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
Somatic or asexual embryogenesis is the production
of embryo-like structures from somatic cells without
gamets fusion. During their development, somatic
embryos pass through stages similar to those observed
in zygotic embryogenesis (Dodeman et al ., 1997).
Somatic embryos are independent of the surrounding
tissues and accumulate embryo-specific proteins and
mRNAs (Zimmermann, 1993). They arise naturally in
some species in a process known as direct somatic
embryogenesis (Williams & Maheswaran, 1986). In
contrast, somatic embryos arise from in vitro cultured
cells in the process called indirect somatic
embryogenesis. This process requires the induction of
embryogenic competence. The most usual method to
induce embryogenic competence consists of exposing
explants to a high auxin concentration during a variable
period of time, and then transfering cells to an auxin-
free medium. Indirect somatic embryogenesis is the most
common method to generate somatic embryos for
practical uses and has been described in hundreds of
species (reviewed by Redenbaugh, 1993; Bajaj,
1995a,b). A special type of indirect somatic
embryogenesis is secondary somatic embryogenesis,
which consists of the production of somatic embryos
using somatic embryos as initial explants. Secondary
somatic embryogenesis have been described in nearly
one hundred species (Raemarkers et al., 1995). Although
secondary embryos frequently show low conversion rates
to plants, they also can be used in practical applications.
The rapid improvement in the somatic embryogenesis
methods allows extensive practical and commercial
applications, par ticularly for in vitro clonal
micropropagation (Bornman, 1993; Vasil, 1994). Of
special interest for agroforestry is the use of somatic
embryos as synthetic seeds (Redenbaugh, 1993; Bajaj,
1995a, b; Litz & Gray, 1995) and their scale-up production
in bioreactors (Onishi et al., 1994). However, somatic
embryogenesis has other practical applications in
Agroforestry, including crop improvement (cell selection,
genetic transformation, somatic hybrid and polyploid plant
production), germplasm preservation, virus elimination,
in vitro metabolite production, and in vitro mycorrhizal
initiation.
USES OF SOMATIC EMBRYOGENESIS IN
CROP IMPROVEMENT
Cell selection
Cell selection consists of the regeneration of plants
from cell populations combined with the selection of
desirable genetic characteristics (Jacobs et al., 1987).
In order to avoid the production of genetic chimeras, it is
necessary to use a plant regeneration system able to
regenerate plants from single selected cells. Somatic
embryogenesis allows regeneration of whole plants from
single cells (Toonen & de Vries, 1996), making possible
its use in cell selection programs.
The initial cell populations in cell selection programs
R. Bras. Fisiol. Veg., 10(1):1-12, 1998.
can be artificially mutagenized but it is easier and cheaper
to use non-mutagenized cells cultured in vitro and to
take advantage of somaclonal variation. Plantlets derived
from in vitro culture may exhibit abnormal phenotypes,
often heritable. This variability is known as somaclonal
variation and has been extensively described in plants
derived from somatic embryos (Amberger et al., 1992;
Fourré et al., 1997; Kuksova et al., 1997). Somaclonal
variations are a problem for in vitro clonal
micropropagation but they are desirable in cell selection
programs. Somaclonal variations can produce desirable
agronomic characteristics such increases in salt
tolerance, resistance to herbicide, diseases, extreme
temperatures, aluminium or desiccation, or can yield
interesting biochemical or ornamental mutations
(Maluszynsky et al., 1995). For example, cell selection
and regeneration via somatic embryogenesis have been
used to improve salt tolerance and disease resistance
in Citrus (Litz et al., 1985), virus resistance in sugarcane
(Oropeza & de Gracia, 1996), germination at low
temperature in melon (Ezura et al., 1995) and resistance
to phytotoxins in coffee (Nyange et al., 1995). The
applications of cell selection and somatic embryogenesis
in obtaining new varieties in floricultural crops have been
reviewed by Hutchinson et al. (1992).
