Revista Brasileira de Fisiologia Vegetal, 10(1):1-12, 1998

REVIEW

THE POTENTIAL USES OF SOMATIC

EMBRYOGENESIS IN AGROFORESTRY ARE NOT
LIMITED TO SYNTHETIC SEED TECHNOLOGY1

Carlos M. Vicient2,3 and F. Xavier Martínez3

Departament d’Agronomia. Escola Superior d’Agricultura de Barcelona. Universitat
Politècnica de Catalunya. Urgell 187, 08036 Barcelona, Spain.

ABSTRACT - Somatic or asexual embryogenesis is the process by which somatic cells develop into
plants. The rapid improvement in somatic embryogenesis methods allow the use of somatic embryos in
plant micropropagation as synthetic seeds. However, practical applications of somatic embryogenesis
are not limited to synthetic seed technology. Somatic embryogenesis can be used in the regeneration of
genetically transformed plants, polyploid plants, or somatic hybrids. Moreover, promising results indicate
the possibility to use somatic embryogenesis in cell selection programs and germplasm cryopreservation.
The application of somatic embryogenesis to plant virus elimination, metabolite production, and in vitro
mychorrhizal initiation has been investigated.
Additional index terms: cell selection, cryopreservation, plant regeneration, somatic hybrid, transgenic
plants, virus elimination.

USOS POTENCIAIS DA EMBRIOGENESE SOMÁTICA EM FLORESTA NÃO ESTA

LIMITADO A APLICAÇÃO DE SEMENTES SINTÉTICAS

RESUMO - A embriogenese somática ou assexual é o processo pelo qual células somáticas se

desenvolvem em plantas. A rápida melhoria nos métodos de embriogenesi tem permitudo o emprego
de embriões somáticos na micropropagação de plantas como as chamadas sementes sintéticas. Além
do uso em sementes sintéticas, a embriogenese somática pode ser usada na regeneração de plantas
geneticamente transformadas, plantas poliplóides ou de híbridos somáticos. Adicionalmente, resulta-
dos promissores inidicam a possibilidade do uso da embriogenesi somática em programas de seleção
de células e na crio-preservação de germoplasmas. A aplicação da embriogenese somática para a
limpeza de viroses, para a produção de metabólitos e para a iniciação de micorrizas in vitro também
tem sido investigada.
Termos adicionais para indexação: criopreservação, híbridos somáticos, limpeza de virus, plantas
trangênicas, regeneração de plantas, seleção de células.

1 Received in 25/06/1997 and accepted in 11/02/1998.

2 Current address and address for reprint request: Institute of
Biotechnology, University of Helsinki, P.O. Box 56, Viikinkaari
9, FIN-00014, Finland.
3 CMV and FXM are Doctors in Biology by the University of

Barcelona and FXM is professor of Agronomy in the Escola

Superior d’Agricultura de Barcelona, Universitat Poletècnica
de Catalunya, Urgell 187, 08036 Barcelona, Spain. Abbreviations : ABA - abscisic acid.

1
2 Vicient & Martínez

INTRODUCTION can be artificially mutagenized but it is easier and cheaper

to use non-mutagenized cells cultured in vitro and to
Somatic or asexual embryogenesis is the production take advantage of somaclonal variation. Plantlets derived
of embryo-like structures from somatic cells without from in vitro culture may exhibit abnormal phenotypes,
gamets fusion. During their development, somatic often heritable. This variability is known as somaclonal
embryos pass through stages similar to those observed variation and has been extensively described in plants
in zygotic embryogenesis (Dodeman et al ., 1997). derived from somatic embryos (Amberger et al., 1992;
Somatic embryos are independent of the surrounding Fourré et al., 1997; Kuksova et al., 1997). Somaclonal
tissues and accumulate embryo-specific proteins and variations are a problem for in vitro clonal
mRNAs (Zimmermann, 1993). They arise naturally in micropropagation but they are desirable in cell selection
some species in a process known as direct somatic programs. Somaclonal variations can produce desirable
embryogenesis (Williams & Maheswaran, 1986). In agronomic characteristics such increases in salt
contrast, somatic embryos arise from in vitro cultured tolerance, resistance to herbicide, diseases, extreme
cells in the process called indirect somatic temperatures, aluminium or desiccation, or can yield
embryogenesis. This process requires the induction of interesting biochemical or ornamental mutations
embryogenic competence. The most usual method to (Maluszynsky et al., 1995). For example, cell selection
induce embryogenic competence consists of exposing and regeneration via somatic embryogenesis have been
explants to a high auxin concentration during a variable used to improve salt tolerance and disease resistance
period of time, and then transfering cells to an auxin- in Citrus (Litz et al., 1985), virus resistance in sugarcane
free medium. Indirect somatic embryogenesis is the most (Oropeza & de Gracia, 1996), germination at low
common method to generate somatic embryos for temperature in melon (Ezura et al., 1995) and resistance
practical uses and has been described in hundreds of to phytotoxins in coffee (Nyange et al., 1995). The
species (reviewed by Redenbaugh, 1993; Bajaj, applications of cell selection and somatic embryogenesis
1995a,b). A special type of indirect somatic in obtaining new varieties in floricultural crops have been
embryogenesis is secondary somatic embryogenesis, reviewed by Hutchinson et al. (1992).
which consists of the production of somatic embryos Regeneration of genetically transformed plants
using somatic embryos as initial explants. Secondary
somatic embryogenesis have been described in nearly Gene transfer methods are an essential part of the
one hundred species (Raemarkers et al., 1995). Although new technologies that are altering conventional plant
secondary embryos frequently show low conversion rates breeding and have become indispensable tools. However,
to plants, they also can be used in practical applications. in some species the lack of an efficient regeneration
The rapid improvement in the somatic embryogenesis method is a great bottleneck in the use of transformation
methods allows extensive practical and commercial technology. For these species the development of
applications, par ticularly for in vitro clonal regeneration methods based on somatic embryogenesis
micropropagation (Bornman, 1993; Vasil, 1994). Of could be the solution as it has been for coffee (Spiral et
special interest for agroforestry is the use of somatic al., 1993) and cassava (Schöpke et al., 1996). However,
embryos as synthetic seeds (Redenbaugh, 1993; Bajaj, the plant transformation techniques do not transform all
1995a, b; Litz & Gray, 1995) and their scale-up production cells, and plants regenerated from transformed tissues
in bioreactors (Onishi et al., 1994). However, somatic via organogenesis are often chimeras (Krastanova et al.,
embryogenesis has other practical applications in 1995). Somatic embryos arise from single cells (Toonen
Agroforestry, including crop improvement (cell selection, & de Vries, 1996) and for this reason regeneration via
genetic transformation, somatic hybrid and polyploid plant somatic embryogenesis reduces the formation of
production), germplasm preservation, virus elimination, chimeras. Nevertheless, somatic embryogenesis seems
in vitro metabolite production, and in vitro mycorrhizal to produce fewer rates of somaclonal variations
initiation. compared with organogenesis (Heinze & Schmidt, 1995).
Several different strategies that have been used in
USES OF SOMATIC EMBRYOGENESIS IN the regeneration of transformed plants using somatic
embryogenesis (Figure 1, Table 1):
CROP IMPROVEMENT A) Transformation of the explant and regeneration via
direct somatic embryogenesis. The explant tissue is
Cell selection
transformed and then regenerated via direct somatic
Cell selection consists of the regeneration of plants embryogenesis. This system is fast, simple and avoids
from cell populations combined with the selection of the problems of somaclonal variations since an in vitro
desirable genetic characteristics (Jacobs et al., 1987). culture phase is absent. This methodology facilitates
In order to avoid the production of genetic chimeras, it is the transformation of some recalcitrant species for
necessary to use a plant regeneration system able to which successful plant regeneration has been
regenerate plants from single selected cells. Somatic reported only via direct somatic embryogenesis (Tetu
embryogenesis allows regeneration of whole plants from et al., 1990). However, direct somatic embryogenesis
single cells (Toonen & de Vries, 1996), making possible have been described in only few species.
its use in cell selection programs. B) Transformation of the explant and regeneration via
The initial cell populations in cell selection programs indirect somatic embryogenesis . This method does

R. Bras. Fisiol. Veg., 10(1):1-12, 1998.

 Somatic embryogenesis 3

FIGURE 1- Schematic representation of the different strategies that have been used in the regeneration of genetically transformed
plants using somatic embryogenesis. A, Transformation of the explant and regeneration via direct somatic embryogenesis; B,
Transformation of the explant and regeneration via indirect somatic embryogenesis; C, Transformation of embryogenic in vitro cell
culture and regeneration via indirect somatic embryogenesis; D, Transformation of somatic embryos and regeneration through
secondary somatic embryogenesis; E, Transformation of somatic embryos, proliferation through secondary embryogenesis and
regeneration through organogenesis. In the squares the phase in which transformation is performed. DSE, direct somatic
embryogenesis; ISE, indirect somatic embryogenesis; SSE, secondary somatic embryogenesis. In white, non-transformed tissues;
in grey, tissues subjected to transformation; in black, stably transformed tissues.
TABLE 1- Species of agroforestry interest in which regeneration of transformed plant has been made using somatic embryogenesis.
S P E C IE S S T R AT E G Y T R A N S F O R M AT I O N REFERENCES

