Plant Cell Reports
Plant Cell Reports (1991) 10:362-365
9 Springer-Verlag 1991
Plant regeneration through somatic embryogenesis from suspension culture-derived protoplasts of Paspalum scrobiculatum L. Pritilata Nayak and S.K. Sen Plant Molecular & Cellular Genetics, Bose Institute Centenary Building, P-1/12, C.I.T. Scheme VII-M, Calcutta - 700 054 India Received February 1, 1990/Revised version received July 2, 1991 - Communicated by C.T. Harms
ABSTRACT
MATERIALS AND METHODS
Prol:oplasts were released from embryogenic suspension culture of Paspalum scrobiculatum and cultured in either liquid or semisolid KM medium supplemented with 2,4-D in the dark al: 24~ wil:h or without a feeder layer. Cell wall formation was observed in 75% of the plated protoplasl:s. Microcolonies developed after l0 d of cull:ure, which in turn formed callus upon l:ransfer to M-2 medium (Nayak and Sen, 1989). The highest plating effeciency (ca 7%) was obtained in l:hin-layer liquid culture. The macrocalli formed somatic embryos which regenerated to plantlets. The plantlel:s were grown to flowering plants upon transfer to soil.
Isolation of protoplasts. Four to six months old embryogenic suspension cultures of pa.spalum scrobiculatum (Nayak and Sen, 1989) were used as source of protoplasts (Fig. IA). Three days after subculture, I g of cells were collected and mixed with 10 ml of enzyme solution consisting of 2% (w/v) cellulase Onozuka R-10 (Yakull: Honsha, Japan), 0.1% (w/v) Pectolyase Y-23 (Kikkoman Co., Japan), 0.65 M mannil:ol, 14 mM CaCI2.2H_O, 0.7 mM NaH2PO4.2H_O , 3 mM MES buffer (p~ 5.6) and incubated in ~ e dark al: 28~ in a roller shaker at 4 rpm. A f t e r incubation for 4 h, the enzyme mixture was passed through a series of nylon meshes (Henri Simons Ltd., England) ranging from 20 ~m to 118 ~ m , centrifuged for 5 rain at 100 x g. The pellet was washed by resuspending in washing medium (the same solution without enzymes) and centrifuged for 3 min. The centrifugation and resuspension was repeated thrice. The isolated protoplasts were tested with FDA staining for viability (Widholm, 1972) and finally suspended in culture medium.
ABBREVIATIONS BAP - 6-benzylaminopurine; 2,4-D - 2,4-dichlorophenoxyacetic acid; FDA Fluoresceine diacetate; MES 2-(N-Morpholino) ethanesulfonic acid; MS Murashige & Skoog medium (1962). INTRODUCTION Members of the f a m i l y Poaceae amongst monocotyledons are not known to be susceptible to A@robacterium tumefaciens infection. One approach to effecting gene transfer in this group of plants is direct gene transfer into protoplasts. In order to translate such a possibility into reality, routine plant regeneration from protoplasts is the first step. It is highly encouraging that protoplasts of Poaceae do respond favourably in culture and their totipol:ency has been realised in a number of cases of which the most recent ones are Kyozuka e t al., 1987,1988; Lee el: al., 1989; Nayak and Sen, 1990 in rice, Harris el: al., 1988; Vasil el: al., 1990 in Tril:icum aesl:ivum, Rhodes el: al., 1988; Prioli and Sbndahl, 1989; Shillito et al., 1989 in maize; Srinivasan and Vasil, 1986 in Sugarcane; Horn el: al., 1988 in Dactylis glomerata; Dalton, 1988 in Fesl:uca and Lolium; Wei and Xu, 1990 in Sorghum vulgare. Inl:erestingly enough, in all cases excepl: in one (Kyozuka el: al., 1987), embryogenic suspension cultures have been the source of protoplasts. We had reported l:hat we could develop totipotent embryogenic cell suspensions of Paspalum sorobiculatum (Nayak and Sen, 1989), a minor millet for semi-arid areas of the Indian subcontinent. This communication reports on our being able to generate plants through somatic embryogenesis from protoplasts of such embryogenic suspension culture lines. Offprint requests to: S.K. Sen