Regeneration of genetically transformed plants
Gene transfer methods are an essential part of the
new technologies that are altering conventional plant
breeding and have become indispensable tools. However,
in some species the lack of an efficient regeneration
method is a great bottleneck in the use of transformation
technology. For these species the development of
regeneration methods based on somatic embryogenesis
could be the solution as it has been for coffee (Spiral et
al., 1993) and cassava (Schöpke et al., 1996). However,
the plant transformation techniques do not transform all
cells, and plants regenerated from transformed tissues
via organogenesis are often chimeras (Krastanova et al.,
1995). Somatic embryos arise from single cells (Toonen
& de Vries, 1996) and for this reason regeneration via
somatic embryogenesis reduces the formation of
chimeras. Nevertheless, somatic embryogenesis seems
to produce fewer rates of somaclonal variations
compared with organogenesis (Heinze & Schmidt, 1995).
Several different strategies that have been used in
the regeneration of transformed plants using somatic
embryogenesis (Figure 1, Table 1):
A) Transformation of the explant and regeneration via
direct somatic embryogenesis. The explant tissue is
transformed and then regenerated via direct somatic
embryogenesis. This system is fast, simple and avoids
the problems of somaclonal variations since an in vitro
culture phase is absent. This methodology facilitates
the transformation of some recalcitrant species for
which successful plant regeneration has been
reported only via direct somatic embryogenesis (Tetu
et al., 1990). However, direct somatic embryogenesis
have been described in only few species.
B) Transformation of the explant and regeneration via
indirect somatic embryogenesis . This method does
 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
A r a c h is h y p o g a e a
A s p a r a g u s o ffic in a lis
C a la d iu m b ic o lo r
C a r y a illin o e n s is
C a r ic a p a p a y a
C h r y s a n te m u m
C ic e r a r ie tin u m
C itr u s s p .
C o ffe a c a n e p h o r a
C u c u m is s a tiv u s
D a tu r a in n o x ia
F e s tu c a s p .
G ly c in e m a x
Ip o m o e a b a ta t a s
J u g la n s s p .
L o liu m s p .
M a n ih o t e s c u le n t a
M e d ic a g o s a t iv a
M e d ic a g o tr u n c u la ta
O e n a n t h e s t o lo n ife r a
O r y z a s a tiv a
P a n a x g in s e n g
P ic e a s p .
P ru n n u s sp.
R o s a h y b r id a
S a c ch a ru m
o f fic in a r u m
S o la n u m m e lo n g e n a
S o rg h u m s p.
V itis s p .
Z ea m ays
C A g r o b a c t e r iu m
B M ic r o b o m b a r d m e n t
D M ic r o b o m b a r d m e n t
C M ic r o b o m b a r d m e n t
B A g r o b a c t e r iu m
D A g r o b a c t e r iu m
B A g r o b a c t e r iu m
C A g r o b a c t e r iu m
D M ic r o b o m b a r d m e n t
B A g r o b a c t e r iu m
A A g r o b a c t e r iu m
C M ic r o b o m b a r d m e n t
D A g r o b a c t e r iu m
B A g r o b a c t e r iu m
C M ic r o b o m b a r d m e n t
A A g r o b a c t e r iu m
C M ic r o b o m b a r d m e n t
B M ic r o b o m b a r d m e n t
C M ic r o b o m b a r d m e n t
B A g r o b a c t e r iu m
C A g r o b a c t e r iu m
D A g r o b a c t e r iu m
C M ic r o b o m b a r d m e n t
C M ic r o b o m b a r d m e n t
D M ic r o b o m b a r d m e n t
E A g r o b a c t e r iu m
D A g r o b a c t e r iu m
B A g r o b a c t e r iu m
D M ic r o b o m b a r d m e n t
C M ic r o b o m b a r d m e n t
B A g r o b a c t e r iu m
C A g r o b a c t e r iu m
D M ic r o b o m b a r d m e n t
B A g r o b a c t e r iu m
C A g r o b a c t e r iu m
B A g r o b a c t e r iu m
C M ic r o b o m b a r d m e n t
B A g r o b a c t e r iu m
B M ic r o b o m b a r d m e n t
C M ic r o b o m b a r d m e n t
C A g r o b a c t e r iu m
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 3
L iv in g t o n e a n d B ir c h , 1 9 9 5
S in g s it e t a l . , 1 9 9 7
C a b r e r a - P o n c e e t a l. , 1 9 9 7
Li and W u, 1990
M c G r a n a h a m e t a l. , 1 9 9 3