A g r o s tis p a lu s tr is C A g r o b a c t e r iu m Z h o n g e t a l., 1 9 9 3
A r a c h is h y p o g a e a B M ic r o b o m b a r d m e n t L iv in g t o n e a n d B ir c h , 1 9 9 5
D M ic r o b o m b a r d m e n t S in g s it e t a l . , 1 9 9 7
A s p a r a g u s o ffic in a lis C M ic r o b o m b a r d m e n t C a b r e r a - P o n c e e t a l. , 1 9 9 7
C a la d iu m b ic o lo r B A g r o b a c t e r iu m Li and W u, 1990
C a r y a illin o e n s is D A g r o b a c t e r iu m M c G r a n a h a m e t a l. , 1 9 9 3
C a r ic a p a p a y a B A g r o b a c t e r iu m C a b r e r a - P o n c e e t a l. , 1 9 9 6 ; Ya n g e t a l . , 1 9 9 6
C A g r o b a c t e r iu m C h e n g e t a l. , 1 9 9 6
D M ic r o b o m b a r d m e n t F it c h e t a l . , 1 9 9 0
C h r y s a n te m u m B A g r o b a c t e r iu m P a v in g e r o v á e t a l . , 1 9 9 4
C ic e r a r ie tin u m A A g r o b a c t e r iu m R a m a n a e t a l. , 1 9 9 6
C itr u s s p . C M ic r o b o m b a r d m e n t Ya o e t a l . , 1 9 9 6
C o ffe a c a n e p h o r a D A g r o b a c t e r iu m S p ir a l e t a l . , 1 9 9 3
C u c u m is s a tiv u s B A g r o b a c t e r iu m R a h a r jo e t a l. , 1 9 9 6
C M ic r o b o m b a r d m e n t S c h u lz e e l a l. , 1 9 9 5
D a tu r a in n o x ia A A g r o b a c t e r iu m D u c r o c q e t a l. , 1 9 9 4
F e s tu c a s p . C M ic r o b o m b a r d m e n t S p a n g e n b e r g e t a l. , 1 9 9 5
G ly c in e m a x B M ic r o b o m b a r d m e n t L iu e t a l. , 1 9 9 6
C M ic r o b o m b a r d m e n t S t e w a r t e t a l. , 1 9 9 6
Ip o m o e a b a ta t a s B A g r o b a c t e r iu m N e w e ll e t a l . , 1 9 9 5
C A g r o b a c t e r iu m G a m a - M ic s e t a l . , 1 9 9 6
J u g la n s s p . D A g r o b a c t e r iu m M c G r a n a h a n e t a l., 1 9 9 0
L o liu m s p . C M ic r o b o m b a r d m e n t Ye e t a l. , 1 9 9 7
M a n ih o t e s c u le n t a C M ic r o b o m b a r d m e n t S c h ö p k e e t a l. , 1 9 9 6
D M ic r o b o m b a r d m e n t R a e m a k e r s e t a l. , 1 9 9 6
E A g r o b a c t e r iu m L i e t a l. , 1 9 9 6
M e d ic a g o s a t iv a D A g r o b a c t e r iu m N in k o v ic e t a l . , 1 9 9 5
M e d ic a g o tr u n c u la ta B A g r o b a c t e r iu m C h a b a u t e t a l., 1 9 9 6
O e n a n t h e s t o lo n ife r a D M ic r o b o m b a r d m e n t B e e n a n d K im , 1 9 9 5
O r y z a s a tiv a C M ic r o b o m b a r d m e n t L i e t a l . , 1 9 9 7 ; N a y a k e t a l. , 1 9 9 7 ; J a in e t a l . , 1 9 9 6 b
P a n a x g in s e n g B A g r o b a c t e r iu m L e e e t a l. , 1 9 9 5
P ic e a s p . C A g r o b a c t e r iu m D r a k e e t a l. , 1 9 9 7
D M ic r o b o m b a r d m e n t C h a r e s t e t a l . , 1 9 9 6 ; B o m m in e n i e t a l . , 1 9 9 7
P ru n n u s sp. B A g r o b a c t e r iu m S c o r z a e t a l., 1 9 8 9
C A g r o b a c t e r iu m D a C a m a r a - M a c h a d o e t a l. , 1 9 9 5
R o s a h y b r id a B A g r o b a c t e r iu m v a n d e r S a lm e t a l . , 1 9 9 7
S a c ch a ru m C M ic r o b o m b a r d m e n t S n y m a n e t a l. , 1 9 9 6
o f fic in a r u m
S o la n u m m e lo n g e n a B A g r o b a c t e r iu m F a r i e t a l., 1 9 9 5
S o rg h u m s p. B M ic r o b o m b a r d m e n t C a s a s e t a l., 1 9 9 3
V itis s p . C M ic r o b o m b a r d m e n t K ik k e r t e t a l . , 1 9 9 6
C A g r o b a c t e r iu m N a k a n o e t a l., 1 9 9 4 ; M a u r o e t a l., 1 9 9 5
Z ea m ays C M ic r o b o m b a r d m e n t Z h o n g e t a l., 1 9 9 6

R. Bras. Fisiol. Veg., 10(1):1-12, 1998.

4 Vicient & Martínez

not eliminate somaclonal variation but could be applied however in some species somatic embryos seem to
in more species. be sensitive to the selective agents usually used to
C) Transformation of embryogenic cell culture and detect transformed cells, as for example in cassava
regeneration via indirect somatic embryogenesis. (Schöpke et al., 1993), and regeneration through
Transformation of cultured cells is easier than of secondary somatic embryogenesis is not easy. Li et
explants and plant regeneration is faster. al. (1996) proposed a method combining somatic
D) Transformation of somatic embryos and regeneration embryogenesis and organogenesis: secondary
through secondar y or indirect somatic somatic embryos were transformed using
embryogenesis. Somatic embryos seem to be easily Agrobacterium infection and transgenic plants were
transformed. Transformed embr yos could be regenerated via organogenesis after cycles of
regenerated by secondary somatic embryogenesis secondary somatic embryogenesis.
or by producing embryogenic calli or cell cultures. A
comparison of both methods in Oenanthe stolonifera Most of the reports on regeneration of genetically
have shown that the second method gives higher transformed plants correspond to tests or optimizations
transformation efficiencies (Been & Kim, 1995). of the methods using either genes conferring resistance
E) Transformation of somatic embryos, proliferation to antibiotics or reporter genes. However, some practical
through secondary embryogenesis and regeneration experiments have been yet made using different types
through organogenesis. Secondary embryogenesis of transgenes, especially with disease resistance genes
provides great quantities of easily transformed cells, (Table 2).

TABLE 2- Species genetically transformed using somatic embryogenesis with genes of agricultural interest.

PLANT GENE REFERENCES

Cucumber Chitinase genes Raharjo et al., 1996
Gene encoding the coat protein of the grapevine somatic chrome
Grapevine Legall et al., 1994
mosaic nepovirus
Grapevine Gene encoding the Grapevine Fan Leaf Virus coat protein Mauro et al., 1995

Papaya Gene encoding the Papaya Ringspot Virus coat protein Cheng et al., 1996

Peanut Bacillus thuringensis insecticide crystal protein (cryIAc) gene Singsit et al., 1997

Rosa hybrida ROL genes van der Salm et al., 1997

Rice LEA3 gene from barley Jain et al., 1996b

Rice Bacillus thuringensis insecticide crystal protein (cryIAc) gene Nayak et al., 1997

Soybean Synthetic Bacillus thuringensis insecticide crystal protein (cryIAc) gene Stewart et al., 1996

Soybean Gene encoding a mammalian stearyl CoA delta 9 desa-turase Liu et al., 1996
Sweet potato Trypsin inhibitor and lectin genes Newell et al., 1995

Somatic hybrid regeneration problems and also allows new combinations of nuclear
Crosses of cultivated species with wild type and organelle DNA, but a plant regeneration system is
relatives are a potential source of genetic variability for first required. The regeneration of fused protoplasts can
plant genetic improvement (Glimelius et al., 1991). be done via organogenesis or via somatic
However, their utilization in many species is limited embryogenesis. As has been mentioned previously,
because such crosses can not be made. Moreover, the somatic embr yogenesis has the advantage of
regeneration of somatic hybrids could be difficult in some regenerating plants from single cells, and for these
species, even using embryo rescue techniques. The reason it is the selected regeneration method in many
protoplast fusion technique overcomes part of these somatic hybridization projects (Table 3).

R. Bras. Fisiol. Veg., 10(1):1-12, 1998.

 Somatic embryogenesis 5

TABLE 3- Somatic hybrids in which somatic embryogenesis has been used in their production.

SPECIES REFERENCES

Actinidia intragenic hybrids Mu et al., 1990

Arachis intragenic hybrids1 Feng et al., 1996
Asparagus intragenic hybrids Kunitake et al., 1996
Citrus intragenic hybrids Grosser et al., 1996
Citrus reticulata x Citropsis gilletiana Grosser et al., 1990
Citrus reticulata x Citropsis gabunensis Ling and Iwamasa, 1994
Citrus sinensis x Murraya paniculata Shinozaki et al., 1992
Citrus sinensis x Atalantia ceylanica Louzada et al., 1993
Cucumis intragenic hybrids Dabauza et al., 1991
Fortunella crassifolia x Citrus sinensis Deng et al., 1992
Helianthus intragenic hybrids Fambrini et al., 1996
Lycopersicon intragenic hybrids Lanzhuang and Adachi, 1996
Medicago intragenic hybrids Mendis et al., 1991
Oryza intragenic hybrids Ozawa et al., 1996
Pennisetum americanum x Saccharum officinarum Tabaeizadeh et al., 1986
Rosa sinensis x Lavatera thuringiaca Vazquez-Thello et al., 1996
1
Hybrids were recovered through tip culture followed by somatic embryogenesis.