Culture of protoplasts. For culture of protoplasts, KM (Kao and Michayluk, 1975) basal medium supplemented with KM vitamins, organic acids, carbohydrates, 200 mg/I casein hydrolysate, 2% (v/v) coconut milk and 1 mg/I 2,4-D (pH 5.6) was used. Protoplasts were cultured either in liquid medium or in medium supplemented with 1.6% Sea-Plaque agarose. For plating in agarose, the double strength culture medium was mixed with equal volume of agarose dissolved in water at 45~ Measured amount of protoplasts (counted through a haemocytometer) were suspended in the medium, mixed thoroughly and plated in petridishes. The protoplast suspension was either plated in thin liquid layer ( I ml per 35 mm plastic petridishes) or onto a feeder-layer. Same suspension culture cell lines were used as feeder-layer 3 days after subculture. 10 ml of suspension was mixed with 10 ml of M-2 mediu~n (Nayak a n d Sen, 1989) with 1.6% noble agar at 45 C and poured into a 90 mm petridish. A nitrocellulose membrane f i l t e r (sertorius) of 47 mm diameter, 0.8 mm pore size was laid on top of this feeder-layer and 200 x~l of protoplasts in liquid medium was layered on top of the membrane. A f t e r 7 d of culture, fresh medium was added to the thin-liquid layer after pipetting off the old medium. In case of the feeder-layer, the membrane f i l t e r with the culture was transferred to a fresh feeder-layer
363
Fig. I. Plant regeneration througil somal:ic embryogenesis from suspension culture derived protoplast~ of Paspalurn scrobiculatum, A. Embryogenic suspension culture cells. B. Isolated protoplasts. C. Ist division. D. Microcolony. E. Microcolonies generated in agar medium, transferred into liquid medium for further growth. F, Embryogenic callus with globular pro-embryos. G. Scanning microscopy showing pro-embryos and globular embryos. H. Somatic embryos with scutellum (s) and well-developed coleoptile (c). I. Regeneration. J. Plantlets. K. Soil growing flowering plant (p - panicle). Bars represent 50/urn in A,D; 25 ~m in B,C; I cm in E,I; 500,um in F.H; 100 ~m in G and 10 cm in K. plate. On the other hand, the plated protoplasts embedded in the agarose medium were transferred as round block to fresh liquid medium in 90 mm plates (Fig. IE). Individual microcolonies, when grown to a suitable size i.e., 300 to 500 /um were transferred to M-2 medium for further growth. Calli were transferred to fresh M-2 medium and then to regeneration medium (MS basal medium with 2 to 5 mg/I BAP and 0.25 to 0.5 mg[I NAA). The regenerated plantlets were maintained in medium containing half strength of the nutrients and vitamins~ solidified with 0.8% agar without growth regulators for one month a n d then transferred to soil. For scanning electron microscopy, the procedure of Nayak and Sen (1989) was followed. RESULTS AND DISCUSSION The results presented here are based on one representative experiment after each of the steps of the method, were standardised through 4 to 15 trials, The essentialities of our observation have helped us to develop a protocol for routine use. Protoplast yield turned out to be 6.8-7.9 x 106/g of cells with ca. 90-95% viability. The yield of proto-
plast was however found to vary with the increase in age of the suspen ion culture (after callus initiation) as well as with the age of the cells in subculture, used for isolation. The purified protoplasts consisted of large ( 25 ~m) and small ( 10 sum) protoplasts with less than 0.05% of undigested cells (Fig. IB). The cell contaminants were elongated or eliptical in shape, thin-walled with or without cytoplasm and such cells were found to be nonviable as determined through FDA staining test. Similar observations were also made by earlier workers (Heyser, 1984; Abdullah et a l . 1986; Yamada et al., 1986), Few multinucleate, large protoplasts could also be observed which might have occurred as the result of spontaneous fusion of the protoplasts. Similar occurrence was reported by Prioli and Sondahl (1989) in case of maize and observed in rice by us (Nayak and Sen, in preparation). Regeneration of cell wall by the protoplasts was achieved in about 75% of the cases after 24 to 48 h of incubation. In thin liquid cultures, the first division took place (Fig. IC) within 2 to 4 d of culture. The subsequent second division occurred during 4 and 8 d of culture. Although the frequency of cell wall regeneration was high, the first divisions occurred at
