available for use in a genetic complementation
selectionsystem, it has been demonstrated that mesophyll protoplasts from donor parents carrying transgenic
antibiotic resistance can be used to produce fertile somatic hybrids selected by dual antibiotic resistance
(Sproule et al., 1991). The fusion of protoplasts from 6-azauracil-resistant cell lines of Solanum melongena
(aubergine) with protoplasts from the wild species yielded hybrid, purple-pigmented cell colonies that
underwent regeneration via organogenesis (Gleddie et al., 1986).

As protoplasts from the parental cell suspension cultures could not be regenerated, hybrids could be
screened by their 6-azauracil resistance, capacity to synthesize anthocyanins (purple pigment) and ability to
undergo shoot organogenesis. The restoration of regeneration ability through complementation has also been
observed in Nicofiana cell-fusion products (Douglas et al., 1981a; Gleddie et al., 1986). The hybrids
resulting from the study were found to be resistant to root knot nematodes and spider mites, important
agricultural traits. However, they were also completely sterile and could not be incorporated into an
aubergine-breeding programme.

Two possible ways of solving this sterility problem, 'back' fusions of somatic hybrids with the cultivated
parents and initiation of suspension cultures of the hybrid cells so that more of the wildspecies chromosomes
can be eliminated, have so far been unsuccessful with these hybrids (S. Gleddie). Selection of hybrids and
use of protoplast fusion for hybridization in crop plants has been reported in Brassicas, citrus, rice, carrot,
canola, tomato, and the forage legumes alfalfa and clover (Akagi et al., 1989; Bajaj, 1989; Tanno- Suenaga
et al., 1988; Vardi et al., 1989, Kao et al., 1991).
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Evans & Bravo (1988) have recommended that production of novel hybrids through protoplast fusion should
focus on four areas: (1) agriculturally important traits; (2) achieving combinations that can only be
accomplished by protoplast fusion; (3) somatic hybrids integrated into a conventional breeding programme;
and (4) the extension of protoplast regeneration to a wider range of crop species. In the case of the above-
mentioned example of Nicotiana, all of these criteria were met although this took 12 years from the isolation
of the fusion product in 1978 to the release of the first variety in 1990; this underlines the often overlooked
fact that it takes 10 to 20 years to take initial research results to the stage of a recognized cultivar
(Kuckucketal, 1991).

TYPES OF SOMATIC HYBRIDS

If the complete genome of two different species are combined parasexually, then an amphiploid somatic
hybrid results. This is the most common occurrence in Somatic hybridization experiments. If one fusion
partner is an unadapted species with some desirable trait, then the resulting somatic hybrids can be expected
to carry many of the undesirable traits of the unadapted species along with the trait of interest. Therefore
plant breeders would indeed be foolishly optimistic to expect table ready germplasm from such effort;
considerable back crossing and ploidy reduction are required to introgress the desirable trait into germplasm
suitable for cultivar release.

Indeed much of the recent effort in Somatic hybridization has been directed at development of somatic
hybrids and/or verification that the trait of interest has been transmitted. Subsequent utilization of somatic
hybrid germplasm in plant breeding has been less frequent (Evans and Bravo, 1988). In an effort to limit the
genetic contribution of an unadapted ‘parent’ to the product of protoplast fusion, some geneticists have
promoted asymmetric Somatic hybridization, where by the genome of the donor species if fractionated by
irradiation prior to fusion.

The resulting asymmetric hybrids retain the complete genome of the recipient (adapted) species and only
fragments of the genome of the donor (unadapted) species. Irradiation is imprecise and damage to the donor
genome is random. Therefore transmission of the trait of interest is not guaranteed and the amount of the
donor genome transmitted is highly variable, depending upon the irradiation dosage and the tolerance of the
recipient genome to chromosome fragments and rearrangements (Evans and Bravo, 1988).

Cybrids or cytopasmic hybrids result from protoplast fusion between a cultivated species and enucleated or
nucleus inactivated protoplasts bearing a different plastome. For plant breeding purposes, cybridization
offers the possibility of developing lines for hybrid breeding via cytoplasmic male sterility systems in a
single step. Such systems are the mainstay of hybrid cultivar production in many crops and availability of
suitable plastome variation has been a limitation in breeding (Gleddieet al., 1983).

METHODS FOR ISOLATION AND FUSION OF PLANT PROTOPLAST

Successful Somatic hybridization was originally obtained through the use of PEG-mediated (polyethylene
glycol) fusion. More recently, electro fusion techniques have become available.

Direct One Step Method

In one step method, the leaf segments are incubated overnight (15-18h) with enzyme mixture at 25°C and
teased gently to liberate the protoplasts. The mixture is filtered through fine wire gauze to remove leaf
debris, transferred to 13 X 1000 mm screw capped tubes and centrifuged at 100g for 1 min. The protoplasts
form a pellet and supernatant removed. The process is repeated three times and protoplasts washed with
13% sorbitol solution, which is later replaced by 20% sucrose solution and centrifuged at a speed of 200g
 Page 55 of 94

for 1 min. The cleaned protoplasts, which will now float (debris settles do), can be pipetted out and bulked
(Gamborg et al., 1981).

Enzymatic Method of Isolation of Protoplast

The enzymatic method is almost invariably used now for the isolation of protoplasts, since it gives large
quantities of protoplasts, where cells are not broken and osmotic shrinkage is minimum.
However,sometimes mechanical and enzymatic methods are combined, where cells are first separated
mechanically and later used for isolation of protoplasts through enzymatic treatment. (Gamborg et al., 1981).

Mechanical Method of Isolation of Protoplast

In mechanical method, cells are kept in a suitable plasmolyticum (in plasmolysed cells, protoplasts shrink
away from cell wall) and cut with a fine knife, so that protoplasts are released from cells cut through the cell
wall, when the tissue is again deplasmolysed.This method is suitable for isolation of protoplasts from
vacuolated cells (e.g. onion bulbs, scales, radish roots). However, this method gives poor yield of protoplasts
and is not suitable for isolating protoplast from meristematic and less vacuolated cells. The mechanical
method, though, was used as early as 1892, is now only rarely used for isolation of protoplasts (Gamborg et
al., 1981).

SELECTION OF FUSION PRODUCTS AND VERIFICATION OF HYBRIDS

Selection Schemes

When protoplast of two different sources are mixed in the hope of obtaining somatic hybrids, a range of
possible products may regenerate, resulting from unfused protoplasts of either”parent” (somaclones
produced), homofusion between genetically similar protoplast of either parent (polyploidy somaclones
produced), heterofusions between single protoplast of each parent (amphidiploids somatic hybrids
produced), or multiple fusions among several protoplasts (highly polyploid somatic hybrids produced). In
addition, aneuploids often arise due to chromosome loss during cell culture or aberrant mitotic cycles within
the hybrid nucleus. Without any selection against the undesirable products, researchers are faced with
distinguishing the desired amphidiploids somatic hybrids from the undesired variable ploidy somaclones of
each parent and highly polyploidy hybrids resulting from multiple protoplast fusions (Evans et al., 1984).

The simplest selection has been to analyze whatever plants may regenerate by morphological or molecular
markers to determine their possible genomic constitution. When heterokaryons regenerate more readily than
homokaryons, this can be effective. Otherwise, many more sophisticated selection schemes have been
developed to encourage regeneration of only the somatic hybrids, but all generally carry some inherent cost.
Selection media can be used if sufficient tissue culture research has been done on the prospective fusion
partners. If each protoplast source can reliably be predicted to regenerate only in the presence of a distinct
unique media component, elimination of both components in the medium used for regeneration following
protoplast fusion should result exclusively in somatic hybrids.

If one parent is known to be non-regenerable, then this procedure can be simplified by omitting only the
medium requirement of the regenerable “parent”. This particular complementation scheme requires the
conduct of rigorous plant tissue culture experimentation on both fusion parents prior to their utilization in
Somatic hybridization schemes in order to predict the outcome with any reliability. In addition, absolute
medium requirements in plant tissue culture are difficult to identify (Evans et al., 1984). The use of
metabolic inhibitors such as iodoacetate (IOA) to prevent regeneration of unfused protoplasts of one fusion
partner has greatly enhanced complementation schemes. Because it is common to find that one protoplast
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source is unregenerable, protoplasts from such a source can be fused with IOA-treated protoplasts of an
ordinarily regenerable source with the expectation that only fusions will regenerate. Introduction of
transgenic antibiotic resistance into one or both fusion partners has also been used to facilitate
complementation schemes.

This provides an easy selection scheme by which protoplasts lacking antibiotic resistance will not
regenerate on medium containing the antibiotic. Antibiotic resistance in an unregenerable protoplast source
combined with antibiotic sensitivity in a regenerable protoplast source then provides a suitable selection
scheme for exclusive regeneration of somatic hybrids. Ishige (1995) used transgenic resistance to kanamycin
in one potato dihaploid and hygromycin in another to regenerate exclusively somatic hybrids that varied for
ploidy. However, the restrictions of this system are the extensive pre-breeding required to introduce
transgenic resistance into the protoplast fusion partner(s) and the limitation of the procedure only to
transgenic plants.

In order to eliminate the necessity of pre-breeding one of the fusion partners, Dorr et al., (1994) developed a
selection method to label protoplasts with superparamagnetic beads mediated by biotinylated lectins.
Electrofusion of these labeled protoplasts with transgenic kanamycin resistant protoplasts was followed by
sorting using a magnetic cell sorter to retain protoplasts labeled with the microbeads. Subsequent culture of
selected cells on kanamycin containing medium resulted in a significant enrichment for somatic hybrids
among the regenerants.

Surface labeling of prospective fusion partners with biotin and avidin has been used to facilitate
heterologous aggregation of fusion partners during electrofusion (Waara et al., 1998) Hoffmann-Tsay et al.,
(1994) identified several surface active chemicals that could be used as adjuvant to treat protoplasts prior to
electrofusion to increase the fusion rate. They concentrated on adjuvants that did not need to be removed
from the culture medium in order to retain one of the advantages of electrofusion over chemical fusion i.e.,
the direct culture of fusion products.

Mollers et al., (1994) likewise studied the possibility of increasing the frequency of somatic hybrid
formation during electrofusion by selectively inactivating one protoplast source with the mitochondrial
inhibitor, nonly-acridine orange (NAO). NAO was considered to be an alternative to IOA that might have
the specific result of predetermining the mitochondrial composition of somatic hybrids. Neither of these two
techniques, i.e. chemical adjuvants or NAO, has been used extensively in somatic hybridization subsequent
to the original reports. Funatsuki eat al., (1994) described a modification of chemical fusion where the
fusion was conducted on a Millicell (Millipore) membrane on a puddle of PEG, this facilitated heterofusions
between protoplasts that differed considerably in size.

Biochemical selection of somatic hybrid

Gamborg et al., (1981) demonstrated the value of biochemically based selection. This selection procedure
was based on a prior knowledge of the differential growth characteristics and nutritional requirements of
unfused and hybrid mesophyll protoplasts isolated from the genetically different Nicotiana glauca and N.
langsdorffii. Protoplasts of the hybrid were able to grow on a defined medium in culture to form calli,
whereas parental types failed to develop into calli.

This selection system has an advantage in that the requirement of a mutant as one of the fusion partners is
totally eliminated. Schieder and Kohn (1986) utilized the differential sensitivity of protoplasts isolated from
Petunia parodii and P. hybrida to the drug actinomycin D. In an MS medium the mesophyll protoplasts of
Petunia hybrid developed up to a macroscopic callus stage and those of P. parodii divided to form only small
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cell colonies. The addition of actinomycin D to the culture medium apparently had a slight effect on the
regeneration potential of P. parodii protoplasts while those of P. hybrida failed to divide. Pandeya et al.,
(1986) dopted a similar procedure for somatic hybrids between Nicotiana sylvestris and N. knightiana. This
selection system makes use of two dominant drug resistant cell lines. Selection of somatic hybrids following
protoplast fusion of two such cells, lines is carried out on, media containing both drugs.

Complementary selection of somatic hybrid

The selection of somatic hybrids as a result of complementation by auxotrophic mutants may be useful as
only the hybrid lines are expected to survive in the minimal medium. Auxotrophs are mutants requiring
specific compounds for their growth. Pandeya et al., (1986) succeeded in selection of numerous somatic
hybrids by utilizing protoplasts of nitrate reductase deficient and chlorate resistant mutant lines of tobacco.
Protoplasts of two genetically different mutants were fused and cultured in a, medium containing nitrate as
the sole nitrogen source. Parental protoplasts did not grow in the presence of nitrate, whereas fusion
products regenerated.

The complementation selection based on auxotrophic mutants, even though desirable and efficient, is very
limited because of the limitation due to the paucity of higher plant auxotroph. Nonallelic albino mutants
were used for selection by Melchers (1992). He fused haploid chlorophyll deficient and light-sensitive
protoplasts of Nicotiana tabacum and cultured them under high intensities of light. After two months green
colonies were observed in culture dishes as a consequence of complementation between the two albino
mutants. On further culturing these green colonies regenerated somatic hybrid plants.

Visual selection of somatic hybrids

In most of the somatic hybridization experiments selection procedures involve fusion of chlorophyll
deficient (non-green) protoplasts of one parent with the green protoplasts of the other parent (wild type)
since this facilitates visual identification of heterokaryons at the light microscope level. Non-green
protoplasts are isolated from cultured cells, epidermal cells, or antibiotic induced albino plantlets. The visual
selection procedure is coupled with complementary natural differences in the sensitivity of parental
protoplasts to media constituents which enable only the hybrid cells to develop in cultures and regenerate
plants.

Wild type protoplasts of Petunia parodii were fused with albino protoplasts isolated from cell suspension
cultures of P. hybrida, P. inflata, and P. parviflora in separate experiments (Atanassov et al., 1995). In all
these combinations P. parodii green protoplasts were eliminated at the small colony stage, while the albino
protoplasts of the other parent developed colorless colonies. Hybrid components proliferated into green calli
and subsequently regenerated somatic hybrid plants. Using this method Evans and Bravo (1983), recovered
somatic hybrids in Datura.

In these systems the protoplasts of a chlorophyll deficient mutant regenerated into shoots on a defined
medium while the wild type protoplasts did not. After fusion of these two types of protoplasts somatic
hybrids were recovered and the intermediate nature of the hybrid could be confirmed. The hybrid thus
recovered demonstrated the potential for shoot production of the chlorophyll deficient mutant plus the
potential for chlorophyll synthesis of the wild type.

In experiments on intergeneric somatic hybridization, Schieder and Kohn (1986) used the scheme in which
the parental protoplasts and heterokaryons were allowed to develop calli in cultures. The morphological
differences in the resultant three types of calli permitted identification of the hybrid tissue, which could then
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bemicroscope, be isolated mechanically by means of Drummond pipette, and can be cloned in microdrop
cultures. This approach suffers from the fact that it requires special culture media for each particular hybrid
cell type to divide and form clusters.

This is also called the fishing method. Somatic hybrid callus has been similarly obtained in fusion between
colorless protoplasts of Glycine max derived from cell cultures with the green mesophyll protoplasts of
Nicotiana glauca. The fusion products can be identified by the presence of chloroplasts in one half of the cell
and starch granules in the other half. Diffusion of chloroplasts throughout the cell occurs shortly after fusion.
Even though the mechanical method of isolation of somatic fusion products is the most tedious method, it
may be the most likely method for recovering osmotic hybrids in a variety of different plants, especially
legumes, cereals, and tree species (Gamborg et al., 1981).

