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Somatic embryo production in bioreactors
Journal of Biotechnology, 26 (1992) 99-109 © 1992 Elsevier Science Publishers B.V. All rights reserved 0168-1656/92/$05.00
99
BIOTEC 00824
Somatic embryo production in bioreactors Plamen D. Denchev
a Alexander I. Kuklin a and Alan H. Scragg b
a Institute of Genetic Engineering, Somatic Embryogenesis Lab, 2232 Kostinbrod-2, Bulgaria b Plant Science Group, The University of the West of England, Frenchay, Bristol, UK
Somatic embryogenesis; Bioreactors; Liquid culture; Alfalfa
Introduction
Within the past ten years much progress has been made with various aspects of plant tissue culture and the outlook for in vitro genetic manipulation is considerably more encouraging. One of the more significant developments has been the demonstration of plant regeneration by somatic organogenesis and embryogenesis from various explants (Williams and Maheswaran, 1986; Tisserat et al., 1979; Sharp et al., 1980; Ammirato, 1989). Two different pathways for morphogenesis in vitro have been recognised, direct and indirect where plants can be obtained from the explant indirectly or directly via organogenesis or embryogenesis (Fig. 1). These pathways differ in the necessity of dedifferentiating the initial explant and differences in identifying the in vitro progeny when compared to the parent plant. Organogenesis is characterized by the production of a unipolar bud primordium with subsequent development of the primordium into a leafy vegetative shoot. The developing shoot induces procambial strands to establish a conducting connection between the young shoot and the maternal tissue. The shoot then becomes rooted via root primordia formation and subsequent root organogenesis. Organogenesis is considered as one of the most widely used commercial method of regeneration (Litz, 1986). The procedures are however, labour-intensive due to the number of manual manipulations involved and the low multiplication rates. Gradual acclimatization of plants to the greenhouse and then to the field is also needed. These numerous steps are accompanied by extensive costs and commercialization has been limited to high unit-value crops. The cost of tissue culture propagation using Correspondence to: A.H. Scragg, The University of the West of England, Coldharbour Lane, Frenchay, Bristol BS16 1QY, UK.
100
MORPHOGENESlS~-~_~
IiNDmEcTI [organogenesis [ embryogenesis l organogenes_s lmb~ yogene isJ
i _I-
IcaHusogenesis] shoot induction t
embryo induction
cfion planHefs [ plantlefs
shoot induction
embryo induction
roof induction p[anflefs p~anHetsI
Fig. 1. General pathways for somatic morphogenesis.
this technology is too expensive for seed propagated crops. What is necessary is a highly mechanised, direct greenhouse or field planting system that would enable production costs to be comparable to that of seeds or transplants derived from seeds (Redenbaugh et al., 1986). Somatic embryogenesis is an apparent candidate for this form of plant propagation. In somatic embryogenesis a new individual with a bipolar structure (i.e. a rudimentary plant with a r o o t / s h o o t axis) arises from a single cell and shows no vascular connections with its maternal tissue (Haccius, 1978). Somatic embryogenesis has been successful in several species and has been the subject of recent reviews: - cereals and grasses (Lorz et al., 1988; Vasil, 1987, 1988); - woody plants such as forest trees, both conifers and angiosperms (Von Arnold and Wallin, 1988); - grain or large seed legumes such as soybean, pea and bean (Ranch et al., 1985); recalcitrant tropical crops such as banana, cassava and mango (Cronauer-Mitra and Krikorian, 1988; Litz, 1986). Currently, somatic embryogenesis is best known as a pathway for induced regeneration from in vitro tissue culture, occurring either indirectly from callus, suspension or protoplast culture, or directly from cells of an organised structure such as a stem, segment or zygotic embryo (Williams and Maheswaran, 1986). The potential applications of somatic embryogenesis for plant improvement depend to a large extent on whether embryos are developed through callus or directly from explant cells. The process of cell dedifferentiation causes a higher frequency of somaclonal variation while direct somatic embryo formation appears to give rise to relatively uniform clonal material (Williams, 1988). -
101
I
c~
/ l
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I
I c
Ill
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~
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e
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Fig. 2. General procedures for direct and indirect somatic embryogenesis. CI, callus; e, explant; p, plant; i, induction of somatic embryos; s, sieving; d, development of somatic embryos; m, maturation of somatic embryos; c, conversion of somatic embryos to plants (redrawn from Redenbaugh et al., 1987a).
The process is affected by several factors such as; initial genotype, type and physiological stage of the explant and cultural conditions which are the focus of our studies (Williams, 1988; Ammirato, 1989). Development of an experimental system for plant regeneration via somatic embryogenesis includes the following steps (Fig. 2): - screening of embryogenic genotypes - induction of somatic embryos - somatic embryo development - maturation of embryos - somatic embryo conversion - plant development.
Indirect embryogenesis This p h e n o m e n o n was first observed in suspension culture of carrot (Daucus carota) by Steward and co-workers (1958) and in carrot callus grown on agar medium by Reinert (1959). Efforts have been made in the recent years to study different explant sources and their effect on somatic embryo production (Ammirato, 1989). Somatic embryos may arise in vitro from several sources of culture,
102 diploid cells, vegetative cells of mature plants, mature, and immature zygotic embryos. Our results (Denchev, 1987) have shown that high embryogenic lines from Medicago falcata and M. sativa T1, M. trautvetteri, M. rigidula in comparison with numerous genotypes selected so far (Atanassov and Brown, 1984; Brown and Atanassov, 1985; Kao and Michayluk, 1981; Bingham et al., 1988) produced somatic embryos on solid induction medium in parallel with callus formation (Denchev and Atanassov, 1988a). This reaction was determined by the age of the explant and the concentration of the auxin employed. A stringent correlation had been established between the stage of development, the initial explant, the concentration of 2,4-D and the process of dedifferentiation and differentiation in vitro (Denchev and Atanassov, 1988b; Denchev et al., 1990a). A step toward the scale-up of the process is the production of somatic embryos in suspension cultures. Cell suspension cultures are generally initiated by transferring fragments of undifferentiated callus to a liquid medium, which is then agitated during the culture period. They are widely used as a model system for studying pathways of secondary metabolism, enzyme induction and gene expression, degradation of xenobiotics and represent a basis for large scale cultivation in bioreactors. There are several problems limiting somatic embryo production in suspension cultures: low mitotic cell index - cellular aggregates embryogenic potential. The somatic embryo yield can be improved by sieving the embryogenic suspension culture for appropriate aggregate sizes (Walker and Sato, 1981). During cultivation the morphogenic potential decreases with time and the number of abnormal structures increases (Street, 1973; Denchev, 1987). One of the ways that this could possibly be overcome is to establish a system for direct somatic embryogenesis.