364 a low frequency (ca 11%). Subsequent divisions took place rapidly resulting in multicellular clumps within 10 d. Microcolonies were visible after about 15 d to the naked eye (Fig. ID). When the microcolonies were transferred to fresh medium, macrocolonies formed within one week. The plating efficiency as estimated after 21 d was found to be ca 7%.6Highest plating efficiency was observed at I-2 x 10 protoplasts/ml plating density. The microcolonies when transferred to solidified M-2 medium developed into two types of calli. Nearly half of the callus population was compact, hard granular and whitish in colour and embryogenic. On the other hand, the mass comprising loose, soft, friable and yellowish-white callus made up of loosely associated elongated, vacuolated cells were non-embryogenic. Upon transfer to MS basal medium containing 2 mg/I 2,4-D, I mg/I NAA, I mg/I BAP and 5% coconut milk, more than 70% of the embryogenic calli developed into hard, compact structures. Globular pro-embryos were formed within 10 d of culture (Fig, IF,G). In presence of 0.5 mg/I 2,4-D in the medium proembryos developed into well organised embryos with scutellum and coleoptile (Fig. IH). Thirty four plantlets could be recovered when 400 embryos were transferred to regeneration medium (Fig. II) of which 31 turned out to be green (Fig. I J). The green plants were transferred to soil (Fig. IK). 17 plants could be raised to maturity in the outdoor condition. Generation of embryogenic suspension culture cells is an essential step for protoplast derived plant production. This has been possible lately for many members of the Poaceae family. Success in being able to develop a protocol for generation of embryogenic suspension culture cell lines not only depends upon the growth conditions, but also on the genotype of the experimental plant material. The embryogenic cells when converted into protoplasts, often maintain the embryogenic potentiality. The passage through a callus phase in such cases does not seem to disturb the embryogenic programming of the cell types. The biology of the suspension derived embryogenic cells may thus form an interesting experimental system in future for identifying developmental factors which contribute towards competency for regeneration. The present success to generate plants from protoplasts technically put us into an advantageous state for adoption to gene transfer techniques. The importance of embryogenic suspension culture protoplasts (Vasil et al., 1990; Wei and Xu, 1990) for their ultimate use in production of transgenic cereals has been established (Datta et al., 1990). However, the account of recent successes (Fromm et al., 1990; Gordon-Kamm el: al., 1990) in being able to transfer foreign genes to corn using Biolistic technology, provides the much needed relief that the plant genetic engineers were looking for. Additionally, the case of transformation in rice through infection by virulent strain of Agrobacterium (Raineri et al., 1990) and the evidence for tranment gene expression in intact and organised rice tissues (Dekeyser et al., 1990) carry additionally fresh hopes for future. ACKNOWLEDGEMENTS Authors are thankful to the CSIR, New Delhi for financial assistance in the form of a fellowship to PN and to American Embassy and I C A R , New Delhi for a grant from the US held Rupee fund (FG-In658/In ARS - 233).