Flow cytometry and sorting selection of somatic hybrids

Various laboratories are using techniques of flow cytometry and fluorescent activated cell sorting for
analysis of plant protoplasts while maintaining their viability. These techniques have also been applied for
sorting and selection of heterokaryons. Galbraith et al., (1989) have described a universally applicable
method for electronic sorting of heterokaryons formed by fusing the protoplasts of two parents labeled with
different vital fluorescent dyes, such as rhodomine isothiocyanate and floreceine isothiocyanate. The fused
and unfused products are sorted in a "cell sorter" machine based on the presence or absence of fluorescence
of both dyes in the fusion products (Gamborg et al., 1981).

METHODS OF DETECTION

Once putative hybrids have been regenerated, an array of techniques is now available for proof of their
hybrid nature. Although intermediate morphology and heteroallelic isozyme patterns are still frequently
employed in recent reports, most authors are now using molecular analysis to demonstrate hybridity. Even if
specific probes for restriction fragment length polymorphisms (RFLPs) have not been developed for the
species under investigation, probes that anneal to universal genes, such as rDNA from other species, will
often reveal differences between the two protoplast sources following restriction enzyme digest and southern
blotting (Harding and Millam, 1999).

As Johnson et al., (2000) reported there in no phylogenetic limitation to protoplast fusion. Even fusions
between plant and animal protoplasts have been conducted. However, regeneration of somatic hybrids has
only been possible when fusion partners are somewhat related. Somatic hybridization has allowed us to
exceed the limits of sexual compatibility and evidence of partial genome transfer has even been presented
after fusion between a monocot (Hordeum vulgare L.) and a dicot (Daucus carota L.) (Kisaka et al., 1997).
However it has been far more common to obtain only unregenerable callus if the fusion partners are too
phylogenetically distant, because such callus has little potential in plant breeding.

APPLICATION OF SOMATIC HYBRIDIZATION

One of the most significant developments in the field of plant tissue culture, Witnessed during the last few
decades, is the isolation, culture and fusion of protoplasts (Gamborg et al., 1981). A more recent
achievement is the manipulation and regeneration of these cultured or fused protoplasts into whole plants.
Since in plant cells, the plasma membrane is bound by a rigid cellulose wall (unlike animal cells), it has
been relatively difficult to handle plant cells.

In 1960, it was demonstrated by Cocking at the University of, Nottingham (U.K.), that naked cells called
protoplasts can be obtained through enzymatic degradation of cell walls. This led to significant
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developments in the field of somatic cell genetics in higher plants. Cultured protoplasts can be used not only
for somatic cell fusions, but also for taking up foreign DNA, cell organelles, bacteria and virus particles. In
view of this, the isolation and culture of protoplasts has become a very important area of research, within the
realm of plant biotechnology (Bajaj, 1990).

The essential ingredients of the technique include isolation of protoplasts, culture of protoplasts,
introduction of foreign DNA into protoplasts, raising whole plants from cultured protoplasts and fusion of
protoplasts leading to somatic hybridization. Somatic hybridization involves fusion of two distantly related,
to closely related plant protoplasts at intraspecific, interspecific, intergeneric, and interfamily levels, with
sub sequent regeneration of hybrid cells into hybrid plants. The term protoplast was first used by Manstein
in 1880. Plastids and mitochondrial genomes (cytoplasmically encoded traits) are inherited maternally in
sexual crossings.

Through the fusion process the nucleus and cytoplasm of both parents are mixed in the hybrid cell
(heterokaryon). This results in various nucleocytoplasmic combinations. Sometimes interactions in the
plastome and genome contribute to the formation of cybrids (cytoplasmid hybrids). Cybrids, in contrast to
conventional hybrids, possess a nuclear genome from only one parent but cytoplasmic genes from both
parents. The process of protoplast fusion resulting in the development of cybrids is known as cybridization.
In cybridization, heterozygosity of extrachromosomal material can be obtained, which has direct application
in plant breeding. According Gamborg et al., (1981) to somatic hybridization involves four major steps:

i. Isolation of protoplasts from different species

ii. Culture of protoplasts

iii. Fusion of protoplasts, and

iv. Identification and selection of hybrid cells and their subsequent regeneration into whole plants Somatic
cell fusion appears to be the only means through which two different parental genomes can be recombined
among plants that cannot reproduce sexually (asexual or sterile).

Protoplasts of sexually sterile (haploid, triploid, and aneuploid) plants can be fused to produce fertile
diploids and polyploidy. Somatic cell fusion overcomes sexual incompatibility barriers. In some cases
somatic hybrids between two incompatible plants have also found application in industry or agriculture.
Somatic cell fusion is useful in the study of cytoplasmic genes and their activities and this information can
be applied in plant-breeding experiments.

Somatic Hybrids for Cytoplasmic Male Sterility

Methods have been developed to substitute the nucleus of one species into the cytoplasm of another species,
whose mitochondria are inactivated. This type of substitution in some cases, led to generation of
cytoplasmic male sterility. For this purpose, a report by Melchers (1992); and his coworkers, the two types
of protoplasts, used for the production of, somatic hybrids, and were treated differently, as follows:

 mesophyll protoplasts of tomato (Lycopersicon esculentum) were treated with iodoacetamide (IDA)
to inactivate mitochondria and
 mesophyll protoplasts of Solanum acaule (or S. tuberosum = potato) were irradiated with γ or x-rays
to inactivate nuclei. The protoplasts were mixed in 1: 1 ratio and induced to fuse using Ca2+ and
PEG, leading to the production of heterologous hybrids. Among the fusion products, some hybrid
tomato plants were indistinguishable from the original cultivars, with respect to morphology,
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physiology and chromosome number (2n=24), but exhibited various degrees of male sterility. The
variation included (Melchers,1992):
 Complete lack of pollen or malformation of anthers;
 shrunken pollen,
 Normal looking stainable pollen that could not germinate. In five tomato cultivars, male sterility
induced in this manner was inherited maternally over several generations. Therefore, it was
obviously cytoplasmic male sterility.

The mitochondrial DNA of these CMS hybrids did not resemble mtDNA of either parent, and was
instead recombinant type, representing a hybrid mitochondrial genome. Therefore, protoplast fusion can
be effectively used for production of CMS lines and has the following advantages:

• Only one step is required;

• The nuclear genotype of the cultivar remains unaffected,

• There are prospects that 100% of the progenies of somatic hybrids will be CMS. The restorer lines for
these CMS lines have also been shown to be available in tomato, so that hybrid seed can be produced
without manual emasculation.

Somatic Hybrids for Gene Transfer

The family Solanaceae contains the most commonly used species for somatic hybridization. The genera
from this family that have been often used for somatic hybridization include Nicotiana, Datura, Petunia,
Solanum, Lycopersicon, etc. In Rutaceae, Citrus has been combined with species from other genera. In
Leguminoseae also, several genera including Medicago, Trifolium and Lotus have been used for somatic
hybridization. Even among cereals, hybrids between rice (Oryza sativa) and Echinochloa oryzicola
(barnyard grass) have been obtained. However, one of the most extensive programmes has been in progress
in the family Brassicaceae at Uppsala, Sweden.

A collaborative programme in Brassicaceae is also underway at, New Delhi, the callaborative institutes
being IARI, TERI and Delhi University. Some of the interspecific somatic hybrids produced. Similarly
intergeneric somatic hybrids and intertribal hybrids within Brassicaceae. The hybrids within Brassicaceae
are also diagrammatically represented.Intergeneric fertile hybrids were obtained in several cases particularly
within the tribe Brassiceae.

These fertile hybrids were back crossed to cultivated species followed by screening for economic traits like
drought and insect resistance (transferred from Eruca), pathogen resistance (transferred from Sinapis),
cytoplasmic male sterility or CMS (transferred from Diplotaxis), cold tolerance (from Barborea), and high
concentration of nervonic acid, a lubricant (from Thalspi). Symmetric hybrids between tomato and potato,
produced by protoplast fusion, have been shown to exhibit intermediate cold tolerance. In., another example,
substitution of Solanum acaule genome into S. tuberosum resulted in an appreciable increase of frost
resistance.

Somatic Hybridization - An Alternative to Sex

Plant breeders through classical sexual hybridization have contributed significantly to agriculture. High-
lysinecorn, high-protein rust-tolerant wheat, and new varieties of fruits are all examples of successful
breeding and selection. Generally, any single genus contains an adequate gene pool which, if in the right
combination(s), could produce nearly any desired quality (qualities). Often however, a combination of
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"qualities" may be found only in species which are not sexually compatible, i.e., neither seeds nor embryos
form (Melchers, 1992). Historically, plant breeding has been restricted to either pollination of one plant with
another or the reciprocal cross, producing seeds which, when sown, germinated and produced plants.
Evaluation of the seedlings could require more than 10 years at considerable expense to the grower.
Occasionally, no seeds resulted from failure of ovule development.

Incompatibility sometimes resulted from ploidy ( chromosome number) where the pollen haploid number
was not the same as the ovule number. Because the gametes were of different ploidy, chromosomes did not
pair off during meiosis and no embryos formed. In other instances pollen tubes (which penetrate the stigma)
did not form sufficiently to travel the distance from the style to the ovary (Melchers, 1992). Investigators
have determined by scanning electron microscopy (SEM) that pollens which are incompatible with another
plant simply lay on the stigma and do not enter the style. It appears that the stigma may recognize given
pollen on a chemical basis, accepting what is "compatible" and rejecting what is "incompatible."

When incompatibility occurs by failure of the pollen tubes to grow to sufficient length, the plant breeder has
several alternatives. One choice is to induce the stigma to "recognize" pollen by treating the stigma with
mixed pollen (including one that is desired in the cross) or by modifying the stigma surface using paraffin
oil (power et al., 1989). Another choice is to practice style amputations close to the ovary, apply stigmatic
fluid, and pollinate. The latter procedure can be applied to any flowering plant with varying degrees of
difficulty. Often, however, seeds produced are devoid ofendosperm, requiring then the application of what is
termed " embryo culture." Embryos are cultivated on sterile nutrient agar which acts as the "endosperm" for
the growing embryo.

This technique has been noted as a means of generating interspecific hybrid crosses of the genus Lilium
(power et al., 1989). Before alternative techniques are attempted in plant breeding, various modifications of
sexual propagation should be used. They still remain the easiest and the most certain to yield results.

PRINCIPLE OF SOMATIC HYBRIDIZATION

Obtaining hybrids from widely differing species within a genus may be difficult if not impossible by sexual
means. An "alternative to sex" is now being acclaimed as a potential solution to disease tolerance, decreased
crop yields, climatic maladjustment, etc. This technique is somatic hybridization. Somatic cells are non-sex
cells. Cells from any organ of the plant theoretically can be used to form somatic hybrids (power et al.,
1989). Somatic hybridization is conducted in a sterile environment.

All reagents are either sterilized in an autoclave or by filtration with a Millipore filter. In theory the
procedures are simple to a plant or animal biochemist. For example, one could attempt to somatically
hybridize a Martagon lily with L. brownii, using either leaf mesophyll cells or cells from scale sections.
Leaves or scales from both species would be completely disinfected using hypochlorite solutions (Chlorox)
followed by thorough washing with sterile distilled water.

The organ materials are then treated separately with an enzyme mixture consisting of pectinase, cellulase,
and hemicellulase to disperse the tissue and remove cell walls. This treatment would be given separately to
both species. Under gentle agitation for 12 hours, the reaction mass becomes a viscous mixture of cell wall
debris, cell organelles, and the desired protoplasts. Under the microscope, protoplasts appear as spheres
devoid of their cell walls.

The fragile protoplasts are purified by a series of centrifugations in media-sugar solutions to remove cell
debris and the degradative enzymes (power et al., 1989). The principle of somatic hybridization is to
 Page 62 of 94

combine the protoplasts of two species and "fuse" the two protoplasts to produce a heterokaryon (a cell
containing genetically different nuclei). The process of fusion is carried out in the presence of calcium ions
either at high pH (10.5) or with the aid of polyethylene glycol (PEG) at a concentration of 25070-30%.
Often a high loss of viable cells occurs, depending upon the genus, species, age of plant materials, and
laboratory procedures, the principle problem being microbial contamination. However, with cell
concentrations (density) at 2 x 105 cells/milliliter, a number of heterokaryons are still expected (Power et al.,
1989). Following removal of the fusion media, the cells are transferred to a new medium for growth, and the
cells can be observed microscopically for fusion of the two species, viability, and for rate of growth. If the
fused cells are viable, the first mitotic division can be observed within 4 days.

If this event does not occur, death of the heterokaryon ensues. Because plants vary widely in their need for
hormones, light, and temperature, every somatic hybrid cross must entail a thorough study of growth
requirements for success. Quite often the new somatic hybrid will develop a callus tissue (undifferentiated
tissue) which must be transferred to a suitable medium for bulb growth, root development, and subsequent
growth (power et al., 1989).

Intraspecific somatic hybrids

Although the greatest efforts in somatic hybridization have been for introgression of alien germplasm into
cropplants through interspecific protoplast fusion, there has also been considerable effort at intraspecific
somatic hybridization for various purposes. Intraspecific somatic hybridization has been applied to potato,
for genetic reconstruction of tetraploids by fusion of selected dihaploids. Chase in 1963 proposed an
analytical breeding

schemes for potato and other polyploidy crops. The tetraploid genome is first reduced to the dihaploid level
by haplodization schemes, then breeding is conducted among dihaploids where segregation ratios and
inheritance of desirable quantitative traits is simpler. Finally, the tetraploid condition is restored through
sexual polyploidization, colchicines doubling or some other means. Wenzel et al., (1979) were quick to
realize that the newly developed technique would facilitate analytic breeding in potato, especially because
experience with dihaploids had revealed them to be reluctant to flower and frequently sterile, there by
prohibiting sexually combining their genomes.

Another purported advantage of this breeding strategy is possibility of eliminating deleterious alleles
harbored in the tetraploid, but revealed at the diploid level through partial breeding. Rasmussen (1995)
electrofused two potato dihaploids that exhibited resistance to the cyst nematode, Globodera pallid.
Although one dihaploid never flowered and the other was male and female fertile. The somatic hybrids also
exhibited a range of resistance to G.pallida, some combining the resistance to separate pathotypes from each
dihaploid fusion partner (Rasmussen et al., 1996).

Employing a similarity strategy, Cooper-Bland et al., (1994) found variable resistance to G.pallida among
tetraploid somatic hybrids between selected dihaploid clones. Considerably variation has been found among
intraspecific somatic hybrids regenerated from a single fusion; however, there appears to be little
cytoplasmic influence on these phenotypes (Frei et al., 1998). In other crop genera, intraspecific somatic
hybridization has been used to over come various breeding barriers.

Koyama et al., (1995) used protoplast fusion to regenerate plants from a carrot (Daucus carota L.) cell line,
selected for utilization of Al-phosphate that had been maintained in vitro for over 10 years and had lost
regenerative capacity. Protoplast fusion of this cell line with IOA-inactivated wild-type carrot resulted in
regenerable somatic calluses that were tolerant of Al-phosphate. Tamura et al., (1998) fused protoplasts of
 Page 63 of 94

two hexaploid (2n=6x=90) cultivars of Japanese persimmon that produced only female flowers to obtain
dodecaploid (2n=12x=180) somatic hybrids. Sexually incompatible cultivars of hexaploid sweet potato
[Ipomoea batatas (L.) Lam.] Were also combined into dodecaploid (2n=12x=180) somatic hybrids
eliminated to have limited pollen fertility (Wang et al., 1997).

