Plant Cell Reports
Plant Cell Reports (1990) 9:216-220
9 Springer-Verlag 1990
Efficient plant regeneration from rice protoplasts in general medium Zhijian Li and Norimoto Murai Department of Plant Pathology and Crop Physiology, College of Agriculture, Louisiana State University, Baton Rouge, LA 70803-1720, USA Received March 9, 1990/Revised version received May 11, 1990
Communicated by G. C. Phillips
S u m m a r y . We e s t a b l i s h e d an efficient and reproducible procedure for protoplast propagation and fertile plant regeneration of rice (Oryza sativa L.) cultivars N i p p o n b a r e and Taipei 309. Selection of scutellum-derived secondary calli, the use of General medium and nurse culture were all found to be critical in the procedure. When 5 basal media (Murashige and Skoog, RY-2, modified R2, Amino Acid and General media) were compared, suspension callus growth rate, protoplast yield and plating efficiency were all about 30% higher in General medium than in the second-best R2 medium. Only one month was required to develop suspension cultures for protoplast isolation using General medium. A plating efficiency as high as 17% and a plant r e g e n e r a t i o n frequency of 67% were achieved by the improved procedure. Agronomic traits of protoplast- and seed-derived plants were found to be similar.
a small genome size (6 x 108 bp; n= 12) of which over 50% is single copy DNA. In addition, rice has a large germplasm collection, well-developed classical genetics, and restriction fragment length polymorphism maps. A n u m b e r of laboratories have reported plant regeneration from rice protoplasts (Fujimura et al. 1985, Coulibaly and Demarly 1986, Kyozuka et al. 1987, Hayashi et al. 1988). However, in most cases the procedures involved a long development period of suspension cell lines extending from six months to two years, and the quality of the resulting protoplasts was unpredictable (Abdullah et al. 1986, Toriyama et al. 1986, Yamada et al. 1986). Thus, the published procedures cannot readily be adopted for developing a reliable system for gene expression analysis in rice. As a first step in e s t a b l i s h i n g a reliable and easilyaccessible gene transfer system, we report here an efficient procedure for rice protoplast culture and fertile plant regeneration using the General medium of Chen (1986).
Abbreviations. AA, Amino Acid medium (Muller and Grafe 1978);
Materials and Methods
MS, Murashige and Skoog (1962) medium.
Plant materials. Seeds of the rice (Oryza sativa L.) japonica cuitivar Nipponbare were generously provided by Dr. K. Okuno, Hokuriku
Introduction
We are interested in studying the regulation of gene expression in rice, a monocotyledonous plant. Our objective is to develop a homologous gene transfer system for rice to c i r c u m v e n t the difficulties encountered in heterologous dicot gene expression systems (Mural et al. 1983). Rice has a number of advantages for studying gene regulation and molecular genetics. Rice is an important cereal crop in which protoplast regeneration and transformation have been demonstrated (Toriyama et al. 1988, Shimamoto et al. 1989, Hayashimoto et al. 1990). Rice is diploid and has Offprint requests to: N. Murai
National Agricultural Experiment Station, Niigata, Japan. The cultivar Taipei 309 seeds were kindly supplied by Dr. M. C. Rush from the LSU Department of Plant Pathology and Crop Physiology who first introduced this cultivar into tissue culture. An Oc cell line was kindly provided by Dr. K. Syono, University of Tokyo. Another Oc cell line was provided by Dr. K. Shimamoto, Plantech Research Institute, Y o k o h a m a . All cell lines were c u l t u r e d in 125 ml E r l e n m e y e r flasks c o n t a i n i n g 25 ml of MS basal medium supplemented with 4.5/zM 2,4-D and 3% (w/v) sucrose. Cultures were gently rotated on a gyratorv shaker (80 rpm) under continuous fluorescent lighting (8.4 #tool m -'2 s "l) at 27~ in a growth chamber. Subcultures were repeated weekly. Ca//us bub~don. Dehulled mature seeds were sterilized in 70% (v/v) aqueous ethanol for 1.5 min then in 2.6% (w/v) sodium hypochlorite-
217 water for 45 rain with vigorous agitation, followed by 3 rinses with sterile distilled water. Sterilized seeds were plated on MS basal medium supplemented with 9 ~M 2,4-D, 3% (w/v) sucrose and 1% (w/v) Difco Bacto-agar, and cultured in the dark at 27~ for 14 d. After removal of e n d o s p e r m s and elongated shoots and roots, embryos were transferred to fresh callus induction medium and grown for another 14 d. Small friable secondary caUi developed from primary scuteUum-derived caUi were the starting material for cell suspension cultures.