Direct
embryogenesis
Direct somatic embryogenesis offers the possibility of obtaining somatic embryos directly from single cells or groups of cells with a minimum or an absence of callus formation (Ammirato, 1989). Direct development is a function of both medium composition and explant source (Tisserat et al., 1979). This process offers a number of potential applications: (1) Direct cloning of commercial F 1 hybrids for species where material can be sold to growers in 'seedling' transplant form. The ideal system might involve continuous direct embryogenesis from F 1 sexual embryos with periodic harvesting of clonal somatic embryos for growing on. (2) Rapid cloning of valuable breeding stocks at the earliest possible stage of the life cycle after crossing.
103
(3) In vitro selection and screening of genotypes for whole-plant characters at the earliest possible stage of the life cycle. (4) Generation of 'seedling' clones in outbreeding species where each seed normally represents a different genotype. (5) Mechanization and automatization of the microclonal propagation by using bioreactor systems. Our results with alfalfa (Denchev, 1987; Denchev and Attanassov, 1988a) showed that the age of the explant is to a certain extent a limiting factor in
A
t
..~ air
C
•
air
D........~ .t
air
a~r
mist ~
,,
.t
air
Fig. 3. Bioreactor configurations suited embryogenic suspension cultures A, stirred-tank; B, bubble column; C, airlift, internal loop; D, external loop airlift; E, stirred-tank with draft tube; F, mist phase bioreactor (redrawn from Moo-Young and Chisti, 1988).
104
dedifferentiation and differentiation in vitro. Proceeding from the idea discussed above we have developed two different procedures for somatic embryo formation: (1) Drop culture. Leaf petioles were separated from the plants and cultured in a drop of induction medium in a petri dish, without shaking. (2) Liquid culture. Young trifoliated leaves from several alfalfa varieties were chopped and directly introduced into liquid induction medium. For both cultures a multistep operation for somatic embryo induction, development and maturation (Fig. 2) based on liquid media was developed. The availability of embryogenic systems based on liquid media makes it possible to produce somatic embryos in large vessels and scale up the whole process to an economically feasible technology. Large-scale production of somatic embryos is essential if micropropagation (seedlings) and artificial seed systems are to compete with natural seeds (Redenbaugh et al., 1986, 1987b; Redenbaugh, 1990). Embryogenic suspension plant cells are usually grown in shake flasks which are quite satisfactory for laboratory scale cultivation. The gentle shaking in a shake flask is very effective for suspending the cells, enhancing oxygen supply and to aid the mass transfer of nutrients without damaging the structure of delicate plant cells. For the large-scale cultivation of plant cells and somatic embryos we can not simply make the flask bigger. It is necessary to select a bioreactor configuration which may be operated at a large-scale which can provide adequate mixing while minimizing the intensity of shear stress. It is known that plant cells are relatively large in size and have relatively weak walls making them highly susceptible to shear damage. When cells in suspension are subjected to moderate levels of shear stress, they will tend to deform or rupture causing cell death (Hooker et al., 1990). In theory there are several bioreactor configurations that should suit shear sensitive and aeration requiring embryogenic suspension cultures (Fig. 3). Choosing an optimal bioreactor configuration for a given process often depends on a number of factors, including oxygen transfer and mixing requirements and the magnitude of acceptable shear rates. Some of these factors can be mutually contradictory (Moo-Young and Chisti, 1988). One of the most versatile bioreactor systems used industrially is the mechanically agitated bioreactor. This type of system is effective in the mixing of the contents, the suspension of cells, the break-up of air bubbles for enhanced aeration and the prevention of large cell aggregates forming (Hooker et al., 1990). Large scale cultivation of plant cells has been examined in several papers (Sahai and Knuth, 1985; Kargi and Rosenberg, 1987). The effect of shear on the viability of plant cell suspensions in a stirred bioreactor has been considered in some recent articles (Scragg, 1988a,b). Different impeller designs have an impact on the behaviour of the culture and the power consumption (Hooker et al., 1990). Gas-lift devices are finding increased use in biotechnology industries in a variety of arrangements and applications. The main advantages of this type of reactor are versatility, simple construction, the absence of regions of high-shear, reasonably high mass and heat transfer, and appreciable bioreactor productivity and yield at low power input rates (Kawase, 1989).
105
An interesting alternative is the nutrient mist bioreactor which uses ultrasonically driven mists to deliver nutrients to plant tissue cultures. The plant tissues are situated on a mesh support in the growth compartment. They are bathed by nutrient mists formed by sonicating a liquid medium in a separate compartment. The mists are wafted from one compartment to another by a stream of gas. Among the advantages of the mist bioreactor are the considerable reduction of nutrient quantities and the reduced chances of contamination, even with medium changes (Fox, 1988; Weathers et al., 1988, 1989; Weathers and Giles, 1989). When considering scale-up of bioreactor production of somatic embryos it should be borne in mind that embryos are counted as individuals, and a bioreactor that could produce 10-100 000 embryos would be practical. This does not necessarily imply the use of the large bioreactors, as used for microbial growth with volume of 1-5000000 (Styer, 1985). Not all of the embryos produced, however, could be converted to vigorous plants. At the same time seed propagated plants would require greater embryo quantities so that the process could be economical. Alfalfa for example is cultivated in Canada at 1.1-1.7 x 106 seeds per acre. Retail seeds only for North America are approx. 300 million. Such quantities of 'convertible' embryos could be obtained not only by volume increase but by optimization of the process. A typical program for plant propagation via direct and indirect somatic embryogenesis is shown in Fig. 4. The first attempt to scale-up somatic embryogenesis was performed using carrot cells in 20 1 carboys (Backs-Husemann and Reinert, 1970) but only a few embryos formed. Kessel and Carr (1972) investigated the effect of aeration on carrot somatic embryogenesis in a 5 1 bioreactor. It was calculated that carrot requires a low dissolved 0 2 tension (16%) for successful somatic embryo differentiation. Ammonium concentrations and pH were studied in carrot cultures undergoing somatic embryogenesis (Dougall and Verma, 1978; Wetherell and Dougall, 1976) and it was shown that carrot could form somatic embryos with ammonium as the sole source of nitrogen (i.e. without nitrate), but only if the pH of the medium was held constant by titration to an acceptable value (Dougall and Verma, 1978). Batch cultures showed that the growth curve was identical to microbial growth curves. The length of the lag phase was influenced by the inoculum concentration (Puhan and Martin, 1971). At higher inoculum concentrations the lag phase could be reduced to less than one day. Batch growth data could be analyzed in several ways (Ricica and Dobersky, 1981) to provide useful information for continuous cultures. Investigations with continuous cultures were carried out in a spin filter bioreactor (Styer, 1985). In this design, there is no cell washout and the cell density continuously increases. If a culture medium that stimulates embryo differentiation rather than cell proliferation is used in the bioreactor the number of cell clusters remains constant. Alfalfa has received a great deal of interest recently due to its importance as a forage for stock breeding. The overall yield of an alfalfa variety is the average of both excellent and poor genotypes. If superior genotypes could be
acre = 0.4047 ha.