Protoplasts of two cultivars of taro (Colocasia esculenta schott) have been electrofused to develop
autotetraploid somatic hybrids (Murakami et al., 1998). The utility of these highly polyploid intraspecific
somatic hybrids has yet to be demonstrated. It may well depend on the efficacy of chromosome reduction
procedures such as anther culture or pseudogamy in these species to generate useful variation for breeding
programs.

Interspecfic somatic hybrids

The majority of somatic hybridization has been conducted to obtain interspecfic somatic hybrids.
Occasionally sexual hybrids are possible, but generally the objective is to transcend breeding barriers (Guo
et al., 1998).

Intergeneric Somatic Hybrids

Intergeneric somatic hybrids generally bridge a much wider taxonomic gap between fusion partners than
interspecific somatic hybrids. In many cases, regeneration of hybrid callus has been impossible or only weak
sterile plants have resulted. However, in other cases, viable hybrids with evidence of at least partial genome
transfer resulted (Guo and Deng, 1999.

ADVANTAGES, LIMITATIONS, AND CONSTRAINTS OF SOMATIC HYBRIDIZATION

While the concept of somatic hybridization (protoplast fusion) is over 80 years old, the method still has not
found widespread application in the development of new plant hybrids. However, Government, university,
and corporate research units are examining the potentials of such methods. Somatic hybridization is not
"genetic engineering." While nuclei fuse, as do the cell walls, the method itself usually does not involve the
infusion of outside genes or genomes into the chromosomes (recombinant DNA).

The method involves changes at the cellular level where chromosomes are either compatible at nuclear
fusion or incompatible (Bajaj, 1989). The method of somatic hybridization is not intended to replace sexual
propagation; it is intended for use when sexual hybrids cannot be made because of the incompatibility of
widely divergent species. Indeed, somatic hybrids would not be expected to be any more fertile than
interspecific hybrid crosses obtained by sexual means.

Moreover, the hybrids may prove to be lacking in utility as garden or commercial plants. Nevertheless, the
need for plants with specific qualities, heretofore unavailable from classical breeding, may be achieved
through somatic hybridization (Bajaj, 1989). It is tempting to speculate what diverse crosses could arise
from heterokaryon. Each genus and each species offers a challenge in basic research to determine specific
growth requirements for the fused cells.

The technique itself, no doubt, depletes the system nutrients aIt may further damage cell components, called
organelles, such that the cells cannot divide normally. As scientific investigation determines the best
possible methods for cell fusion, nutrition, and subsequent growth, somatic hybridization will take off as a
method. In the meantime, the plant breeder is admonished to rely on standard breeding methods, with some
modification, and develop plants which have useful qualities—disease tolerance, flower colour and form,
glasshouse forcing capability, etc.
 Page 64 of 94

CONCLUSION

Most of somatic hybrids must be backcrossed to the adapted parent in order to contribute cultivar
development. Of critical importance, therefore, is the elucidation of somatic hybrid chromosomal behavior
during sexual crosses. Due to rapid elimination of chromosomes through backcrossing, it is important that
homologous paring occurs in somatic hybrids so that opportunities for intergenomic recombination arise.
Such paring is likely to happen in interspecific and even intergeneric somatic hybrids.

However, combinations of more phylogenetically distant plant species (intertribal somatic hybrids) may not
be useful for transferring nuclear traits due to a lack of homologous paring. Such fusion may be more useful
for creating novel nuclear-cytoplasmic combinations often important for the development of CMS systems.
Transgenic plant production has certainly overwhelmed somatic hybridization as a technique heavily utilized
by plant breeders and geneticists over the past decade.

This is understandable considering that transgenic modification theoretically affects only a single trait of
interest without introducing innumerable extraneous genes of uncertain agronomic influence. The
opportunity of genetic revision of existing cultivars is afforded by genetic engineering. Conversely, somatic
hybridization involves introgressing traits of interest into crop plants without any prior knowledge of their
genetic control. Resulting somatic hybrids are generally not expected to be ready for cultivar release without
additional breeding effort to acquire the trait of interest in a more acceptable genetic background resembling
current cultivars.

However, an advantage of somatic hybridization compared to genetic engineering concerns the controversy
surrounding genetically modified organisms, a stigma that does not apply somatic hybrids. Current
restrictions that hinder the release of transgenic crops are avoided using somatic hybrid plant material. The
impact of somatic hybridization can be expected to be much quieter than that of genetic engineering because
traditional breeding must intervene between the biotechnological events (protoplast fusion) and the release
of a product for market.

Valuable resistances to various pathogens or environmental stresses have been identified in direct somatic
hybrids and many have been entered into current plant breeding programs and plant hormones necessary for
meiosis and subsequent mitosis.
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Androgenic haploids: Factors controlling development and its application in crop

Improvement
Androgenesis in flowering plants is a unique biological process. It provides an understanding of the
biological basis of single-cell microspore embryogenesis to the production of a dihaploid plant. This system
provides an unparalleled opportunity to shorten the breeding cycle and fix agronomic traits in the
homozygous state, such as recessive genes for disease resistance. The most desirable dihaploid variation in
all the major crops including rice, wheat, barley, maize, rape, cotton, sunflower, coffee, etc. has already been
developed and utilized in modern crop breeding. Many known and a few unknown factors are involved in
such development. A few noteworthy factors are donor plants, genotypic variation, media composition, and
handling of cultures, which may have a greater influence on the response of androgenesis. A further
opportunity has arisen to use a pollen-specific gene, promoter and transgenic dihaploid (homozygous), gene
expression, proteomics, translational regulation and post-translational modification of genes to widen the
scope of crop improvement. The homozygous (isogenic) lines will provide unique genetic material for
mapping populations for use in functional genomics and molecular breeding.

MALE reproductive processes take place in the stamens in flowering plants. The diploid cells undergo
meiosis and produce haploid male spores or microspores. In general, microspores divide mitotically and
differentiate into multicellular male gametophytes or pollen grains. The principle of androgenesis is to arrest
the development of the pollen grains (male gametophytes) and to force them towards a somatic pathway
(Figure 1). In vitro androgenesis can be achieved from the microspores, leading to the formation of haploids
either by direct embryogenesis or via callus formation. The callus-derived plants are generally undesirable
as they exhibit genetic variation and polysomy. Anther culture is the main technique for haploid induction in
crop improvement. Culture of whole or parts of inflorescences has helped simplify the technique1–4.
Another alternative is to culture isolated or shed microspores. However the reports on isolated microspore
culture are rather limited5–9 in majority cases, the in vitro response of microspores is observed within the
anthers. Since the beginning of modern plant breeding practices, intensive efforts have been made to speed
up the production of homozygous lines, which normally requires at least six inbreeding generations. The
starting material for the production of homozygous lines in just one generation is the haploid gametes. From
the time of accidental but immensely valuable discovery of androgenic haploidy in 1964 by Guha and
Maheshwari10 and production of rice haploids in 1968 by Niizeki and Oono11, impressive advances have
been made in several laboratories throughout the world. The main advantage of using haploids is the rapid
and complete homozygosity of the offspring, which allows an easy selection of phenotypes for quantitative
characters. As it is impossible to cover the entire work on advances in research on haploidy in crops, I have
considered summarizing some important factors that influence the process of androgenesis and give some
examples from major crops and from our own research dealing with androgenesis in cereals.

Donor plants

The unknown quality of donor plants decisively influences androgenesis. The sample of microspores, the
release of microspores from the anther and their subsequent divisions leading to plant regeneration often
depend on the conditions under which the donor plants grow in a particular environment. Donor plants of
wheat and barley grown during October–December provided an excellent microspore response (personal
experience during my work at Grünbach, Germany, 1985–86). In the rice crop, plants grown during the dry
season have provided the best microspore response (personal experience at IRRI during 1993– 2000). Under
optimized conditions of a phytotron with controlled light, temperature, and humidity, which enable plants to
 Page 66 of 94

maintain a healthy growth with disease and pestfree status, rice, wheat, and barley plants yield a high degree
of success of anther culture response with reproducible results.

Figure 1. Schematic representation of microspore androgenesis

Genotype response and environmental effect

A genotype grown in a particular environment plays an important role in androgenic response. Many crop
genotypes are quite recalcitrant in their in vitro response. Several studies indicate that such a response is
influenced by gene combinations, which will be discussed later. A few detailed studies have been made on
the genetic control of microspore response of wheat12, barley13, rice14–16, and maize17–19 (Figure 2).

Microspore stage

In most cases, the early uni- to mid-uninucleate stage of microspores is the most suitable for androgenic
response4,6,8,20,21. The anthers of maize containing microspores in the late uni- to early-binucleate stage
have been found to be most responsive22. In dicot species, unicellular to early bi-cellular pollen stage is
suitable for microspore embryogenesis (e.g. Brassica napus)23.

Pre culture treatment

The induction of microspores to sporophytic instead of gametophytic pathway is strongly influenced by

some kind of stress treatment of the anthers before culture. The response to chilling or heat treatment is also
genotypedependent. However, a temperature shock has been reported to improve the androgenetic response
in many plant species24,25. Starvation of anther culture in sugar-free medium before release of microspores
induces better anther culture response26. Nevertheless, such procedures have to be optimized for each plant
species. For example, some indica rice cultivars do not require any cold or hot treatment prior to culture to
induce androgenesis (unpublished data), whereas 10 days, cold treatment at 8oC for wheat, and 8–28 days,
cold treatment at 40°C for some genotypes of barley are very useful. However, genotype is the most critical
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factor in obtaining good microsporogenesis irrespective of cultivars/varieties used under certain culture
conditions27.

Culture media

The nutrient medium not only provides nutrition to the microspores but also directs the pathway of embryo
development. It is critical to change the composition of the media or replenish them to keep the balance of
micronutrients and maintain the pH. The pH of the media, particularly liquid media, changes dramatically
with time at the onset of embryo development7. Two familiar basal media, the chemically defined N6
medium28 and the MS medium29, have been generally used with modifications. Anthers (ca. 30) were
floated on the surface of 10-ml aliquots of media in 50-mm Sterilin plastic dishes or 10–15 anthers were
cultured in 24 wells containing 1.5-ml of the media. It has been established that the nitrogen composition of
the culture medium plays a significant role in androgenesis30. Increasing glutamine and decreasing
ammonium nitrate enhance embryo development in many cereal species6,21,27,31–33. Higher concentration
of sucrose showed better microspore-embryogenesis responses in wheat6. Addition of Ficol in the liquid
medium improved plant regeneration of barley34. Further modification of the media and use of Ficol 400
were shown to promote the rate of haploid induction in barley and wheat6,7,35. The use of maltose instead
of sucrose has dramatically enhanced embryo induction and plant regeneration in cereals. However, the
concentration needs to be suitably adjusted for each crop. Since the use of maltose in the liquid medium of
barley by Hunter31, several groups to improve cereal androgenesis, protoplast culture, and plant
regeneration have advocated the advantage of using maltose. The use of abscisic acid in the media36 or use
of potato media37 could induce greater incidence of androgenesis in rice than standard media. Osmotic
pressure of the medium may play an important role in the maturation of microspore-derived embryos.

Methods

The procedures for treating microspores in the medium for inducing androgenesis can be broadly classified

into four categories. The simplest one is to culture the inflorescences containing mid- to uninucleate
microspores in the liquid or solid medium. A few such reports, including on barley1,4, indicate that direct
embryos can be obtained from the microspores originated from the anthers of the spikelets. The second
method refers to the shed pollen culture, in which anthers are cultured on the liquid/agar medium. After a
few days, microspores are shed freely from the anthers and they divide and develop further in major cereals.
Often such microspores start division within the anthers even before they are released. On the top of the
liquid medium containing Ficol, such embryos float and often produce direct plant regeneration6–8,33–35.
 Page 68 of 94

The third method involves mechanical isolation of the microspores, which can be cultured as protoplasts.
However, the response of plant regeneration is limited in this procedure9,22,38– 41. The fourth method is to
culture the anther on the surface of the agar medium. Orientation of anthers is not important in regulating
embryo development. Anthers may be removed by hand individually or by using a suction pump and plated
on the agar medium42.

Ploidy level

The haploid set of chromosomes in microspore-derived plants becomes spontaneously doubled under culture
conditions. However, the percentage of doubling varies among the crops, including different genotypes of a
cultivar. So far, barley showed the highest incidence of spontaneous doubling (up to 87%)38, followed by
rice (up to 72%)43, wheat (up to 50%)44, and maize (6.3% and quite unpredictable)45. In general,
colchicine is used for chromosome doubling at the whole-plant level for barley, rice, and maize. Sometimes,
colchicine is used in the medium for chromosome doubling in wheat21. Colchicine employed in the medium
before the first microspore mitosis can contribute to a significant increase in gametophytic chromosome
number. The microspore undergoes its first mitotic division, followed by endomitosis or nuclear fusion,
resulting in dihaploid plants20,33,46,47 (Figure 1). Direct microspore embryogenesis leading to fertile plant
regeneration in barley and wheat is obviously influenced by the specific pathway combining with
spontaneous doubling of the chromosomes. Microspore-derived calli and embryoids often show aneuploids,
dihaploids, and polyploids48.

Plant regeneration

Many factors are involved in obtaining regeneration of fertile green plants from cultured microspores
(Figure 2). The kind of nutrient media, genotype, culture vessels, condition of donor plants, carbohydrate
sources, phytohormones, reduced nitrogen (glutamine), and handling of cultures are some of the important
factors that influence microspore embryogenesis. Individual factors become critical in the induction of
microspore embryogenesis, such as co-culture of the ovary or ovary-conditioned media for wheat6.
However, such factors may not influence genotypes having an inherent potential for high-frequency plant
regeneration, such as in vitro friendly barley genotype ‘igri’, which yields 50 green plants per cultured
anther38. However, it is to be emphasized that one noteworthy concept of embryogenic culture developed
from immature embryos popularized by Indra Vasil which is very helpful in looking forward to the objective
of cell culture development49. Many changes occur in the culture from day 1 to subsequent subculture. A
thorough observation of the cultures, from induction to embryo development is critical. Replenishing the
media to avoid depletion of some essential micronutrients and balancing the pH often helps in the
conditioning of the cultures and their further development. Embryo maturation is another critical stage, as
the developing cereal embryos must be transferred to a regeneration medium at the right time, lowering the
carbohydrate concentration and increasing relative cytokinins levels to auxins. A simple regeneration
medium works very well for wheat and barley as is evident from the germination of encapsulated
microspore-derived embryos of barley50 and wheat33,35.

Molecular understanding of albinism

Albinism is a common feature of microspore-derived plantlets. What factors affect the extent of albinism?
Genetic background of the donor plants is an important factor. Cold pretreatment in general and the use of
Ficol in the liquid medium may delay or arrest nuclear synchronization and help in producing green plants.
It is evident that albino rice plants devoid of 23S and 16S rRNA51 and albino barley plants do not contain
mature chloroplasts52. In general, albino plants (e.g. wheat, barley, and rice) contain deleted forms of the
plastid genome53–55. The size and location of the deletions differ among plants. The results indicated that
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some albino plants lack the region coding the rbcL gene in the plastid genome. However, a more detailed
study is required to elucidate the actual cause of albinism. The use of a modified medium containing barley
starch-melibiose has resulted in considerably fewer albino barley plants56 and cold treatment also favoured
more DH-green plants6–8,31.

