Plant Cell Reports
Plant Cell Reports (1985) 4: 344- 347
© Springer-Verlag 1985
Somatic embryogenesis and plantlet regeneration in the soybean Giycine max B.J. Li, W. H. R. Langridge, and A. A. Szalay Boyce Thompson Institute, Cornell University, Ithaca, NY 14853, USA Received September 9, 1985 / Revised version received November 7, 1985 - Communicated by O. L Gamborg
ABSTRACT A tissue culture procedure for the regeneration of somatic embryos and plantlets from somatic cells of the soybean Glycine max is described. Bean pods of soybean cv. ~M11Twere immersed in liquid nitrogen for 20 minutes. Young embryos were excised from the immature seeds and cultured to form c a l l i . Calli grown from the young embryos were incubated in liquid culture for two weeks. The liquid suspension culture was f i l t e r e d to obtain single cells. The soybean cells were cultured for one month in a liquid medium in hanging d r o p cultures for development into proembryoids. The proembryoids were maintained on a solid growth medium for 40 days. The resultant callus tissue was transferred into MS media containing selected combinations and concentrations of 2,4-Dichlorophenoxyacetic acid, Naphthaleneacetic acid, Kinetin, Benzyladenine and Indoleacetic acid. In the presence of Benzyladenine (0.2 mg/l) and Indoleacetic acid (0.01 mg/l), globular and heart shaped somatic embryos were formed on the surface of the c a l l i . Calli containing somatic embryos were transferred into liquid medium and incubated under low l i g h t conditions. After six months further incubation, more than 1,000 plantlets and a large number of somatic embryoids at various developmental stages were obtained per flask. ABBREVIATIONS KT = kinetin; benzyladenine; NAA = indole acetic acid; acetic acid; MS =
CM = coconut milk; BA = napthalene acetic acid; IAA = 2,4-D = 2,4 dichlorophenoxy Murashige and Skoog medium
INTRODUCTION Plant regeneration from somatic cells has been obtained in many species. However, the successful regeneration of legumes from single cells has been accomplished with only a few species (Gresshoff, 1980; Kao et a l . , 1980; Dos Santos et a l . , 1980; Arciom et--aTT, 1982; Ahuja et --al-/-~, 1983). Regenera~nTf Glycine soja and-G~ycTne tabacina from callus tissue to s-~omat1~ embryos was r e p ~ by Gamborg et al. (1983a). The same author Offprint requests to: B. J. Li
obtained young somatic embryos f r o m domestic soybean varieties (1983b). Christianson et al. (1983) subcultured c a l l i of Glycine max cv. Mitchell on N-amended medium deveT6-~y-C-h-eng et al. (1980), and obtained occasional callus t i s s ~ ~vered with small embryos, which were capable of completing plantlet development. However, until the present there has been no documentation of a procedure for the complete regeneration of domestic soybean plants from single cells. Developmentof a technique for regeneration of soybean plantlets from single cells via somatic embryos is desirable not only for providing new ways to produce soybean varieties, but also for the improvement of existing soybean varieties via modern genetic engineering techniques. We describe here a method for the regeneration of Glycine max plantlets from single cells. "" MATERIALS AND METHODS Preparation of immature embryos from soybean for tissue culture. Immature soybean (Glycine max cv. TGM119) embryos were selected ast ~ i t l - f a l material for culture. Soybean pods were harvested 20 days after f e r t i l i z a t i o n , wrapped in three or four layers of aluminum f o i l and immersed in liquid nitrogen for 20ominutes. The frozen pods were transferred to a 60 C water bath for 20 minutes. The unbroken pods were surface sterilized with 70% ethanol for 2-3 minutes, 20% bleach for 20 minutes, and then washed 4 times in 500 ml of s t e r i l e d i s t i l l e d water. The entire procedure was done in a vertical laminar flow hood. Immature embryos were disse2cted from the seeds, cut into approximately 1-2 mm segments and placed into a variety of culture media. Calli induced from leaves, young stem, and immature embryos without cold treatment were included as control tissues. The formation of soybean callus tissue. The most rapidly growing callus tissue was obtained on MS medium (Murashige and Skoog, 1962) containing 2.0% sucrose, 2,4-D (i-2 mg/l) and Gelrite (Kelco Inc.) 0(2.2 g / l ) , pH 5.8. The c a l l i were cultured at 28 C with a 12 hr photoperiod (19.5uE), and subcultured at two week intervals. The total time of culture on this medium was 40 days.