106
Z
7
ct
/
tFF-J
[~ianfif°aml m
& --~
Ce
i
e
FIELD Fig. 4. Tentative protocol for somatic embryo production in bioreactors via indirect and direct somatic embryogenesis. CI, callus; csn, cell suspension; e, explant; ce, chopped explant; i, induction of somatic embryos; d, development of somatic embryos; m, maturation; tr, transplants; stt, seed tape technology; fd, fluid drilling; en, encapsulation of somatic embryos.
propagated asexually significant improvements in alfalfa production could be achieved (Chen et al., 1987). Different types of growth systems for alfalfa embryo production via indirect somatic embryogenesis were examined (Chen et al., 1987; Stuart et al., 1987). The air-lift bioreactors were expected to give higher yields because of the low shear force they exert. The cell viability remained constant but there were no embryos formed in the experiments carried out by Chen et al. (1987). The authors explain this to either a too high DO level or removal of volatile compounds from the liquid medium by the air throughput. Stuart et al. (1987) obtained a slightly higher yield of embryos in the air-lift than in flasks or propeller stirred bioreactors. The quality of the somatic embryos produced in the stirred bioreactor was similar to that produced in large 2 1 flasks on a shaker grown at 110 rpm. They germinated normally on hormone free B 5 agar medium and developed into plantlets (Chen et al., 1987). The conversion results of Stuart et al. (1987) were, however, not so optimistic. The germination of somatic embryos produced in bioreactors is very low compared to the conversion in agar based-systems and flask cultures. Aeration seems to be a parameter that will vary significantly from species to species (Stuart et al., 1987). Alfalfa apparently requires high levels of aeration (90-100% dO 2) when compared to carrot. An experiment was designed to estimate the effect of daily pH titration to 5.5
107 on embryogenic alfalfa cultures. T h e results showed that embryogenic plant cell cultures possess a large capacity to acidify the external medium. Bioreactor studies of growth and nutrient utilization in alfalfa suspension cultures were p e r f o r m e d by M c D o n a l d and J a c k m a n (1989). T h e authors observed two-phase growth for batch suspension cultures in Schenk and Hildebrant medium. This growth behaviour may be attributed to direct metabolism of a m m o n i u m during the first growth phase, induction of nitrate reduction enzymes during the second lag phase and metabolism of the a m m o n i u m as it is g e n e r a t e d during the second growth phase. High sodium levels due to p H control to 5.5 resulted in growth decrease. O u r preliminary results using bioreactors for large scale somatic embryo production via direct embryogenesis showed that it is possible to p r o d u c e embryos in stirred bioreactors as well as in airlifts. Using our multistep p r o c e d u r e we observed that the induction in the present status of our studies should be d o n e in flasks. According to o u r previous results ( D e n c h e v et al., 1990a) induction of somatic embryos in liquid cultures, the concentrations of inoculum should be 1 mg of c h o p p e d explant per ml medium. If the particle size of the c h o p p e d explant is too small or when using bubble columns, air-lift or stirred bioreactors they a d h e r e to the glass vessel, further progress is strongly depressed. T h e next developmental step d e m a n d s a 20-fold increase in volume ( D e n c h e v et al., 1990b) which makes bioreactor cultivation very suitable. This p r o c e d u r e makes it possible to select the flasks with the best induced material and to eliminate c o n t a m i n a t e d explants. In conclusion, it could be stated that large-scale cultivation of somatic embryos not only from high-unit-value crops is possible but m o r e efforts should be m a d e to elucidate the theoretical and practical problems, so that the technology can b e c o m e commercially competitive.