Homozygous and polyploid line production problems, for example by maintaining long-term
embryogenic calli or cell-suspension cultures. However,
Somatic embryogenesis can produce spontaneous maintenance of cell cultures by subculture is time
polyploidy in the regenerated plants, as has been consuming and expensive, and involves the risk of loss
observed in melon ( Cucumis melo) and Iochroma of valuable materials through contaminations or human
warscewiczii (Canhoto et al., 1990; Ezura and Oosawa, or technical errors. Additional problems of long-term cell
1994). The application of colchicine or the cultures include the risk of genetic changes by somaclonal
anti-microtubular drug amiprophos-methyl in the variation and decreases in the plant regeneration capacity
embryogenic medium enhances the number of polyploid and in the viability of the regenerated plants (Fitch &
embryos produced (Gmitter et al., 1991; Binarova and Moore, 1993). The frequent initiation of new embryogenic
Dolezel, 1993). Triploid plants can be regenerated when cell cultures can reduce the adverse effects on genetic
somatic embryogenesis is performed using endosperm fidelity and regeneration potential but is expensive and
as explant as have been observed in Prunnus persica time consuming. Moreover, the initiation of new cultures
(Liu and Liu, 1980), Citrus (Gmitter et al., 1990) and could be particularly difficult in some species in which
Acacia nilotica (Garg et al., 1996). the establishment of embryogenic cell cultures have
It is also possible to regenerate plants from haploid proven to be complicated or in which the embryogenic
explants (microspores) via somatic embryogenesis. explants are only available during short periods of the
However, haploid plant regeneration from anthers year.
(androgenesis) or ovules (gynogenesis) through embryo However, some alternatives exist. The manipulation
formation is not considered as somatic embryogenesis required for cell culture maintenance can be reduced by
in sensu stricto (Raghavan, 1983) and for this reason maintaining the cultures at low temperatures (0-10°C)
we are not going to treat this subject. The combination (Attree & Fowke, 1993; Janeiro et al., 1995). It is also
of haploid plant production with chromosome doubling possible to reduce manipulation by growing cell cultures
techniques has been used to obtain homozygous plants in hermetic flasks at room temperature (Joy et al., 1991).
in a single generation (Gudu et al., 1993; Zhao & The loss of embryogenic potential can be partially
Simmonds, 1995). reduced by the application of exogenous spermidine
(Bajaj & Rajam, 1995). In addition, the establishment of
Germplasm preservation long-term cell cultures in hormone-free media could
The storage of seeds in dry and cold conditions reduce the risk of somaclonal variations. Long-term
offers a convenient method for long-term storage of the embryogenic cell-lines cultured on hormone-free medium
germplasm. However, the seeds of some species not have been described in some species, including
can not be stored at low temperatures or are not asparagus (Delbreil et al ., 1994), carrot (Smith &
desiccation-tolerant. Moreover, some crop species are Krikorian, 1989), grape (Gray, 1992) and Hevea
only propagated vegetatively, causing additional brasilensis (Cailloux et al., 1996). However, cell culture
preservation problems. Plant cell in vitro culture on hormone-free medium reduces somaclonal variation
technology provides a potential solution for these but not the amont of manipulation or the loss of

R. Bras. Fisiol. Veg., 10(1):1-12, 1998.

6 Vicient & Martínez

embryogenic potential. of the plant regeneration capacities of frozen asparagus

Cryopreservation (storage at -196°C in liquid nitrogen) somatic embryos, calli, cell suspensions, and meristems,
of embryogenic cell cultures, explants or somatic embryos the best results were obtained from embryogenic calli
is a good alternative to cell culture maintenance (Kartha, (Uragami, 1991). The use of embryogenic calli in
1987). Cryopreservation minimizes the necessity of the cryopreservation experiments has been described also
establishment and maintenance of the embryogenic cell in cotton (Rajasekaran, 1996) and barley (Fretz & Lörz,
cultures, reducing manipulations, genetic variation and 1995), but most experiments have been made using
risks of loss. The possibility of combining the preservation suspension cultured cells because it is very much easier
potential of cryopreservation with the amplification to make uniform and effective cryoprotectant treatments
potential of somatic embryogenesis makes this technique in liquid media. Embryogenic cell culture explants and
very interesting for germplasm conservation institutes. somatic embryos have been succesfully used in Larix
Moreover, in some cases the embryogenic potential and/ and carrot, respectively (Tessereau et al., 1994; Bonga,
or regeneration capacity of the embryogenic cell culture 1996).
is improved after cryopreservation (Bercetche et al, 1990; Many factors in the freezing procedure can influence
Lynch et al., 1994). This phenomena may be explained freezing tolerance: pre-treatments, cryoprotection agents,
by a selective survival of the embryogenic cells with freezing and thawing procedures, and post-thawing
respect to the non-embryogenic ones. Regeneration of treatments. Freezing tolerance can be improved in plant
frozen explants, cell cultures or somatic embryos through tissues by use of pre-treatments. A relationship seems
somatic embryogenesis have been shown to be possible to exist between tolerance to desiccation and tolerance
in many species (Table 4). to freezing, and treatments increasing drought tolerance
also increase freezing tolerance. The reason is probably
TABLE 4- Species of agroforestry interest in which plant that the treatments improving desiccation tolerance
regeneration from frozen somatic embryos, or from produce a reduction in the free cell water contents,
preventing intracellular ice crystal formation. Some of the
SPECIES REFERENCES components successfully used in the pre-treatments
medium are:
Asparagus officinalis Nishizawa et al., 1993 - Abscisic acid. Abscisic acid (ABA) increases cultured
Coffea sp. Mycock, et al., 1995 cell freezing tolerance, probably preparing cells for
Daucus carota Tessereau et al., 1994 desiccation (Uragami, 1991; Tessereau et al., 1994). A
Elaeis guineensis Dumet et al., 1993 treatment of 2 µM ABA increased freezing tolerance in
Gossypium hirsutum Rajasekaran, 1996 barley and wheat cultures (Fretz and Lörz, 1995).
Hordeum vulgare Fretz et al., 1992 - Osmoticum. Pre-culture in a medium of high osmotic
Ipomoea batatas Blakesley et al., 1996 concentration increases freezing tolerance and survival
Larix sp. Bonga, 1996 rates. For example, a 0.75 M sucrose pre-treatment
Manihot esculenta Mycock, et al., 1995 increases the freezing tolerance of oil palm somatic
Oryza sativa Jain et al., 1996a embryos (Dumet et al., 1993). A treatment with 0.4-0.7
M sucrose increases freezing tolerance in embryogenic
Panicum maximum Gnanapragasam and Vasil, 1992
explants of sweet potato (Blakesley et al., 1996), while
Pennisetum glaucum Gnanapragasam and Vasil, 1992 0.3 M sorbitol in suspension cells of barley (Fretz & Lörz,
Phoenix dactylifera Mycock et al., 1995 1995). High molecular mass substances like PEG or
Picea abies Bercetche et al., 1990 dextrans are more effective in improving freezing
Picea glauca Attree et al., 1995 tolerance than are carbohydrates (Attree et al., 1995).
Pisum sativum Mycock et al., 1995 - Partial desiccation. Partial desiccation reduces
Saccharum officinarum Gnanapragasan and Vasil, 1992 intracellular water content, reducing ice formation and
Setaria italica Lu and Sun, 1992 increasing freezing tolerance. For example, desiccation
Triticum aestivum Kendall et al., 1993 of sweet potato embryogenic explants prior to freezing
improved the freezing tolerance (Blakesley et al., 1996)
Zea mays Gordon-Kamm et al., 1990
and desiccated white spruce somatic embryos survived
frozen explants or cell cultures through somatic embryogenesis, freezing treatment at higher frequencies than non-
have been shown to be possible. desiccated ones (Attree et al., 1995).
- Cold. In some cases, cold pre-treatment improves
Many factors influence freezing tolerance. A very freezing tolerance (Dallaire et al., 1994). However, this
important factor is the plant material used. It is possible depends on the species and tissue. For example, cold
to freeze many different tissues, including various treatment improves freezing tolerance in barley calli but
explants, embryogenic cell cultures (suspended cells or not in barley suspended cells (Fretz & Lörz, 1995).
calli) and somatic embryos at different stages of Plant material must be frozen in the presence of a
development. The selection of the tissue to be frozen is cryoprotectant solution. The optimal composition of the
critical because freezing tolerance depends on the water solution depends on the plant material and species. In
content of the tissue and on the number and size of the general, complex mixtures of cryoprotecting agents work
cell vacuoles, in cells with few and small vacuoles being better than single compound solutions probably because
more freezing tolerant (Uragami, 1991). In a comparison in the mixtures each compound is present a lower