345 Establishment of a single cell l i q u i d c u l t u r e system. Prior to t r a n s f e r into l i q u i d medium, the callus tissue was pressed with a spatula to separate c e l l s from the tissue. The macerated callus tissue was transferred to a 200 ml f l a s k containing 40 ml of MS medium containing 2.0% sucrose, 2,4-D (0.5 mg/l) and 5.0% CM, pH 5.8. The tissue WaSoincubated on a gyrorotatory shaker (I00 rpm) at 22 C with a 12 hr photoperiod under d i f f u s e l i g h t (4.0~E). A f t e r 14 days of incubation the soybean c e l l s were f i l t e r e d through two layers of polyester cloth (45 threads/cm). The f i l t r a t e s contained approximately 95% single c e l l s . The soybean c e l l s (20 ul a l i q u o t s ) were transferred to the cover of a 60xl5mm p l a s t i c p e t r i dish. Each drop contained 200-300 c e l l s . Culture medium (8.0 ml) was added to the bottom of each dish to maintain humidity. At 3-4 day i n t e r v a l s , 5.0 ul of c u l t u r e medium was added to each hanging drop. Cell d i v i s i o n and development of cell colonies and small somatic embryos were observed and recorded under a Zeiss inverted phase contrast microscope.
1980; Lu and V a s i l , 1981; Vasil and V a s i l , 1981a,b). The a b i l i t y to regenerate p l a n t l e t s from c e l l s of immature embryos may depend on the maintenance of the t o t i p o t e n t stage of the embryonic tissues. Low temperature treatment has been used to stimulate the regeneration of differentiated cells. For example, low temperature treatment of mononucleate microspores or pollen (I0°-14°C f o r 10-15 days) can increase the rate of regeneration of plants from single c e l l s . (Nitsch et a l . , 1973; Duncan et a l . , 1976; Sunderland et a--T.~--1977; Chen Ying ei- a-l-., 1981). However t ~ m-echanism of enhancement of regeneration due to cold treatment i s , at t h i s time, unclear. A f t e r u l t r a low temperature treatment of immature embryos, we obtained soybean callus tissue capable of f u r t h e r regeneration. Approximately 80-90% of the embryo fragments transferred to MS medium produced abundant regenerative callus tissue. The callus tissue maintained on MS medium containing 2,4-D (2.0 mg/l) appeared e a r l i e r and grew more abundantly than callus maintained on MS media containing different concentrations of
Procedures f o r d i f f e r e n t i a t i o n of embryoids from callus tissue. A f t e r one month of incubation, the small embryos that formed in the hanging drops were transferred to a s o l i d growth medium composed of MS medium containing 2.0% sucrose, 2,4-D ( i m g / l ) , KT ( I . 0 m g / l ) , CM (5.0%) and G e l r i t e (~.2 g / l ) pH 5.8. A f t e r one month of incubation at 28UCwith a 12 hr photoperiod (19.5~E), calli (0.8-1.0 cm in diameter) were transferred to a v a r i e t y of d i f f e r e n t i a t i o n media. M o r e than 60 d i f f e r e n t media compositions were used to induce f u r t h e r soybean embryogenesis. The basic medium (MS medium) contained 1-4 d i f f e r e n t plant hormones. The ranges of the hormone concentrations used were: 2,4-D (0.2-2.0 m9/l), NAA (0.2-4.0 mg/l), KT ( 0 . I - 2 . 0 mg/l), BA (0.1-2.0 mg/l), IAA (0.005-0.2 mg/l). In a d d i t i o n , many of the media contained B5 vitamins with or without alanine (50-200 mg/l). In several media, the inorganic content of nitrogen was reduced by replacing the MS nitrogen salts with 20 mM ammonium c i t r a t e (N amended medium) (Cheng, 1973). In some media, maltose (2-3%) was substituted f o r sucrose as the carbon source. The c a l l i were subcultured once every two weeks for a period of 10 to 12 months. To f u r t h e r induce d i f f e r e n t i a t i o n , callus tissue was transferred to MS medium containing 2.0% sucrose, BA (0.2 - 0.5 mg/l), IAA (0.01 - 0.1 mg/1) and G e l r i t e (3.0 g / l ~ . The incubation temperature was maintained at 28vC and the i l l u m i n a t i o n increased to 35-39uE with a 12 hr photoperiod. Liquid c u l t u r e procedure f o r the regeneration of mature somatic embryos and p l a n t l e t s from p a r t i a l l y differentiated callus. The p a r t i a l l y d i f f e r e n t i a t e d soybean c a l l i were transferred to MS l i q u i d medium or N-amended medium containing 2,4-D (0.5 mg/l) and 5.0% CM, pH 5.8. The c u l t u r e flasks were sealed with two layers of parafilm and incubated on a gyrorotatory shaker at 100 rpm under d i f f u s e l i g h t (4.0~E). Every week, 20 ml of media was removed from each f l a s k , and replaced with an equal volume of ofresh medium. The cultures were incubated at 22 C f o r s i x months with shaking. RESULTS AND DISCUSSION Immature embryos are an ideal starting material f o r the regeneration of somatic embryos and p l a n t l e t s from cereal species (Vasil and V a s i l ,
Figure I. The development of d i f f e r e n t i a t e d structures on the surface of soybean c a l l u s on solid culture. Globular shaped somatic embryos were detected on the surface of the callus a f t e r 50 days in culture on MS s o l i d medium containing 2.0% sucrose + BA (0.2 mg/l) + IAA (0.01 mg/l) (Figure 2.A). Two weeks l a t e r the globular shaped embryos developed into heart shaped embryos (Figure 2.B).
