Androgenesis and

Gynogenesis

Plant Biotechnology II
Semester : VII
B. Sc. Biotechnology
Shambhu P. Dhital, PhD
2079/07/28/ and 08/18
Androgenic Methods:
• haploid production of plants through anther or microspore
culture has been referred to as androgenesis

Gynogenic methods:
• haploid production of plants from ovary or ovule culture has
been referred to as gynogenesis
Androgenesis

• It is the formation of sporophyte from the male gametophyte on

artificial medium
• It is most commonly found in family solanaceae and poaceae
• In androgenesis immature anther or pollen grains are induced to
follow the sporophytic mode of development by various physical and
chemical stimuli.
There are two methods for in vitro production of
androgenic haploids
They are:
• Anther culture
• Pollen [microspore] culture.
Introduction
The male reproductive part of a flower is called the stamen.
It is composed of a long tube called a filament and has a pollen-producing structure on the end
This oval-shaped structure is called the anther, it produces the male gametophyte known as
pollen.

• In anther culture, using anthers to culture haploid plantlets.

• Haploid plants are characterized by possessing only a single set of
chromosomes in the sporophyte (diploids which contain two sets (2n) of chromosomes).
• Haploid plants are of great significance for the production of homozygous plants and for
the improvement of plants in plant breeding programmes.
• The technique was discovered in 1964 by Guha and Maheshwari in Datura.
• This technique can be used in over 200 species, including tomato, rice, tobacco, barley,
datura, brassica etc.
 Types of androgenesis
• Direct androgenesis
• Indirect androgenesis

Direct androgenesis:
• It is also called pollen derived embryogenesis
• Here pollen grains directly acts as a zygote and
passes through various embryogenic stages
similar to zygotic embryogenesis.
• When the pollen grains has reached globular stage
of embryo, the wall of the pollen is broken
and embryo is released
The released embryo develop cotyledons, which
ultimately give rise to plantlets
Eg: Datura, Brassica campestris
 Indirect androgenesis:-
• In indirect androgenesis, the anther or pollen grains,
instead of normal embryogenesis, divide erratically to
develop Callus.
• Callus tissue which is finally redifferentiates and forms
haploid plantlets
E.g.: rice , wheat, tomato
Androgenesis:
In androgenesis, the male gametophyte (microspore or immature pollen) produces
haploid plant.
• The basic principle is to stop the development of pollen cell into a gamete (sex cell)
and force it to develop into a haploid plant.
• There are two approaches in androgenesis—