R. Bras. Fisiol. Veg., 10(1):1-12, 1998.

 Somatic embryogenesis 7

concentration, reducing the possible toxicity of any one anthers and ovaries of plants infected with GFLV and
of them (Fretz et al., 1992; Wang et al., 1994). A typical various GLR viruses. These calli were heat shock treated
cryoprotectant solution contains DMSO (5-10 %), sorbitol (35°C) and somatic embryos were successfully produced
or sucrose (0.33-0.5 M), and glycerol (2 M). and converted into plants. Regenerated plants were
During cryopreservation, free radicals and activated tested using immunosorbent electron microscopy (ISEM)
oxygen can be generated (Benson & Noroncha-Dutra, and serological tests (ELISA) and the results indicated
1988), both highly toxic for the cells. The presence during that somatic embryogenesis in combination with heat
freezing of anti-oxidants such as ascorbic acid enhances therapy is an effective procedure to eliminate viruses.
the rates of cell survival (Fuller et al., 1988; Fretz & Lörz, Curiously, sometimes elimination success depended on
1995). the explant used. For example, using the same somatic
The conventional freezing method for cryopreserved embryogenesis/heat shock treatment, GFLV virus is
plant material is a two-step procedure in which a first eliminated if the somatic embryos were originated from
step consists in a slow cooling (0,5-1°C/min) to an ovary explants but not if they were originated from
intermediate temperature (usually about -40°C), and a anthers.
second step consisting in a rapid cooling in liquid Strandberg (1993) compared the efficiency in the
nitrogen. This system has been successfully applied to elimination of the yellow-flecked mottled leaves virus in
many species including Panicum maximum, Penisetum Ophiopogon japonicus using meristem and somatic
maximum and maize (Shillito et al ., 1989; embryo plant regeneration without heat shock treatment.
Gnanapragasam and Vasil, 1992). However, this system All plants regenerated from somatic embryos appeared
has an important problem: the necessity of an expensive to be virus-free. In contrast, only about one-third of the
controlled programmable freezing machine. Some plants regenerated from meristems appeared to be virus-
alternative method have been developed succesfully as, free.
for example, the one-step vitrification method (Reinhoud This method has also been succesfully used in Citrus.
et al., 1995; Ishikawa et al., 1996), or a rapid freezing at Somatic embryos were obtained from stylar tissues of
-25°C and transference to liquid nitrogen (Jain et al., lemon plants of different cultivars affected by various
1996a). virus-like diseases. Plants regenerated from the somatic
The post-thaw treatment of cryopreserved cells is also embryos gave negative results when assayed for the
important for the survival and re-growth of the plant presence of viruses, virus-like agents, and viroids
material (Lynch et al., 1994). A rapid thawing at 40-45°C (d‘Onghia et al., 1997).
is usually used. Not removing the cryoprotectant solution
Metabolite Production
does not seem to affect recovery rates significantly
(Gnanapragasam & Vasil, 1990; Wang et al., 1994). The production of metabolites in traditional agriculture
Supporting thawed cells on filter paper has been shown is based on the culture of large quantities of plants,
to increase re-growth frequencies (Meijer et al., 1991; collection of certain organs or tissues that accumulate
Benson et al., 1992; Lynch et al, 1994). The time required the product, and a chemical extraction. Sometimes the
to recover cryopreserved cells depends on the material product is only accumulated in very specific parts of the
and method used. For example, a 2 month period have plants such fruits, seeds, flowers or embryos and
been reported for cultured cells of Festuca, Lolium, and sometimes very large quantities of plants are necessary
maize (Shillito et al., 1989; Wang et al., 1994), and 8-35 for the production of small quantities of metabolite.
days for rice (Meijer et al., 1991). Metabolite production in cell cultures has been proposed
Using a suitable method, it was possible to recover as an alternative for traditional methods. Some success
plant cells with nearly the same regeneration rates than has been reported with these techniques (Fujita & Tabata,
for non-frozen controls, independently of the time of 1987) but sometimes the undifferentiated cultured cells
storage in liquid nitrogen (Nishizawa et al., 1993). For does not accumulate the metabolite, or the metabolite is
example, after more than 4 years, frozen suspended cells accumulated only in low quantities. The addition of
and cotton callus showed normal morphology after precursors or elicitors can increase metabolite production
thawing and high growth and regeneration rates (Luckner & Diettrich, 1987; Eilert et al., 1987), but not
(Rajasekaran et al., 1996). always. Alternatively, organ culture or somatic
embryogenesis, when the products accumulate in the
Virus elimination
embryo, can be used. There are several factors that affect
Somatic embryos have a well-developed vascular the viability of these processes. The most important is
system but this system is not connected to the explant economics. Due to the high price of in vitro culture, the
tissue (Newton & Goussard, 1990). This makes possible metabolites to be produced by plant cell culture must be
the use of somatic embryogenesis as a method to of high economic value. Janick (1993) suggested that
eliminate viruses, combined or not with heat shock suitable metabolites are products of pharmaceutical
treatments (Goussard & Wiid, 1992; Strandberg, 1993). interest such as alkaloids and certain lipids, some flavor
Grapevine GFLV (Grapevine Fan Leaf Virus) and A-, compounds and food additives, and pigments (reviewed
2-, and 3-GLR viruses (Grapevine Leafroll associated by Janick, 1991). Some examples are:
viruses) were eliminated using somatic embryogenesis - Caffeine and theobromine from somatic embryos of
associated with thermotherapy (Goussard et al., 1991; cacao (Janick et al., 1982; Paiva & Janick, 1983).
Goussard & Wiid, 1992). Calli were produced from - Celery flavor compounds from celery somatic embryos

R. Bras. Fisiol. Veg., 10(1):1-12, 1998.

8 Vicient & Martínez

(Al-Abta et al., 1979). 607, 1992.

- Gamma-linolenic acid from borage (Borago officinalis) ATTREE, S.M. & FOWKE, L.C. (1993) Embryogeny of gymnosperms:
advances in synthetic seed technology. Plant Cell, Tissue and Organ
somatic embryos (Janick et al., 1987; Whipkey et al., Culture, 35:1-35, 1993.
1988; Quinn et al., 1989). ATTREE, S.M.; POMEROY, M.K. & FOWKE L.C. Development of white
- Jojoba liquid wax from jojoba somatic embryos (Gabr, spruce (Picea glauca (Moench) Voss) somatic embryos during
1993). culture with abscisic acid and osmoticum, and their tolerance to
drying and frozen storage. Journal of Experimental Botany,
- Taxoids from Taxus species (Lee and Son, 1995; Wann 46:433-439, 1995.
and Goldner, 1994). BAJAJ, Y.P.S. (Ed.) Somatic embryogenesis and synthetic seed I.
For industrial use, in vitro extraction must be cheaper Biotechnology in Agriculture and Forestry. New York, Springer-
compared with traditional methods. Due to the economic Verlag, 1995a, v.30.
limitations, mass somatic embryo production in BAJAJ, Y.P.S. (Ed.) Somatic embryogenesis and synthetic seed II.
Biotechnology in Agriculture and Forestry. New York, Springer-
bioreactors is essential. The composition of the media Verlag, 1995b, v.31.
and the problems associated with mass production in BAJAJ, S. & RAJAM, M.V. Efficient plant regeneration from long-term
bioreactors are especially important due to culture factors callus cultures of rice by spermidine. Plant Cell Reports, 14:717-
which can affect metabolite production. With Brassica 720, 1995.
BEAUBAIRE, N.A.; DANIELSON, D.; KLENBERG, D. &
napus microspore-derived somatic embryos, exogenous SAMUELSSON, B. Production of Borago officinalis L. Acta
ABA produced an increase in the total fatty acid content Horticulturae, 208:101-113, 1988.
(Pomeroy et al., 1994). Another example is gamma- BEEN, C.G. & KIM, B.D. Transformation through somatic embryo of
linolenic acid production from borago somatic embryos Oenanthe stolonifera with Agrobacterium. Journal of The Korean
(Quinn et al., 1989). The gamma-linolenic acid is a pre- Society for Horticultural Science, 36:792-798, 1995.
BENSON, E.E. & NORONCHA-DUTRA, A.A. Chemi-luminescence in
cursor in the prostaglandin synthesis. Borago seeds cryopreserved plant- tissue cultures.- The possible involvement
contain about 7% gamma-linolenic acid but large-scale of singlet oxygen in cryoinjury. Cryo-Letters, 9:120-131, 1988
borago cultures are problematic (Beaubaire et al., 1988). BENSON, E.E.; LYNCH, P.T. & JONES, J. The detection of lipid
Gamma-linolenic acid in the seed accumulates mainly peroxidation products in cryo-protected and frozen rice cells:
consequences for post-thaw survival. Plant Science, 85:107-114,
in the cotyledons, so it is necessary to obtain somatic 1992
embryogenesis systems producing good rates of BERCETCHE, J.; GALERNE, M. & DEREUDDRE, J. Augmentation
conversion to cotyledonary-stage embryos. In the optimal des capacités de régénération de cals embryogènes de Picea
conditions described by Quinn et al (1989), the gamma- abies (L.) Karst après congélation dans l’azote liquide. Comptes
linolenic acid content in somatic embryo was only 22.5% Rendus a l’Academie de Sciences Paris Série III, 310:357-363,
1990.
compared with 56% observed in zygotic cotyledon and BINAROVA, P. & DOLEZEL, J. Effect of anti-microtubular drug
33% in zygotic hypocotyl. amiprophos-methyl on somatic embryogenesis and DNA ploidy
levels in alfalfa and carrot cell suspension cultures. Biologia
Mycorrhizal initiation Plantarum, 35:329-339, 1993.
BLAKESLEY, D.; ALMAZROOEI, S.; BHATTI, M.H. & HENSHAW, G.G.
The combination of somatic embryogenesis and in Cryopreservation of non-encapsulated embryogenic tissue of
vitro mycorrhizal production was first proposed by sweet potato (Ipomaea batatas). Plant Cell Reports, 15:873-876,
Redenbaugh et al., in 1987. The application of somatic 1996.
embryo technology in forestry has been studied BOMMINENI, V.R.; CHIBBAR, R.N.; BETHUNE, T.D.; TSANG, E.W.T.
extensively, specially the application of synthetic seed & DUNSTAN, D.I. The sensitivity of transgenic spruce (Picea
glauca (Moench) Voss) cotyledonary somatic embryos and
technology in forest regeneration. The combination of somatic seedlings to kanamycin selection. Transgenic Research,
synthetic seed technology with mycorrizal initiation is very 6:123-131, 1997.
attractive. However, very little work has been done on BONGA, J.M. Frozen storage stimulates the formation of embryo-like
this subject. Some experiments have been made with structures and elongating shoots in explant from mature Larix
decidua and L. x eurolepsis trees. Plant Cell, Tissue and Organ
hybrid larch (Larix x eurolepis Henry) in which the in vitro Culture, 46:91-101, 1996.
inoculation with various ectomycorrhizal fungi enhanced BORNMAN, C.H. Micropropagation and somatic embryogenesis. In:
the development of somatic embryo-derived plantlets HAYWARD, M.D.; BOSEMARK, N.O. & ROMAGOSA, I. (Eds.).
(Piola et al., 1995). Plant Breeding: principles and prospects . Cambridge,
Chapman & Hall, 1993. p. 246-260.
CABRERA-PONCE, J.L.; VEGAS-GARCÍA, A. & HERRERA-
ACKNOWLEDGEMENTS ESTRELLA, L. Regeneration of transgenic papaya plants via
somatic embryogenesis induced by Agrobacterium rhizogenes.
We thank Michel Delseny, Yves Meyer and Martine In Vitro Cellular and Developmental Biology, 32:86-90, 1996.
Devic for critically reading the manuscript and Tom CABRERA-PONCE, J.L.; LOPEZ, L.; ASSAD-GARCIA, N.; MEDINA-
AREVALO, C.; BAILEY, A.M. & HERRERA-ESTRELLA, L. An
Roscoe and Alan H. Schulman for carefully correcting efficient par ticle bombardment system for the genetic
the manuscript and their critical reading. transformation of asparagus (Asparagus officinalis L). Plant Cell
Reports, 16:255-260, 1997.
CAILLOUX, F.; JULIEN-GUERRIER, J.; LINOSSIER, L. & COUDRET,
REFERENCES A. Long-term somatic embryogenesis and maturation of somatic
embryos in Hevea brasilensis. Plant Science, 120:185-196, 1996.
AL-ABTA, S.; GALPIN, I.J. & COLLIN, H.A. Flavour compounds in CANHOTO, J.M.; LUDOVINA, M.; GIMARAES, S. & CRUZ, G.S. In
tissue cultures of celery. Plant Science Letters, 16:129-134, 1979. vitro induction of haploid, diploid and triploid plantlets by anther
AMBERGER, L.A.; SHOEMAKER, R.C. & PALMER, R.G. Inheritance culture of Iochroma warscewiczii Regel. Plant Cell, Tissue and
of two independent isozyme variants in soybean plants derived Organ Culture, 21:171-177, 1990.
from tissue culture. Theoretical and Applied Genetics, 84:600- CASAS, A.M.; KONONOWICZ, A.K.; ZEHR, U.B.; TOMES, D.T.;