Suspension culture. Five basal media tested for their effect on rapid initiation of cell suspension culture were MS, General (Chen 1986), RY-2 (Yamada eta/. 1986), modified R2 (Ohira eta/. 1973, Kyozuka et al. 1987), and AA media. The composition of General medium in mg p e r l i t e r m e d i u m is 3000 KNO3, 400 N H . H A P O 4 , 166 C a C I 2 " 2 H ~ O , 185 M g S O 4 - 7 H ~ O , 42.1 NabSeE/'DTA, 4.4 M n S O 4 - 4 I ~ O , 1.5 Z n S O 4 * 7 H _ t ~ , 1.6 H3BO3, 0.8 KI, 0.025 CuSO4"5HA~3, 0.25 NaMoO.-2~LLO, 0.025 COC12.6H20 , 1.0 thiamine.H~l, 0.5 pyridoxine./~CI, 0~ nicotinic acid, 2.0 glycine, 1.0 2,4-D and 30000 sucrose. Secondary scutellum callus (1 g) was transferred to 25 ml of suspension medium in a 125 ml Erienmeyer flask. Two-thirds of the original medium was replaced weekly with fresh medium for three weeks. Protoplast isolation. Suspension cells (3 g) were placed in a Petri dish (100 x 15 ram) with 20 ml of filter-sterilized enzyme solution containing 4% (w/v) Cellulase RS (Yakult), 1% (w/v) Macerozyme R10 and 0.4 M mannitol. After 3 h of incubation in the dark at 30~ without shaking, the enzymatic digests were passed through 40/zm and 20 t~m nylon meshes to remove undigested cell clumps and debris. Undigested cell clumps were washed twice with 20 ml of KMC solution (Harms and Potrykus 1978) to liberate protoplasts from cell dumps. The combined filtrates were centrifuged at 130 x g for 8 min. The protoplast pellets were washed 3 times by suspending in KMC solution and centrifuging at 130 x g for 4 rain. Purified protoplasts were suspended in a protoplast medium consisting of General or one of the other four basal media supplemented with 4.5 btM 2,4-D and 0.4 M sucrose. Protoplast density was determined using a . h e m a c y t o m e t e r u n d e r an inverted microscope at 200 x magnification. Protoplast culture. Protoplasts were cultured using the agarose bead method of Shillito eta/. (1983) and the mixed nurse culture method of Kyozuka et al. (1987). One ml of 2 x 106 protoplasts in suspension was mixed gently with a n equal volume of prewarmed protoplast medium containing 2.5% (w/v) SeaPlaque agarose (FMC). The protoplast mixture was then poured into a Petri dish (60 x 15 ram). The solidified agarose sheet was cut into 10 mm square blocks and was suspended in 5 ml of protoplast medium. About 100 mg of vigorously-growing nurse cells were added to each plate. The plates were rotated gently on a gyratory shaker (30 rpm) in the dark at 25~ for 10 d. At the end of the nurse culture period, agarose blocks were washed twice in protoplast medium to remove nurse cells, transferred to fresh protoplast medium and maintained for an additional 4 d. Agarose blocks were transferred to fresh protoplast medium and maintained for another 14 d, at which time the plating efficiency was determined. The plating efficiency of protoplasts was defined as a percentage of the number of dividing ~olonies per total number of protoplasts originally plated in a 1 mm agarose block. The average numl~r of dividing protoplasts was obtained by counting 10 blocks of 1 mm size. F o r plant regeneration, agarose blocks containing 14-d old protoplast colonies were t r a n s f e r r e d to a soft agarose medium c o n t a i n i n g G e n e r a l o r o n e of t h e o t h e r four basal media supplemented with 4.5 tzM 2,4-D, 3% (w/v) sucrose and 0.25% (w/v)
Sigma type I agarose. Protoplast colonies were propagated on a sof~ a~arose medium under continuous fluorescent lighting (56/zmol m s ) at 27~ for 2 to 4 weeks or until the colony size reached 2 mm in diameter.