References Ammirato, Ph.V. (1989) Recent progress in somatic embryogenesis. Newsl. IAAPTC 57, 2-16. Atanassov, A. and Brown, D. (1984) Plant regeneration from suspension culture and mesophyll protoplasts of Medicago sativa L. Plant Cell Tissue Organ Culture 3, 149-162. Backs-Husemann, D. and Reinert, J. (1970) Embryobildung durch isolierte Einzelzellen aus Gewebekulturen von D. carota. Protoplasma 70, 36-90. Bingham, E.T., McCoy, T.J. and Walker, K.A. (1988) Alfalfa tissue culture. In: Alfalfa and Alfalfa Improvement. Agronomy monograph, ASA-CSSA-SSSA USA 29, pp. 903-929. Brown, D. and Atanassov, A. (1985) Role of genetic background in somatic embryogenesis in Medicago. Plant Cell Tissue Organ Culture 4, 111-122. Chen, T.H.H., Thompson, B.G. and Gerson, D.F. (1987) In vitro production of alfalfa somatic embryos in fermentation systems. J. Ferment. Technol. 65, 353-357. Cronauer-Mitra, S.S. and Krikorian, A.D. (1988) Plant regeneration via somatic embryogenesis in the seeded diploid banana Musa ornata Roxb. Plant Cell Rep. 7, 23-25. Denchev, P.D. (1987) Development of an experimental model for in vitro selection of herbicide resistance in alfalfa Medicago falcata. Ph.D. thesis, Inst. Genetics, ANS, Moscow. Denchev, P.D. and Atanassov, A. (1988a) Effect of atrazine on the viability and embryo formation of
108 alfalfa M. falcata L. In: 6th Congr. Federation of European Societies of Plant Physiology, Yugoslavia, 1407. Denchev, P.D. and Atanassov, A. (1988b) Using in vitro somatic embryogenesis for mass clonal propagation of alfalfa. In: Staszewski, Z. and Utrata, A. (Eds.), Unconventional Methods in Lucerne Breeding, Poland, pp. 17-21. Denchev, P.D., Velcheva, M., Dragijska, R., Kuklin, A. and Atanassov, A. (1990a) Somatic embryogenesis in Medicago. Biotechnology (Bulgaria) 5-6, 66-70. Denchev, P.D., Velcheva, M. and Atanassov, A. (1990b) A system for direct somatic embryogenesis in Medicago. Abstract 7th IAPTC Congress, Amsterdam, p. 249. Dougall, D.K. and Verma, D.C. (1978) Growth and embryo formation in wild carrot suspension cultures with ammonium as a sole nitrogen source. In vitro 14, 180-182. Fox, J. (1988) Plants thrive in ultrasonic nutrient mists. Biotechnology 6, 3. Haccius, B. (1978) Question of unicellular origin of non-zygotic embryos in callus cultures. Phytomorphology 28, 74-81. Hooker, B.S., Lee, J.M. and An, G. (1990) Cultivation of plant cells in a stirred vessel: Effect of impeller design. Biotechnol. Bioeng. 35, 296-304. Kao, K. and Michayluk, M. (1981) Embryoid formation in alfalfa cell suspension cultures from different plants. In vitro 17, 645-648. Kargi, F. and Rosenberg, M.Z. (1987) Plant cell bioreactors: present status and future trends. Biotechnol. Prog. 3(1), 1-8. Kawase, Y. (1989) Liquid circulation in external-loop airlift bioreactors. Biotechnol. Bioeng. 33. 540-546. Kessel, R.H.J. and Carr, A.H. (1972) The effect of dissolved oxygen concentration on growth and differentiation of carrot (D. carota) tissue. J. Exp. Bot. 23, 996-1007. Litz, R.E. (1986) Mango. In: Evans D.A. et al. (Eds.), Handbook of Plant Cell Culture 4, 612-625. Lorz, H., Goebel, E. and Brown, P. (1988) Advances in tissue culture and progress towards genetic transformation of cereals. Plant Breed. 100, 1-25. McDonald, K.A. and Jackman, A.P. (1989) Bioreactor studies of growth and nutrient utilization in alfalfa suspension cultures. Plant Cell Rep. 8, 455-458. Moo-Young, M. and Chisti, Y. (1988) Considerations for designing bioreactors for shear-sensitive culture. Bio/Technology 6, 1291-1296. Puhan, Z. and Martin, S.M. (1971) The industrial potential of plant cell culture. Prog. Ind. Microbiol. 9, 13. Ranch, J.P., Oglesby, L. and Zielinski, A.C. (1985) Plant regeneration from embryo derived cultures of soybeans. In vitro Cell Dev. Biol. 21,653-658. Redenbaugh, K. (1990) Application of artificial seed to tropical crops. Hortic. Sci. 25(3), 251-255. Redenbaugh, K., Paasch, B.D., Nichol, J.W. and Kossler, M.E. (1986) Somatic seeds: encapsulation of asexual plant embryos. Bio/Technology 4, 797-801. Redenbaugh, K., Slade, D. Viss, P. and Fujii, J.A. (1987a) Encapsulation of somatic embryos in synthetic seed coats. Hortic. Sci. 22, 803-809. Redenbaugh, K., Viss, P., Slade, D. and Fujii, J.A. (1987b) Scale-up: artificial seeds. In: Green, C.E., Somers, D.A., Hackett, W.P. and Biesboer, D.D. (Eds.), Plant Tissue and Cell Culture, pp. 473-493. Reinert, J. (1959) ~/ber die Kontrolle der Morphogenese und Adventivembryonen an Gewebekulturen aus Karotten. Planta 53, 318-333. Ricica, J. and Dobersky, P. (1981) Complex systems. In: Calcott, C. (Ed.), Continuous Cultures of Cells, CRC Press, Boca Raton, FL, pp. 63-96. Sahai, O. and Knuth, M. (1985) Commercializing plant tissue culture processes: economics, problems and prospects. Biotechnol. Prog. 1, 1-9. Scragg, A.H., Allan, E.J. and Leckie, F. (1988a) Effect of shear on the viability of plant cell suspensions. Enzyme Microbiol. Technol. 10, 361-367. Scragg, A.H., Bond, P.A. and Fowler, M.W. (1988b) Bioreactor performance, mixing and shear in the large scale growth of plant cells. In: 6th Eur. Conf. Mixing, pp. 457-464. Sharp, W., Sondhi, M. and Maraffa, S. (1980) The physiology of in vitro asexual embryogenesis. Hortic. Rev. 2, 268-310.
109 Steward, F.C., Mapes, M.O. and Mears, K. (1958) Growth and organised development of cultured cells II. Organizations in cultures grown from freely suspended cells. Am. J. Bot. 445, 705-708. Street, H. (1973) Plant cell cultures: their potential for metabolic studies. In: Milborrow B. (Ed.), Biosynthesis and its Control in Plants. Academic Press, London, pp. 93-125. Stuart, D.A., Strickland, S.G. and Walker, J.A. (1987) Bioreactor production of alfalfa somatic embryos. Hortic. Sci. 22, 800-803. Styer, D.J. (1985) Bioreactor technology for plant propagation. In: Henke, R.R. and Hughes, K.W. (Eds.), Tissue Culture in Forestry and Agriculture, Plenum, New York, pp. 117-130. Tisserat, B., Esan, E. and Murashige, T. (1979) Somatic embryogenesis in angiosperms. Agric. Rev. 1, 1-78.