Haploid artificial seeds

Artificial or synthetic seeds consist of somatic embryos in a protective coating (calcium alginate). The main
purpose is to utilize the somatic embryos efficiently for conversion of plants. Production of perfect somatic
embryos is a prerequisite for the development of artificial seeds. Calcium alginate made from brown algae is
used for the gel encapsulation system. The selected embryos were mixed with sodium alginate, single
embryos were dropped into a bath of calcium salts, resulting in single somatic embryos encased in a clear,
hydrated bead. The rigidity of the gel beads protects the fragile embryo during handling. The capsule gel can
also potentially serve as a reservoir of nutrients just like an artificial endosperm. Microspore-derived
artificial seeds of barley and wheat were developed and germinated to normal plants after storing them in a
cold room35,50. Somatic embryogenesis has been reported in nearly all-major monocot and dicot species
and a few gymnosperms49,56–58.

Marker-assisted selection of dihaploids

Biotechnological tools complement breeding programmes in many ways, one of which is to be able to
identify target genes (or mapped gene of agronomic importance) with the assistance of DNA markers, a
process called markerassisted selection or MAS59. Anther culturability is a quantitative trait controlled by
nuclear-encoded genes60–62. However, earlier genetic studies on haploidy merely determined whether there
are differences in response among varieties, and whether the traits such as callus induction and plant
regeneration are heritable. With the development of MAS system, these characteristics can now be detected
at the molecular level. Quantitative trait loci responsible for culturability of anthers were surveyed and
analysed with the molecular map constructed from a population resulting from anther culture of a DH
line63,64. Parameters for four traits were callus induction, green plant differentiation frequency, albino plant
differentiation frequency and green plantlet yield frequency. All four traits displayed continuous distribution
among the DH lines. For callus induction frequency, five QTLs were identified on chromosomes 6, 7, 8, 10
and 12. Two QTLs for green plantlet differentiation frequency were located on chromosomes 1 and 9
whereas there was a major QTL for albino plantlet differentiation on chromosome 9. No independent QTL
was found for green plantlet yield frequency. These results may be useful in the selection of parents with
high response to anther culture for rice haploid breeding and in the establishment of permanent DH
populations for molecular mapping. To clarify the association between chromosomal regions showing
distorted segregation and anther culturability, the anther culturability of DH lines derived from a Japonica/
Indica cross having distorted segregation on chromosomes 1, 3, 7, 10 and 11 was examined62. One region
on chromosome 1 was found to control callus formation from microspores, and another region on
chromosome 10 appeared to control the ratio of green to albino regenerated plants. In both regions, the
Nipponbare (Japonica parent) allele had a positive effect. Three regions on chromosomes 3, 7, and 11,
however, showed no significant effect on anther culturability. Likewise, using recombinant inbred lines from
a cross between Milyang 23 and Gihobleo, QTL associated with green plant regeneration located on
chromosomes 3 and 10 were mapped65,66. The QTL on chromosome 10 was detected repeatedly using
three AC methods and was tightly linked to three markers. One of these three markers, RZ400, was able to
effectively identify genotypes with good (>10%) and poor (<3%) regenerability based on the cultivars and
two F2 populations. This marker enables the screening of rice germplasm for anther culturability and
introgression into elite lines in breeding programmes. The growth and development of plants from callus
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cultures is influenced by genes controlling the production of certain enzymes essential for the metabolism of
differentiating cells, tissues and organs. Peroxidases are metalloprotein enzymes containing porphyrin-
bound iron and are found to be associated with many physiological processes including morphogenesis. The
amount of peroxidase present in four indica cultivars and all possible F1 combinations was quantified67.
These authors found that calli with high regeneration capacity showed high values of peroxidases while
those with low amounts gave only albinos or predominant albinos with few green plants, suggesting the role
played by peroxidases in morphogenesis of anther calli. By employing isozyme markers like peroxidase, the
embrogenic as well as regeneration potentials of calli can be identified and utilized for selecting high
regenerating calli.

Dihaploids in genomics

Genomics implies DNA sequencing, the routine use of DNA microarray technology to analyse the gene
expression profile at the mRNA level, and improved informatic tools to organize and analyse such data68.
Doubled haploid (DH) lines are useful for genetic analysis, particularly quantitative traits69. QTLs affect
some important agronomic traits in cultivated rice. QTL studies have been facilitated by the development of
molecular markers using segregating populations, F2 or backcross populations. However, these studies are
difficult to replicate to obtain accurate phenotypic values for precise QTL mapping. The use of recombinant
inbred lines (RILs) provides many advantages in QTL studies but it will take a long time to develop such
populations. Recently, many studies have employed DH populations to construct genetic maps and locate
QTLs. Because DH lines are homozygous, they can be propagated without further segregation. This
characteristic feature allows for the precise measurement of quantitative traits by repeated trials and for a
reduction in the environmental component of the total phenotypic variance70. One caution though in the use
of anther cultured (AC)-derived materials is the possible distorted segregation of RFLP-markers derived DH
populations. Yamagishi and colleagues62 observed that ten and eleven of the 50 markers in two ACderived
populations showed distorted segregation ratios from the theoretical ratio of 1 : 1. Parental alleles were not
randomly transmitted from the F1 plant to the AC-derived plants. Additionally, the segregation ratios of
seven and six RFLP markers, respectively, were distorted both from the 1 : 1 ratios and from the observed
ratios in the F2 population62. The chromosomal regions involving these markers were on chromosomes 1, 3,
7, 10, 11 and 12. The percentage of the markers showing segregation distortion in the AC-derived
populations was similar to that in the F2 population. Thus, distortion in segregation does not appear to be a
major drawback in the use of AC populations for rice breeding and genetics. The importance of doubled
haploid populations in the study of quantitative traits is confirmed by Chen et al.71 demonstrating that most
gametoclonal variations among DH plants involve quantitative traits and the frequency of distinct variations
is not high. Biochemical and molecular analysis proved high degree of genetic stability of gametoclones
concluding that although AC may, to some extent, modify the performance of microspore-derived plants, it
will not dramatically affect their utilization in plant breeding and genetic engineering programmes. A good
deal of research involving dihaploids in the study QTLs can be found in the literature. One such trait is tiller
angle, which has great significance in the high yield breeding of rice; too small tiller angle reduces the
resistance to disease while big tiller angle is undesirable for high yield72. Based on the constructed linkage
map of a DH population from a female parent, which has a spreading plant type and a male parent having a
compact plant type, two major QTLs were detected on chromosomes 9 and 11, and one minor QTL on
chromosome 9. Similar studies using doubled haploids have been used in the study of QTLs for length of
top internodes, plant height and days to heading73, ratooning ability and grain yield traits74, and cold
tolerance of seedlings75. Employment of molecular genetic markers is particularly useful as an alternative
strategy to phenotype selection for rice root traits. Breeding varieties with increased root penetration ability
through hardpans and other root traits is difficult since screening numerous genotypes under field conditions
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is laborious and time-consuming. Further, soil compaction being not uniform and inconsistent throughout
rice fields makes evaluation of root traits difficult76. For studies on QTLs for rice root characteristics such
as root vitality77, constitutive root morphology such as deep root morphology and root thickness78, osmotic
adjustment, root penetration index, basal root thickness, penetrated root thickness, root pulling force, total
root dry weight, penetrated root dry weight and penetrated root length79, doubled haploid populations have
been used. A DH population is a kind of permanently stable population. Its genetic structure is fixed so it
can be grown at different times and locations for detecting QTLs and evaluating the interactions between
genotypes and environment, i.e. the phenotypic expression level of QTLs in different environments. This
technique has been applied in identification of 22 QTLs for six agronomic traits of rice in three different
locations (environments). QTLs for spikelets and grains per panicle were common across environment,
while traits like heading date and plant height were more sensitive to environment70. Doubled haploid rice
populations have also been used in the QTL studies on rice grain quality80, grain shape81, paste viscosity
characteristic82, aromatic traits83, and brown planthopper resistance81. Accumulation and fixation of
marker genes using genetic male sterile composite crosses and later on employing anther culture technique
was done by Suh and Song84. The dihaploid plants induced from the AC of the composite crossed plants
showed the segregation ratio for male sterility as well as five or six marker genes generated through this
method. Aneuploids (plants with extra chromosomes in addition to the normal haploid chromosome
complement) are usefulfor genetic research; for example, for investigating genic imbalance caused by extra
chromosomes at the haploid and diploid levels and for studying chromosome behavior in meiosis and rice
genome construction85. It has been difficult to produce aneuploids in rice, but through anther culture,
haploid plants with one extra chromosome (n + 1) have been obtained. Like-wise, aneuploids and
tetrasomics have been derived from anther culture of trisomic rice plants85,86. These aneuploids could be
used to assign DNA markers to individual chromosomes. Meiotic behaviour and morphological features of
auto-pentaploid rice plant derived from anther culture have also been investigated for genetic and
cytological studies87. The new tools of marker assisted breeding such as Restriction Fragment Length
Polymorphisms (RFLP) and Random Amplified Polymorphic DNA (RAPD) have been used very effectively
in combination with DH-lines for molecular genome analysis for major crop species. RFLP-markers for
different resistance genes have been identified in barley88 including the resistance gene ym4 (ref. 89).
Furthermore, concerning ym4 an isozyme as well as different RAPD markers including the very tightly
linked marker OP-Z04H660 is known. However, this primer exhibiting an additional band of about 660 bp
in susceptible lines shows a quite complex banding pattern. Furthermore, as OP-Z04H660 is inherited in a
dominant manner and linked to the resistance allele in repulsion phase, it does not facilitate the identification
of heterozygous susceptible plants in F2 which will segregate resistant plants in the offspring. Therefore,
although OP-Z04H660 has to be considered well suited for marker-assisted selection in DH lines,
experiments were conducted in order to convert it in a more specific marker discriminating between
homozygous and heterozygous genotypes.

Genes controlling androgenesis and pollen-specific genes

Regeneration of cultured microspores of crops is a heritable characteristic. Genetic determination of in vitro

regenerability appears complex. Additive gene effects explain most of the variations observed in different
genotypes but cytoplasmic influences and non-additive gene actions also play significant roles90. Most
cereal genotypes are recalcitrant in androgenesis with a few exceptions. This suggests that the genetic
factors govern the potential degree of in vitro androgenesis. Several groups were working in maize to map
the genes affecting a high response to androgenesis, using RFLP analysis of the microspore-derived cultures
of several F1 hybrids. Certain regions of the chromosomes appear to be associated with the formation of
embryo-like structures91. All limited attempts determining gene number have concluded that only a few
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genes are involved91–94. Further studies are required to finely map the regions that are highly associated
with the anther culture response. Many DH lines, at least in barley, rice, and wheat, are available. Genetic
and molecular maps including quantitative trait loci of major cereals (rice is in the advanced level) are
becoming available. More precise location of such genes and their cloning could lead to further use in cereal
breeding. The most intensively studied pollenspecific gene is Zm-13 from maize, which is shown to be
expressed in the late stages of microspore development95. A similar gene, PS1, has been cloned from rice
and is shown to be expressed in rice96.

Transgenesis

Dividing microspores can be used to develop an embryogenic cell suspension that is eventually used as a
source of totipotent protoplast for genetic transformation. The first homozygous transgenic indica rice was
reported to use such haploid microspore culture97. A few more reports are now available in rice98 and
barley99. Microinjection could be a powerful tool for introducing DNA into the nucleus of a potential
microspore. Transgenic plants could be obtained as shown in Brassica100. However, except for transgene
expression, this method did not produce any transgenic cereals. The present author, along with Gunther
Neuhaus, German Spangenberg, Karabi Datta, and Ingo Potrykus at ETH-Zürich, Switzerland, worked
intensively for 3 years using thousands of potential dividing microspores of cereals without success.
However, rice androgenesis has been successfully exploited through anther culture of primary transgenic
plants, thus attaining homozygosity of the transgene locus in one generation101,102 (Figure 2). The biolistic
system has been used to produce fertile transgenic barley plants using microspore cultures99. Protein
synthesis during microspore embryogenesis Biochemical analyses have been reported in several plant
species like barley, wheat, rape, rice, etc. aiming to identify the markers for embryogenesis103. Six proteins
were found differentially expressed during the later stage of pollen embryo development104. High
throughput protein sequence analysis (proteomics) using mass spectrometry may provide new insights into
protein profiling linked with the coding genes. Efficient microspore embryogenesis in Brassica napus makes
it possible to study gene expression using mRNA differential display PCR (DD-PCR) and microarray. DD-
PCR is a sensitive technique that can distinguish differentially expressed multiple gene families. Several
laboratories are now involved in identifying those genes including transcription factor genes regulating
microspore embryogenesis.

Applications

Androgenesis provides the most commonly used method for the doubled-haploid production that was
eventuallyapplied in breeding and crop improvement. Today, many improved DH cultivars have been
reported with several improved agronomic characteristics. Many improved crops including rice varieties,
especially salt-tolerant ones have been developed105–109, along with the development of other improved
cereals such as barley110, wheat44, maize111 and rape23,108.

Conclusion

Homozygous lines are of utmost importance in breeding programmes. Androgenesis supports the
development of such valuable DH lines. Recent developments in functional genomics, such as the fine
mapping of DH populations, will help elucidate the genes that confer agronomic characters as well as in
vitro responses. We should be able to use these genes in improving tissue culture regenerability of elite
desirable cultivars along with the novel traits. The ability to transform and regenerate plants represents the
most powerful tool and advancement in plant biotechnology. This process also provides identification and
greater use of recessive genes for resistance. Transgenic homozygous crops are now available using this
system. Homozygous lines provide uniform agronomic characters that can follow the stringent regulations
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required for registration under biosafety regulations of National Programmes. Dihaploids combining with
value added transgenes or using MAS favour the development of new tailor made improved crops.

Anther/Pollen Culture for Production of Haploids/Doubled Haploids

In self-pollinated crops, an inordinately long period is required to assemble desirable gene combinations
from different sources in homozygous form. Generally, it takes 8-10 years to develop stable, homozygous
and ready-to-use materials from a fresh cross of two or more parental lines. In cross-pollinated crops,
because of inbreeding depression, it becomes difficult to develop vigorous inbreds for hybrid seed
production programs. In this regard, haploids possessing a gametic chromosome number are very useful for
producing instant homozygous true-breeding lines. In addition, haploids constitute an important material for
induction and selection of mutants, particularly for recessive genes. In conventional breeding, the early
segregating-generation populations involve variation attributable to both additive and non-additive genetic
effects (Khush & Virk 2002), whereas doubled haploid (DH) lines exhibit variation only of additive genetic
nature, including additive x additive type of epistatis, which can be easily fixed through a single cycle of
selection. The elimination of dominance effects leads to high narrow-sense heritability, and availability of
sufficient seed of each DH line allows for replicated testing. Thus, in contrast to relatively 158 S. S. Gosal et
al. large segregating populations in conventional genetic studies, fewer DH lines are required for the purpose
of selection of desired recombinants. For instance, in rice about 150 DH lines derived from F1, instead of
4,000-5,000 F2 plants, are sufficient for selecting desirable genotypes. Production of haploids has also been
exploited during wide hybridization for the development of addition and substitution lines. Anther/pollen
culture is an attractive alternative for developing haploids (sporophytes with gametophytic chromosome
number). One of the very popular methods for production of haploids is anther or microspore culture.
Incubation of cultures under optimum conditions leads to growth of microspores into sporophytes. The
following parameters have been recognized as particularly important for successful anther/microspore
culture:

(i) growth conditions of donor plant,

(ii) genotype of donor plant,

(iii)pretreatment of anthers,

(iv) developmental stage of anthers/microspores,

(v) composition of culture medium, and

(vi) physical conditions during culture growth.