Plant regeneration. Protoplast-derived calli were transferred onto a plant regeneration medium containing N6 basal medium (Chu et aL 1975), 2% (w/v) sucrose, 3% (w/v) sorbitol, and 0.7% (w/v) Sigma type I agarose. CaUi were maintained under continuous fluorescent fighting at 27~ and subcultured every 21 d. The number of calli producing regenerated plants and the total number of regenerated plants were counted after 50 d in N6 medium. Regenerated plants were transferred to 20era plastic pots containing rice field soil (Crowley silt loam). The pots were placed in the greenhouse bench flooded with tap water 5 cm above the soil surface.
Results and Discussion Five basal media were selected for comparison. MS, RY-2 (Yamada et al. 1986), modified R2 (Fujimura et al. 1985, Kyozuka et al. 1987) and AA media (Abdullah et al. 1986, Toriyama et al. 1986) have been used successfully for rice protoplast culture. General medium (Chen 1986) was derived from N6 medium (Chu et al. 1975) for improvement of callus induction from anther culture of both japonica and indica rice cultivars, but had not been tested previously for protoplast culture. Explant source for suspension culture.
The scutellum-derived primary calli of Nipponbare formed small friable secondary calli upon one subculture in callus induction medium. Careful selection of these friable secondary calli was found essential in establishing rapidly-growing suspension cultures. Suspension callus growth rate.
A significant difference among 5 basal media in the growth rate of suspension cultures was observed after weekly subculture for 3 successive passages. General medium supported the highest cell growth rate (2.87 g/flask), almost tripling the initial fresh weight during the 3 week culture period. The value represents the average fresh weights of 3 flasks in 3 independent experiments. Modified R2 medium ranked second in promoting cell growth (2.13 g/flask), while callus weight increases (1.71, 1.65 and 1.51 g/flask) were less pronounced in RY, MS and AA suspension media, respectively. The appearances of cell aggregate calli in the 5 media were distinct from each other. Suspensions in General medium readily produced light yellow and finely-dispersed cell aggregates. A similar appearance of the cell aggregates was observed in suspension cultures in modified R2 medium. However, calli inoculated in RY and MS suspension media did not
218
Fig. 1. Plant regeneration from rice protoplasts cultured in General medium. (a) Protoplasts isolated from Nipponbaresuspension calli in General medium. Bar represents 10/,m. (b and c) Aggregates of 4 to 20 cells at the end of 10-d nurse culture. Bar represents 10/,m. (d) Protoplastderived colonies in agarose beads after 14 d in soft agarose medium. Bar represents 1 era. (e) Fertile protoplast-derived plants in the greenhouse.
grow noticeably during the first week and instead turned slightly brown. A f t e r subculturing these aggregates produced new tissue very slowly. Calli in AA suspension medium soon became white, formed small callus clumps and grew very slowly.