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 h e n g e t a l. , 1 9 9 6
F it c h e t a l . , 1 9 9 0
P a v in g e r o v á e t a l . , 1 9 9 4
R a m a n a e t a l. , 1 9 9 6
Ya o e t a l . , 1 9 9 6
S p ir a l e t a l . , 1 9 9 3
R a h a r jo e t a l. , 1 9 9 6
S c h u lz e e l a l. , 1 9 9 5
D u c r o c q e t a l. , 1 9 9 4
S p a n g e n b e r g e t a l. , 1 9 9 5
L iu e t a l. , 1 9 9 6
S t e w a r t e t a l. , 1 9 9 6
N e w e ll e t a l . , 1 9 9 5
G a m a - M ic s e t a l . , 1 9 9 6
M c G r a n a h a n e t a l., 1 9 9 0
Ye e t a l. , 1 9 9 7
S c h ö p k e e t a l. , 1 9 9 6
R a e m a k e r s e t a l. , 1 9 9 6
L i e t a l. , 1 9 9 6
N in k o v ic e t a l . , 1 9 9 5
C h a b a u t e t a l., 1 9 9 6
B e e n a n d K im , 1 9 9 5
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
L e e e t a l. , 1 9 9 5
D r a k e e t a l. , 1 9 9 7
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
S c o r z a e t a l., 1 9 8 9
D a C a m a r a - M a c h a d o e t a l. , 1 9 9 5
v a n d e r S a lm e t a l . , 1 9 9 7
S n y m a n e t a l. , 1 9 9 6
F a r i e t a l., 1 9 9 5
C a s a s e t a l., 1 9 9 3
K ik k e r t e t a l . , 1 9 9 6
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 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
in more species.
C) Transformation of embryogenic cell culture and
regeneration via indirect somatic embryogenesis.
Transformation of cultured cells is easier than of
explants and plant regeneration is faster.
D) Transformation of somatic embryos and regeneration
through secondar y or indirect somatic
embryogenesis. Somatic embryos seem to be easily
transformed. Transformed embr yos could be
regenerated by secondary somatic embryogenesis
or by producing embryogenic calli or cell cultures. A
comparison of both methods in Oenanthe stolonifera
have shown that the second method gives higher
transformation efficiencies (Been & Kim, 1995).
E) Transformation of somatic embryos, proliferation
through secondary embryogenesis and regeneration
through organogenesis. Secondary embryogenesis
provides great quantities of easily transformed cells,
however in some species somatic embryos seem to
be sensitive to the selective agents usually used to
detect transformed cells, as for example in cassava
(Schöpke et al., 1993), and regeneration through
secondary somatic embryogenesis is not easy. Li et
al. (1996) proposed a method combining somatic
embryogenesis and organogenesis: secondary
somatic embryos were transformed using
Agrobacterium infection and transgenic plants were
regenerated via organogenesis after cycles of
secondary somatic embryogenesis.
Most of the reports on regeneration of genetically
transformed plants correspond to tests or optimizations
of the methods using either genes conferring resistance
to antibiotics or reporter genes. However, some practical
experiments have been yet made using different types
of transgenes, especially with disease resistance genes
(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
Crosses of cultivated species with wild type
relatives are a potential source of genetic variability for
plant genetic improvement (Glimelius et al., 1991).
However, their utilization in many species is limited
because such crosses can not be made. Moreover, the
regeneration of somatic hybrids could be difficult in some
species, even using embryo rescue techniques. The
protoplast fusion technique overcomes part of these
problems and also allows new combinations of nuclear
and organelle DNA, but a plant regeneration system is
first required. The regeneration of fused protoplasts can
be done via organogenesis or via somatic
embryogenesis. As has been mentioned previously,
somatic embr yogenesis has the advantage of
regenerating plants from single cells, and for these
reason it is the selected regeneration method in many
somatic hybridization projects (Table 3).

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

 Somatic embryogenesis
TABLE 3- Somatic hybrids in which somatic embryogenesis has been used in their production.