Vasil, 1.K. (1987) Developing cell and tissue culture systems for the improvement of cereal and grass crops. J. Plant Physiol. 128, 193-218. Vasil, I.K. (1988) Progress in the regeneration and genetic manipulation of cereal crops. Bio/Technology 6, 397-402. Von Arnold, S. and Wallin, A. (1988) Tissue culture methods for clonal propagation of forest trees. Newsl. IAPTC 56, 2-13. Walker, K. and Sato, S. (1981) Morphogenesis in callus tissues of M. satitga: the role of ammonium ion in somatic embryogenesis. Plant Cell Tissue Organ Culture 1, 109-121. Weathers, P.J., Cheetham, R.D. and Giles, K.L. (1988) Dramatic increases in shoot number and lengths for Musa, Cordyline and Nephrylepsis using nutrient mists. Acta Hortic. 230, 39-44. Weathers, P.J., Dilorio A. and Cheetham, R.D. (1989) A bioreactor for differentiated plant tissues. Proc. BIOTECH USA Conf., pp. 247-256. Weathers, P.J. and Giles, K.L (1989) Mist cultivation of cells. U.S. Patent 4,857,464. Wetherell, D.F. and Dougall, D.K. (1976) Sources of nitrogen supporting growth and embryogenesis in cultivated wild carrot tissue. Physiol. Plant. 37, 97-103. Williams, E.G. and Maheswaran, G. (1986) Somatic embryogenesis: factors influencing coordinated behaviour of cells as an embryogenic group. Ann. Bot. 57, 443-462. Williams, E.G. (1988) Somatic embryogenesis as a tool in plant improvements. In: Natesh, S., Chopra, V.L., Ramachandran, S. and Balkema, A.A. (Eds.), Biotechnology in Agriculture, A.A. Balkema, Rotterdam, pp. 179-184.
99
BIOTEC 00824
Somatic embryo production in bioreactors Plamen D. Denchev
a Alexander I. Kuklin a and Alan H. Scragg b
a Institute of Genetic Engineering, Somatic Embryogenesis Lab, 2232 Kostinbrod-2, Bulgaria b Plant Science Group, The University of the West of England, Frenchay, Bristol, UK
Somatic embryogenesis; Bioreactors; Liquid culture; Alfalfa
Introduction
Within the past ten years much progress has been made with various aspects of plant tissue culture and the outlook for in vitro genetic manipulation is considerably more encouraging. One of the more significant developments has been the demonstration of plant regeneration by somatic organogenesis and embryogenesis from various explants (Williams and Maheswaran, 1986; Tisserat et al., 1979; Sharp et al., 1980; Ammirato, 1989). Two different pathways for morphogenesis in vitro have been recognised, direct and indirect where plants can be obtained from the explant indirectly or directly via organogenesis or embryogenesis (Fig. 1). These pathways differ in the necessity of dedifferentiating the initial explant and differences in identifying the in vitro progeny when compared to the parent plant. Organogenesis is characterized by the production of a unipolar bud primordium with subsequent development of the primordium into a leafy vegetative shoot. The developing shoot induces procambial strands to establish a conducting connection between the young shoot and the maternal tissue. The shoot then becomes rooted via root primordia formation and subsequent root organogenesis. Organogenesis is considered as one of the most widely used commercial method of regeneration (Litz, 1986). The procedures are however, labour-intensive due to the number of manual manipulations involved and the low multiplication rates. Gradual acclimatization of plants to the greenhouse and then to the field is also needed. These numerous steps are accompanied by extensive costs and commercialization has been limited to high unit-value crops. The cost of tissue culture propagation using Correspondence to: A.H. Scragg, The University of the West of England, Coldharbour Lane, Frenchay, Bristol BS16 1QY, UK.
100
MORPHOGENESlS~-~_~
IiNDmEcTI [organogenesis [ embryogenesis l organogenes_s lmb~ yogene isJ
i _I-
IcaHusogenesis] shoot induction t
embryo induction
cfion planHefs [ plantlefs
shoot induction
embryo induction
roof induction p[anflefs p~anHetsI
Fig. 1. General pathways for somatic morphogenesis.
this technology is too expensive for seed propagated crops. What is necessary is a highly mechanised, direct greenhouse or field planting system that would enable production costs to be comparable to that of seeds or transplants derived from seeds (Redenbaugh et al., 1986). Somatic embryogenesis is an apparent candidate for this form of plant propagation. In somatic embryogenesis a new individual with a bipolar structure (i.e. a rudimentary plant with a r o o t / s h o o t axis) arises from a single cell and shows no vascular connections with its maternal tissue (Haccius, 1978). Somatic embryogenesis has been successful in several species and has been the subject of recent reviews: - cereals and grasses (Lorz et al., 1988; Vasil, 1987, 1988); - woody plants such as forest trees, both conifers and angiosperms (Von Arnold and Wallin, 1988); - grain or large seed legumes such as soybean, pea and bean (Ranch et al., 1985); recalcitrant tropical crops such as banana, cassava and mango (Cronauer-Mitra and Krikorian, 1988; Litz, 1986). Currently, somatic embryogenesis is best known as a pathway for induced regeneration from in vitro tissue culture, occurring either indirectly from callus, suspension or protoplast culture, or directly from cells of an organised structure such as a stem, segment or zygotic embryo (Williams and Maheswaran, 1986). The potential applications of somatic embryogenesis for plant improvement depend to a large extent on whether embryos are developed through callus or directly from explant cells. The process of cell dedifferentiation causes a higher frequency of somaclonal variation while direct somatic embryo formation appears to give rise to relatively uniform clonal material (Williams, 1988). -
101
I
c~
/ l
[
I
I c
Ill
4q Dt30 o
P
~
O
e
/
i
d
rn
Fig. 2. General procedures for direct and indirect somatic embryogenesis. CI, callus; e, explant; p, plant; i, induction of somatic embryos; s, sieving; d, development of somatic embryos; m, maturation of somatic embryos; c, conversion of somatic embryos to plants (redrawn from Redenbaugh et al., 1987a).
The process is affected by several factors such as; initial genotype, type and physiological stage of the explant and cultural conditions which are the focus of our studies (Williams, 1988; Ammirato, 1989). Development of an experimental system for plant regeneration via somatic embryogenesis includes the following steps (Fig. 2): - screening of embryogenic genotypes - induction of somatic embryos - somatic embryo development - maturation of embryos - somatic embryo conversion - plant development.