1. Anther culture and Pollen (microspore) culture.

• Young plants, grown under optimal conditions of light, temperature and humidity, are
suitable for androgenesis.
1. Anther culture (protocol)
1. Selected plants are cultivated until they reach flower bud stage.
2. In some cases flower buds are chilled few days prior to culture.
3. Flower buds of proper size and developmental stage are taken and surfaces
sterilized ( alcohol or hypochlorite solution for 10—20 minutes and rinsed several
times in SDH2O.
4. The anthers are carefully excised from flower buds.
5. Filaments must be removed prior to culture; otherwise callus may be formed at the
cut ends.
6. Anthers may be cultured either on agar medium or on a filter paper bridge liquid
medium.
7. Anthers are cultured at 25° C in presence or absence of light. Light is essential after
plantlets are formed. Continuous illumination from cool white fluorescent lamp of
300 lux is satisfactory.
8. After 4—5 weeks plantlets are formed.
9. These plantlets are carefully separated quite early and cultured on a fresh root-
inducing medium (0.5% agar and half-strength to that of the anther culture medium).
10. After formation of proper root system they are transplanted to pots.
 2. Pollen /microspore culture (protocol)
1. Selected plants are cultivated until they reach flower bud stage.
2. In some cases flower buds are chilled few days prior to culture.
3. Flower buds of proper size and developmental stage are taken and surfaces sterilized
(alcohol or hypochlorite solution for 10—20 minutes and rinsed w i t h S D H 2 O ) .
4. The anthers are carefully excised from flower buds (Filaments must be removed prior to
culture; otherwise callus may be formed at the cut ends).
5. Anthers are placed in liquid medium in a petridish.
6. Pollen grains are removed from the anthers either mechanically or by natural dehiscence.
7. To obtain pollen grains from the anthers, Sunderland and Roberts developed ‘float
culture’ technique (in this method whole anthers are taken from cold treated buds and are
allowed to float on the surface of the liquid medium).
6. Culture media containing pollen grain are sealed with para-film and incubated at 28°C in
dark for fourteen days.
7. After 14 days the culture is kept in an illuminated chamber at 25° C and a day length of 12
hours.
8. 3-8 weeks may be required to obtain haploid plantlets.
Pollen culture
technique
Methods of pollen culture
1. Nitsch’s culture technique.
2. Nurse culture technique.
3. Float culture technique.
Method I:- Nitsch’s culture technique
This is considered as the basic protocol for pollen culture
1. Selection of suitable unopened flower bud, sterilization, excision of anther
without filaments are the same as described previously in anther culture.
2. About 50 anthers are placed in small sterile beaker containing 20 ml of liquid
basal medium (MS or White or Nitsch and Nitsch).
3. Anthers are then pressed against the side of beaker with the sterile glass piston of
a syringes to squeeze out the pollens.
4. The homogenized a n t h e r are then filtered through a nylon sieve to remove
that the anther tissue debris.
5. The filtrate or pollen suspension is then centrifuged at low speed ( 500-800
rpm/min) for five minutes.
6. The supernant debris is discarded and pallet (pollen) is suspended in fresh
liquid medium and washed twice by repeated centrifugation and resuspension in
fresh liquid medium.
6. Pollens are mixed finally with measured volume of liquid basal medium so that
it makes the density of 1:3-1: 4 pollen/ml.
7. A 2.5 ml of pollen suspension is pipetted off and is spread in 5 cm
petridish. Pollens are best grown in liquid medium, but if necessary, they can
be grown by plating very soft agar added medium. Each dish is sealed with
cello tape to avoid dehydration.
8. Petri dishes are incubated at 27-30 0C under low intensity of white cool
light (500 lux,16 hrs).
Method II :- Nurse culture technique:-
The nurse culture technique, first introduced this method by Sharp et.al. ( 1972).
1. Selection of flower bud sterilization excision of anther, isolation of suitable pollen
is the same as described previously.
2. In this method, the intact anthers are placed horizontally on the top of solid or
semisolid basal medium in a conical flask.
3. A small filter paper disc is placed over the intact anther and about 10 pollen grains
in the suspension are then placed on the filter paper disc. Here the intact anthers are
considered as the nurse tissue.
4. The pollen gains kept on nurse tissue grow and form a culture of green
parenchymatous tissue in two weeks, such tissue ultimately form the haploid callus
tissue.
Method III :- Float culture technique
• Anthers are excised.
• Those are placed in petridishes containing liquid medium.
• Anthers float in liquid medium.
• The anthers release their pollen grains into the
medium in a few days.
Factors influencing anther culture

1. Physiological status of donor plant

2. Effect of temperature
3. Culture medium
4. Anther wall factor
5. Stages of microspore
6. Stages of embryo sac
7. Genotype of donor plants
8. Effect of female flower position
9. Pretreatment of anthers
1) Genotype of donor plants:-
• The genotype of the donor plant plays a significant role in
determining the frequency of pollen production.
Example :- Horedum of each genotype differs with respect to
androgenic response in anther culture.

2) Anther wall factor:-

• The anther wall provide the nourishment in the development of isolated pollen of a
number of species.
• There are reports that glutamine alone or in combination with serine and myo
inositol could replace the anther wall factor for isolated cultures.
3) Culture medium:-
• For anther culture, medium requirements vary with genotype and the age of the
anther as well as condition under which donor plants are grown.
• Incorporation of activated charcoal into the medium has stimulated the induction of
androgenesis.
• The iron in the medium plays a very important role for the induction of haploids.
• Potato extracts, coconut milk and growth regulators like auxin and cytokinin are
used for anther and pollen culture.