346
f
O,Smm
C
Imrn
E
~m--2
other selected phytohormones. After 40 days in culture on MS medium containing 2,4-D (2.0 mg/l), the size of the callus was 0.3 to 0.5 cm in diameter. Callus tissue was obtained from embryos which were not cold treated, however 80-90% of the c a l l i did not survive long term subculture in the d i f f e r e n t i a t i o n media used in the experiment. No mature somatic embryos were detected on any of the surviving c a l l i . Tissues from the other soybean v a r i e t i e s used did not survive the u l t r a low temperature treatment. A study (Tu and L i , 1984) documented the inhibitory effects of high concentration of 2,4-D on cell d i f f e r e n t i a t i o n . To avoid early loses of embryogenic c a p a b i l i t y caused by superoptimal concentrations of 2,4-D, we used
Figure 2. Somatic embryogenesis and p l a n t l e t formation in l i q u i d culture. C a l l i containing globular and heart shaped embryoids were transferred to l i q u i d medium, and cultured for six months as previously described. (A) Globular shaped somatic embryo. (B) Oval shaped embryos. (C) Torpedo shaped embryo. (D) Further d i f f e r e n t i a t i o n of embryoid into a polarized structure. (E) Mature somatic embryo. (F) R o o t d i f f e r e n t i a t i o n from mature somatic embryo. (G) Plantlet developed from a mature somatic embryo. (H) Green leaves on p l a n t l e t s r i s i n g above the surface of the medium (arrows). ( I ) Close view of regenerating p l a n t l e t s in the culture flask. (J) Culture flask with large numbers of soybean somatic embryos and p l a n t l e t s .
347 only calli-grown in the presence of less than 2.0 mg/l 2,4-D for f u r t h e r regeneration studies. The early developmental pathway of single cells to somatic embryoids was extremly polymorphic. Many small somatic embryos established p o l a r i t y at this stage. Further development of somatic embryoids was not detected in hanging drop cultures. In order to induce further development, the proembryoids were transferred onto growth medium f o r one month and then onto more than 60 d i f f e r e n t media formulations and cultured for I0 to 12 months. In several media, for example MS supplemented with KT (2.0 mg/l), BA (2.0 mg/l), NAA (0.5 mg/l), maltose 2% and G e l r i t e (2.2 g / l ) , callus tissue grew rapidly. The callus tissue remained f r i a b l e and l i g h t green in color, however, no further d i f f e r e n t i a t i o n of the embryoids was observed. This callus tissue was transferred to MS solid medium containing 2.0% sucrose, BA (0.2 mg/l) and IAA (0.01 mg/l). After 50 days in culture, globular and heart shaped embryoids were detected on the surface of the c a l l i (Figure I.A,B). Callus tissue orginating from stem or l e a f tissue grown on the same medium did not d i f f e r e n t i a t e . Several authors have reported that reducing hormone levels or optimizing the r a t i o of phytohormones in the medium at c r i t i c a l times was important f o r continued d i f f e r e n t i a t i o n (Ammirato, 1977; Kochba et a l . , 1978; Ammirato, 1983). Even with reduced concentrations of phytohormones, further d i f f e r e n t i a t i o n of the heart shaped embryoids to mature somatic embryos and p l a n t l e t s was not obtained. To stimulate further development, calli containing globular and heart shaped embryoids were transfered from solid medium to l i q u i d culture medium as described above. A large number of somatic embryos at d i f f e r e n t stages of development were detected a f t e r 6 months incubation in l i q u i d medium. Small proembryoids (consisting of 30 to several hundred c e l l s ) , globular shaped embryoids (Figure 2.A,B), heart shaped embryoids, torpedo shaped embryoids (Figure 2.C), mature somatic embryos (Figure 2.D,E,F) and small p l a n t l e t s were a l l found in the same culture flask (Figure 2.G,H). A maximum of 1,484 p l a n t l e t s , many having a primary root and well developed green l e a f l e t s (Figure 2.H) were detected