Indirect embryogenesis This p h e n o m e n o n was first observed in suspension culture of carrot (Daucus carota) by Steward and co-workers (1958) and in carrot callus grown on agar medium by Reinert (1959). Efforts have been made in the recent years to study different explant sources and their effect on somatic embryo production (Ammirato, 1989). Somatic embryos may arise in vitro from several sources of culture,
102 diploid cells, vegetative cells of mature plants, mature, and immature zygotic embryos. Our results (Denchev, 1987) have shown that high embryogenic lines from Medicago falcata and M. sativa T1, M. trautvetteri, M. rigidula in comparison with numerous genotypes selected so far (Atanassov and Brown, 1984; Brown and Atanassov, 1985; Kao and Michayluk, 1981; Bingham et al., 1988) produced somatic embryos on solid induction medium in parallel with callus formation (Denchev and Atanassov, 1988a). This reaction was determined by the age of the explant and the concentration of the auxin employed. A stringent correlation had been established between the stage of development, the initial explant, the concentration of 2,4-D and the process of dedifferentiation and differentiation in vitro (Denchev and Atanassov, 1988b; Denchev et al., 1990a). A step toward the scale-up of the process is the production of somatic embryos in suspension cultures. Cell suspension cultures are generally initiated by transferring fragments of undifferentiated callus to a liquid medium, which is then agitated during the culture period. They are widely used as a model system for studying pathways of secondary metabolism, enzyme induction and gene expression, degradation of xenobiotics and represent a basis for large scale cultivation in bioreactors. There are several problems limiting somatic embryo production in suspension cultures: low mitotic cell index - cellular aggregates embryogenic potential. The somatic embryo yield can be improved by sieving the embryogenic suspension culture for appropriate aggregate sizes (Walker and Sato, 1981). During cultivation the morphogenic potential decreases with time and the number of abnormal structures increases (Street, 1973; Denchev, 1987). One of the ways that this could possibly be overcome is to establish a system for direct somatic embryogenesis.
Direct
embryogenesis
Direct somatic embryogenesis offers the possibility of obtaining somatic embryos directly from single cells or groups of cells with a minimum or an absence of callus formation (Ammirato, 1989). Direct development is a function of both medium composition and explant source (Tisserat et al., 1979). This process offers a number of potential applications: (1) Direct cloning of commercial F 1 hybrids for species where material can be sold to growers in 'seedling' transplant form. The ideal system might involve continuous direct embryogenesis from F 1 sexual embryos with periodic harvesting of clonal somatic embryos for growing on. (2) Rapid cloning of valuable breeding stocks at the earliest possible stage of the life cycle after crossing.
103
(3) In vitro selection and screening of genotypes for whole-plant characters at the earliest possible stage of the life cycle. (4) Generation of 'seedling' clones in outbreeding species where each seed normally represents a different genotype. (5) Mechanization and automatization of the microclonal propagation by using bioreactor systems. Our results with alfalfa (Denchev, 1987; Denchev and Attanassov, 1988a) showed that the age of the explant is to a certain extent a limiting factor in
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t
..~ air
C
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air
D........~ .t
air
a~r
mist ~
,,
.t
air
Fig. 3. Bioreactor configurations suited embryogenic suspension cultures A, stirred-tank; B, bubble column; C, airlift, internal loop; D, external loop airlift; E, stirred-tank with draft tube; F, mist phase bioreactor (redrawn from Moo-Young and Chisti, 1988).
104
dedifferentiation and differentiation in vitro. Proceeding from the idea discussed above we have developed two different procedures for somatic embryo formation: (1) Drop culture. Leaf petioles were separated from the plants and cultured in a drop of induction medium in a petri dish, without shaking. (2) Liquid culture. Young trifoliated leaves from several alfalfa varieties were chopped and directly introduced into liquid induction medium. For both cultures a multistep operation for somatic embryo induction, development and maturation (Fig. 2) based on liquid media was developed. The availability of embryogenic systems based on liquid media makes it possible to produce somatic embryos in large vessels and scale up the whole process to an economically feasible technology. Large-scale production of somatic embryos is essential if micropropagation (seedlings) and artificial seed systems are to compete with natural seeds (Redenbaugh et al., 1986, 1987b; Redenbaugh, 1990). Embryogenic suspension plant cells are usually grown in shake flasks which are quite satisfactory for laboratory scale cultivation. The gentle shaking in a shake flask is very effective for suspending the cells, enhancing oxygen supply and to aid the mass transfer of nutrients without damaging the structure of delicate plant cells. For the large-scale cultivation of plant cells and somatic embryos we can not simply make the flask bigger. It is necessary to select a bioreactor configuration which may be operated at a large-scale which can provide adequate mixing while minimizing the intensity of shear stress. It is known that plant cells are relatively large in size and have relatively weak walls making them highly susceptible to shear damage. When cells in suspension are subjected to moderate levels of shear stress, they will tend to deform or rupture causing cell death (Hooker et al., 1990). In theory there are several bioreactor configurations that should suit shear sensitive and aeration requiring embryogenic suspension cultures (Fig. 3). Choosing an optimal bioreactor configuration for a given process often depends on a number of factors, including oxygen transfer and mixing requirements and the magnitude of acceptable shear rates. Some of these factors can be mutually contradictory (Moo-Young and Chisti, 1988). One of the most versatile bioreactor systems used industrially is the mechanically agitated bioreactor. This type of system is effective in the mixing of the contents, the suspension of cells, the break-up of air bubbles for enhanced aeration and the prevention of large cell aggregates forming (Hooker et al., 1990). Large scale cultivation of plant cells has been examined in several papers (Sahai and Knuth, 1985; Kargi and Rosenberg, 1987). The effect of shear on the viability of plant cell suspensions in a stirred bioreactor has been considered in some recent articles (Scragg, 1988a,b). Different impeller designs have an impact on the behaviour of the culture and the power consumption (Hooker et al., 1990). Gas-lift devices are finding increased use in biotechnology industries in a variety of arrangements and applications. The main advantages of this type of reactor are versatility, simple construction, the absence of regions of high-shear, reasonably high mass and heat transfer, and appreciable bioreactor productivity and yield at low power input rates (Kawase, 1989).