REFERENCES Abdullah R, Cocking EC, Thompson JA (1986) Efficient plant regeneration from rice protoplasts through somatic embryogenesis. Bio/Technol 4:1087-1090 Dalton SJ (1988) Plant regeneration from cell suspension protoplasts of Festuca arundinacea Schreb. (tall fescue) and Lolium perenne L. (perennial ryegrass). J. Plant Physiol 132:170-175 Datta SK, Peterhans A, Datta K, Potrykus I (1990) Genetically engineered fertile indica rice recovered from protoplasts. Bio/Technol 8:736-740 Dekeyser RA, Claes B, De Rycke RMU, Habets ME, Van Montagu MC, Caplan AB (1990) Transient gene expression in intact and organised rice tissues. The Plant Cell 2:591-602 Fromm ME, Morrish F, Armstrong C, Williams R, Thomas J, Klein TM (1990) Inheritance and expression of chimeric genes in the progeny of transgenic maize plants. Bio/Technol 8:833-839 Gordon-Kamm WJ, Spencer TM, Mangano ML, Adams TR, Daines RJ, Start WG, O'Brien JV, Chambers SA, Adams WR, Willetts NG, Rice TB, Mackey CJ, Krueger RW, Kausch AP, Lemaux PG (1990) Transformation of maize cells and regeneration of fertile transgenic plants. The Plant Cell 2:603-618 Harris R, Wright M, Byrne M, Varnum J, Brightwell B, Schubert K (1988) Callus formation and plant:let regeneration from protoplasts derived from suspension cultures of wheat (Triticum aestivum L.). Plant Cell Rep 7:337-340 Heyser JW (1984) Callus and shoot regeneration from protoplasts of p r o s o millet (Panicum miliaceum L.) Z Pflanzenphysiol 113:293-299 Horn ME, Conger BV, Harms CT (1988) Plant regeneration from protoplasts of embryogenic suspension cultures of orchardgrass (Dactylis glomerata L.). Plant Cell Rep 7:371-374 Kao KN, Michayluk MR (1975) Nutritional requirements for growth of Vicia hajastana cells and protoplasts at very low population density in liquid media. Planta 126:105-110 Kyozuka J, Hayashi Y, Shimamoto K (1987) High frequency plant regeneration from rice protoplasts by novel nurse culture methods. Mol Gen Genet 206:408-413 Kyozuka J, Otoo E, Shimamoto K (1988) Plant regeneration from protoplasts of indica rice : genotypic differences in culture response. Theor Appl Genet 76:887-890 Lee L, Schroll RE, Grimes HD, Hodges TK (1989) Plant regeneration from indica rice (Oryza saliva L.) protoplasts. Planta 178:325-333 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473-497 Nayak P, Sen SK (1989) Plant regeneration through somatic embryogenesis from suspension cultures of a minor millet, Paspalum scrobiculatum. Plant Cell Rep 8:296-299 Nayak P, Sen SK (1990) Plant regeneration from protoplasts of indica rice. In: 4th Annual Meeting of The Rockefeller Foundations International Programme on Rice Biotechnology, IRRI, Manila May, 1990 Potrykus I (1990) Gene transfer to cereals : An assessment. Bio/Technol 8:532-542 Prioli LM, S~ndahl MR (1989) Plant regeneration and recovery of fertile plants from protoplasts of maize (Zee mays L.) Bio/Technol 7:589-594
365 Raineri DM, Bot:tino P, Gordon MP, Nester EW (1990) Agrobacterium mediated transformation of rice (Oryza sat:iva L.). Bio/Technol 8:33-38 Rhodes CA, Lowe KS, Ruby KL (1988) Plant: regenerat:ion from protoplasts isolated from embryogenic maize cell cultures. Bio/Technol 6:56-60 Shillito RD, Carswell GK, Johnson CM, DiMaio J J, Harms CT (1989) Regeneration of fertile plants from protoplasts of elite inbred maize. Bio/Technol 7:581-587 Srinivasan C, Vasil IK (1986) Plant regeneration from protoplasts of sugarcane. J Plant Physiol 126:41-48
Vasil V, Redway F, Vasil IK (1990) Regeneration of plants from embryogenic suspension culture prot:oplasts of wheat (Triticum aest:ivum L.). Bio/Technol 8:429-434 Wei Z, Xu Z (1990) Regeneration of fertile plants from embryogenio suspension culture protoplasts of Sorghum ~ . Plant: Cell Rep 9 : 5 1 - 5 3 Widholm JM (1972) The use of fluorescein diacetat:e and phenosafranine for determining viability of cultured plant ceils. Stain Technol 47:189-191 Yamada Y, Yang ZQ, Tang DT (1986) Plant regeneration from protoplast derived callus of rice (Oryza sativa L.). Plant Cell Rep 4:85-88
Plant Cell Reports (1991) 10:362-365
9 Springer-Verlag 1991
Plant regeneration through somatic embryogenesis from suspension culture-derived protoplasts of Paspalum scrobiculatum L. Pritilata Nayak and S.K. Sen Plant Molecular & Cellular Genetics, Bose Institute Centenary Building, P-1/12, C.I.T. Scheme VII-M, Calcutta - 700 054 India Received February 1, 1990/Revised version received July 2, 1991 - Communicated by C.T. Harms
ABSTRACT
MATERIALS AND METHODS
Prol:oplasts were released from embryogenic suspension culture of Paspalum scrobiculatum and cultured in either liquid or semisolid KM medium supplemented with 2,4-D in the dark al: 24~ wil:h or without a feeder layer. Cell wall formation was observed in 75% of the plated protoplasl:s. Microcolonies developed after l0 d of cull:ure, which in turn formed callus upon l:ransfer to M-2 medium (Nayak and Sen, 1989). The highest plating effeciency (ca 7%) was obtained in l:hin-layer liquid culture. The macrocalli formed somatic embryos which regenerated to plantlets. The plantlel:s were grown to flowering plants upon transfer to soil.