Anthers are cultured in liquid or on semisolid agar medium (Gill et al. 2003), where they may directly give
rise to embryoids or may lead to callus formation before differentiation. The embryoids develop into haploid
plantlets or doubled haploids in some crops (because of spontaneous doubling of chromosomes during callus
proliferation). Haploids may be treated with colchicine to obtain fertile, doubled haploid homozygous plants
for field testing and selection. Production of haploids/doubled haploids through anther culture from F1 rice
plants results in true-breeding plants in less than one year, which otherwise takes 7 to 8 generations through
conventional methods (Gosal et al. 1996). Several cultivars are either in test or have been released in rice,
wheat, maize, rapeseed, and mustard in China, Canada, Denmark, the United States and France (Guzman &
Zapata-Arias 2000; see Table 1). But in several instances, poor androgenesis, occurrence of mixoploids, and
albino plants have been the recurring problems. Attempts have also been made to produce haploids in grain-
legumes like pigeon pea (Bajaj, Singh, & Gosal 1980) and vegetable crops, including tomato (Segui-Simarro
 Page 74 of 94

& Nuez 2007). In the case of tomato, both gametophytic and sporophytic calli were produced from cultured
anthers. In vitro-induced disturbance of cytokinesis and subsequent fusion of daughter nuclei may be the
reason for mixoploidy and genome doubling during tetrad compartmentalization and callus proliferation
(Segui-Simarro & Nuez 2007). After the first report on androgenesis (Guha & Maheshwari 1964) following
this approach, haploids have been produced in more than 247 plant species and hybrids belonging to 38
genera and 34 families of dicots and monocots (http://www.biotechnology4u.com/). Hundreds of varieties
have been developed through anther culture in rice, brassica, barley, and wheat. The doubled-haploid
approach is also being used for the rapid development of QTL-mapping populations, construction of genetic
linkage maps for traits of interest, and rapid fixing of transgenes.
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Role of doubled haploids in vegetable crop improvement

Introduction

The first ambitious objective, among the millennium development goals, consists in the eradication of
extreme poverty and food shortage by the 2015 target date. Now-a-days, for fighting hunger and
malnutrition using a sustainable and low-input farming system, plant breeding rather than agro-chemistry
and mechanization seems to be able to more efficiently increase food and feed production on less land and
often in a more environment-friendly way. Particularly, recent advances in biotechnology represent a
valuable and powerful tool to enhance the efficiency and shorten the time required to reach the fixed
purposes in a breeding programme, as well as to address economic and ecological goals. Among the
biotechnological methods, haploid (H) and doubled haploid (DH) technology has long been recognized as a
valuable tool to help plant improvement. Double haploid means a plant or line obtained by doubling the
chromosome number of a haploid plant or individual. The genetic upgradation of crops through
conventional breeding approaches require longer time so there is a need to assist these methods following
certain biotechnological tools to shorten the breeding cycle and Double Haploid (DH) breeding is one such
tool which has been widely used in breeding programmes. The potential of haploidy for plant breeding arose
in 1964 with the achievement of haploid embryo formation from in vitro culture of Datura anthers (Guha
and Maheshwari, 1964, 1966), which was followed by successful in vitro haploid production in tobacco
(Nitsch and Nitsch, 1969). Many attempts have been made since then, resulting in published protocols for
over 250 plant species belonging to almost all families of the plant kingdom (reviewed in Maluszynski et al.,
2003). Double haploid technique is a valuable method for genetic cartography of complex traits viz. yield,
transgenesis and genomics. In order to obtain a DH, two main steps should be usually considered

1. Induction of haploid

2. Doubling of chromosome number of the haploid individual

Haploid cells or the plants that contain a single complete set of chromosomes or individuals having
gametophytic chromosome number in its sporophyte.

Why there is need to develop doubled haploids?

1. For development of homozygous lines which are used in hybrid seed production.

2. For fixation of heterosis:-doubled haploids are required because are more homozygous as compared to
conventional breeding methods.

3. For mutational studies and easy to induce mutation by chromosome doubling which can be done by
colchicine treatment so there is induction of mutation.

4. For production of biotic and abiotic stress resistant plants.

Heterosis can be fixed. So by choosing parents of different biotic and abiotic stress resistance we can select
resistant plants.

5. For cytogenetical research.

6. For induction of genetic variability at haploid level.

7. For evolutionary studies.

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8. For genome mapping as genetic maps are very important to understand the structure and organization of
genomes from which evolution patterns and syntenic relationships between species can be deduced. DH
populations have become standard resources in genetic mapping for species in which DHs are readily
available. Doubled haploid populations are ideal for genetic mapping. It is possible to produce a genetic map
within two years of the initial cross regardless of the species. Map construction is relatively easy using a DH
population derived from a hybrid of two homozygous parents as the expected segregation ratio is simple, i.e.
1:1. Doubled haploid plant is a plant in which the plant cells contain two gene sets which are exactly
identical. But in case of other plants cells contain two gene sets which are almost identical (but not exactly).
Doubled haploids helps in accelerating breeding as plants which are selected from a doubled haploid
population always breed true whereas those plants which are developed through conventional breeding
method donot breed true. In case of conventional breeding method there is production of disease resistant
high quality & poor quality, disease susceptible high quality & poor quality plants from disease resistant
high quality plants. It takes 10-12 generations until all off-springs breed true to type whereas in case of
doubled haploid plants there is production of disease resistant high quality plants and it takes only 3-5 years
earlier than conventional breeding method.

Methods for haploid production

1. In vivo method

2. In vitro method

In vivo method: This includes other methods like distant hybridization crosses followed by chromosome
elimination, bulbosum technique, parthenogenesis and Inducer based approach. Again parthenogenesis
include pseudogamy, semigamy and apogamy.The possibilities to use interspecific hybridisation between
Brassica napus and some wild species in the family of Brassicaceae for introduction of resistance to the
important fungal pathogens is presented. A large collection of wild relatives as resources of resistance to
Leptosphaeri amaculans, Alternaria brassicola, A. raphanin and Plasmodiophora brassicae has been
screened. Successful hybridisation with Brassica napus has been achieved using Brassica elongata, B.
fructiculosa, B. souliei, Diplotaxis tenuifolia, Hirschfeldia incana, Coincya monensis and Sinapis arvensis.
The ‘bulbosum’ method was the first haploid induction method to produce large numbers of haploids across
most genotypes and quickly entered into breeding programmes. Inducer based approach means the haploid
inducing lines have been used in maize to produce haploids by development of unfertilized egg cells.
Haploid seed induction rate of Inducer lines – 8-12 %.

Apogamy: Development of sporophyte directly from gametophyte, without fusion of gametes; such
sporophytes have the same chromosome number as the gametophyte from which they have been derived
(apospory, diplospory).
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Parthenogenesis: A haploid embryo develops from the haploid egg.

Pseudogamy: Pollination serves as a stimulus for embryo development but the egg and sperm nuclei do not
fuse. Fusion of the polar nuclei with one of the sperm nuclei may occur to produce endosperm.

Semigamy: The haploid sperm nucleus enters the egg but does not fuse with the haploid egg nucleus. Each
nucleus divides independently creating a haploid embryo that contains sectors of male and female origin.

In vitro method: This includes androgenesis and gynogenesis. Androgenesis is the process of induction and
regeneration of haploids and double haploids originating from male gametic cells. Due to its high
effectiveness and applicability in numerous plant species, it has outstanding potential for plant breeding and
commercial exploitation of DH. For example, Brassica spp. In vitro induction of maternal haploids, so-
called gynogenesis, is another pathway to the production of haploid embryos exclusively from a female
gametophyte. It can be achieved with the in vitro culture of various un-pollinated flower parts, such as
ovules, placenta attached ovules, ovaries or whole flower buds. Although gynogenetic regenerants show
higher genetic stability and a lower rate of albino plants compared to androgenetic ones, gynogenesis is used
mainly in plants in which other induction techniques, such as androgenesis and the pollination methods
above described, have failed. Gynogenic induction using unpollinated flower parts has been successful in
several species, such as onion, sugar beet, cucumber, squash, gerbera, sunflower, wheat, barley etc. but its
application in breeding is mainly restricted to onion and sugar beet. The success of the method and its
efficiency is greatly influenced by several biotic and abiotic factors. The genotype of donor plants,
combined with growth conditions, is the crucial factor. In onion, for example, pronounced differences in
embryo yields have been recorded among accessions and among plants within accessions.

Factors affecting haploid induction and subsequent regeneration of embryos

1. The genotype of the donor plants affects the haploid induction and also the subsequent regeneration
of embryos.

2. Physiological condition of donor plants that is growth at lower temperature and high illumination.

3. Developmental stage of gametes, microspores and ovules.

4. Pre-treatment that is cold treatment of inflorescences prior to culture, hot treatment of cultured
microspores.

5. Composition of the culture medium including culture on “starvation” medium low with carbohydrates
and/or macro elements followed by transfer to normal regeneration medium specific to the species.

6. Physical factors during tissue culture like light and temperature.

Chromosome Doubling

Double haploids can occur spontaneously, but in most cases chromosome doubling of haploids is required to
restore fertility. This is achieved by the use of antimicrotubuleagents. Haploid plant may grow up to a
flowering stage, but viable gametes cannot be formed due to lack of pairing partner of homologous
chromosomes in meiosis. Consequently there is no seed formation. Mechanisms of spontaneous doubling
differ, with nuclear fusion being the most common cause. As first described by Sunderland et al. (1974)
[12], synchronous division of two or more nuclei in early stages of embryo development might develop a
common spindle. The nuclear fusion theory is supported by the frequent occurrence of a small proportion of
triploid regenerants. Nuclear fusions might be associated with delayed cell wall formation, which, as
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reviewed by Kasha (2005). Chemical treatment might be avoided by using in vitro adventitious somatic
regeneration, which itself frequently leads to increased ploidy. Such an approach was efficient in onion
(Alan et al., 2007) [1]. The method has two advantages: the first being that no potentially damaging
chemicals are used in the process and the second that regenerants do not for the most part show a mixoploid
character. Up to 100% doubling efficiency in relation to individual line treatment can be achieved using this
method (Jakse et al., 2010).

Methods of chromosome doubling (Diploidization)

1. Endomitosis: Endomitosis is described as chromosome multiplication and seperation but failure of spindle
leads to one restitution nucleus with chromosome number doubled. It has also been called ‘Nuclear
Restitution’.

2. Endoreduplication: Endoreduplication is a phenomenon of DNA or Chromosome doubling without

Cytokinesis.

3. C-mitosis: C-mitosis is nothing but endomitosis under the influence of colchicine.

4. Nuclear fusion: it occurs when two or more nuclei divide synchronously and develop a common spindle.
Thus, two or more nuclei could result with doubled, polyploid or aneuploid chromosome number.

Other chromosome doubling agents

1. Acenanaphthene
2. Chloramphenicol
3. Nitrous oxide
4. Parafluorophenyl alanine
5. 8- hydroxyquinone
6. Colchicine

Colchicine: It is analkaloid isolated by French chemists P.S. Pelletier and J. Caventon in 1820. It is a toxic
natural alkaloid and secondary metabolite, extracted from plants of the genus Colchicum (autumn crocus,
Colchicum autumnale, also known as "meadow saffron"). It is extracted from seeds and corms of
Colchicum. Increase fertile plant and reduce albinism in anther culture. The Systematic (IUPAC) name for
colchicine is C22H25NO6.Colchicine inhibits microtubule polymerization by binding to tubulin.
Availability of tubulin is essential to mitosis, and therefore colchicine effectively functions as a "mitotic
poison" or “spindle poison”.

Methods of colchicine application:

1. Seed treatment (0.001 to 1 %; 0.2 % is more common)

2. Germinating seed treatment
3. Growing shoot apex (0.1 to 1% Colchicine)
4. Treatment of growing point in the cotyledonary stage
5. Colchicine in glycerine (0.2 - 0.4% colchicine in 10% glycerine)
6. Colchicine in emulsion (0.2 - 0.4% colchicine)
7. Colchicine in agar (1% colchicine and 2% agar mixed in equal parts)
8. Among all methods of colchicine application, shoot apex treatment at the seedling stage is most
effective.

Checking of ploidy level

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After treating with colchicine checking the ploidy level of colchicine treated plant is important. This can be
achieved by using ploidyanalyser, flow cytometry, morphological observation, indirect method based on
guard cells. Several direct and indirect approaches are available for determining the ploidy level of
regenerated plants. Indirect approaches are based on comparisons between regenerated and donor plants in
terms of plant morphology (plant height, leaf dimensions and flower morphology), plant vigour and fertility,
number of chloroplasts and their size in stomatal guard cells. They are fairly unreliable and subject to
environmental effects but do not require costly equipment. Direct methods for ploidy determination are
more robust and reliable and include conventional cytological techniques, such as counting the chromosome
number in root tip cells (for a protocol, Maluszynska, 2003) [9] and measurement of DNA content using
flow cytometry (for a protocol, Bohanec, 2003) [2]. The latter provides a rapid and simple option for large-
scale ploidy determination as early as in the in vitro culturing phase. It also enables detection of mixoploid
regenerants (having cells with different ploidy) and the determination of their proportion. Flow cytometry is
a laser-based, biophysical technology employed in cell counting, cell sorting, biomarker detection and
protein engineering, by suspending cells in a stream of fluid and passing them by an electronic detection
apparatus. It allows simultaneous multiparametric analysis of the physical and chemical characteristics of up
to thousands of particles per second.The latter provides a rapid and simple option for large-scale ploidy
determination as early as in the in vitro culturing phase. It also enables detection of mixoploid regenerants
(having cells with different ploidy) and the determination of their proportion. In the past, evaluation of
regenerants mainly relied on phenotypic markers, progeny testing after self-pollination and isozyme
analysis. Nowadays, DNA molecular markers, such as AFLP (Amplified Fragment Length Polymorphism),
RAPD (Random Amplified Polymorphic DNA), SCAR (Sequence Characterized Amplified Regions) or
SST (Simple Sequence Repeat), are commonly used for homozygosity testing and assessment of plant
origin. There is a considerable difference in interpretation between dominant or co-dominant electrophoretic
profiles. Co-dominant molecular markerwell as isozyme markers, have the advantage that a single locus,
when heterozygous in donor plants, might be used for homozygosity determination. In contrast, a more
complex profile is analyzed with dominant markers. In such a case, bands missing from the donor profile
indirectly indicate homozygosity. An approach is used in potato, in which selection is based on a
homozygous dominant colour marker gene carried by the pollinator line (Maine, 2003) [8]. The purple spot
embryo marker shows up on seeds whose embryos possess a genome from the pollinator. Those hybrid
seeds are discarded, while spotless dihaploid seeds are included in breeding process. Selection can be
repeated at the seedling stage, when a purple nodal band can be detected on the hybrid’s stem. In the case of
both maize and potato selectable markers, it is not possible to distinguish hybrid seeds resulting from
unintentional self-pollination of donor plants. Selection has to be supplemented with other morphological or
molecular markers. A fast and reliable haploid identification method is needed for large scale production of
DHs. Morphological markers expressed at the embryo, seed or early seedling stages are preferentially used.
Genetics of DH population

In DH method only two types of genotypes occur for a pair of alleles, A and a, with the frequency of ½ AA
and ½ aa, while in diploid method three genotypes occur with the frequency of ¼ AA, ½ Aa, ¼ aa. Thus, if
AA is desirable genotype, the probability of obtaining this genotype is higher in haploid method than in
diploid method. If n loci are segregating, the probability of getting the desirable genotype is (1/2) n by the
haploid method and (1/4) n by the diploid method. Hence the efficiency of the haploid method is high when
the number of genes concerned is large. Studies were conducted comparing DH method and other
conventional breeding methods and it was concluded that adoption of doubled haploidy does not lead to any
bias of genotypes in populations, and random DHs were even found to be compatible to selected line
produced by conventional pedigree method.
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Applications of DH in plant breeding

1. Development of homozygous inbred line and cultivars in self-pollinated crops it can be released directly
as a cultivar and in a cross pollinated crop it can be used as an inbred line.