4) Stages of microspores:-
• Anther are most productive when cultured at the uninucleate microspore stage.
Example: barley, wheat, rice etc
• Anther of some species give the best response if pollen is cultured at first mitosis or
later stage. Example:- datura, tobacco
6) Physiological status of donor plant:-
• Physiological status of donor plant such as water stress, nitrogen requirement and age
of donor plant highly affect the pollen embryogenesis.
• Plants starved of nitrogen may give more responsive anthers compared to those that
are well fed with nitrogenous fertilizers.
7) Pretreatment of anthers:
• Appropriate treatment of anthers is required for good success of haploid
production.
• Certain chemicals are known to induce parthenogenesis e.g. 2-
chloroethylphosphonic acid (ethrel).
• When plants are treated with ethreal, multinucleated pollens are produced. These
pollens when cultured may form embryos.
8. Temperature influence:
• In general, when the buds are treated with cold temperatures (3-6°C) for about 3 days,
induction occurs to yield pollen embryos in some plants e.g. Datura, Nicotiana.
• Further, induction of androgenesis is better if anthers are stored at low
temperature, prior to culture e.g. maize, rye.
• There are also reports that pretreatment of anthers of certain plants at higher
temperatures (35°C) stimulates androgenesis e.g. some species of Brassica and
Capsicum.
9. Effect of light:
• In general, the production of haploids is better in light.
• There are however, certain plants which can grow well in both light and dark.
• Isolated pollen appears to be sensitive to light.
• Thus, low intensity of light promotes development of embryos in pollen cultures
e.g. tobacco.
10. Effect of culture medium:
• The success of another culture and androgenesis is also dependent on the
composition of the medium.
• There is, however, no single medium suitable for anther cultures of all plant species.
• The commonly used media for anther cultures are MS, White’s, Nitsch and Nitsch,
N6 and B5.
• These media in fact are the same as used in plant cell and tissue cultures.
• Sucrose, nitrate, ammonium salts, amino acids and minerals are
essential for androgenesis.
• In some species, growth regulators — auxin and/or cytokinin are required for
optimal growth.
• When the anther culture medium is supplemented with activated charcoal,
enhanced androgenesis is observed.
• It is believed that the activated charcoal removes the inhibitors from the medium
and facilitates haploid formation.
Anther and Pollen culture
Pathway of
development
Pathway of cell development
Pathway I
The uninucleate pollen grains may divide symmetrically to yield two equal daughter cells, both of
which undergo further divisions it will contribute to sporophyte development. Vegetative and
generative cells are not distinctly formed in this path Example: Datura innoxia
Pathway II
In this case . the uninucleate pollen divides unequally which will result in the formation of
vegetative and generative cells The sporophyte or plantlet arises through further division in the
vegetative cell while the generative cell does not divide. Examples: Nicotiana tabacum , Hordeum
vulgare , Triticum aestivum
Pathway III
The uninucleate pollen undergoes a normal division but pollen embryos are predominantly formed
from generative cell alone. The generative cell either does not divide at all or does so only to
limited extent The vegetative cell does not divide. Examples: Hyoscyamus niger
Pathway IV
In some species, the uninucleate pollen grains divide unequally, producing generative and
vegetative cell but both these cells divide repeatedly to contribute to the development of
sporophyte. Examples: Datura metal
Importance and Implications of Anther and Pollen Culture

1. Haploids derived from anther and pollen culture are useful in cytogenic studies.
2. Recessive phenotypic characters can be identified easily by comparing
heterozygous diploid with haploid or homozygous diploid population.
3. Double haploid that are homozygous and fertile, are readily obtained, enabling
the selection of desirable gene combination.
4. Culture of isolated pollen provides a novel experimental system for the study of
factor controlling pollen embryogenesis of higher plants.
5. It also provides information for the interpretation of chromosome homology.
6. Genetic analysis could be performed on haploid population to establish
inheritance patterns.
 Advantages of Pollen Culture over Anther Culture
• Overcrowding of pollen grains in anther is eliminated and isolated pollen grains are equally
exposed to nutrient medium.
• Unwanted growth of diploid cells of anther wall and other associated tissue is eliminated.
• The stage of androgenesis can be observed starting from single cell.
• Various factors governing androgenesis can be better regulated.
• Pollen is deal for uptake, transformation and mutagenic studies as pollen can be uniformly
exposed to chemicals and physical mutagens.
• Pollen may be directly transformed into an embryoid. So, it is very suitable for
understanding biochemistry and physiology of androgenesis.
• Higher yield of haploid plants per anther could be expected in pollen
culture than the anther culture.
Application of Haploids in Plant Breeding

1. Releasing new varieties through F1 Double - haploid System.

2. Selection of mutants resistance to diseases.
3. Developing asexual lines of tree perennial species.
4. Transfer of desired alien gene.
5. Establishment of haploids and diploid cell lines of pollen plant.
1. Releasing new varieties through F1 Double – haploid System:-
• Haploid breeding technique usually involve only one cycle of meiotic recombination.
• The anthers of the hybrid (F1) progeny are excellent breeding material for raising
pollen-derived homozygous plants (Double –haploids) in which
complementary parental characteristics are combined in one generation.
• Double –haploids are also useful in studies related to inheritance of quantitative traits.
• Using double –haploid technique new varieties have been developed in barley, Brassica,
rice, maize , rye, potato, pepper and asparagus.
2. Selection of Mutants Resistance to Diseases:

• Mutants with resistance to disease is of prime importance in crop improvement.

• Haploids provide a relatively easier system for the induction of mutations.
• Some examples of using anther culture technique in mutant successfully are
tobacco mutants resistant to black shank disease and wheat lines resistant to
scab. (Fusarium graminearum).