Protoplast yield. The basal media employed in suspension cultures also significantly influenced the protoplast yield and plating efficiency. To compare the medium effect, suspensions were subcultured weekly for 4 weeks in the 5 separate liquid m e d i a and h a r v e s t e d cells w e r e d i g e s t e d enzymatically to release protoplasts. Cells cultured in General m e d i u m released the largest n u m b e r of protoplasts, p r o d u c i n g an average of 8.0 million protoplasts per g fresh weight of suspension in 4 independent experiments. Cultures in modified R2 medium produced about 40% fewer protoplasts (5.1 x 106/g) than those in General medium. Consistent p r o t o p l a s t yields w e r e d i f f i c u l t to o b t a i n from suspension cultures in RY, AA and MS media (3.1, 3.0 and 2.3 x 106/g, respectively) for the short culture
period. The low protoplast yield of these cultures is probably a reflection of the slow growth rate of these suspensions. The average protoplast yields obtained from R2 and AA cultures in this study were similar to the protoplast yields in previous reports (Kyozuka et al. 1987, Abdullah et al. 1986). M o r e than 95% of protoplasts isolated from suspension cultures in General medium exhibited a typical spherical shape with dense cytoplasm (Fig. 1, a). Little contamination by intact single cells and fewer than 5% s p o n t a n e o u s l y - f u s e d p r o t o p l a s t s were observed in these protoplasts. In contrast, protoplasts isolated from suspension cultures in the other four media contained single elongated cells and small cell clumps to various extents.
Protoplast plating efficiency. To evaluate the medium effect, protoplasts isolated from suspensions in a given basal m e d i u m were propagated in the same basal m e d i u m plus 0.4 M sucrose and 4.5 tzM 2,4-D. Cell wall regeneration and the first cell divisions were observed after 3 d of nurse
219 culture in all cases. However, subsequent active cell divisions were more evident in protoplast cultures in General and modified R2 media than in other media. Compact colonies of protoplasts (visible to the naked eye) in General and modified R2 media were observed at the 16-cell stage within. 10 d (Fig. 1, b and c). These colonies grew vigorously and developed calli of about 1 mm in diameter after 4 weeks of protoplast culture (Fig. 1, d). Smaller, slowly-growing colonies were obtained from protoplast cultures in other media. The protoplast plating efficiency in the 5 basal media essentially correlated with the suspension growth rate and protoplast yield. The highest plating efficiency was obtained in protoplasts maintained in General medium (7.3%), the second highest in R2 medium (4.6%), then followed by RY (2.1%), MS (2.0%) and AA (1.9%). The protoplast plating efficiency represents an average of 2 independent experiments. The use of General medium in protoplast cultures appeared to promote the highest plating efficiency of protoplasts isolated from suspension culture in General medium. When protoplasts derived from suspension cells in General medium were cultured in the 5 separate protoplast media, the plating efficiency was 10.4 % in General medium, 7.4 % in R2 medium, 2.2 % in MS medium, 1.2 % in R Y m e d i u m and 0.5 % in A A medium. Thus, the high plating efficiency in G e n e r a l m e d i u m a p p e a r e d to be due to the combination of the superior quality of protoplasts isolated from suspension culture in General medium, as well as the growth promotion effect of General medium in protoplast culture. The superior effect of General medium may in part be due to the use of (NH4)H2PO 4 as the sole source of ammonium and phosphorus elements. Effects of nitrogen sources on rice suspension cell growth and p r o t o p l a s t yield have b e e n p r e v i o u s l y s t u d i e d (Fukunaga and King, 1978). A high concentration of ammonium ion appeared to inhibit rice protoplast growth (Yamada et al. 1986). General m e d i u m contains a low concentration of ammonium ion (3.5 raM) compared to R2 and MS media.
Nurse cell quantity and culture duration. The addition of nurse cells in protoplast culture has been shown to be critical in p r o m o t i n g vigorous p r o t o p l a s t g r o w t h ( K y o z u k a et al. 1987). We determined the nurse cell quantity and nurse culture duration that produced the highest protoplast division f r e q u e n c y in G e n e r a l m e d i u m . The m a x i m u m protoplast plating efficiency was found at nurse cell concentrations between 100 and 200 mg per 5 ml of protoplast medium (Fig. 2). Less than 100 mg of nurse cells may not supply e n o u g h p u t a t i v e growthstimulating substances, whereas 200 mg or more of nurse cells may compete for nutrients with protoplasts.