Isolation of protoplasts. Four to six months old embryogenic suspension cultures of pa.spalum scrobiculatum (Nayak and Sen, 1989) were used as source of protoplasts (Fig. IA). Three days after subculture, I g of cells were collected and mixed with 10 ml of enzyme solution consisting of 2% (w/v) cellulase Onozuka R-10 (Yakull: Honsha, Japan), 0.1% (w/v) Pectolyase Y-23 (Kikkoman Co., Japan), 0.65 M mannil:ol, 14 mM CaCI2.2H_O, 0.7 mM NaH2PO4.2H_O , 3 mM MES buffer (p~ 5.6) and incubated in ~ e dark al: 28~ in a roller shaker at 4 rpm. A f t e r incubation for 4 h, the enzyme mixture was passed through a series of nylon meshes (Henri Simons Ltd., England) ranging from 20 ~m to 118 ~ m , centrifuged for 5 rain at 100 x g. The pellet was washed by resuspending in washing medium (the same solution without enzymes) and centrifuged for 3 min. The centrifugation and resuspension was repeated thrice. The isolated protoplasts were tested with FDA staining for viability (Widholm, 1972) and finally suspended in culture medium.
ABBREVIATIONS BAP - 6-benzylaminopurine; 2,4-D - 2,4-dichlorophenoxyacetic acid; FDA Fluoresceine diacetate; MES 2-(N-Morpholino) ethanesulfonic acid; MS Murashige & Skoog medium (1962). INTRODUCTION Members of the f a m i l y Poaceae amongst monocotyledons are not known to be susceptible to A@robacterium tumefaciens infection. One approach to effecting gene transfer in this group of plants is direct gene transfer into protoplasts. In order to translate such a possibility into reality, routine plant regeneration from protoplasts is the first step. It is highly encouraging that protoplasts of Poaceae do respond favourably in culture and their totipol:ency has been realised in a number of cases of which the most recent ones are Kyozuka e t al., 1987,1988; Lee el: al., 1989; Nayak and Sen, 1990 in rice, Harris el: al., 1988; Vasil el: al., 1990 in Tril:icum aesl:ivum, Rhodes el: al., 1988; Prioli and Sbndahl, 1989; Shillito et al., 1989 in maize; Srinivasan and Vasil, 1986 in Sugarcane; Horn el: al., 1988 in Dactylis glomerata; Dalton, 1988 in Fesl:uca and Lolium; Wei and Xu, 1990 in Sorghum vulgare. Inl:erestingly enough, in all cases excepl: in one (Kyozuka el: al., 1987), embryogenic suspension cultures have been the source of protoplasts. We had reported l:hat we could develop totipotent embryogenic cell suspensions of Paspalum sorobiculatum (Nayak and Sen, 1989), a minor millet for semi-arid areas of the Indian subcontinent. This communication reports on our being able to generate plants through somatic embryogenesis from protoplasts of such embryogenic suspension culture lines. Offprint requests to: S.K. Sen
Culture of protoplasts. For culture of protoplasts, KM (Kao and Michayluk, 1975) basal medium supplemented with KM vitamins, organic acids, carbohydrates, 200 mg/I casein hydrolysate, 2% (v/v) coconut milk and 1 mg/I 2,4-D (pH 5.6) was used. Protoplasts were cultured either in liquid medium or in medium supplemented with 1.6% Sea-Plaque agarose. For plating in agarose, the double strength culture medium was mixed with equal volume of agarose dissolved in water at 45~ Measured amount of protoplasts (counted through a haemocytometer) were suspended in the medium, mixed thoroughly and plated in petridishes. The protoplast suspension was either plated in thin liquid layer ( I ml per 35 mm plastic petridishes) or onto a feeder-layer. Same suspension culture cell lines were used as feeder-layer 3 days after subculture. 