2. It gives an immediate product of stable recombinants from species crosses or fixation of heterotic
combination.

3. There is no masking effects because of high homogenity.

4. High efficiency is seen in stacking specific targeted genes in homozygous line.

5. There is an increased performance per se due to selection pressure in the haploid phase or during first
generation of DHs.

6. Simplified logistics for seed exchange between main and off season programmes since each line is fixed
and can be represented by a single plant.

7. Development of substitution and addition lines.

The induction and regeneration of haploids followed by spontaneous or induced doubling of chromosomes
are widely used techniques in advanced breeding programs of several agricultural species. They have been
successfully used for commercial cultivar production of species such as asparagus, eggplant, melon, pepper
and more than 290 varieties have already been released. Using DH technology, completely homozygous
plants can be established in one generation thus saving several generations of selfing comparing to
conventional methods, by which also only partial homozygosity is obtained. It should be noted that,
following chromosome doubling, DH plants are normally selfed for maintenance and for further
multiplication. In cross-pollinated species with strongly expressed self-incompatibility, various techniques
are used to overcome the incompatibility reaction. For instance in Brassicas, bud pollination is enhanced by
treatment in a CO2 enriched atmosphere (Nakanishi & Hinata, 1973) [10] or by application of gibberelic
acid, sodium chloride, urea or ammonium sulphate on stigmas. Alternatively, DH lines might be clonally
propagated, in which case micropropagation is often the best choice. Mutation breeding is another area of
plant improvement for which doubled haploid techniques can help to accelerate the process. Homozygosity
of regenerants and true breeding propagation enables the fixation of mutations in the first generation after
mutagenic treatment. All mutated traits are immediately expressed, allowing screening for both recessive
and dominant mutants in the first generation without the need for self-pollination. The first option is, that
mutagenic treatment is applied to dormant seeds that, on germination and flowering, produce M1 gametes,
which are used as donor material for haploid culture. The second option relies on mutagenic treatment of
haploid cells in vitro. The mutagenic agent is usually applied soon after microspore isolation at the
uninucleate stage, before the first nuclear division in order to avoid heterozygosity and chimerism caused by
spontaneous diploidization through nuclear fusion. In vitro mutagenic treatment can be followed by in vitro
selection of desired traits, such as disease and herbicide resistance.

Applications of DH in genomics

1. DHs serve to recover recessives.

2. DHs ideal for the study of mutation frequency and spectra.

3. It helps in permanent mapping population.

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4. It can be repeated at any time in different laboratories by different researchers.

5. It can be used to enhance the expression level of transgene.

6. Construction of genetic maps or Gene tagging / locating genes.

7. Identification of molecular markers for trait selection.

8. QTL analysis is facilitated by using DH mapping populations and enable accurate phenotyping.

9. In DH populations, Dominant Markers are as efficient as Co-Dominant Markers.

Advantages of DHs

1. The ability to produce homozygous lines after a single round recombination saves a lot of time for the
plant breeders.

2. Studies conclude that random DH’s are comparable to the selected lines in pedigree inbreeding.

3. The other advantages include development of large number of homozygous lines, efficient genetic
analysis and development of markers for useful traits in much less time.

4. More specific benefits include the possibility of seed propagation as an alternative to vegetative
multiplication in ornamentals, and in species such as trees in which long life cycles and inbreeding
depression preclude traditional breeding methods, doubled haploidy provides news, asalternatives.

5. The induction of DH lines in dioecious plants, in which sex is determined by a regulating gene, has an
additionaladvantage. Such a case is well studied in Asparagus, in which sex dimorphism is determined by a
dominant gene M. Female plants are homozygous for the recessive alleles (mm), while male plants are
heterozygous (Mm). Androgenically produced DH lines are therefore female (mm) or 'supermale' (MM). An
advantage of supermales is that, when used as the pollinating line, all hybrid progeny are male.

Disadvantages of DH breeding technique

1. Frequency of haploid occurrence is low.

2. Success of DH method is genotype dependent.

3. Some techniques (e.g. Inducers line in maize) are proprietary and not available to all interested breeders.

4. Success is unpredictable and can consume valuable resources.

5. Health and legal concerns related to handling the doubling chemical agent.

6. Haploids from polyploid species have more than one set of chromosomes and are polyhaploids. For
example dihaploids(2n=2x) from tetraploid potato (Solanum tuberosum ssp. tuberosum, 2n=4x), trihaploids
(2n=3x) from hexaploid kiwifruit (Actinidia deliciosa, 2n=6x) etc. Dihaploids and trihaploids are not
homozygous like doubled haploids, because they contain more than one set of chromosomes. They cannot
be used as true-breeding lines but they enable the breeding of polyploid species at the diploid level and
crossings with related cultivated or wild diploid species carrying genes of interest.

Approaches for DH production: There are two approaches for double haploid production. They are anther
culture and ovary culture.
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Anther culture: The in vitro culturing of anthers containing microspores or immature pollen grains on a
nutrient medium for the purpose of generating haploid plantlets. Culturing anthers for the purpose of
obtaining Double Haploid is not easy with many field crop species, particularly with the cereals, cotton, and
grain legumes.

Procedure for Anther Culture: The immature anthers should be collected during morning time, then
inoculation can be done in protected condition (laminar air flow). After inoculation, there is proliferation of
anthers occur which results in formation of callus. Then there is development of embryo which leads haploid
plant production. After treating with colchicine transplanting of plants can be done. It results in doubled
haploid plant production.

Ovary Culture: Production of haploid individual by culture of unfertilized ovaries to obtain haploid plants
from egg cells or other haploid cells of the embryosac. The plants produced are referred as Gynogenic
Haploids. In vitro fertilization for the production of distant hybrids avoiding style and stigmatic
incompatibility that inhibit pollen germination and pollen tube growth.

Novel approaches combining DHs and molecular genetics

A simplified scheme for backcrossing has been proposed (Forster et al., 2007), aimed at shortening the
period needed for the introduction of a particular trait from donor to recipient germplasm. According to the
scheme, DHs are produced from the BC1 generation. Segregation of parental chromosomes into the filial
generation is followed by molecular markers to identify lines with only recipient chromosomes. The gene of
interest should thus be introduced into the recipient chromosome by a random crossing over event in the
BC1 generation. A protocol for “reverse breeding” was proposed by Wijnker et al. (2007). According to this
invention, superior hybrid genotypes are first identified among the segregating population. Using genetic
transformation, a gene for induced suppression of meiotic recombination is then introduced, and several DH
lines are produced. Segregation of chromosomes is followed by chromosome specific molecular markers
and a final combination of two lines represents complementary sets constituting the original heterozygous
superior hybrid.

Doubled haploid production in fruit crops

Introduction

The most widely cultivated temperate and subtropical fruit trees in the world are citrus, bananas, grapes,
apples, peaches, pears, plums, apricot and kiwis. World fruit production amounts to 497.4 million metric
tons in 2004 (FAOSTAT, Database). The main goals of research on fruit breeding are: to obtain new
varieties with a shorter juvenile non-fruiting period, an increased yield, a longer ripening season, regular
bearing, seedlessness and improved external and internal quality of the fruits. Another important aim in fruit
tree improvement research is to make available new scions and rootstocks selected for resistance or
tolerance to biotic and abiotic stresses. Fruit species breeding is based on either conventional (hybridization,
selection, mutation) or biotechnological methods employing embryo culture, regeneration from protoplasts,
somatic hybridization, in vitro mutant selection, genetic transformation and haploid production. Using an
integrated approach with both biotechnological tools and conventional ones it is possible to obtain good
results in a short time.

Importance of haploids and DH

Haploids are sporophytic plants with the gametophytic chromosome number because they originate from a
single gamete. The importance of haploids in plant breeding and genetic research was recognized with the
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discovery of the first natural haploid in Datura stramonium and Nicotiana (Blakeslee et al., 1922; Blakeslee
and Belling, 1924; Kostoff, 1929), but long before techniques for producing haploids by in vitro gametic
embryogenesis became available. The discovery by Guha and Maheshwarim (1964, 1966) that, by in vitro
culture of immature anthers of Datura innoxia, a change in the normal gametophytic development into
sporophytic development can be induced and embryos with a haploid chromosome number can be obtained,
led to further and extensive research on androgenesis. The interest of breeders in haploids or, by doubling
the chromosome numbers, DH, lies in the possibility of shortening the time needed to produce completely
homozygous lines compared to conventional breeding. In fact, haplo-diploidization through gametic
embryogenesis allows the single-step development of complete homozygous lines from heterozygous
parents. In a conventional breeding programme, pure lines are developed after several generations of selfing
and still may not be 100% homozygous. In woody plants, generally characterized by a long reproductive
cycle, a high degree of heterozygosity, large size, and, sometimes, self-incompatibility, it is not possible to
obtain haploidization through conventional methods. Actually, the absence of pure lines in woody plants
makes genetic studies rather difficult to conduct. New superior cultivars produced via gametic
embryogenesis (above all from the male gametes) have been reported for several genotypes (Evans, 1989),
and DH are being routinely used in breeding programs for new cultivar development in many crops
(Veilleux, 1994). Often in vitro regenerated plants show differences in their morphological and biochemical
characteristics, as well in chromosome number and structure. ‘‘Gametoclonal variation’’, the variation
observed among plants regenerated from cultured gametic cells (Evans et al., 1984; Morrison and Evans,
1987), is another opportunity to use haploids in crop improvement. Unlike ‘‘somaclonal variation’’ which is
related to the variation among plants regenerated from cultured cells or tissue (Larkin and Scowcroft, 1981),
gametoclonal variation results from both meiotic and mitotic division. Moreover, because of their
homozygosity, in the gametoclones it is possible to observe the direct expression of both dominant and
recessive mutations. Several different sources of variation have to be considered in order to explain
gametoclonal variation including new genetic variation induced by the cell culture procedures, new variation
resulting from segregation and independent assortment, new variation induced by the chromosome doubling
procedure and new variation induced at diploid level, resulting in heterozygosity (Morrison and Evans,
1987; Huang, 1996). Double haploids can also increase the efficiency of crop breeding programmes,
particularly of genome mapping. They, in fact, provide excellent material to obtain reliable information on
the location of major genes and QTLs for economically important traits (Khush and Virmani, 1996).

Haploids and DH production in fruit crops

Since 1970s, extensive research has been carried out to obtain haploids for fruit tree breeding through
gametic embryogenesis (Chen, 1986; Ochatt and Zhang, 1996). However, as reviewed by Ochatt and Zhang
(1996), this has not always given satisfactory results. Generally, haploids can mainly be induced by two
strategies i.e. by regeneration from the female gamete or from the male gamete.

Haploids from the female gametes

Development of spontaneous haploids

Spontaneous haploids can occur either due to parthenogenesis, i.e. the production of an embryo from an egg
cell without the participation of the male gamete, or due to apogamy, which means the production of an
embryo from a gametophytic cell other than the ovum. In 1974, Kasha reported spontaneously developed
haploids in over 100 angiosperm species. According to Zhang et al. (1990), there are also several fruit tree
species such as apple, pear, peach, plum, apricot, citrus, etc. capable to produce spontaneous haploids, but
generally in very low numbers and with low viability. In situ parthenogenesis induced by irradiated pollen
 Page 84 of 94

followed by in vitro embryo culture Parthenogenesis induced in vivo by irradiated pollen, followed by in
vitro culture of embryos, can be an alternative method of obtaining haploids in fruit crops. Gynogenesis by
in situ pollination with irradiated pollen has been successfully used for Malus domestica (L.) Borkh (Zhang
and Lespinasse, 1991; Ho¨ fer and Lespinasse, 1996), Pyrus communis L. (Bouvier et al., 1993), Actinidia
deliciosa (A. Chev) (Pandey et al., 1990; Chalak and Legave, 1997). The method is based on the in vitro
culture of immature seeds or embryos obtained as a result of pollination with pollen irradiated by gamma
rays from cobalt 60, and it should be tested in those species in which in vitro anther culture has not been
successfully applied. Irradiation does not hinder pollen germination, but prevents pollen fertilization,
stimulating the development of haploid embryos from ovules. The success of this technique is dependent on
the choice of radiation dose, the developmental stage of the embryos at the time of culture, culture
conditions and media requirements. In situ or in vitro parthenogenesis induced by pollen from a triploid
plant followed by in vitro embryo culture Pollen from a triploid plant, like irradiated pollen, germinates, but
does not fertilize and stimulates the development of haploid embryos from ovules. For example, three
haploid plants were obtained from in vivo crosses of two monoembryonic diploids (clementine and ‘‘Lee’’)a
triploid hybrid of ‘‘Kawano natsudaidai’’ (Citrus natsudaidai Hayata) (Oiyama and Kobayashi, 1993).
Haploid and diploid embryos did not show any difference in their size, however, haploid seedlings grew
very slowly in the soil. Restriction endonuclease analyses of both nuclear and chloroplast ribosomal DNA
were used to determine the maternal origin of these haploids. The in vitro stigmatic pollination technique
consists of applying pollen to the apical part of the stigma of an excised gynoecium implanted in solid
culture medium. This method was successfully applied in Citrus clementina Hort. ex Tan. (Germana` and
Chiancone, 2001). Some ovaries were transformed into brownish and friable callus, sometimes breaking to
reveal ovules. From this kind of ovary the gynogenic embryos emerged 4 or 5 months after in vitro
pollination, which is practically the same time required for regeneration from anther culture. The pollination
and mature stage of pistils were necessary for gynogenic embryo regeneration.

Haploids from the male gametes

Both basic and applied studies have improved the knowledge of pollen biology and pollen biotechnology,
making the manipulation of pollen development and function, a reliable tool for crop improvement
(Mulcahy, 1986). The most important application of pollen biotechnology in breeding and genetic studies is
the ability to obtain haploids and DH. In vitro anther or isolated microspore culture, are usually the most
effective and widely used methods of producing haploids and DH. Regeneration from male gametes has
been reported in about 200 species belonging to some families, such as Solanaceae, Cruciferae and
Gramineae (Dunwell, 1986; Hu and Yang, 1986). On the other hand, some of the members of Leguminosae
family and many woody plants are more recalcitrant (Sangwan-Norrel et al., 1986; Bajaj, 1990; Raghavan,
1990; Wenzel et al., 1995). The cellular, biochemical and molecular bases for the transformation of
microspores into pollen embryos are not yet been completely understood. However, it is already possible to
report some findings. For example, it is known that the androgenic character is genetic and inheritable, and
that the stage of microspore development is critical for induction. Usually in the period around the first
haploid mitosis (late uninucleate or early bicellular pollen stage), male gametes become competent to
differentiate in a different way from the gametophytic pathway with continued growth and division.
Moreover, external stresses need to be present to enable competent microspores to undergo androgenic
development. The stress can be physical (wounding connected to the anther excision and culture), thermal
(heat, cold) or chemical (water stress, starvation). The induced microspores are characterized by an altered
synthesis and an accumulation of RNA and proteins, and it seems that the genes involved in this
reprogramming are stress-related and/or associated with the zygotic embryogenesis.