R. Bras. Fisiol. Veg., 10(1):1-12, 1998.

 Somatic embryogenesis 9

AXTELL, J.D.; BUTLER, L.G.; BRESSAN, R.A. & HASEGAWA, Helianthus annuus x Helianthus tuberosus . Plant Science,
P.M. Transgenic sorghum plants via microprojectile bombardment. 114:205-214, 1996.
Proceedings of the National Academy of Sciences of USA, FARI, M.; NAGY, I.; CSANYI, M.; MITYKO, J. & ANDRASFALVY, A.
90:11212-11216, 1993. Agrobacterium mediated genetic transformation and plant
CHABAUT, M.; LARSONNEAU, C.; MARMOUGET, C. & HUGUET, T. regeneration via organogenesis and somatic embryogenesis from
Transformation of barrel medic ( Medicago trunculata Gaertn.) by cotyledon leaves in eggplant ( Solanum melongena L. cv.
Agrobacterium tumefaciens and regeneration via somatic ‘Kecskemeti lila’). Plant Cell Reports, 15:82-86, 1995.
embryogenesis of transgenic plants with the MtENOD12 nodulin FENG, Q.; STALKER, H.T. & PATTEE, H.E. Plant recovery of self and
promoter fused to the gus reporter gene. Plant Cell Reports, interspecific hybrids of Arachis by in vitro culture of peg tips. Crop
15:305-310, 1996. Science, 36:1660-1666, 1996.
CHAREST, P.J.; DEVANTIER, Y. & LACHANCE, D. Stable genetic FITCH, M.M.M.; MANSHARDT, R.M.; GONSALVES, D. & SLIGHTOM,
transfor mation of Pice marina (black spruce) via par ticle J.L. Stable transformation of papaya via microprojectile
bombardment. In Vitro Cellular and Developmental Biology, bombardment. Plant Cell Reports, 9:189-194, 1990.
32:91-99, 1996. FITCH, M.M.M.; MANSHARDT, R.M.; GONSALVES, D. & SLIGHTOM,
CHENG, Y.H.; YANG, J.S. & YEH, S.D. Efficient transformation of J.L. Transgenic papaya plants from Agrobacterium mediated
papaya by coat protein gene of papaya ringspot virus mediated transformation of somatic embryos. Plant Cell Reports,
by Agrobacterium following liquid-phase wounding by 12:245-249, 1993.
embryogenic tissues with carborundum. Plant Cell Reports, FOURRÉ, J.L.; BERGER, P.; NIQUET, L. & ANDRE, P. Somatic
16:127-132, 1996. embryogenesis and somaclonal variation in Norway spruce:
DABAUZA, M., ROIG, L.A. & MORENO, V. Selective methods for the morphogenetic, cytogenetic and molecular approaches.
recovery of somatic hybrids of Cucumis melo x C. metuliferus and Theoretical and Applied Genetics, 94:159-169, 1997.
C. sativus x C. metuliferus. Reports on Cucurbitaceae Genetics FRETZ, A.; JAHNE, J. & LÖRZ, H. Cryopreservation of embryogenic
Cooperation, 14:81-84, 1991. suspension cultures of barley ( Hordeum vulgare L.) Botanica
DA CAMARA MACHADO, A.; PUSCHMANN, M.; PUHRINGER, H.; Acta, 105:140-145, 1992.
KREMEN, R.; KATINGER, H. & LAIMIER DA CAMARA FRETZ, A. & LORZ, H. Cryopreservation of in vitro cultures of barley
MACHADO, M. Somatic embryogenesis of Prunnus subhirtella (Hordeum vulgare L. and H. murinum L.) and transgenic cells of
autumno rosa and regeneration of transgenic plants after wheat (Triticum aestivium L.). Journal of Plant Physiology,
Agrobacterium -mediated transformation. Plant Cell Reports, 146:489-496, 1995.
14:333-340, 1995. FUJITA, Y. & TABATA, M. Secondary metabolites from plant cells.
DALLAIRE, S.; HOUDE, M.; GAGNE, Y.; SAINI, S.; BOILEAU, N.; Pharmaceutical applications and progress in commercial
CHEVRIER, N. & SARHAN, F. ABA and low temperature induces production. In: GREEN, C.E.; SOMERS, D.A.; HACKETT, W.P. &
freezing tolerance via distinct regulatory pathways in wheat. Plant BIESBOER, D.D. (Eds.) Plant Tissue and Cell Culture. New York,
Cell Reports, 35:1-9, 1994. Alan R Liss, 1987. p.169-185.
DELBREIL, B.; GOEBEL-TOURAND, I.; LEFRANÇOIS, C. & JULLIEN, FULLER, B.J.; GOWER, J.D. & GREEN, C.J. Free radical damage
M. Isolation and characterisation of long-term embryogenic lines and organ preservation: fact or fiction. Cryobiology, 25:377-393,
in Asparagus officinalis L. Journal of Plant Physiology, 144:194- 1988.
200, 1994. GABR, M.F. In vitro production of jojoba liquid wax from somatic
DENG, X.X.; GROSSER, J.W. & GMITTER, F.G. Intergeneric somatic embryos proliferated via vegetative tissues. Egyptian Journal of
hybrid plants from protoplast fusion of Fortunella crassifolia cultivar Horticulture, 20:135-144, 1993.
‘Meiwa’ with Citrus sinensis cultivar ‘Valencia’. Scientia GAMA-MICS; LEITE, R.P.; CORDEIRO, A.R. & CANTLIFFE, D.J.
Horticulturae, 49:55-62, 1992. Transgenic sweet potato plants obtained by Agrobacterium
DODEMAN, V.L.; DUCREUX, G. & KREIS, M. Zygotic embryogenesis tumefaciens mediated transformation. Plant Cell Tissue and
versus somatic embryogenesis. Journal of Experimental Botany, Organ Culture, 46:237-244, 1996.
48:1493-1509, 1997. GARG, L.; BHANDARI, N.N.; RANI, V. & BHOJWANI, S.S. Somatic
D’ONGHIA, A.M.; DE PASCUALE, F.; CARIMI, F.; SAVINO, V. & embryogenesis and regeneration of triploid plants in endosperm
CRESCIMANNO, F.G. Somatic embryogenesis from style culture cultures of Acacia nilotica. Plant Cell Reports, 15:855-858, 1996.
as a possible means for virus elimination in Citrus. Journal GLIMELIUS, K.; FAHLESSON, J.; LANDGREN, M.; SJÖDIN, C. &
Phytopathology, 145:77-79, 1997. SUNDBERG, E. Gene transfer via somatic hybridisation in plants.
DRAKE, P.M.W.; JOHN, A.; POWER, J.B. & DAVEY, M.R. Expression Trends in Biotechnology, 9:24-30, 1991.
of the gus a gene in embryogenic cell lines of sitka spruce following GMITTER, F.G. jr.; LING, X.B. & DENG, X.X. Induction of triploid Citrus
Agrobacterium-mediated transformation. Journal of Experimental plants from endosperm calli in vitro. Theoretical and Applied
Botany, 48:151-155, 1997. Genetics, 80:785-790, 1990.
DUCROCQ, C.; SANGWAN, R.S. & SANGWAN-NORREEL, B.S. GMITTER, F.G. jr.; LING, X.B.; CAI, C.Y. & GROSSER, J.W.
Production of Agrobacterium-mediated transgenic fertile plants Colchicine-induced polyploidy in Citrus embryogenic cultures,
by direct somatic embryogenesis from immature zygotic embryos somatic embryos, and regenerated plantlets. Plant Science,
of Datura innoxia. Plant Molecular Biology, 25:995-1009, 1994. 74:135-141, 1991.
DUMET, D.; ENGELMANN, F.; CHABRILLANGE, N. & DUVAL, Y. GNANAPRAGASAM, S. & VASIL, I.K. Plant regeneration from a
Cryopreservation of oil palm (Elaeis guineensis Jacq.) somatic cryopreserved embryogenic cell suspension of a commercial
embryos involving a desiccation step. Plant Cell Reports, sugarcane hybrid ( Saccharum sp.). Plant Cell Reports,
12:352-355, 1993 9:419-423, 1990.
EILERT, U.; KURZ, W.G.W. & CONSTABEL, F. Alkaloid accumulation GNANAPRAGASAM, S. & VASIL, I.K. Cryopreservation of immature
in plant cell cultures upon treatment with elicitors. In: GREEN, C.E.; embryos, embryogenic callus and cell suspension cultures of
SOMERS, D.A.; HACKETT, W.P. & BIESBOER, D.D. (Eds.) Plant gramineous species. Plant Science, 83:205-215, 1992.
Tissue and Cell Culture. New York, Alan R Liss, 1987. p.213- GORDON-KAMM, W.J.; SPENCER, T.M.; MANGANO, M.L.; ADAMS,
219. T.R. & LEMAUX, P.G. Transformation of maize cells and
EZURA, H. & OOZAWA, K. Ploidy of somatic embryos and the ability regeneration of fertile transgenic plants. Plant Cell, 2:603-618,
to regenerate plantlets in melon (Cucumis melo L). Plant Cell 1990.
Reports, 14:107-111, 1994. GOUSSARD, P.G.; WIID, J. & KASDORF, G.G.F. The effectiveness of
EZURA, H.; AMAGAI, H.; KIKUTA, I.; KUBOTA, M. & OOSAWA, K. in vitro somatic embryogenesis in eliminating fanleaf virus and
Selection of somaclonal variants with low-temperature leafroll associated viruses from grapevines. South African
germinability in melon (Cucumis melo L.) Plant Cell Reports, Journal of Enololgy and Viticulture, 12:77-81, 1991.
14:684-688, 1995. GOUSSARD, P.G. & WIID, J. The elimination of fanleaf virus from
FAMBRINI, M.; CIONINI, G. & PUGLIESI, C. Development of somatic grapevines using in vitro somatic embryogenesis combined with
embryos from morphogenetic cells of the interspecific hybrid heat therapy. South African Journal of Enololgy and Viticulture,