per flask. Development of p l a n t l e t s was not detected in N-amended medium. During long term tissue culture, a few c a l l i w i l l occasionally d i f f e r e n t i a t e into mature somatic embryos, and even into small p l a n t l e t s , as reported by Cao et a l . (1978). These authors obtained c a l l i from mononucleate maize microspores, a f t e r 7-8 months on solid medium, a large number of somatic embryos d i f f e r e n t i a t e d from several of the c a l l i . After successive subcultures, the a b i l i t y to induce somatic embryos from these c a l l i was retained. To obtain a greater understanding of the phenomenon of occasional somatic embryogenesis from callus tissue, the d e t a i l s of d i f f e r e n t i a t i o n callus were observed with a dissecting microscope. When attempts were made to separate c e l l s of soybean c a l l i , in several cases, d i f f e r e n t i a t e d structures were observed inside the callus. For example, when the callus was separated with dissecting needles, many individual proembryoids at d i f f e r e n t early stages of development were observed within the i n t e r i o r of the callus tissue, and under certain culture conditions these d i f f e r e n t i a t e d structures developed further. We have found that
" w e l l - d i f f e r e n t i a t e d " c a l l i and appropriate culture conditions were both required for successful p l a n t l e t regeneration from soybean callus tissue. The l i q u i d culture conditions described above permit the regeneration of soybean somatic c e l l s into p l a n t l e t s . In summary, immature soybean embryos were subjected to extremely cold temperature to reverse the direction of development of c e l l s in the embryo. A liquid suspension culture was established from the frozen tissue to obtain single cells with an increased capability for regeneration. These c e l l s were placed on solid medium to induce growth and d i f f e r e n t i a t i o n into heart shape embryoids. C a l l i containing embryoids in many stages of early development were returned to liquid culture conditions which induced d i f f e r e n t i a t i o n into p l a n t l e t s . ACKNOWLEDGEMENTS This research was supported by National Science Foundation Grant PCM 8410753 to A.A. Szalay and p a r t i a l l y funded by the Boyce Thompson Endowment. We would l i k e to thank Dr. D. Dudits, Dr. D. Golemboski, and Dr. J. Noti for c r i t i c a l reading the manuscript. Special thanks to Ms. D. Bridwell for typing the manuscript. REFERENCES Ahuja PS, Hadiuzzaman S, Davey MR, Cocking EC (1983) Plant Cell Reports 2:101-104. Ammirato PV (1977) Plant Physiol. 59:579-586. Ammirato PV (1983) Biotechnology March:68-74. Arciom MR, Davey AVP, Dos Santos, Cocking EC (1982) Z. Pflanzenphysiol. 106:105-110. Cao Z, Guo C, Hao J (1981) Acta Genetica Sinica 8(3):269-274. Cheng TY, Saka H, Voqui-Dinh TH (1980) Plant Sci. Lett. 19:91-99. Chen Ying, Zuo Qiuxian, Li Shuyuan, L@ Deyang, Zheng Shiwen (1981) Acta Genetica Sinica 8(2):158-163. Christianson ML, Warnick DA, Carlson PS (1983) Science 222:632-634. Duncan EJ (1976) Protoplasma 90:173-177. Gamborg OL, Davis BP, Stahlhut RW (1983a) Plant Cell Reports 2:209-212. Gamborg OL, Davis BP, Stahlhut RW (1983b) Plant Cell Reports 2:213:215. Gresshoff P (1980) Bot. Gaz. 141:157-164. Kao KN, Michayluk MR (1980) Z. Pflanzenphysiol. 96:135-141. Kamada H, Harada H (1981) Plant Cell Physiol. 22:1423-1429. Kochba J, Spiegel-Roy P, Neumann H, Soad S (1978) Z. Pflanzenphysiol. 89:427-436. Lu CY, Vasil IK (1981) Ann. Bot. 48:543-548. Murashige T, Skoog F (1962) Physiol. Plant. 15:473-476. Nitsch C, Norrell B (1973) CR. Acad. Sci. 276D:303-306. Dos Santos AVP, Outka DE, Cocking EC, Davey MR (1980) Z. Pflanzenphysiol. 99:261-270. Sunderland N (1977) Nature 270(5634):236-238. Tu GH, Li BJ (1984) Journal of Cell Biology (China) 6(4):164-168. Vasil V, Vasil IK (1980) Theor. Appl. Genet. 56:97-99. Vasil V, Vasil IK (1981a) Amer. J. Bot. 68(6):864-872. Vasil V, Vasil IK (1981b) Ann. Bot. 47:669-678.