Haploidization by anther culture

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Research on haploidization by anther culture has been carried out on several fruit trees (Ochatt and Zhang,
1996). Floral buds, with the pollen grains at a specific stage of development, are collected from the donor
plant, usually from field trees. After pre-treatment, the buds are surface sterilized usually by immersion in
70% (v/v) ethyl alcohol, followed by immersion in sodium hypochlorite solution (about 1.5% active chlorine
in water) containing a few drops of Tween 20, and finally rinsed three times for 5 min with sterile distilled
water. Petals are aseptically removed with small forceps, and anthers are carefully dissected and placed into
the medium. The stage of pollen development is commonly determined by staining one or more anthers per
bud with acetocarmine, Schiff’s reagent, or DAPI stain.

Haploidization by isolated microspore culture

Pollen culture is performed by removing somatic anther tissue. This technique, although more difficult and
laborious, is ideal for studying the mechanism of pollen embryogenesis, because it eliminates the unknown
effects of the sporophytic anther tissue, thereby allowing a greater control over the culture process. The few
reports about this method in fruit crops are regarding apple (Oldani, 1993; Ho¨ fer et al., 1999; Ho¨ fer,
2004), citrus (Germana` et al., 1996) and olive (Bueno et al., 2004). Research is in progress on microspore
culture of several genotypes: prickly pear, cherry, olive, loquat, etc. (Germana` et al., unpublished).
Investigation of isolated microspore culture of several Citrus species (lemon, orange, clementine, sour
orange, grapefruit) and a related genus (Poncirus) has been carried out (Germana` et al., 1996). After various
periods of time (1–4 months), the isolated microspores of almost all investigated. Citrus species produced
multinucleated structures and developed into small proembryos, which failed to develop any further.
Formation of ‘‘pseudobulbils’’, white or green spherical bodies, described in Citrus by Button and Kochba
(1977), has been obtained only in those genotypes (clementine and lemon) that had also produced haploids
by anther culture. Ho¨ fer et al. (1999) for the first time reported the induction of embryogenesis and plant
formation from isolated microspore of the apple cultivar ‘Rene’. In a succesive report (Ho¨ fer, 2004), the
improvement of the induction phase through the study of the pre-treatment, the concentration of carbon
source and the microspore density, has been achieved. Recently, Bueno et al. (2004) obtained sporophytic
division, multinucleate microspores and multicellular structures in isolated microspore culture of olive
cultivars Arbequina and Picual.

Haploids and DH in the main fruit crops

Citrus

Citrus species represent the largest production of fruits worldwide, with over 105.4 million tons produced
during 2005 (FAOSTAT database). All cultivated forms of Citrus and related genera (Poncirus, Fortunella,
etc.) are diploid with a monoploid number of chromosomes (n=x=9) (Frost, 1925). Triploid and tetraploid
forms of Citrus also exist. In Citrus natsudaidai haploid seedlings were first obtained by the application of
gamma rays (Karasawa, 1971). One haploid embryo was obtained in an immature seed from a diploid
(Clementine mandarin) *diploid (Pearl tangelo) cross (Esen and Soost, 1972). The production of nine
haploid plantlets, which did not survive, and two embryogenic callus lines have been obtained in clementine
(Citrus clementina Hort. ex Tan.), cv. SRA 63 after in situ parthenogenesis induced by pollen of Meyer
lemon (Citrus meyeri Y. Tan.) irradiated at 300, 600 and 900 Gray (Gy) from a cobalt 60 source (Ollitrault
et al., 1996). Flowers of clementine SRA 63 were pollinated in the field with the irradiated pollen; fruits
were picked at maturity and embryos were cultivated in vitro. Three haploid plants were obtained from in
vivo crosses of two monoembryonic diploids (clementine and ‘‘Lee’’)* a triploid hybrid of ‘‘Kawano
natsudaidai’’ (Citrus natsudaidai Hayata) (Oiyama and Kobayashi, 1993). Haploid plantlet regeneration
through gynogenesis in Citrus clementina Hort. ex Tan., cv. Nules, has been induced by in vitro pollination
 Page 86 of 94

with pollen from a triploid plant (Gemana` and Chiancone, 2001). The pollen source chosen was
‘Oroblanco’, a triploid grapefruit- type citrus obtained in 1958 through a cross between an acidless pummelo
(Citrus grandis Osbeck) and a seedy, tetraploid grapefruit (Citrus paradisi Macf.) (Soost and Cameron,
1980). With regards to Citrus and their relatives (Germana` , 2003), haploid plantlets have been recovered,
by anther culture, from Poncirus trifoliate L. Raf. (Hidaka et al., 1979) and C. madurensis Lour. (Chen et al.,
1980); one doubled haploid plantlet has been obtained from the hybrid No. 14 of C. ichangensis* C.
reticulata (Deng et al., 1992a); haploid plantlets and highly embryogenic haploid calli of C. clementina Hort.
ex Tan. (Germana` et al. 1994, 2000a, 2005; Germana` and Chiancone, 2003); haploid, but albino embryos
of ‘Mapo’ tangelo (C. deliciosa* C. paradisi) (Germana` and Reforgiato, 1997); haploid and diploid calli,
embryos and leafy structures but no green plants of C. limon L. Burm. f. (Germana` et al., 1991); haploid
embryos of Clausena excavate (Froelicher and Ollitrault, 2000) have been also achieved. In vitro pollen
embryogenesis is affected by numerous factors: genotype, the pre-treatment applied to anthers or to floral
buds, pollen developmental stage, donor plant growth conditions, culture media (macro and microelements,
carbon source, and plant growth regulators), and conditions of incubation.

Culture medium in Citrus anther culture

The diverse genotypes show very different basal medium, different carbon sources and plant growth
regulators requirements to induce pollenderived plant formation (Germana` , 1997). Usually, anther culture
media are solidified by adding agar. Other gelling agents can be potato starch (Germana` , 1997; Germana`
et al., 2000a, unpublished), gelrite (Froelicher and Ollitrault, 2000), agarose (Kadota et al., 2002; Assani et
al., 2003) and gellan gum (Kadota et al., 2002). There are different opinions regarding the use of liquid
media rather than solid media. Chaturvedi and Sharma (1985) obtained diploid plantlet regeneration by
floating C. aurantifolia anthers on a liquid medium, then embedding them in a semisolid medium. Germana`
et al. (unpublished) obtained better results in Citrus using a solid medium rather than a liquid one: in liquid
medium, citrus anthers initially swell, and later turn brown and sometimes shrivell. Activated charcoal was
beneficial to androgenesis induction of P. trifoliata (Deng et al., 1992a). However, no positive effect of
activated charcoal addition has been observed in anther culture of several Citrus species (Germana` et al.,
1994; unpublished).

Conditions of incubation of Citrus anther culture

Regarding the conditions of incubation, Chen (1985) observed that temperature seems to be more important
than light in Citrus anther culture, and they obtained embryos at 20–25 °C, especially under dark conditions.
The temperatures usually used in citrus anther culture are 25– 28 °C (Germana` , 1997). After dark
incubation, Petri dishes are usually placed under cool white fluorescent lamps with a photosynthetic photon
flux density of 27–60 lmol m)2 s)1 and a 16–18 h light photoperiod (Germana` et al., 1994, 2000a;
Germana` and Reforgiato, 1997; Germana` and Chiancone, 2003).

Embryo development from Citrus microspores and the origin of haploids

Regarding the origin of haploids, Hidaka and Omura (1989) described cytologically the development of
embryos from microspores in C. aurantium and P. trifoliata. When the nucleus divides without cell division,
a multinucleate pollen grain is initially formed which later gives rise to a multicellular structure, that
develops into a proembryo and finally into an embryo, until the exine rupture. Moreover, nuclear fusion
among vegetative and generative nuclei has been observed, and this might explain an increase in ploidy
level. Morphological and ultrastructural studies, at cellular and sub-cellular levels, of early microspore
embryogenesis in several embryogenic varieties of Citrus clementina revealed very important aspects of this
embryogenic process, indicating differences between Citrus microspores derived embryos and those derived
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from other embryogenic species, such as starch accumulation during the first embryonic stages. Moreover,
different cellular types have been observed in these embryos after the exine breakdown (Ramirez et al.,
2003). The developmental process of a plant from a single microspore is referred to as microspore
embryogenesis, although the route of regeneration may be via direct embryogenesis (Figure 1A), secondary
embryogenesis or, organogenesis. In other cases microspores in culture produce undifferentiated calli,
instead of embryos. Usually, after 1 week of culture, most of the anthers are swollen and then they start to
produce calli. Anther-derived calli can be non-morphogenic, or highly embryogenic, and they can maintain
embryogenic potential for a long time. The morphogenic calli in citrus appear friable (Figure 1B) and white.
Sometimes calli develop from two different lobes of an anther. The
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embryogenic calli differentiate into a clump of embryos (Figure 1C). Plant recovery, hardening and
characterization of regenerants obtained by Citrus anther culture Plantlet formation from cultured anthers
may occur either directly through embryogenesis of microspores or indirectly through organogenesis or
embryogenesis of microspore-derived callus. The embryogenic haploid callus is multiplied and, as the
embryos appear, they are germinated in Petri dishes. They are later transferred to Magenta boxes (Sigma
V8505) or to test tubes (Figure 1D). The well-structured pollen-derived citrus embryos develop normally
like zygotic embryos, through the globular, the heart, the torpedo and the cotyledonary stages and often
produce secondary embryos. In fruit crop anther culture, often teratomatal structures, cotyledonary-fused,
pluricotyledonary and thickened embryos are observed (Ho¨ fer, 1995; Germana` , 1997). Green, compact
and non-morphogenic calli emerging from anthers were also observed. Haploid embryos often germinate
vigorously in vitro; by contrast, haploid plantlets grow slowly in soil, presumably due to harmful recessive
genes expressed in homozygosity. These plantlets, when transplanted in vivo, frequently die as a result of
fungal contamination. In Citrus, better results have been obtained by grafting in vitro homozygous small
shoots (2–3 mm) onto etiolated 20-dayold Troyer citrange seedlings. After 3–4 months, the grafted plantlets
obtained were washed with sterile water to remove the medium from their roots and then transferred to
sterilized pots containing peat moss, sand and soil in the ratio 1:1:1 or to Jiffy pots for the acclimation phase
(Figure 1E). The new scions obtained were later grafted onto 2-year-old sour orange seedlings (Figure 1F
and G). They showed a more compact habit and a decrease in vigour, with significantly smaller leaves,
shorter internodes and more thorns when compared to the heterozygous parent of the same age of grafting
(Germana` et al., 2000b). Isozyme analyses have been employed to confirm the gametic origin of calluses
and plantlets in citrus (Germana` et al., 1991, 1994, 2000a, b; Deng et al., 1992a; Ollitrault et al., 1996;
 Page 89 of 94

Germana` and Reforgiato, 1997; Germana` and Chiancone, 2001). Isozyme techniques allow us to
distinguish between androgenetic and somatic tissue when the enzyme is heterozygotic in the diploid
condition of the donor plant and the regenerants show a lack of an allele. For example, Citrus clementina is
heterozygous for PGI-1 and PGM. To identify the origin of calli, embryos and plantlets obtained, their crude
extracts are analysed using two enzyme systems: phosphoglucoisomerase (PGI) and phosphoglucomutase
(PGM), as reported by Grosser et al. (1988). Numbering for isozymes (PGI-1) and lettering for different
allozymes are the same as those used by Torres et al. (1978). According to Torres et al. (1978), the
heterozygous clementine parent is FI (F=allele which specifies fast migration toward the anode enzyme;
I=intermediate) in PGM, and WS (W=allele which specifies an enzyme migrating faster than F; S=allele
which specifies a slowly migrating enzyme) in PGI. For analysis of calli and leaves obtained from anther
culture, the presence of a single band was retained as the homozygous state (Figure 2) and both enzyme
systems confirmed the androgenic nature of regenerants because of the lack of an allele.

The aberrant transmission of random amplified polymorphic DNA (RAPD) markers, due to the presence of
a band found in DH and not present in the parental, has been observed in homozygous clementine
(Germana` et al., 2000b) as well as in peach (Pooler and Scorza, 1995a). Microsatellites have been also
employed to characterize regenerants obtained from citrus anther culture (Germana` and Chiancone, 2003;
Germana` et al., 2005).

Production of triploids in Citrus

The importance of triploids in some fruit crops improvement, like Citrus or table grape, derives from the
seedlessness of their fruits. This is a desirable trait of commercial importance and one of the main goals in
many breeding programmes. Triploids can be conventionally produced by 2x* 4x and 4x* 2x crosses.
Triploid plants can also be obtained through in vitro culture of endosperm, which, being the fusion of three
haploid nuclei, is triploid. Triploid hybrid Citrus plants were recovered by in vitro embryogenesis from
endosperm-derived calli (Gmitter et al., 1990). One of the most interesting applications of haploids in Citrus
breeding is the possibility of obtaining triploid somatic hybrids by fusion between haploid and diploid
protoplasts (Kobayashi et al., 1997; Ollitrault et al., 2000). Recently (Germana` et al., 2005), ploidy analysis
by flow cytometry of 94 regenerants from clementine anther culture, showed as many as 82% of them were
tri-haploids, rather than haploids or doubledhaploids as expected. Regeneration from anther culture was
therefore proposed as a rapid, and attractive method of obtaining new triploid varieties in clementine, which
could be of great interest for the fresh fruit market that now requires fruit to be seedless.

Gametosomatic hybridization in Citrus

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A preliminary study on gametosomatic fusion between Poncirus trifoliata tetrads and somatic protoplasts of
Citrus sinensis cv ‘Jincheng’ was reported by Deng et al. (1992b). One chimeric plantlet was regenerated,
but not further reports on this subject have been published indicating that this method is probably not useful
for citrus breeding. Malus domestica (L.) Borkh Apples, with over 63.4 million tons produced during 2005
(FAOSTAT database), are after citrus, bananas and grapes, the most produced fruits in the world. Several
methods have been set up to obtain haploid plants in Malus domestica (L.) Borkh, 2n=2x=34. Induction of
embryogenesis and regeneration of pollen-derived plants from anther culture in this species has been
reported by several authors (Fei and Xue, 1981; Xue and Niu, 1984; Zarsky et al., 1986; Zhang et al., 1987;
Ho¨ fer and Hanke, 1990, 1994; Verdoodt et al., 1998; Ho¨ fer, 2003). The induction of embryogenesis from
cultured apple anthers is still low and highly genotype- dependent (Ho¨ fer, 1995, 1997). Zhang and
Lespinasse (1988) reported the induction of gynogenesis through in vitro culture of unpollinated ovaries and
ovules, without plant regeneration. Haploid plant have been obtained through in situ parthenogenesis
induced by pollination of cv. Erovan with pollen irradiated at 500– 1000 Gy, followed by in vitro embryo
culture (Zhang, 1988). This technique has been successfully applied to other apple cultivars also with
different c-rays from Cobalt 60 (Zhang et al., 1987; Zhang and Lespinasse, 1991; Zhang et al., 1992; De
Witte and Keulemans, 1994). The induction of embryogenesis and plant formation from isolated apple
microspores has been reported in the cultivar ‘Rene’ (Ho¨ fer et al., 1999). Further improvement of the
induction phase allowed to obtain induction of androgenic embryos in the following cultivars: ‘Alkmene’,
‘Remo’, ‘Rene’ and ‘Realka’; for all of them, except for ‘Alkemene’, the results obtained from microspore
culture were up to 10 times better than those from anther culture (Ho¨ fer, 2004).