R. Bras. Fisiol. Veg., 10(1):1-12, 1998.

10 Vicient & Martínez

13:81-83, 1992. Asparagus officinalis and A. macowanii through electrofusion.

GRAY, D.J. Somatic embryogenesis and plant regeneration from immature Plant Science, 116:213-222, 1996.
zygotic embryos of muscadine grape (Vitis rotundifolia) cultivars. LANZHUANG, C. & ADACHI, T. Efficient hybridisation between
American Journal of Botany, 79:542-546, 1992. Lycopersicon esculentum and L. peruvianum via ‘embryo rescue’
GROSSER, J.W.; GMITTER, F.G.; TUSA, N. & CHANDLER, J.L. and in vitro propagation. Plant Breeding, 115: 251-256, 1996.
Somatic hybrid plants from sexually incompatible woody species: LEE, B.S. & SON, S.H. A method for producing taxol and taxanes
Citrus reticulata and Citropsis gilletiana. Plant Cell Reports, 8:656- from embryo cultures of Taxus species. WO Patent N°95/02063,
659, 1990. 1995.
GROSSER, J.W.; GMITTER, F.G.; TUSA, N.; REFORGIATO-RECU- LEE, H.S.; KIM, S.W.; LEE, K.W.; ERIKSSON, T. & LIU, J.R.
PERO, G. & CUCINOTTA, P. Further evidence of a cybridization Agrobacterium-mediated transformation of ginseng ( Panax
requirement for plant regeneration from Citrus leaf protoplasts ginseng) and mitotic stability of the inserted beta-glucurodinase
following somatic fusion. Plant Cell Reports, 15: 672-676, 1996. gene in regenerants from isolated protoplasts. Plant Cell Reports,
GUDU, S.; PROCUNIER, J.D.; ZIAUDDIN, A. & KASHA, K.J. Anther 14:545-549, 1995.
culture derived homozygous lines in Hordeum bulbosum . Plant LEGALL, O.; TORREGROSA, Y.; DANGLOT, T.; CANDRESSE, T. &
Breeding, 110:109-115, 1993. BOUQUET, A. Agrobacterium-mediated genetic transformation of
HEINZE, B. & SCHMIDT, J. Monitoring genetic fidelity vs somaclonal grapevine somatic embryos and regeneration of transgenic plants
variation in Norway spruce (Picea abies) somatic embryogenesis expressing the coat protein of the grapevine somatic chrome
by RAPD analysis. Euphytica, 85:341-345, 1995. mosaic nepovirus (GCMV). Plant Science, 102:161-170, 1994.
HUTCHINSON, J.F.; KAUL, V.; MAHESWARAN, G.; MORAN, J.R.; LI, B:J: & WU, M. Transformation of foreign genes into the
GRAHAM, M.W. & RICHARDS, D. Genetic improvement of monocotyledonous plant Caladium bicolor mediated by
floricultural crops using biotechnology. Australian Journal of constructed Ti-plasmid of Agrobacterium tumefaciens. Chinese
Botany, 40:765-787, 1992. Science Bulletin, 35:52-55, 1990.
ISHIKAWA, M.; TANDOM, P.; SUZUKI, M. & YAMAGUISHI-CIAMPI, A. LI, H.Q.; SAUTTER, C.; POTRYKUS, I. & PUONTI-KAERLAS, J.
Cryopreservation of bromegrass ( Bromus inermis Leyss) Genetic transformation of cassava (Manihot esculenta Crantz).
suspension cultured cells using slow prefreezing and vitrification Nature Biotechnology, 14:736-740, 1996.
procedures. Plant Science, 120:81-88, 1996. LI, Z.; UPADHYAYA, N.M.; MEENA, S.; GIBBS, A.J. & WATERHOUSE,
JACOBS, M.; NEGRUTIU, I.; DIRKS, R. & CAMMAERTS, D. Selection P.M. Comparison of promoters and selectable marker genes for
programmes for isolation and analysis of mutants in plant cell use in Indica rice transformation. Molecular Breeding, 3:1-14,
cultures. In: GREEN, C.E.; SOMERS, D.A.; HACKETT, W.P. & 1997.
BIESBOER, D.D. (Ed.) Plant Tissue and Cell Culture. New York, LING, J.T. & IWAMASA, M. Somatic hybridisation between Citrus
Alan R Liss, 1987. p.243-264. reticulata and Citropsis gabunensis through electrofusion. Plant
JAIN, S.; JAIN, R.K. & WU, R. A simple and efficient procedure for Cell Reports, 13:493-497, 1994.
cryopreservation of embryogenic cells of aromatic indica rice LITZ, R.E.; MOORE, G.A. & SRINIVASAN, C. In vitro systems for
varieties. Plant Cell Reports, 15:712-717, 1996a. propagation and improvement of tropical fruits and palms.
JAIN, R.K.; JAIN, S.; WANG, B. & WU, R. Optimisation of biolistic Horticultural Reviews, 7:157-200, 1985.
method for transient gene expression and production of LITZ, R.E. & GRAY, D.J. Somatic embryogenesis for agricultural
agronomically useful transgenic Basmati rice plants. Plant Cell improvement. World Journal of Microbiology and
Reports, 15:963-968, 1996b. Biotechnology, 11:416-425, 1995.
JANEIRO, L.V.; BALLESTER, A. & VIEITEZ, A.M. Effect of cold storage LIU, S.Q. & LIU, J.Q. Callus induction and embryo formation in
on somatic embryogenesis systems of Camellia . Journal of endosperm culture of Prunnus persica. Acta Botanica Sinica,
Horticultural Science, 70:665-672, 1995. 22:198-199, 1980.
JANICK, J.; WRIGHT, D.C. & HASEWAGA, P.M. In vitro production of LIU, W.N.; TORISKY, R.S.; McALLISTER, K.P.; AVDIUSHKO, S.;
cacao seed lipids. Journal of The American Society for HILDEBRAND, D. & COLLINS, G.B. Somatic embryo cycling:
Horticultural Science, 107:919-922, 1982. evaluation of a novel transformation and assay system for seed
JANICK, J.; SIMON, J.E. & WHIPKEY, A. In vitro propagation of borage. specific gene expression in soybean. Plant Cell Tissue and Organ
HortScience 22:493-495, 1987. Culture, 47:33-42, 1996.
JANICK, J. Production of seed lipids via culture of somatic embryos. LIVINGTONE, D.M. & BIRCH, R.G. Plant regeneration and
In: RATTRAY, J. (Ed.) Biotechnology of Plant Fats and Oils. microprojectile-mediated gene transfer in embryonic leaflets of
Champaign, American Oil Chemistry Society, 1991, p.76-104. peanut ( Arachis hypogaea L.) Australian Journal of Plant
JANICK, J. Agricultural uses of somatic embryos. Acta Horticulturae, Physiology, 22:585-591, 1995.
136: 207-215, 1993. LOUZADA, E.S.; GROSSER, J.W. & GMITTER, F.G. Intergeneric
JOY, R.W.; KUMAR, P.P. & THORPE, T.A. Long term storage of somatic somatic hybridization of sexually incompatible parents: Citrus
embryogenic white spruce tissue culture at ambient temperature. sinensis and Atalantia ceylanica. Plant Cell Reports, 12:687-690,
Plant Cell, Tissue and Organ Culture, 25:53-60, 1991. 1993.
KARTHA, K.K. Advances in the cryopreservation technology of plant LU, T.G. & SUN, C.S. Cryopreservation of millet (Setaria italica L.)
cells and organs. In: GREEN, C.E.; SOMERS, W.P., HACKETT, Journal of Plant Physiology, 139:295-298, 1992.
A.R. & BIERSBOER, D.D. (Eds.) Plant Tissue and Cell Culture. LUCKNER, M. & DIETTRICH, B. Biosynthesis of cardenolides in cell
New York, A.R. Liss, 1987. p. 447-458. cultures of Digitalis lanata. The result of a new strategy. In: GREEN,
KENDALL, E.J.; KARTHA, K.K.; QURESHI, J.A. & CHERMAK, P. C.E.; SOMERS, D.A.; HACKETT, W.P. & BIESBOER, D.D. (Ed)
Cryopreservation of immature spring wheat zygotic embryos using Plant Tissue and Cell Culture. New York, Alan R Liss, 1987,
an abscisic acid pretreatment. Plant Cell Reports, 12:89-94, 1993. p.187-197.
KIKKERT, J.R.; HEBERT-SOULE, D.; WALLACE, P.G.; STRIEM, M.J. LYNCH, P.T.; BENSON, E.E.; JONES, J.; COCKING, E.C.; POWER,
& REISCH, B.I. Transgenic plantlets of ‘Chancellor’ grapevine (Vitis J.B. & DAVEY, M.R. Rice cell cryopreservation: the influence of
sp) from biolistic transformation of embryogenic cell suspensions. culture methods and the embryogenic potential of cell suspensions
Plant Cell Reports, 15:311-316, 1996. on post-thaw recovery. Plant Science, 98:185-192, 1994.
KRASTANOVA, S.; PERRIN, M.; BARBIER, P.; DEMANGEAT, P.; MALUSZYNSKY, M.; AHLOOWALIA, B.S. & SIGURBJORNSSON, B.
CORNUET, N.; BARDONNET, L.; OTTEN, L.; PINCK L. & WALTER Application of in vivo and in vitro mutation techniques for crop
B. Transformation of grapevine roostocks with the coat protein gene improvement. Euphytica, 85:303-315, 1995.
of grapevine fanleaf nepovirus. Plant Cell Reports, 13:357-360, MAURO, M.C.; TOUTANIN, S.; WALTER, B.; PINCK, L.; OTTEN, L.;
1995. COUTOS-THEVENOT, P.; DELOIRE, A. & BARBIER, P. High
KUKSOVA V.B. ; PIVEN, N.M. & GLEBA, Y.Y. Somaclonal variation efficiency regeneration of grapevine plants transformed with the
and in vitro induced mutagenesis in grapevine. Plant Cell Tissue GFLV coat protein gene. Plant Science, 112:97-106, 1995.
and Organ Culture, 49:17-27, 1997. McGRANAHAN, G.H.; LESLIE, C.A.; URATSU, S.L. & DANDEKAR,
KUNITAKE, H.; NAKASHIMA, T.; MORI, K.; TANAKA, M.; SAITO, A. & A.M. Improved efficiency of the walnut somatic embryo gene
MII, M. Production of interspecific somatic hybrid plants between transfer system. Plant Cell Reports, 8:512-516, 1990.