3. Developing Asexual Lines of Tree Perennial Species:

• Chinese workers obtained pollen –derived tall rubber tree which could be
multiplied by asexual propagation to raise several clones.
4. Transfer of Desired Alien Gene:

• In rice , developing a resistance to blast requires about 12 years by conventional

breeding through back crossing.
• Through hybridization and anther culture, this can be achieved in two years
(Examples: Rice cv . Zhonghua No.8 and 9 released by the institute of crop Breeding
and cultivation in china.
5. Establishment of Haploids and Diploid Cell Lines:
• The anther culture technique was used to establish both haploid and diploid
somatic cell lines in wheat and maize.
Gynogenesis: The process of development of haploid plants by in vitro culture
of unfertilized eggs cells or other haploid cells of female gametophytic is
called as in vitro gynogenesis.

There are two methods for in vitro production of gynogenic haploids:

Ovary culture
Ovule culture

• Haploid plants can be developed from ovary or ovule cultures. It is possible to

trigger female gametophytes (megaspores) of angiosperms to develop into a
sporophyte. The plants so produced are referred to as gynogenic haploids.
• Gynogenic haploids were first developed by San Noem (1976) from the ovary
cultures of barley (Hordeum vulgare). This technique was later applied for raising
haploid plants of rice, wheat, maize, sunflower, sugar beet and tobacco.
• In vitro culture of un-pollinated ovaries (or ovules) is usually employed when the
anther cultures give unsatisfactory results for the production of haploid plants.
Procedure :
• The flower buds are excised 24-48 hr. prior to
anthesis from un-pollinated ovaries.
• After removal of calyx, corolla and stamens, the ovaries
are subjected to surface sterilization.
• The ovary, with a cut end at the distal part of pedicel, is
inserted in the solid culture medium.
• Whenever a liquid medium is used, the ovaries are placed
on a filter paper or allowed to float over the medium.
• The culture is incubated at 25 C for 16 h light and 2000 lux
light intensity
• The commonly used media are MS, White’s, N6 and
Nitsch, supplemented growth factors.
• Production of gynogenic haploids is particularly useful in
plants with male sterile genotype.
• For such plant species, this technique is superior to
another culture technique.
Steps for gynogenesis
Two stages :
Induction: The ovaries are cultured on a liquid MS medium having low
auxin and kept in dark
Regeneration: Ovaries are transferred into agar medium with high auxin and kept
in light

The major limitations of gynogenesis are;

1. The dissection of unfertilized ovaries and ovules is rather difficult.
2. The presence of only one ovary per flower is another disadvantage
(there are a large number of microspores in one another).
Identification of Haploids:
Two approaches based on morphology and genetics are commonly used to detect or
identify haploids.
• The vegetative and floral parts and the cell sizes of haploid plants are relatively
reduced when compared to diploid plants. By this way haploids can be detected in a
population of diploids. Morphological approach, however, is not as effective as genetic
approach.
• Genetic markers are widely used for the specific
identification of haploids. Several markers are in use.
Production of homozygous plants

• Haploid plants are obtained either by androgenesis or gynogenesis.

• These plants may grow up to a flowering stage, but viable gametes
cannot be formed due to lack of one set of homologous chromosomes.
• Consequently, there is no seed formation.
• Haploids can be diploidized (by duplication of chromosomes) to
produce homozygous plants.
There are mainly two approaches for diploidization
Colchine treatment and endomitosis
 Colchicine Treatment:
Colchicine is very widely used for diploidization of homologous chromosomes. It acts as an
inhibitor of spindle formation during mitosis and induces chromosome duplication. There are
many ways of colchicine treatment to achieve diploidization for production of
homozygous plants.
1. When the plants are mature, colchicine in the form of a paste is applied to the axils of leaves.
Now, the main axis is decapitated. This stimulates the axillary buds to grow into diploid and
fertile branches.
2. The young plantlets are directly treated with colchicine solution, washed thoroughly
and replanted. This results in homozygous plants.
3. The axillary buds can be repeatedly treated with colchicine cotton wool for about 2-3
weeks.
 Endomitosis:
• Endomitosis is the phenomenon of doubling the number of
chromosomes without division of the nucleus.
• The procedure involves growing a small segment of haploid plant
stem in a suitable medium supplemented with growth regulators (auxin and cytokinin).
• This induces callus formation followed by differentiation.
• During the growth of callus, chromosomal doubling occurs by endomitosis. This
results in the production of diploid homozygous cells and ultimately plants.
Application of Haploids in Plant Breeding

1. Releasing new varieties through F1 Double - haploid System.

2. Selection of mutants resistance to diseases.
3. Developing asexual lines of tree perennial species.
4. Transfer of desired alien gene.
5. Establishment of haploids and diploid cell lines.
Thank
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