Plant Cell Reports (1985) 4: 344- 347
© Springer-Verlag 1985
Somatic embryogenesis and plantlet regeneration in the soybean Giycine max B.J. Li, W. H. R. Langridge, and A. A. Szalay Boyce Thompson Institute, Cornell University, Ithaca, NY 14853, USA Received September 9, 1985 / Revised version received November 7, 1985 - Communicated by O. L Gamborg
ABSTRACT A tissue culture procedure for the regeneration of somatic embryos and plantlets from somatic cells of the soybean Glycine max is described. Bean pods of soybean cv. ~M11Twere immersed in liquid nitrogen for 20 minutes. Young embryos were excised from the immature seeds and cultured to form c a l l i . Calli grown from the young embryos were incubated in liquid culture for two weeks. The liquid suspension culture was f i l t e r e d to obtain single cells. The soybean cells were cultured for one month in a liquid medium in hanging d r o p cultures for development into proembryoids. The proembryoids were maintained on a solid growth medium for 40 days. The resultant callus tissue was transferred into MS media containing selected combinations and concentrations of 2,4-Dichlorophenoxyacetic acid, Naphthaleneacetic acid, Kinetin, Benzyladenine and Indoleacetic acid. In the presence of Benzyladenine (0.2 mg/l) and Indoleacetic acid (0.01 mg/l), globular and heart shaped somatic embryos were formed on the surface of the c a l l i . Calli containing somatic embryos were transferred into liquid medium and incubated under low l i g h t conditions. After six months further incubation, more than 1,000 plantlets and a large number of somatic embryoids at various developmental stages were obtained per flask. ABBREVIATIONS KT = kinetin; benzyladenine; NAA = indole acetic acid; acetic acid; MS =
CM = coconut milk; BA = napthalene acetic acid; IAA = 2,4-D = 2,4 dichlorophenoxy Murashige and Skoog medium
INTRODUCTION Plant regeneration from somatic cells has been obtained in many species. However, the successful regeneration of legumes from single cells has been accomplished with only a few species (Gresshoff, 1980; Kao et a l . , 1980; Dos Santos et a l . , 1980; Arciom et--aTT, 1982; Ahuja et --al-/-~, 1983). Regenera~nTf Glycine soja and-G~ycTne tabacina from callus tissue to s-~omat1~ embryos was r e p ~ by Gamborg et al. (1983a). The same author Offprint requests to: B. J. Li
obtained young somatic embryos f r o m domestic soybean varieties (1983b). Christianson et al. (1983) subcultured c a l l i of Glycine max cv. Mitchell on N-amended medium deveT6-~y-C-h-eng et al. (1980), and obtained occasional callus t i s s ~ ~vered with small embryos, which were capable of completing plantlet development. However, until the present there has been no documentation of a procedure for the complete regeneration of domestic soybean plants from single cells. Developmentof a technique for regeneration of soybean plantlets from single cells via somatic embryos is desirable not only for providing new ways to produce soybean varieties, but also for the improvement of existing soybean varieties via modern genetic engineering techniques. We describe here a method for the regeneration of Glycine max plantlets from single cells. "" MATERIALS AND METHODS Preparation of immature embryos from soybean for tissue culture. Immature soybean (Glycine max cv. TGM119) embryos were selected ast ~ i t l - f a l material for culture. Soybean pods were harvested 20 days after f e r t i l i z a t i o n , wrapped in three or four layers of aluminum f o i l and immersed in liquid nitrogen for 20ominutes. The frozen pods were transferred to a 60 C water bath for 20 minutes. The unbroken pods were surface sterilized with 70% ethanol for 2-3 minutes, 20% bleach for 20 minutes, and then washed 4 times in 500 ml of s t e r i l e d i s t i l l e d water. The entire procedure was done in a vertical laminar flow hood. Immature embryos were disse2cted from the seeds, cut into approximately 1-2 mm segments and placed into a variety of culture media. Calli induced from leaves, young stem, and immature embryos without cold treatment were included as control tissues. The formation of soybean callus tissue. The most rapidly growing callus tissue was obtained on MS medium (Murashige and Skoog, 1962) containing 2.0% sucrose, 2,4-D (i-2 mg/l) and Gelrite (Kelco Inc.) 0(2.2 g / l ) , pH 5.8. The c a l l i were cultured at 28 C with a 12 hr photoperiod (19.5uE), and subcultured at two week intervals. The total time of culture on this medium was 40 days.