SPECIES
Actinidia intragenic hybrids
Arachis intragenic hybrids1
Asparagus intragenic hybrids
Citrus intragenic hybrids
Citrus reticulata x Citropsis gilletiana
Citrus reticulata x Citropsis gabunensis
Citrus sinensis x Murraya paniculata
Citrus sinensis x Atalantia ceylanica
Cucumis intragenic hybrids
Fortunella crassifolia x Citrus sinensis
Helianthus intragenic hybrids
Lycopersicon intragenic hybrids
Medicago intragenic hybrids
Oryza intragenic hybrids
Pennisetum americanum x Saccharum officinarum
Rosa sinensis x Lavatera thuringiaca
1
Hybrids were recovered through tip culture followed by somatic embryogenesis.
Homozygous and polyploid line production
Somatic embryogenesis can produce spontaneous
polyploidy in the regenerated plants, as has been
observed in melon ( Cucumis melo) and Iochroma
warscewiczii (Canhoto et al., 1990; Ezura and Oosawa,
1994). The application of colchicine or the
anti-microtubular drug amiprophos-methyl in the
embryogenic medium enhances the number of polyploid
embryos produced (Gmitter et al., 1991; Binarova and
Dolezel, 1993). Triploid plants can be regenerated when
somatic embryogenesis is performed using endosperm
as explant as have been observed in Prunnus persica
(Liu and Liu, 1980), Citrus (Gmitter et al., 1990) and
Acacia nilotica (Garg et al., 1996).
It is also possible to regenerate plants from haploid
explants (microspores) via somatic embryogenesis.
However, haploid plant regeneration from anthers
(androgenesis) or ovules (gynogenesis) through embryo
formation is not considered as somatic embryogenesis
in sensu stricto (Raghavan, 1983) and for this reason
we are not going to treat this subject. The combination
of haploid plant production with chromosome doubling
techniques has been used to obtain homozygous plants
in a single generation (Gudu et al., 1993; Zhao &
Simmonds, 1995).
Germplasm preservation
The storage of seeds in dry and cold conditions
offers a convenient method for long-term storage of the
germplasm. However, the seeds of some species not
can not be stored at low temperatures or are not
desiccation-tolerant. Moreover, some crop species are
only propagated vegetatively, causing additional
preservation problems. Plant cell in vitro culture
technology provides a potential solution for these
R. Bras. Fisiol. Veg., 10(1):1-12, 1998.
5
REFERENCES
Mu et al., 1990
Feng et al., 1996
Kunitake et al., 1996
Grosser et al., 1996
Grosser et al., 1990
Ling and Iwamasa, 1994
Shinozaki et al., 1992
Louzada et al., 1993
Dabauza et al., 1991
Deng et al., 1992
Fambrini et al., 1996
Lanzhuang and Adachi, 1996
Mendis et al., 1991
Ozawa et al., 1996
Tabaeizadeh et al., 1986
Vazquez-Thello et al., 1996
problems, for example by maintaining long-term
embryogenic calli or cell-suspension cultures. However,
maintenance of cell cultures by subculture is time
consuming and expensive, and involves the risk of loss
of valuable materials through contaminations or human
or technical errors. Additional problems of long-term cell
cultures include the risk of genetic changes by somaclonal
variation and decreases in the plant regeneration capacity
and in the viability of the regenerated plants (Fitch &
Moore, 1993). The frequent initiation of new embryogenic
cell cultures can reduce the adverse effects on genetic
fidelity and regeneration potential but is expensive and
time consuming. Moreover, the initiation of new cultures
could be particularly difficult in some species in which
the establishment of embryogenic cell cultures have
proven to be complicated or in which the embryogenic
explants are only available during short periods of the
year.
However, some alternatives exist. The manipulation
required for cell culture maintenance can be reduced by
maintaining the cultures at low temperatures (0-10°C)
(Attree & Fowke, 1993; Janeiro et al., 1995). It is also
possible to reduce manipulation by growing cell cultures
in hermetic flasks at room temperature (Joy et al., 1991).