R. Bras. Fisiol. Veg., 10(1):1-12, 1998.

 Somatic embryogenesis 11

McGRANAHAN, G.H.; LESLIE, C.A.; DANDEKAR, A.M.; URATSU, S.L. somatic embryogenesis and applications in plant breeding.
& YATES, I.E. Transformation of pecan and regeneration of transgenic Euphytica, 81:93-107, 1995.
plants. Plant Cell Reports, 12:634-638, 1993. RAEMAKERS, C.J.J.M.; SOFIARI, E.; TAYLOR, N.; HENSHAW, G.;
MEIJER, E.G.M.; van IREN, F.; SCHRIJNEMAKERS, E.; HENSGENS, JACOBSEN, E. & VISSER, R.G.F. Production of transgenic cas-
L.A.M.; van ZIJDERVELD, M. & SCHILPEROORT, R.A. Retention sava (Manihot esculenta Crantz) plants by particle bombardment
of the capacity to produce plants from protoplasts in cryopreserved using luciferase activity as selection marker. Molecular Breeding,
cell lines of rice ( Oryza sativa L.). Plant Cell Reports, 10:171- 2:339-349, 1996.
174, 1991. RAGHAVAN, V. Biochemistry of somatic embryogenesis. In: EVANS,
MENDIS, M.H.; POWER, J.B. & DAVEY, M.R. Somatic hybrids of the D.A.; SHARP, W.R.; AMMIRATO, P.V. & YAMADA, Y. (Eds.)
forage legumes Medicago sativa L. and M. falcata L. Journal of Handbook of Plant Cell Culture: Techniques for propagation and
Experimental Botany, 42:1565-1573, 1991. Breeding. New York, Macmillan, 1983. v.1, p.655-671.
MU, S.K.; FRASSER, L.G. & HARVEY, C.F. Initiation of callus and RAHARJO, S.H.T.; HERNANDEZ, M.O.; ZHANG, Y.Y. & PUNJA, Z.K.
regeneration of plantlets from endosperm of Actinidia interspecific Transformation of pickling cucumber with chitinase-encoding
hybrids. Scientia Horticulturae, 44:107-117, 1990. genes using Agrobacterium tumefacienns. Plant Cell Reports,
MYCOCK, D.J.; WESLEY-SMITH, J. & BERJAK, P. Cryopreservation 15:591-596, 1996.
of somatic embryos of four species with and without cryoprotectant RAJASEKARAN, K. Regeneration of plants from cryopreserved
pre-treatment. Annals of Botany, 75:331-336, 1995. embryogenic cell suspension and callus cultures of cotton
NAKANO, M.; HOSHINO, Y. & MII, M. Regeneration of transgenic plants (Gossypium hirsutum L). Plant Cell Reports, 15:859-864, 1996.
of grapevine (Vitis vinifera L.) via Agrobacterium rhizogenes- RAMANA, R.V.; VENU, C.; JAYASREE, T. & SADANADAM, A. Direct
mediated transformation of embryogenic calli. Journal of somatic embryogenesis and transformation in Cicer arietinum L.
Experimental Botany, 45:649-656, 1994. Indian Journal of Experimental Biology, 34:716-718, 1996.
NAYAK, P.; BASU, D.; DAS, S.; BASU, A.; GHOSH, D.; REDENBAUGH, K.; VISS, P.; SLADE, D. & FUJII, J.A. Scale-up: artifi-
RAMAKRISHNAN, N.A.; GHOSH, M. & SEN S.K. Transgenic elite cial seeds. In: GREEN, C.E.; SOMERS, D.A.; HACKET, W.P. &
indica rice plants expressing CryIAc ð-endotoxin of Bacillus BIESBOER, D.D. (Eds.) Plant Tissue and Cell Culture. New York,
thuringiensis are resistant against yellow stem borer (Scirpophaga Alan R. Liss Inc., 1987a, p.473-493.
incertulas). Proceedings of the National Academy of Sciences REDENBAUGH, K. (Ed.) Synseeds. Boca Raton FL, CRC Press, 1993
of the USA, 94:2111-2116, 1997. REINHOUD, P.J.; SCHRIJNEMAKERS, E.W.M.; van IREN, F. & KIJNE,
NEWELL, C.A.; LOWE, J.M.; MERRYWEATHER, A.; ROOKE, L.M. & J.W. Vitrification and heat-shock treatment improve
HAMILTON, W.D.O. Transformation of sweet potato (Ipomoea ba- cryopreservation of tobacco cell suspensions compared to two-
tatas (L.) Lam.) with Agrobacterium tumefaciens and regeneration step freezing. Plant Cell, Tissue and Organ Culture, 42:261-267,
of plants expressing cowpea trypsin inhibitor and drop lectin. Plant 1995.
Science, 107:215-227, 1995. SCHÖPKE, C.; FRANCHE, C.; BOGUSZ, D.; CHAVARRIAGA, P.;
NEWTON, D.J. & GOUSSARD, P.G. The ontogeny of somatic embryos FAUQUET, C.M. & BEACHY, R.N. Transformation in cassava
from in vitro cultured grapevine anthers. South African Journal (Manihot esulenta Crantz). In: BAJAJ, Y.P.S. (Ed.) Biotechnology
of Enology and Viticulture, 11:70-75, 1990. in Agriculture and Forestry. Berlin, Springer-Verlag, 1993, p.273-
NINKOVIC, S.; MILJUS-DJUKIC, J. & NESKOVIC, M. Genetic 289.
transformation of alfalfa somatic embryos and their clonal SCHÖPKE, C.; TAYLOR, N.; CÁRCAMO, R.; DA KOFFI-KONAN, N.;
propagation through repetitive somatic embryogenesis. Plant Cell, MARMEY, P.; HENSHAW, G.G.; BEACHY, R.N. & FAUQUET, C.
Tissue and Organ Culture, 42:255-260, 1995. Regeneration of transgenic cassava plants (Manihot esculenta
NISHIZAWA, S.; SAKAI, A.; AMANO, Y. & MATSUZAWA, T. Crantz) from microbombarded embryogenic suspension cultures.
Cryopreservation of asparagus ( Asparagus officinalis L.) Nature Biotechnology, 14:731-735, 1996.
embryogenic suspension cells and subsequent plant regeneration SCHULZE, J.; BALKO, C.; ZELLNER, B.; KOPREK, T.; HANSCH, R.;
by vitrification. Plant Science, 91:67-73, 1993. NERLICH, A. & MENDEL, R.R. Biolistic transformation of cucumber
NYANGE, N.E.; WILLIAMSON, B. ; McNICOL, R.J. ; LYON, G.D. & using embryogenic suspension cultures: Long-term expression
HACKETT, C.A. In vitro selection of Coffea arabica callus for of reporter genes. Plant Science, 112:197-206, 1995.
resistance to partially purified phytotoxic culture filtrates from SCORZA, R.; CORDTS, J.M.; MANTE, S.; CALLAHAN, A.M.;
Colletotrichum kahawae. Annals of Applied Biology, 127:425- MORGENS, P. & COHEN, R. The development of regeneration
439, 1995. and transformation systems for peach. Acta Horticulturae 254:19-
ONISHI, N.; SAKAMOTO, Y. & HIROSAWA, T. Synthetic seed as an 23, 1989.
application of mass production of somatic embryos. Plant Cell, SHILLITO, R.O.; CARSWELL, G.K.; JOHNSON, C.M. & DiMAIO, J.J.
Tissue and Organ Culture, 39:137-145, 1994. Regeneration of fertile plants from protoplasts of elite inbred plants.
OROPEZA, M. & De GRACIA, E. (1996) Somaclonal variants resistant Bio/Technology, 7:581-587, 1989
to sugarcane mosaic virus and their agronomic characterisation. SHINOZAKI, S.; FUJITA, K.; HIDAKA, T. & OMURA, M. Plantlet
In Vitro Cellular and Developmental Biology, 32:26-30, 1996. formation of somatic hybrids of sweet orange (Citrus sinensis)
OZAWA, K.; LING, D.H. & KOMAMINE, A. High frequency somatic and its wild relative, orange Jessamine (Murraya paniculata), by
embryogenesis from small suspension cultured clusters of cells electrically-induced protoplast fusion. Japanese Journal of
of an interspecific hybrid of Oryza. Plant Cell Tissue and Organ Breeding, 42:287-295, 1992.
Culture, 46:157-159, 1996. SINGSIT, C.; ADANG, M.J.; LYNCH, R.E.; ANDERSON, W.F.; WANG,
PAIVA, M. & JANICK, J. In vivo and in vitro production of alkaloids in A.; CARDINEAU, G. & OZIAS-AKINS, P. Expression of a Bacillus
Theobroma cacao L. Acta Horticulturae, 131:249-273, 1983. thuringensis cryIA(c) gene in transgenic peanut plants and its
PAVINGEROVA, D.; DOSTAL, J.; BISKOVA, R. & BENETKA, V. Somatic efficacy against lesser cornstalk borer. Transgenic Research,
embryogenesis and Agrobacterium -mediated transformation of 6:169-176, 1997.
chrysanthemum. Plant Science, 97: 95-101, 1994. SMITH, D.L. & KRIKOVIAN, A.D. Release of somatic embryogenic
PIOLA, F.; ROHR, R. & VONADERKAS, P. Controlled mycorrhizal potential from excised zygotic embryos of carrot and maintenance
initiation as a means to improve root development in somatic of proembryogenic cultures in hormone-free medium. American
embryo plantlets of hybrid larch (Larix x eurolepis). Physiologia Journal of Botany, 76:1832-1843, 1989.
Plantarum, 95:575-580, 1995. SNYMAN, S.J.; MEYER, G.M.; CARSON, D.L. & BOTHA, F.C.
POMEROY, K.; BROWN, D.C.W. & TAKAHATA, Y. Response of Brassica Establishment of embr yogenic callus and transient gene
napus L. microspore-derived embryos to exogenous abscisic acid expression in selected sugarcane varieties. South African
desiccation. In Vitro Cellular and Developmental Biology, Journal of Botany, 62:151-154, 1996.
30:196-203, 1994. SPANGENBERG, G.; WANG, Z.Y.; WU, X.L.; NAGEL, J.; IGLESIAS,
QUINN, J.; SIMON, J.E. & JANICK, J. Recovery of gamma-linolenic V.A. & POTRYKUS, I. Transgenic tall fescue (Festuca arundinacea)
acid from somatic embryos of borage. Journal of the American and red fescue (F. rubra) plants from microprojectile bombardment
Society of Horticultural Sciences, 114:511-515, 1989. of embryogenic suspension cells. Journal of Plant Physiology,
RAEMAKERS, C.J.J.M.; JACOBSEN, E. & VISSER, R.G.F. Secondary 145:693-701, 1995.