345 Establishment of a single cell l i q u i d c u l t u r e system. Prior to t r a n s f e r into l i q u i d medium, the callus tissue was pressed with a spatula to separate c e l l s from the tissue. The macerated callus tissue was transferred to a 200 ml f l a s k containing 40 ml of MS medium containing 2.0% sucrose, 2,4-D (0.5 mg/l) and 5.0% CM, pH 5.8. The tissue WaSoincubated on a gyrorotatory shaker (I00 rpm) at 22 C with a 12 hr photoperiod under d i f f u s e l i g h t (4.0~E). A f t e r 14 days of incubation the soybean c e l l s were f i l t e r e d through two layers of polyester cloth (45 threads/cm). The f i l t r a t e s contained approximately 95% single c e l l s . The soybean c e l l s (20 ul a l i q u o t s ) were transferred to the cover of a 60xl5mm p l a s t i c p e t r i dish. Each drop contained 200-300 c e l l s . Culture medium (8.0 ml) was added to the bottom of each dish to maintain humidity. At 3-4 day i n t e r v a l s , 5.0 ul of c u l t u r e medium was added to each hanging drop. Cell d i v i s i o n and development of cell colonies and small somatic embryos were observed and recorded under a Zeiss inverted phase contrast microscope.
1980; Lu and V a s i l , 1981; Vasil and V a s i l , 1981a,b). The a b i l i t y to regenerate p l a n t l e t s from c e l l s of immature embryos may depend on the maintenance of the t o t i p o t e n t stage of the embryonic tissues. Low temperature treatment has been used to stimulate the regeneration of differentiated cells. For example, low temperature treatment of mononucleate microspores or pollen (I0°-14°C f o r 10-15 days) can increase the rate of regeneration of plants from single c e l l s . (Nitsch et a l . , 1973; Duncan et a l . , 1976; Sunderland et a--T.~--1977; Chen Ying ei- a-l-., 1981). However t ~ m-echanism of enhancement of regeneration due to cold treatment i s , at t h i s time, unclear. A f t e r u l t r a low temperature treatment of immature embryos, we obtained soybean callus tissue capable of f u r t h e r regeneration. Approximately 80-90% of the embryo fragments transferred to MS medium produced abundant regenerative callus tissue. The callus tissue maintained on MS medium containing 2,4-D (2.0 mg/l) appeared e a r l i e r and grew more abundantly than callus maintained on MS media containing different concentrations of
Procedures f o r d i f f e r e n t i a t i o n of embryoids from callus tissue. A f t e r one month of incubation, the small embryos that formed in the hanging drops were transferred to a s o l i d growth medium composed of MS medium containing 2.0% sucrose, 2,4-D ( i m g / l ) , KT ( I . 0 m g / l ) , CM (5.0%) and G e l r i t e (~.2 g / l ) pH 5.8. A f t e r one month of incubation at 28UCwith a 12 hr photoperiod (19.5~E), calli (0.8-1.0 cm in diameter) were transferred to a v a r i e t y of d i f f e r e n t i a t i o n media. M o r e than 60 d i f f e r e n t media compositions were used to induce f u r t h e r soybean embryogenesis. The basic medium (MS medium) contained 1-4 d i f f e r e n t plant hormones. The ranges of the hormone concentrations used were: 2,4-D (0.2-2.0 m9/l), NAA (0.2-4.0 mg/l), KT ( 0 . I - 2 . 0 mg/l), BA (0.1-2.0 mg/l), IAA (0.005-0.2 mg/l). In a d d i t i o n , many of the media contained B5 vitamins with or without alanine (50-200 mg/l). In several media, the inorganic content of nitrogen was reduced by replacing the MS nitrogen salts with 20 mM ammonium c i t r a t e (N amended medium) (Cheng, 1973). In some media, maltose (2-3%) was substituted f o r sucrose as the carbon source. The c a l l i were subcultured once every two weeks for a period of 10 to 12 months. To f u r t h e r induce d i f f e r e n t i a t i o n , callus tissue was transferred to MS medium containing 2.0% sucrose, BA (0.2 - 0.5 mg/l), IAA (0.01 - 0.1 mg/1) and G e l r i t e (3.0 g / l ~ . The incubation temperature was maintained at 28vC and the i l l u m i n a t i o n increased to 35-39uE with a 12 hr photoperiod. Liquid c u l t u r e procedure f o r the regeneration of mature somatic embryos and p l a n t l e t s from p a r t i a l l y differentiated callus. The p a r t i a l l y d i f f e r e n t i a t e d soybean c a l l i were transferred to MS l i q u i d medium or N-amended medium containing 2,4-D (0.5 mg/l) and 5.0% CM, pH 5.8. The c u l t u r e flasks were sealed with two layers of parafilm and incubated on a gyrorotatory shaker at 100 rpm under d i f f u s e l i g h t (4.0~E). Every week, 20 ml of media was removed from each f l a s k , and replaced with an equal volume of ofresh medium. The cultures were incubated at 22 C f o r s i x months with shaking. RESULTS AND DISCUSSION Immature embryos are an ideal starting material f o r the regeneration of somatic embryos and p l a n t l e t s from cereal species (Vasil and V a s i l ,
Figure I. The development of d i f f e r e n t i a t e d structures on the surface of soybean c a l l u s on solid culture. Globular shaped somatic embryos were detected on the surface of the callus a f t e r 50 days in culture on MS s o l i d medium containing 2.0% sucrose + BA (0.2 mg/l) + IAA (0.01 mg/l) (Figure 2.A). Two weeks l a t e r the globular shaped embryos developed into heart shaped embryos (Figure 2.B).