Culture medium in apple anther culture

In fruit tree anther culture, the pH of the media is usually adjusted to 5.7–5.8 before autoclaving. A higher
pH (6.2) has been employed for gametic embryogenesis in apple isolated microspore culture (Ho¨ fer et al.,
1999; Ho¨ fer, 2004). Activated charcoal was beneficial to anther culture of apple (Johansson et al., 1987;
Zhang et al., 1990).

Conditions of incubation of apples anther culture

Temperatures used for apple anther culture are 23–30 °C (Ho¨ fer and Lespinasse, 1996; Kadota et al.,
2002). In apple anther culture, Ho¨ fer and Hanke (1990) obtained better results in the dark, while Fei and
Xue (1981) achieved embryo induction under continuous light. Dark was used also in apple isolated
microspore culture (Ho¨ fer et al., 1999; Ho¨ fer, 2004).

Embryo development from apple microspores and the origin of haploids

In apple, the androgenic plants are usually obtained not via direct gamete-derived embryo germination, but
through the occurrence of adventitious shoots also from secondary embryos (Ho¨ fer and Lespinasse, 1996).
Regarding the origin of haploids, Zhang (1988) observed three different routes in apple androgenesis:
formation of two identical nuclei after an abnormal pollen mitosis; division of the vegetative nucleus after a
normal pollen mitosis; and division of the generative nucleus after a normal pollen mitosis. Characterization
and propagation of regenerants obtained by apple gamete embryogenesis Triploids regenerated from anther
culture have been reported in apple (Ho¨ fer, 1994; Ho¨ fer et al., 2002). Isozyme analyses have been
employed to confirm the gametic origin of calluses and plantlets in apple (Ho¨ fer and Grafe, 2000).
Homozygous lines of apple have been analysed by the sequence characterized amplified region (SCAR)
marker ALO7 linked to the Vf gene for scab resistance from Malus floribunda (Ho¨ fer and Grafe, 2000) and
by simple sequence repeats (SSRs) (Kenis and Keulemans, 2000; Ho¨ fer et al., 2002). The single
multiallelic self-incompatibility gene has been used in apple by Verdoodt et al. (1998) to discriminate
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homozygous from heterozygous individuals obtained by parthenogenesis in situ or by anther culture.

Protoplasts were isolated from the stem and leaf of a haploid golden delicious apple clone and protoplast-
derived shoots were successfully propagated in vitro via organogenesis (Patat-Ochatt et al., 1993). Pyrus
communis L. Haploids and DH have been obtained by in situ parthenogenesis induced by irradiated pollen
or by seedling selection (Bouvier et al., 1993) from three pear cultivars: ‘Doyenne´ du Comice’, ‘Williams’
and ‘Harrow Sweet’. Doubled haploids were obtained by either spontaneous chromosome doubling or by
oryzalin treatment (200–300 lM) (Bouvier et al., 2002). Two embryos were produced by pear anther culture,
cv. Le Lectier, but their origin was not established and plant regeneration was not obtained (Kadota et al.,
2002). Temperature used during anther culture is 25 °C for pear (Kadota et al., 2002). In pear, as well as in
apple, regeneration from embryos was obtained after a cold treatment (12 weeks) through rooting of shoots
developed from embryos (Kadota et al., 2002). The adventitious shoots lacked vigour and for the most part
died. In some genotypes hyperhydricity was observed. Isozyme analyses have been employed to confirm the
gametic origin of calluses and plantlets in pear (Bouvier et al., 2002). Microsatellites have been also
employed to assess homozygosity in pear (Bouvier et al., 2002), confirming the achievement of true DH
clones of pear. Pyrus pyrifolia Nakai Triploid plants were obtained by anther culture from the diploid
Japanese pear Pyrus pyrifolia Nakai cultivar Shinko (2n=2x=34) (Kadota and Niimi, 2004). In previous
anther culture experiments carried out on three Japanese pear cultivars, nine embryos were obtained from
cultivar Gold Nijisseiki (at 0.12% rate) and 10 from cv. Shinko (at 0.13% rate), but plant regeneration was
not established (Kadota et al., 2002). No positive effect of activated charcoal addition has been observed in
anther culture of Japanese pear (Kadota and Niimi, 2004). Temperature used for Japanese pear anther
culture is 24 °C (Kadota and Niimi, 2004). Triploids regenerated from anther culture have also been reported
in Japanese pear (Kadota and Niimi, 2004). Prunus persica (L.) Batsch Haploids (1x=1n=8) reported in
peach [Prunus persica (L.) Batsch] arose spontaneously (Pratassenja, 1939; Hesse, 1971; Toyama, 1974).
They show the typical haploid traits of thin shoots, narrow leaves, weak vegetative growth and small non-
fertile or less fertile flowers (Hesse, 1971; Toyama, 1974; Pooler and Scorza, 1995a). Haploids, DH and F1
hybrids recovered by Toyama were the object of a study by Pooler and Scorza (1995a, b), who observed
unreduced gamete occurrence in haploid trees and aberrant transmission of RAPD markers, likely due to
somatic rearrangements (bud sports), often observed in tree fruit species. A study carried out by Scorza and
Pooler (1999) on the growth and yield of F1 hybrid peaches developed from DH demonstrated that their
productivity is similar to those of standard cultivars, but F1 hybrids offer advantages in the production of
uniform seedling scion cultivars. F1 hybrids can be also useful in high-density production (HDP) systems
where the cost of the trees proves to be a limiting factor. In fact, they eliminate the necessity of grafting and
permit the direct sale of seeds or non-grafted seedlings. This can make HDP attractive by reducing
production costs, especially where there is no need for a specific rootstock. Michellon et al. (1974), Seirlis et
al. (1979) and Hammerschlag (1983) obtained haploid callus from peach anther culture, but no plant
regeneration. Prunus avium L. The regeneration of four homozygous lines in sweet cherry (Prunus avium L.)
has been obtained by in situ parthenogenesis induced by pollination with irradiated pollen, followed by
embryo and cotyledon culture in the cultivar ‘Altenburger’ (Ho¨ fer and Grafe, 2003). Production of haploid
callus from anther culture has been also reported (Seirlis et al., 1979; Ho¨ fer and Hanke, 1990; Germana` et
al., unpublished). Isozyme analyses have been employed to confirm the gametic origin of calluses in cherry
(Ho¨ fer and Grafe, 2003). Prunus domestica L. Studies carried out by Peixe et al. (2000) in European plum
(Prunus domestica L., cv. ‘‘Rainha Cla` udia Verde’’), showed that gamma-irradiated (200 Gy) pollen can
induce the formation of 2n endosperm and abnormal embryo development showing the possibility of haploid
embryo formation in this genotype by in situ parthenogenesis. Unfortunately, only heart-shape embryos
were obtained, because no further development was observed. Prunus armeniaca L. The formation of
calluses on cultured anthers of apricot ‘Harcot’, as well as the differentiation of nodular structures have been
 Page 92 of 94

reported by Peixe et al. (2004). A heat pre-treatment of 28 °C for 8 days provided the best results when
compared with 36 or 24 °C (Peixe et al., 2004). After initiation, temperature used was 24/22 °C day/night
(Peixe et al., 2004). The ploidy of calluses, evaluated by flow cytometry, ranged from haploid to octoploid.
Previously, Harn and Kim (1972) obtained callus formation from apricot anther culture, but ploidy level was
not reported. Vitis vinifera L. For grapevine, (2n=2x=38), one of the most cultivated plants in the world,
haploids would be a powerful tool for increasing knowledge about the species and for dealing with the
difficult task of its genetic improvement. One case of haploid was reported by Zou and Li (1981) and
haploid callus line production has been reported by Gresshoff and Doy (1974), Kim and Peak (1981) and
Cersosimo (1986). Regeneration of plants has been obtained by Rajasekaran and Mullins (1979), Bouquet et
al. (1982), Hirabayashi and Akihama (1982), Mauro et al. (1986) and Cersosimo et al. (1990). Anther
culture (Figure 1H) is usually employed to establish diploid somatic embryogenic cultures of Vitis (Mauro
et al., 1986; Cersosimo et al., 1990). Embryogenic callus is valuable for propagation or genetic improvement
and can be used for somatic hybridization by protoplast fusion, genetic transformation, synthetic seed
production and germplasm storage.

A histological study on callused anthers of Vitis rupestris du Lot showed androgenic development of the
microspores (Altamura et al., 1992). These embryos did not develop further to plants, probably due to the
many deleterious genes leading to genetic disorders (Cersosimo, 1996). In grape, Cersosimo (1987) obtained
better results in a solid medium rather than liquid, while Rjasekaran and Mullins (1979) obtained
embryogenic callus production in continuously agitated liquid medium. Temperatures used during anther
culture were 24–26 °C for grape (Cersosimo, 1996). Olea europaea L.

Olive is among the most typical crops and the most important oil-producing plants of the Mediterranean
basin, characterized by a very long juvenile phase, a large plant size and often by self-incompatibility. It is a
diploid (2n=2x=46), outcrossing long-living species. Sporophytic division, multinucleate microspores and
multicellular structures have been successfully induced in isolated microspore culture of two olive cultivars
(Arbequina and Picual) (Bueno et al., 2004). Morus alba L. Because of the dioecious nature of mulberry,
inbreeding to obtain haploids and homozygous plants is not applicable. Gynogenic haploids of a female
clone of mulberry (Morus alba L. Cv.K-2) were obtained by in vitro culture of unpollinated ovaries from in
vitro developed inflorescences (Dennis Thomas et al., 1999). Anther culture has not been successful in
producing haploids of this tree crop (Sethi et al., 1992; Jain et al., 1996). Actinidia deliciosa (A. Chev)
Parthenogenetic tri-haploids were induced in kiwifruit, cv Hayward, an hexaploid species (2n=6x=174), by
irradited pollen. The best results were obtained with a dosage of 500– 1500 Gy and the genotype of the
pollen parent greatly influenced the ability to obtain both seedlings and tri-haploids (Chalak and Legave,
1997). Spontaneous doubling was also observed. Pandey et al. (1990) previously induced parthenogenesis
by pollination with irradiated pollen. Kiwifruit anther culture produced only somatic embryogenesis (Fraser
and Harvey, 1988). [Musa balbisiana (BB)] The production of 41 haploid (n=x=11) plantsfrom anther
culture of banana [Musa balbisiana (BB)], was reported by Assani et al. (2003): 18 from the genotype
Pisang klutuk, 12 from Pisang batu, 7 from Pisang klutuk wulung and 4 from Tani. The frequency of callus
induction was 77% and about 8% of anthers developed embryos after 6 months of culture. The frequency of
embryo formation was genotype-dependent. Temperature used during anther culture was 27 °C under dark
conditions (Assani et al., 2003). Previously, Kerbellec (1996) reported successful haploid plant regeneration
in banana [Musa acuminate (AA)]. Carica papaya L. The main breeding systems of papaya, a polygamous
species, using true-bred lines, benefit greatly from haploid induction through anther culture.

Haploid plantlets and pollen-derived embryos were obtained from papaya anthers cultured at the uninucleate
stage (Litz and Conover, 1978; Tsay and Su, 1985). Rimbeira et al. (2005) greatly increased up to about
4.0% the embryo induction rate (rate of anthers forming embryos) by investigating the effects of pre-cultural
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conditions. For example, the pre-treatment of 35 °C was more efficient for embryo induction than 25 °C and
liquid media were more effective as pre-treatment than solid media in papaya anther culture (Rimbeira et al.,
2005). They also applied a papaya sexdiagnostic PCR technique to plantlets obtained. Haploid plantlets were
induced through anther culture in a medium without any growth regulators and under dark conditions (Tsay
and Su, 1985). Annona squamosa L. Haploid embryos from male gametes were produced through anther
culture of Annona squamosal L. (Nair et al., 1983). Feijoa sellowiana Berg. Multinucleated pollen grains
were obtained in anther culture of feijoa, but attempts to regenerate pollen plants were unsuccessful
(Canhoto and Cruz, 1993). Opuntia ficus-indica (Mill.) Opuntia ficus-indica (Mill.) breeding to obtain new
cultivar development has been hampered by some reproductive aspects such as cleistogamy, nucellar
embryony and low seed germination (Chessa et al., 2000; Chessa and Nieddu, 2002). Research has been
carried out to study the correlation of sequential floral and male gametophyte development and to
investigate the response to in vitro culture of anthers collected from flower buds of two different stages of
development of prickly pear, Opuntia ficus-indica L. Mill. (Gonza´ les-Melendi et al., 2005) Eriobotrya
japonica Lindl. Loquat, originated in China, has adapted well to the Mediterranean climate and grows in the
same areas where citrus species are cultivated. Very often, current varieties are selected as seedling
variations resulting from natural hybridization and not very much attention has been paid to use of
biotechnology as a tool to create new variability in this species. Preliminary research is in progress to apply
anther culture and haploid production to loquat, resulting in callus production and multinucleated pollen
grains (Figure 1I).

Conclusions

The great potential of employing haploidy, doubled haploidy and gametic embryogenesis in fruit crop
breeding is clearly evident. Haploids can improve the efficiency and the speed of the usually cumbersome,
time-consuming, laborious and sometimes rather inefficient conventional breeding methods. Although in
vitro culture of gametes is more or less a standard tool for plant breeders in many crops, particularly
Brassicaceae and cereals, this has yet to be achieved in fruit crop breeding since the deployment of gametic
embryogenesis in fruit crops improvement is still hampered by low frequencies of embryo induction,
albinism, plant regeneration, plant survival and the genotype dependent response. A better understanding of
the gametic embryogenesis process, the improvement of currently available techniques and the development
of new technologies could make haploid production a powerful fruit crop breeding tool in the future,
enabling in these genotypes the effective exploitation of the potential of gamete biotechnology. In order to
make this possible, the fundamental goals are: to enlarge the number of respondent genotypes, to improve
the induction rate (the frequency with which gametes form embryos) and to increase the survival rate (the
percentage of regenerated haploid and doubled haploid plants successfully transferred from in vitro to in
vivo culture conditions). Further goals are to characterize and to deploy haploids and DH in fruit crop
breeding (protoplast fusion, triploid production, transformation, etc.). A better knowledge of the gametic
embryogenesis process in fruit crops is needed to transform this frontier of plant biotechnology into practical
applications in these crops, as has already been achieved in some families (Cruciferae, Gramineae and
Solanaceae).

Doubled haploidy is and will continue to be a very efficient tool for the production of completely
homozygous lines from heterozygous donor plants in a single step. Since the first discovery of haploid
plants in 1920 and in particular after the discovery of in vitro androgenesis in 1964, techniques have been
gradually developed and constantly improved. The method has already been used in breeding programs for
several decades and is currently the method of choice in all species for which the technique is sufficiently
elaborated. Species for which well-established protocols exist predominantly belong to field crops or
vegetables, but the technique is gradually also being developed for other plant species, including fruit and
 Page 94 of 94

ornamental plants and other perennials. It should be mentioned that, in addition to breeding, haploids and
doubled haploids have been extensively used in genetic studies, such as gene mapping, marker/trait
association studies, location of QTLs, genomics and as targets for transformations. Furthermore the haploid
induction technique can now-a-days be efficiently combined with several other plant biotechnological
techniques, enabling several novel breeding achievements, such as improved mutation breeding,
backcrossing, hybrid breeding and genetic transformation