10 ml of suspension was mixed with 10 ml of M-2 mediu~n (Nayak a n d Sen, 1989) with 1.6% noble agar at 45 C and poured into a 90 mm petridish. A nitrocellulose membrane f i l t e r (sertorius) of 47 mm diameter, 0.8 mm pore size was laid on top of this feeder-layer and 200 x~l of protoplasts in liquid medium was layered on top of the membrane. A f t e r 7 d of culture, fresh medium was added to the thin-liquid layer after pipetting off the old medium. In case of the feeder-layer, the membrane f i l t e r with the culture was transferred to a fresh feeder-layer
363
Fig. I. Plant regeneration througil somal:ic embryogenesis from suspension culture derived protoplast~ of Paspalurn scrobiculatum, A. Embryogenic suspension culture cells. B. Isolated protoplasts. C. Ist division. D. Microcolony. E. Microcolonies generated in agar medium, transferred into liquid medium for further growth. F, Embryogenic callus with globular pro-embryos. G. Scanning microscopy showing pro-embryos and globular embryos. H. Somatic embryos with scutellum (s) and well-developed coleoptile (c). I. Regeneration. J. Plantlets. K. Soil growing flowering plant (p - panicle). Bars represent 50/urn in A,D; 25 ~m in B,C; I cm in E,I; 500,um in F.H; 100 ~m in G and 10 cm in K. plate. On the other hand, the plated protoplasts embedded in the agarose medium were transferred as round block to fresh liquid medium in 90 mm plates (Fig. IE). Individual microcolonies, when grown to a suitable size i.e., 300 to 500 /um were transferred to M-2 medium for further growth. Calli were transferred to fresh M-2 medium and then to regeneration medium (MS basal medium with 2 to 5 mg/I BAP and 0.25 to 0.5 mg[I NAA). The regenerated plantlets were maintained in medium containing half strength of the nutrients and vitamins~ solidified with 0.8% agar without growth regulators for one month a n d then transferred to soil. For scanning electron microscopy, the procedure of Nayak and Sen (1989) was followed. RESULTS AND DISCUSSION The results presented here are based on one representative experiment after each of the steps of the method, were standardised through 4 to 15 trials, The essentialities of our observation have helped us to develop a protocol for routine use. Protoplast yield turned out to be 6.8-7.9 x 106/g of cells with ca. 90-95% viability. The yield of proto-
plast was however found to vary with the increase in age of the suspen ion culture (after callus initiation) as well as with the age of the cells in subculture, used for isolation. The purified protoplasts consisted of large ( 25 ~m) and small ( 10 sum) protoplasts with less than 0.05% of undigested cells (Fig. IB). The cell contaminants were elongated or eliptical in shape, thin-walled with or without cytoplasm and such cells were found to be nonviable as determined through FDA staining test. Similar observations were also made by earlier workers (Heyser, 1984; Abdullah et a l . 1986; Yamada et al., 1986), Few multinucleate, large protoplasts could also be observed which might have occurred as the result of spontaneous fusion of the protoplasts. Similar occurrence was reported by Prioli and Sondahl (1989) in case of maize and observed in rice by us (Nayak and Sen, in preparation). Regeneration of cell wall by the protoplasts was achieved in about 75% of the cases after 24 to 48 h of incubation. In thin liquid cultures, the first division took place (Fig. IC) within 2 to 4 d of culture. The subsequent second division occurred during 4 and 8 d of culture. Although the frequency of cell wall regeneration was high, the first divisions occurred at