346
f
O,Smm
C
Imrn
E
~m--2
other selected phytohormones. After 40 days in culture on MS medium containing 2,4-D (2.0 mg/l), the size of the callus was 0.3 to 0.5 cm in diameter. Callus tissue was obtained from embryos which were not cold treated, however 80-90% of the c a l l i did not survive long term subculture in the d i f f e r e n t i a t i o n media used in the experiment. No mature somatic embryos were detected on any of the surviving c a l l i . Tissues from the other soybean v a r i e t i e s used did not survive the u l t r a low temperature treatment. A study (Tu and L i , 1984) documented the inhibitory effects of high concentration of 2,4-D on cell d i f f e r e n t i a t i o n . To avoid early loses of embryogenic c a p a b i l i t y caused by superoptimal concentrations of 2,4-D, we used
Figure 2. Somatic embryogenesis and p l a n t l e t formation in l i q u i d culture. C a l l i containing globular and heart shaped embryoids were transferred to l i q u i d medium, and cultured for six months as previously described. (A) Globular shaped somatic embryo. (B) Oval shaped embryos. (C) Torpedo shaped embryo. (D) Further d i f f e r e n t i a t i o n of embryoid into a polarized structure. (E) Mature somatic embryo. (F) R o o t d i f f e r e n t i a t i o n from mature somatic embryo. (G) Plantlet developed from a mature somatic embryo. (H) Green leaves on p l a n t l e t s r i s i n g above the surface of the medium (arrows). ( I ) Close view of regenerating p l a n t l e t s in the culture flask. (J) Culture flask with large numbers of soybean somatic embryos and p l a n t l e t s .
347 only calli-grown in the presence of less than 2.0 mg/l 2,4-D for f u r t h e r regeneration studies. The early developmental pathway of single cells to somatic embryoids was extremly polymorphic. Many small somatic embryos established p o l a r i t y at this stage. Further development of somatic embryoids was not detected in hanging drop cultures. In order to induce further development, the proembryoids were transferred onto growth medium f o r one month and then onto more than 60 d i f f e r e n t media formulations and cultured for I0 to 12 months. In several media, for example MS supplemented with KT (2.0 mg/l), BA (2.0 mg/l), NAA (0.5 mg/l), maltose 2% and G e l r i t e (2.2 g / l ) , callus tissue grew rapidly. The callus tissue remained f r i a b l e and l i g h t green in color, however, no further d i f f e r e n t i a t i o n of the embryoids was observed. This callus tissue was transferred to MS solid medium containing 2.0% sucrose, BA (0.2 mg/l) and IAA (0.01 mg/l). After 50 days in culture, globular and heart shaped embryoids were detected on the surface of the c a l l i (Figure I.A,B). Callus tissue orginating from stem or l e a f tissue grown on the same medium did not d i f f e r e n t i a t e . Several authors have reported that reducing hormone levels or optimizing the r a t i o of phytohormones in the medium at c r i t i c a l times was important f o r continued d i f f e r e n t i a t i o n (Ammirato, 1977; Kochba et a l . , 1978; Ammirato, 1983). Even with reduced concentrations of phytohormones, further d i f f e r e n t i a t i o n of the heart shaped embryoids to mature somatic embryos and p l a n t l e t s was not obtained. To stimulate further development, calli containing globular and heart shaped embryoids were transfered from solid medium to l i q u i d culture medium as described above. A large number of somatic embryos at d i f f e r e n t stages of development were detected a f t e r 6 months incubation in l i q u i d medium. Small proembryoids (consisting of 30 to several hundred c e l l s ) , globular shaped embryoids (Figure 2.A,B), heart shaped embryoids, torpedo shaped embryoids (Figure 2.C), mature somatic embryos (Figure 2.D,E,F) and small p l a n t l e t s were a l l found in the same culture flask (Figure 2.G,H). A maximum of 1,484 p l a n t l e t s , many having a primary root and well developed green l e a f l e t s (Figure 2.H) were detected