105
An interesting alternative is the nutrient mist bioreactor which uses ultrasonically driven mists to deliver nutrients to plant tissue cultures. The plant tissues are situated on a mesh support in the growth compartment. They are bathed by nutrient mists formed by sonicating a liquid medium in a separate compartment. The mists are wafted from one compartment to another by a stream of gas. Among the advantages of the mist bioreactor are the considerable reduction of nutrient quantities and the reduced chances of contamination, even with medium changes (Fox, 1988; Weathers et al., 1988, 1989; Weathers and Giles, 1989). When considering scale-up of bioreactor production of somatic embryos it should be borne in mind that embryos are counted as individuals, and a bioreactor that could produce 10-100 000 embryos would be practical. This does not necessarily imply the use of the large bioreactors, as used for microbial growth with volume of 1-5000000 (Styer, 1985). Not all of the embryos produced, however, could be converted to vigorous plants. At the same time seed propagated plants would require greater embryo quantities so that the process could be economical. Alfalfa for example is cultivated in Canada at 1.1-1.7 x 106 seeds per acre. Retail seeds only for North America are approx. 300 million. Such quantities of 'convertible' embryos could be obtained not only by volume increase but by optimization of the process. A typical program for plant propagation via direct and indirect somatic embryogenesis is shown in Fig. 4. The first attempt to scale-up somatic embryogenesis was performed using carrot cells in 20 1 carboys (Backs-Husemann and Reinert, 1970) but only a few embryos formed. Kessel and Carr (1972) investigated the effect of aeration on carrot somatic embryogenesis in a 5 1 bioreactor. It was calculated that carrot requires a low dissolved 0 2 tension (16%) for successful somatic embryo differentiation. Ammonium concentrations and pH were studied in carrot cultures undergoing somatic embryogenesis (Dougall and Verma, 1978; Wetherell and Dougall, 1976) and it was shown that carrot could form somatic embryos with ammonium as the sole source of nitrogen (i.e. without nitrate), but only if the pH of the medium was held constant by titration to an acceptable value (Dougall and Verma, 1978). Batch cultures showed that the growth curve was identical to microbial growth curves. The length of the lag phase was influenced by the inoculum concentration (Puhan and Martin, 1971). At higher inoculum concentrations the lag phase could be reduced to less than one day. Batch growth data could be analyzed in several ways (Ricica and Dobersky, 1981) to provide useful information for continuous cultures. Investigations with continuous cultures were carried out in a spin filter bioreactor (Styer, 1985). In this design, there is no cell washout and the cell density continuously increases. If a culture medium that stimulates embryo differentiation rather than cell proliferation is used in the bioreactor the number of cell clusters remains constant. Alfalfa has received a great deal of interest recently due to its importance as a forage for stock breeding. The overall yield of an alfalfa variety is the average of both excellent and poor genotypes. If superior genotypes could be
acre = 0.4047 ha.
106
Z
7
ct
/
tFF-J
[~ianfif°aml m
& --~
Ce
i
e
FIELD Fig. 4. Tentative protocol for somatic embryo production in bioreactors via indirect and direct somatic embryogenesis. CI, callus; csn, cell suspension; e, explant; ce, chopped explant; i, induction of somatic embryos; d, development of somatic embryos; m, maturation; tr, transplants; stt, seed tape technology; fd, fluid drilling; en, encapsulation of somatic embryos.
propagated asexually significant improvements in alfalfa production could be achieved (Chen et al., 1987). Different types of growth systems for alfalfa embryo production via indirect somatic embryogenesis were examined (Chen et al., 1987; Stuart et al., 1987). The air-lift bioreactors were expected to give higher yields because of the low shear force they exert. The cell viability remained constant but there were no embryos formed in the experiments carried out by Chen et al. (1987). The authors explain this to either a too high DO level or removal of volatile compounds from the liquid medium by the air throughput. Stuart et al. (1987) obtained a slightly higher yield of embryos in the air-lift than in flasks or propeller stirred bioreactors. The quality of the somatic embryos produced in the stirred bioreactor was similar to that produced in large 2 1 flasks on a shaker grown at 110 rpm. They germinated normally on hormone free B 5 agar medium and developed into plantlets (Chen et al., 1987). The conversion results of Stuart et al. (1987) were, however, not so optimistic. The germination of somatic embryos produced in bioreactors is very low compared to the conversion in agar based-systems and flask cultures. Aeration seems to be a parameter that will vary significantly from species to species (Stuart et al., 1987). Alfalfa apparently requires high levels of aeration (90-100% dO 2) when compared to carrot. An experiment was designed to estimate the effect of daily pH titration to 5.5
107 on embryogenic alfalfa cultures. T h e results showed that embryogenic plant cell cultures possess a large capacity to acidify the external medium. Bioreactor studies of growth and nutrient utilization in alfalfa suspension cultures were p e r f o r m e d by M c D o n a l d and J a c k m a n (1989). T h e authors observed two-phase growth for batch suspension cultures in Schenk and Hildebrant medium. This growth behaviour may be attributed to direct metabolism of a m m o n i u m during the first growth phase, induction of nitrate reduction enzymes during the second lag phase and metabolism of the a m m o n i u m as it is g e n e r a t e d during the second growth phase. High sodium levels due to p H control to 5.5 resulted in growth decrease. O u r preliminary results using bioreactors for large scale somatic embryo production via direct embryogenesis showed that it is possible to p r o d u c e embryos in stirred bioreactors as well as in airlifts. Using our multistep p r o c e d u r e we observed that the induction in the present status of our studies should be d o n e in flasks. According to o u r previous results ( D e n c h e v et al., 1990a) induction of somatic embryos in liquid cultures, the concentrations of inoculum should be 1 mg of c h o p p e d explant per ml medium. If the particle size of the c h o p p e d explant is too small or when using bubble columns, air-lift or stirred bioreactors they a d h e r e to the glass vessel, further progress is strongly depressed. T h e next developmental step d e m a n d s a 20-fold increase in volume ( D e n c h e v et al., 1990b) which makes bioreactor cultivation very suitable. This p r o c e d u r e makes it possible to select the flasks with the best induced material and to eliminate c o n t a m i n a t e d explants. In conclusion, it could be stated that large-scale cultivation of somatic embryos not only from high-unit-value crops is possible but m o r e efforts should be m a d e to elucidate the theoretical and practical problems, so that the technology can b e c o m e commercially competitive.