364 a low frequency (ca 11%). Subsequent divisions took place rapidly resulting in multicellular clumps within 10 d. Microcolonies were visible after about 15 d to the naked eye (Fig. ID). When the microcolonies were transferred to fresh medium, macrocolonies formed within one week. The plating efficiency as estimated after 21 d was found to be ca 7%.6Highest plating efficiency was observed at I-2 x 10 protoplasts/ml plating density. The microcolonies when transferred to solidified M-2 medium developed into two types of calli. Nearly half of the callus population was compact, hard granular and whitish in colour and embryogenic. On the other hand, the mass comprising loose, soft, friable and yellowish-white callus made up of loosely associated elongated, vacuolated cells were non-embryogenic. Upon transfer to MS basal medium containing 2 mg/I 2,4-D, I mg/I NAA, I mg/I BAP and 5% coconut milk, more than 70% of the embryogenic calli developed into hard, compact structures. Globular pro-embryos were formed within 10 d of culture (Fig, IF,G). In presence of 0.5 mg/I 2,4-D in the medium proembryos developed into well organised embryos with scutellum and coleoptile (Fig. IH). Thirty four plantlets could be recovered when 400 embryos were transferred to regeneration medium (Fig. II) of which 31 turned out to be green (Fig. I J). The green plants were transferred to soil (Fig. IK). 17 plants could be raised to maturity in the outdoor condition. Generation of embryogenic suspension culture cells is an essential step for protoplast derived plant production. This has been possible lately for many members of the Poaceae family. Success in being able to develop a protocol for generation of embryogenic suspension culture cell lines not only depends upon the growth conditions, but also on the genotype of the experimental plant material. The embryogenic cells when converted into protoplasts, often maintain the embryogenic potentiality. The passage through a callus phase in such cases does not seem to disturb the embryogenic programming of the cell types. The biology of the suspension derived embryogenic cells may thus form an interesting experimental system in future for identifying developmental factors which contribute towards competency for regeneration. The present success to generate plants from protoplasts technically put us into an advantageous state for adoption to gene transfer techniques. The importance of embryogenic suspension culture protoplasts (Vasil et al., 1990; Wei and Xu, 1990) for their ultimate use in production of transgenic cereals has been established (Datta et al., 1990). However, the account of recent successes (Fromm et al., 1990; Gordon-Kamm el: al., 1990) in being able to transfer foreign genes to corn using Biolistic technology, provides the much needed relief that the plant genetic engineers were looking for. Additionally, the case of transformation in rice through infection by virulent strain of Agrobacterium (Raineri et al., 1990) and the evidence for tranment gene expression in intact and organised rice tissues (Dekeyser et al., 1990) carry additionally fresh hopes for future. ACKNOWLEDGEMENTS Authors are thankful to the CSIR, New Delhi for financial assistance in the form of a fellowship to PN and to American Embassy and I C A R , New Delhi for a grant from the US held Rupee fund (FG-In658/In ARS - 233).