per flask. Development of p l a n t l e t s was not detected in N-amended medium. During long term tissue culture, a few c a l l i w i l l occasionally d i f f e r e n t i a t e into mature somatic embryos, and even into small p l a n t l e t s , as reported by Cao et a l . (1978). These authors obtained c a l l i from mononucleate maize microspores, a f t e r 7-8 months on solid medium, a large number of somatic embryos d i f f e r e n t i a t e d from several of the c a l l i . After successive subcultures, the a b i l i t y to induce somatic embryos from these c a l l i was retained. To obtain a greater understanding of the phenomenon of occasional somatic embryogenesis from callus tissue, the d e t a i l s of d i f f e r e n t i a t i o n callus were observed with a dissecting microscope. When attempts were made to separate c e l l s of soybean c a l l i , in several cases, d i f f e r e n t i a t e d structures were observed inside the callus. For example, when the callus was separated with dissecting needles, many individual proembryoids at d i f f e r e n t early stages of development were observed within the i n t e r i o r of the callus tissue, and under certain culture conditions these d i f f e r e n t i a t e d structures developed further. We have found that
" w e l l - d i f f e r e n t i a t e d " c a l l i and appropriate culture conditions were both required for successful p l a n t l e t regeneration from soybean callus tissue. The l i q u i d culture conditions described above permit the regeneration of soybean somatic c e l l s into p l a n t l e t s . In summary, immature soybean embryos were subjected to extremely cold temperature to reverse the direction of development of c e l l s in the embryo. A liquid suspension culture was established from the frozen tissue to obtain single cells with an increased capability for regeneration. These c e l l s were placed on solid medium to induce growth and d i f f e r e n t i a t i o n into heart shape embryoids. C a l l i containing embryoids in many stages of early development were returned to liquid culture conditions which induced d i f f e r e n t i a t i o n into p l a n t l e t s . ACKNOWLEDGEMENTS This research was supported by National Science Foundation Grant PCM 8410753 to A.A. Szalay and p a r t i a l l y funded by the Boyce Thompson Endowment. We would l i k e to thank Dr. D. Dudits, Dr. D. Golemboski, and Dr. J. Noti for c r i t i c a l reading the manuscript. Special thanks to Ms. D. Bridwell for typing the manuscript. REFERENCES Ahuja PS, Hadiuzzaman S, Davey MR, Cocking EC (1983) Plant Cell Reports 2:101-104. Ammirato PV (1977) Plant Physiol. 59:579-586. Ammirato PV (1983) Biotechnology March:68-74. Arciom MR, Davey AVP, Dos Santos, Cocking EC (1982) Z. Pflanzenphysiol. 106:105-110. Cao Z, Guo C, Hao J (1981) Acta Genetica Sinica 8(3):269-274. Cheng TY, Saka H, Voqui-Dinh TH (1980) Plant Sci. Lett. 19:91-99. Chen Ying, Zuo Qiuxian, Li Shuyuan, L@ Deyang, Zheng Shiwen (1981) Acta Genetica Sinica 8(2):158-163. Christianson ML, Warnick DA, Carlson PS (1983) Science 222:632-634. Duncan EJ (1976) Protoplasma 90:173-177. Gamborg OL, Davis BP, Stahlhut RW (1983a) Plant Cell Reports 2:209-212. Gamborg OL, Davis BP, Stahlhut RW (1983b) Plant Cell Reports 2:213:215. Gresshoff P (1980) Bot. Gaz. 141:157-164. Kao KN, Michayluk MR (1980) Z. Pflanzenphysiol. 96:135-141. Kamada H, Harada H (1981) Plant Cell Physiol. 22:1423-1429. Kochba J, Spiegel-Roy P, Neumann H, Soad S (1978) Z. Pflanzenphysiol. 89:427-436. Lu CY, Vasil IK (1981) Ann. Bot. 48:543-548. Murashige T, Skoog F (1962) Physiol. Plant. 15:473-476. Nitsch C, Norrell B (1973) CR. Acad. Sci. 276D:303-306. Dos Santos AVP, Outka DE, Cocking EC, Davey MR (1980) Z. Pflanzenphysiol. 99:261-270. Sunderland N (1977) Nature 270(5634):236-238. Tu GH, Li BJ (1984) Journal of Cell Biology (China) 6(4):164-168. Vasil V, Vasil IK (1980) Theor. Appl. Genet. 56:97-99. Vasil V, Vasil IK (1981a) Amer. J. Bot. 68(6):864-872. Vasil V, Vasil IK (1981b) Ann. Bot. 47:669-678.