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108 alfalfa M. falcata L. In: 6th Congr. Federation of European Societies of Plant Physiology, Yugoslavia, 1407. Denchev, P.D. and Atanassov, A. (1988b) Using in vitro somatic embryogenesis for mass clonal propagation of alfalfa. In: Staszewski, Z. and Utrata, A. (Eds.), Unconventional Methods in Lucerne Breeding, Poland, pp. 17-21. Denchev, P.D., Velcheva, M., Dragijska, R., Kuklin, A. and Atanassov, A. (1990a) Somatic embryogenesis in Medicago. Biotechnology (Bulgaria) 5-6, 66-70. Denchev, P.D., Velcheva, M. and Atanassov, A. (1990b) A system for direct somatic embryogenesis in Medicago. Abstract 7th IAPTC Congress, Amsterdam, p. 249. Dougall, D.K. and Verma, D.C. (1978) Growth and embryo formation in wild carrot suspension cultures with ammonium as a sole nitrogen source. In vitro 14, 180-182. Fox, J. (1988) Plants thrive in ultrasonic nutrient mists. Biotechnology 6, 3. Haccius, B. (1978) Question of unicellular origin of non-zygotic embryos in callus cultures. Phytomorphology 28, 74-81. Hooker, B.S., Lee, J.M. and An, G. (1990) Cultivation of plant cells in a stirred vessel: Effect of impeller design. Biotechnol. Bioeng. 35, 296-304. Kao, K. and Michayluk, M. (1981) Embryoid formation in alfalfa cell suspension cultures from different plants. In vitro 17, 645-648. Kargi, F. and Rosenberg, M.Z. (1987) Plant cell bioreactors: present status and future trends. Biotechnol. Prog. 3(1), 1-8. Kawase, Y. (1989) Liquid circulation in external-loop airlift bioreactors. Biotechnol. Bioeng. 33. 540-546. Kessel, R.H.J. and Carr, A.H. (1972) The effect of dissolved oxygen concentration on growth and differentiation of carrot (D. carota) tissue. J. Exp. Bot. 23, 996-1007. Litz, R.E. (1986) Mango. In: Evans D.A. et al. (Eds.), Handbook of Plant Cell Culture 4, 612-625. Lorz, H., Goebel, E. and Brown, P. (1988) Advances in tissue culture and progress towards genetic transformation of cereals. Plant Breed. 100, 1-25. McDonald, K.A. and Jackman, A.P. (1989) Bioreactor studies of growth and nutrient utilization in alfalfa suspension cultures. Plant Cell Rep. 8, 455-458. Moo-Young, M. and Chisti, Y. (1988) Considerations for designing bioreactors for shear-sensitive culture. Bio/Technology 6, 1291-1296. Puhan, Z. and Martin, S.M. (1971) The industrial potential of plant cell culture. Prog. Ind. Microbiol. 9, 13. Ranch, J.P., Oglesby, L. and Zielinski, A.C. (1985) Plant regeneration from embryo derived cultures of soybeans. In vitro Cell Dev. Biol. 21,653-658. Redenbaugh, K. (1990) Application of artificial seed to tropical crops. Hortic. Sci. 25(3), 251-255. Redenbaugh, K., Paasch, B.D., Nichol, J.W. and Kossler, M.E. (1986) Somatic seeds: encapsulation of asexual plant embryos. Bio/Technology 4, 797-801. Redenbaugh, K., Slade, D. Viss, P. and Fujii, J.A. (1987a) Encapsulation of somatic embryos in synthetic seed coats. Hortic. Sci. 22, 803-809. Redenbaugh, K., Viss, P., Slade, D. and Fujii, J.A. (1987b) Scale-up: artificial seeds. In: Green, C.E., Somers, D.A., Hackett, W.P. and Biesboer, D.D. (Eds.), Plant Tissue and Cell Culture, pp. 473-493. Reinert, J. (1959) ~/ber die Kontrolle der Morphogenese und Adventivembryonen an Gewebekulturen aus Karotten. Planta 53, 318-333. Ricica, J. and Dobersky, P. (1981) Complex systems. In: Calcott, C. (Ed.), Continuous Cultures of Cells, CRC Press, Boca Raton, FL, pp. 63-96. Sahai, O. and Knuth, M. (1985) Commercializing plant tissue culture processes: economics, problems and prospects. Biotechnol. Prog. 1, 1-9. Scragg, A.H., Allan, E.J. and Leckie, F. (1988a) Effect of shear on the viability of plant cell suspensions. Enzyme Microbiol. Technol. 10, 361-367. Scragg, A.H., Bond, P.A. and Fowler, M.W. (1988b) Bioreactor performance, mixing and shear in the large scale growth of plant cells. In: 6th Eur. Conf. Mixing, pp. 457-464. Sharp, W., Sondhi, M. and Maraffa, S. (1980) The physiology of in vitro asexual embryogenesis. Hortic. Rev. 2, 268-310.
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Vasil, 1.K. (1987) Developing cell and tissue culture systems for the improvement of cereal and grass crops. J. Plant Physiol. 128, 193-218. Vasil, I.K. (1988) Progress in the regeneration and genetic manipulation of cereal crops. Bio/Technology 6, 397-402. Von Arnold, S. and Wallin, A. (1988) Tissue culture methods for clonal propagation of forest trees. Newsl. IAPTC 56, 2-13. Walker, K. and Sato, S. (1981) Morphogenesis in callus tissues of M. satitga: the role of ammonium ion in somatic embryogenesis. Plant Cell Tissue Organ Culture 1, 109-121. Weathers, P.J., Cheetham, R.D. and Giles, K.L. (1988) Dramatic increases in shoot number and lengths for Musa, Cordyline and Nephrylepsis using nutrient mists. Acta Hortic. 230, 39-44. Weathers, P.J., Dilorio A. and Cheetham, R.D. (1989) A bioreactor for differentiated plant tissues. Proc. BIOTECH USA Conf., pp. 247-256. Weathers, P.J. and Giles, K.L (1989) Mist cultivation of cells. U.S. Patent 4,857,464. Wetherell, D.F. and Dougall, D.K. (1976) Sources of nitrogen supporting growth and embryogenesis in cultivated wild carrot tissue. Physiol. Plant. 37, 97-103. Williams, E.G. and Maheswaran, G. (1986) Somatic embryogenesis: factors influencing coordinated behaviour of cells as an embryogenic group. Ann. Bot. 57, 443-462. Williams, E.G. (1988) Somatic embryogenesis as a tool in plant improvements. In: Natesh, S., Chopra, V.L., Ramachandran, S. and Balkema, A.A. (Eds.), Biotechnology in Agriculture, A.A. Balkema, Rotterdam, pp. 179-184.