The loss of embryogenic potential can be partially
reduced by the application of exogenous spermidine
(Bajaj & Rajam, 1995). In addition, the establishment of
long-term cell cultures in hormone-free media could
reduce the risk of somaclonal variations. Long-term
embryogenic cell-lines cultured on hormone-free medium
have been described in some species, including
asparagus (Delbreil et al ., 1994), carrot (Smith &
Krikorian, 1989), grape (Gray, 1992) and Hevea
brasilensis (Cailloux et al., 1996). However, cell culture
on hormone-free medium reduces somaclonal variation
but not the amont of manipulation or the loss of
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
concentration, reducing the possible toxicity of any one
of them (Fretz et al., 1992; Wang et al., 1994). A typical
cryoprotectant solution contains DMSO (5-10 %), sorbitol
or sucrose (0.33-0.5 M), and glycerol (2 M).
During cryopreservation, free radicals and activated
oxygen can be generated (Benson & Noroncha-Dutra,
1988), both highly toxic for the cells. The presence during
freezing of anti-oxidants such as ascorbic acid enhances
the rates of cell survival (Fuller et al., 1988; Fretz & Lörz,
1995).
The conventional freezing method for cryopreserved
plant material is a two-step procedure in which a first
step consists in a slow cooling (0,5-1°C/min) to an
intermediate temperature (usually about -40°C), and a
second step consisting in a rapid cooling in liquid
nitrogen. This system has been successfully applied to
many species including Panicum maximum, Penisetum
maximum and maize (Shillito et al ., 1989;
Gnanapragasam and Vasil, 1992). However, this system
has an important problem: the necessity of an expensive
controlled programmable freezing machine. Some
alternative method have been developed succesfully as,
for example, the one-step vitrification method (Reinhoud
et al., 1995; Ishikawa et al., 1996), or a rapid freezing at
-25°C and transference to liquid nitrogen (Jain et al.,
1996a).
The post-thaw treatment of cryopreserved cells is also
important for the survival and re-growth of the plant
material (Lynch et al., 1994). A rapid thawing at 40-45°C
is usually used. Not removing the cryoprotectant solution
does not seem to affect recovery rates significantly
(Gnanapragasam & Vasil, 1990; Wang et al., 1994).
Supporting thawed cells on filter paper has been shown
to increase re-growth frequencies (Meijer et al., 1991;
Benson et al., 1992; Lynch et al, 1994). The time required
to recover cryopreserved cells depends on the material
and method used. For example, a 2 month period have
been reported for cultured cells of Festuca, Lolium, and
maize (Shillito et al., 1989; Wang et al., 1994), and 8-35
days for rice (Meijer et al., 1991).
Using a suitable method, it was possible to recover
plant cells with nearly the same regeneration rates than
for non-frozen controls, independently of the time of
storage in liquid nitrogen (Nishizawa et al., 1993). For
example, after more than 4 years, frozen suspended cells
and cotton callus showed normal morphology after
thawing and high growth and regeneration rates
(Rajasekaran et al., 1996).
Virus elimination
Somatic embryos have a well-developed vascular
system but this system is not connected to the explant
tissue (Newton & Goussard, 1990). This makes possible
the use of somatic embryogenesis as a method to
eliminate viruses, combined or not with heat shock
treatments (Goussard & Wiid, 1992; Strandberg, 1993).
Grapevine GFLV (Grapevine Fan Leaf Virus) and A-,
2-, and 3-GLR viruses (Grapevine Leafroll associated
viruses) were eliminated using somatic embryogenesis
associated with thermotherapy (Goussard et al., 1991;
Goussard & Wiid, 1992). Calli were produced from
R. Bras. Fisiol. Veg., 10(1):1-12, 1998.
7
anthers and ovaries of plants infected with GFLV and
various GLR viruses. These calli were heat shock treated
(35°C) and somatic embryos were successfully produced
and converted into plants. Regenerated plants were
tested using immunosorbent electron microscopy (ISEM)
and serological tests (ELISA) and the results indicated
that somatic embryogenesis in combination with heat
therapy is an effective procedure to eliminate viruses.
Curiously, sometimes elimination success depended on
the explant used. For example, using the same somatic
embryogenesis/heat shock treatment, GFLV virus is
eliminated if the somatic embryos were originated from
ovary explants but not if they were originated from
anthers.
Strandberg (1993) compared the efficiency in the
elimination of the yellow-flecked mottled leaves virus in
Ophiopogon japonicus using meristem and somatic
embryo plant regeneration without heat shock treatment.