R. Bras. Fisiol. Veg., 10(1):1-12, 1998.

 Vicient & Martínez 12

SPIRAL, J.; THIERRY, C.; PAILLARD, M. & PETIARD, V. Regeneration by double-antibiotic resistance. Plant Cell Reports, 15:506-511,
of plantlets of Coffee canephora Pierre (Robusta) transformed by 1996
Agrobacterium rhizogenes. Comptes Rendus a l’Academie des WANG, Z.Y.; LEGRIS, G.; NAGEL, J.; POTRYKUS, I. &
Sciences, Série III, 316:1-6, 1993. SPANGENBERG, G. Cryopreservation of embryogenic cell
STEWART, C.N.Jr.; ADANG, M.J.; ALL, J.N.; BOERMA, H.R.; suspension in Festuca and Lolium species. Plant Science,
CARDINEAU, G.; TUCKER, D. & PARROT, W.A. Genetic 103:903-106, 1994.
transformation, recovery, and characterisation of fertile soybean WANN, S.R. & GOLDNER, W.R. Induction of somatic embryogenesis
transgenic for a synthetic Bacillus thuringensis cryIAc gene. Plant in Taxus, and the production of taxane-ring containing alkaloids
Physiology 112:121-129, 1996. thereform. WO Patent N° 93/19585, 1994.
STRANDBERG, J.O. Meristem culture of Ophiopogon japonicus and WHIPKEY, A.; SIMON, J.E. & JANICK, J. In vivo and in vitro lipid
production of embryo-like structures. Plant Cell, Tissue and Organ accumulation in Borago officinalis L. Journal of the American
Culture, 32:277-282, 1993. Oil Chemistry Society, 65:979-984, 1988.
TABAEIZADEH, Z.; FERL, R.J. & VASIL, I.K. Somatic hybridization in WILLIAMS, E.G. & MAHESWARAN G. Somatic embryogenesis:
the Gramineae: Saccharum officinarum L. (sugarcane) and factors influencing coordinated behaviour of cells as an
Pennisetum americanum (L.) K. Schum. Proceedings of the embryogenic group. Annals of Botany, 57:443-462, 1986
National Academy of Sciences of the USA, 83:5616-5619, 1986. YANG, J.S.; YU, T.A.; CHENG, Y.H. & YEH, S.D. Transgenic papaya
TESSEREAU, H.; FLORIN, B.; MESCHINE, M.C.; THIERRY, C. & plants from Agrobacterium-mediated transformation of petioles of
PETIARD, V. Cryopreservation of somatic embryos: A tool for in vitro propagated multishoots. Plant Cell Reports, 15:459-464,
germplasm storage and commercial delivery of selected plants. 1996.
Annals of Botany, 74:547-555, 1994. YAO, J.L.; WU, J.H.; GLEAVE, A.P. & MORRIS, B.A.M. Transformation
TETU, T.; SANGWAN, R.S. & SANGWAN-NORREEL, B.S. Direct of citrus embryogenic cells using particle bombardment and
somatic embryogenesis and organogenesis in cultured immature production of transgenic embryos. Plant Science, 113:175-183,
zygotic embryos of Pisum sativum L. Journal of Plant Physiology, 1996.
137:102-109, 1990. YE, X.; WANG, Z.Y.; WU, X.; POTRYKUS, I. & SPANGENBERG, G.
TOONEN, M.A.J. & DE VRIES, S.C. Initiation of somatic embryos from Transgenic italian ryegrass ( Lolium multiflorum ) plants from
single cells. In: WANG, T.L. & CUMING, A. (Eds.) Embryogenesis: microprojectile bombardment of embryogenic suspension cells.
the generation of a plant. Oxford UK, Bios Scientific , 1996. p.173- Plant Cell Reports, 16:379-384, 1997.
189. ZHAO, J.P. & SIMMONDS, D.H. Application of trifluralin to embryogenic
URAGAMI, A. Cryopreservation of asparagus (Asparagus officinalis microspore cultures to generate doubled haploid plants in Brassica
L.) cultured in vitro. Research Bulletin of the Hokkaido National napus. Physiologia Plantarum, 95:304-309, 1995.
Agricultural Experimental Station, 156:1-37, 1991. ZHONG, H.; BOLYARD, M.G.; SRINIVASAN, C. & STICKLEM, M.B.
VAN DER SALM, T.P.M.; VAN DER TOORN, C.J.G.; BOUWER, R.; Transgenic plants of turfgrass (Agrostis palustris Huds.) from
TENCATE, C.H.H. & DONS, H.J.M. Production of rol gene microprojectile bombardment of embryogenic callus. Plant Cell
transformed plants of Rosa hybrida L and characterisation of their Reports, 13:1-6, 1993.
rooting ability. Molecular Breeding, 3:39-47, 1997. ZHONG, H.; ZHANG, S.; WARKENTIN, D.; SUN, B.; WU, T.; WU, R. &
VASIL, I. Automation of plant propagation. Plant Cell, Tissue, Organ STICKLEN, M.B. Analysis of the functional activity of the 1.4-kb
Culture, 39:105-108, 1994. 5'-region of the rice actin 1 gene in stable transgenic plants of
VAZQUEZ-THELLO, A.; YANG, L.J.; HIDAKA, M. & UOZUMI, T. maize (Zea mays L.). Plant Science, 116:73-84, 1996.
Inherited chilling tolerance in somatic hybrids of transgenic ZIMMERMANN, J.L. Somatic embryogenesis: a model for early
Hibiscus Rosa sinensis x transgenic Lavatera thuringiaca selected development in higher plants. The Plant Cell, 5:1411-1423, 1993.

R. Bras. Fisiol. Veg., 10(1):1-12, 1998.