REFERENCES Abdullah R, Cocking EC, Thompson JA (1986) Efficient plant regeneration from rice protoplasts through somatic embryogenesis. Bio/Technol 4:1087-1090 Dalton SJ (1988) Plant regeneration from cell suspension protoplasts of Festuca arundinacea Schreb. (tall fescue) and Lolium perenne L. (perennial ryegrass). J. Plant Physiol 132:170-175 Datta SK, Peterhans A, Datta K, Potrykus I (1990) Genetically engineered fertile indica rice recovered from protoplasts. Bio/Technol 8:736-740 Dekeyser RA, Claes B, De Rycke RMU, Habets ME, Van Montagu MC, Caplan AB (1990) Transient gene expression in intact and organised rice tissues. The Plant Cell 2:591-602 Fromm ME, Morrish F, Armstrong C, Williams R, Thomas J, Klein TM (1990) Inheritance and expression of chimeric genes in the progeny of transgenic maize plants. Bio/Technol 8:833-839 Gordon-Kamm WJ, Spencer TM, Mangano ML, Adams TR, Daines RJ, Start WG, O'Brien JV, Chambers SA, Adams WR, Willetts NG, Rice TB, Mackey CJ, Krueger RW, Kausch AP, Lemaux PG (1990) Transformation of maize cells and regeneration of fertile transgenic plants. The Plant Cell 2:603-618 Harris R, Wright M, Byrne M, Varnum J, Brightwell B, Schubert K (1988) Callus formation and plant:let regeneration from protoplasts derived from suspension cultures of wheat (Triticum aestivum L.). Plant Cell Rep 7:337-340 Heyser JW (1984) Callus and shoot regeneration from protoplasts of p r o s o millet (Panicum miliaceum L.) Z Pflanzenphysiol 113:293-299 Horn ME, Conger BV, Harms CT (1988) Plant regeneration from protoplasts of embryogenic suspension cultures of orchardgrass (Dactylis glomerata L.). Plant Cell Rep 7:371-374 Kao KN, Michayluk MR (1975) Nutritional requirements for growth of Vicia hajastana cells and protoplasts at very low population density in liquid media. Planta 126:105-110 Kyozuka J, Hayashi Y, Shimamoto K (1987) High frequency plant regeneration from rice protoplasts by novel nurse culture methods. Mol Gen Genet 206:408-413 Kyozuka J, Otoo E, Shimamoto K (1988) Plant regeneration from protoplasts of indica rice : genotypic differences in culture response. Theor Appl Genet 76:887-890 Lee L, Schroll RE, Grimes HD, Hodges TK (1989) Plant regeneration from indica rice (Oryza saliva L.) protoplasts. Planta 178:325-333 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473-497 Nayak P, Sen SK (1989) Plant regeneration through somatic embryogenesis from suspension cultures of a minor millet, Paspalum scrobiculatum. Plant Cell Rep 8:296-299 Nayak P, Sen SK (1990) Plant regeneration from protoplasts of indica rice. In: 4th Annual Meeting of The Rockefeller Foundations International Programme on Rice Biotechnology, IRRI, Manila May, 1990 Potrykus I (1990) Gene transfer to cereals : An assessment. Bio/Technol 8:532-542 Prioli LM, S~ndahl MR (1989) Plant regeneration and recovery of fertile plants from protoplasts of maize (Zee mays L.) Bio/Technol 7:589-594
365 Raineri DM, Bot:tino P, Gordon MP, Nester EW (1990) Agrobacterium mediated transformation of rice (Oryza sat:iva L.). Bio/Technol 8:33-38 Rhodes CA, Lowe KS, Ruby KL (1988) Plant: regenerat:ion from protoplasts isolated from embryogenic maize cell cultures. Bio/Technol 6:56-60 Shillito RD, Carswell GK, Johnson CM, DiMaio J J, Harms CT (1989) Regeneration of fertile plants from protoplasts of elite inbred maize. Bio/Technol 7:581-587 Srinivasan C, Vasil IK (1986) Plant regeneration from protoplasts of sugarcane. J Plant Physiol 126:41-48
Vasil V, Redway F, Vasil IK (1990) Regeneration of plants from embryogenic suspension culture prot:oplasts of wheat (Triticum aest:ivum L.). Bio/Technol 8:429-434 Wei Z, Xu Z (1990) Regeneration of fertile plants from embryogenio suspension culture protoplasts of Sorghum ~ . Plant: Cell Rep 9 : 5 1 - 5 3 Widholm JM (1972) The use of fluorescein diacetat:e and phenosafranine for determining viability of cultured plant ceils. Stain Technol 47:189-191 Yamada Y, Yang ZQ, Tang DT (1986) Plant regeneration from protoplast derived callus of rice (Oryza sativa L.). Plant Cell Rep 4:85-88