All plants regenerated from somatic embryos appeared
to be virus-free. In contrast, only about one-third of the
plants regenerated from meristems appeared to be virus-
free.
This method has also been succesfully used in Citrus.
Somatic embryos were obtained from stylar tissues of
lemon plants of different cultivars affected by various
virus-like diseases. Plants regenerated from the somatic
embryos gave negative results when assayed for the
presence of viruses, virus-like agents, and viroids
(d‘Onghia et al., 1997).
Metabolite Production
The production of metabolites in traditional agriculture
is based on the culture of large quantities of plants,
collection of certain organs or tissues that accumulate
the product, and a chemical extraction. Sometimes the
product is only accumulated in very specific parts of the
plants such fruits, seeds, flowers or embryos and
sometimes very large quantities of plants are necessary
for the production of small quantities of metabolite.
Metabolite production in cell cultures has been proposed
as an alternative for traditional methods. Some success
has been reported with these techniques (Fujita & Tabata,
1987) but sometimes the undifferentiated cultured cells
does not accumulate the metabolite, or the metabolite is
accumulated only in low quantities. The addition of
precursors or elicitors can increase metabolite production
(Luckner & Diettrich, 1987; Eilert et al., 1987), but not
always. Alternatively, organ culture or somatic
embryogenesis, when the products accumulate in the
embryo, can be used. There are several factors that affect
the viability of these processes. The most important is
economics. Due to the high price of in vitro culture, the
metabolites to be produced by plant cell culture must be
of high economic value. Janick (1993) suggested that
suitable metabolites are products of pharmaceutical
interest such as alkaloids and certain lipids, some flavor
compounds and food additives, and pigments (reviewed
by Janick, 1991). Some examples are:
- Caffeine and theobromine from somatic embryos of
cacao (Janick et al., 1982; Paiva & Janick, 1983).
- Celery flavor compounds from celery somatic embryos
8 Vicient & Martínez
(Al-Abta et al., 1979).
- Gamma-linolenic acid from borage (Borago officinalis)
somatic embryos (Janick et al., 1987; Whipkey et al.,
1988; Quinn et al., 1989).
- Jojoba liquid wax from jojoba somatic embryos (Gabr,
1993).
- Taxoids from Taxus species (Lee and Son, 1995; Wann
and Goldner, 1994).
For industrial use, in vitro extraction must be cheaper
compared with traditional methods. Due to the economic
limitations, mass somatic embryo production in
bioreactors is essential. The composition of the media
and the problems associated with mass production in
bioreactors are especially important due to culture factors
which can affect metabolite production. With Brassica
napus microspore-derived somatic embryos, exogenous
ABA produced an increase in the total fatty acid content
(Pomeroy et al., 1994). Another example is gamma-
linolenic acid production from borago somatic embryos
(Quinn et al., 1989). The gamma-linolenic acid is a pre-
cursor in the prostaglandin synthesis. Borago seeds
contain about 7% gamma-linolenic acid but large-scale
borago cultures are problematic (Beaubaire et al., 1988).
Gamma-linolenic acid in the seed accumulates mainly
in the cotyledons, so it is necessary to obtain somatic
embryogenesis systems producing good rates of
conversion to cotyledonary-stage embryos. In the optimal
conditions described by Quinn et al (1989), the gamma-
linolenic acid content in somatic embryo was only 22.5%
compared with 56% observed in zygotic cotyledon and
33% in zygotic hypocotyl.
Mycorrhizal initiation
The combination of somatic embryogenesis and in
vitro mycorrhizal production was first proposed by
Redenbaugh et al., in 1987. The application of somatic
embryo technology in forestry has been studied
extensively, specially the application of synthetic seed
technology in forest regeneration. The combination of
synthetic seed technology with mycorrizal initiation is very
attractive. However, very little work has been done on
this subject. Some experiments have been made with
hybrid larch (Larix x eurolepis Henry) in which the in vitro
inoculation with various ectomycorrhizal fungi enhanced
the development of somatic embryo-derived plantlets
(Piola et al., 1995).
ACKNOWLEDGEMENTS
We thank Michel Delseny, Yves Meyer and Martine
Devic for critically reading the manuscript and Tom
Roscoe and Alan H. Schulman for carefully correcting
the manuscript and their critical reading.
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