20
r
"2 r
10
5-
0
it 0
200
t 400
i 600
t 800
Milligram nurse cells per 5 ml Fig. 2. Protoplast plating efficiency dependent on nurse cell quantity.
Nipponbare protoplasts isolated from suspension cells in General medium were cultured in the presence of the specified amount of Oc cells for 10 days. The values represent an average of 3 independent experiments except those for 25 and 50 mg which were obtained from a single experiment. The bar represents a standard error.
Nurse culture for 8 d supported the highest plating efficiency of b o t h Nipponbare and Taipei 309 protoplasts, 13.7 and 17.3% respectively, in the presence of 100 mg of Oc cells per 5 ml medium. The plating efficiencies of Nipponbare and Taipei 309 protoplasts were 0 and 0.7% without nurse culture, 4.3 and 3.5% after 4 d of nurse culture, 13.8 and 17.3% after 8 d, 12.8 and 13.4% after 12 d, and 6.5 and 14.4% after 16 d of nurse culture, respectively. Two successive 4- or 8-d cultures had no greater protoplast plating efficiency, 4.2 and 9.3% for Nipponbare, and 6.8 and 15.5% for Taipei 309, respectively, than single 4- or 8-d cultures. The plating efficiency of Nipponbare p r o t o p l a s t s was an a v e r a g e of 2 i n d e p e n d e n t experiments while that of Taipei 309 was from a single experiment.
Plant regeneration. After transfer to hormone-free N6 medium, protoplastderived calli initially grew slowly with browning in older callus tissue. Subcultures at shorter intervals, such as 2 to 3 weeks, ensured consistent growth and subsequent differentiation of cultured callus. The formation of roots and shoots occurred within 20 d after transfer. Complete plants were regenerated after 50 d of culture (Fig. 1, e). General medium was not effective in promoting plant regeneration. A total of 141 regenerated plants (plant regeneration frequency = 67%) on N6 medium were obtained from 30 calli derived from protoplasts cultured in General medium. The a v e r a g e n u m b e r of plants per regenerating callus was 7.0, with some calli producing more than 15 plants. One advantage of the improved procedure is that it shortens to five months the time required from the
220
initiation of callus induction to c o m p l e t e plant regeneration. Only one month was required to develop suspension cultures from which protoplasts were isolated. In addition, one month was required for callus induction, one month for protoplast propagation and two months for plant regeneration. Thus, gene expression in transgenic rice plants may be analyzed three months after protoplast transformation, when suspension cells are available.
Agronomic traits. Protoplast-derived plants grown in the greenhouse exhibited no apparent phenotypic variation when compared to seed-derived plants of Nipponbare. The mean plant height and panicle length of 17 protoplastderived plants were 74.5 ___ 1.9 and 15.8 __. 0.4 cm, respectively ( m e a n __. SE). The average number of tillers per plant and spikelets per panicle were 8.7 _ 0.5 and 64.3 _ 3.3, respectively. Seed fertility of these plants ranged from 3.4 to 93.7%, with an average of 65.4 %. A low frequency of somaclonal variation among protoplast-derived rice plants in the field has been reported previously (Ogura et aL 1987). Acknowledgmnent~ We wish to thank Andreana Lisca and Christian Knaak for excellent technical assistance; Dr. Ko Shimamoto for advice in protoplast culture; Drs. David J. Longstreth, Mary E. Musgrave, Milton C. Rush and Mark D. Burow for critical reading of the manuscript; and all members of the Plant Molecular Biology Laboratory for discussion and encouragement. This work was supported in part by grants from the Louisiana Education Quality Support Fund (1987-90)-RD-A-6 and from the Louisiana State University System BiotechnologyProgram to N. M.
References Abdullah R, Cocking EC, Thompson JA (1986) Bio/Technol 4: 10871090 Chen Y (1986) In: Hu H, Yang HY (eds) Haploids of Higher Plants in vitro, China Academic Publishers and Springer-Verlag, Germany, pp 1-25 CoulibalyMY, Demarly Y (1986) Z Pflanzenzuchtg96: 79-81. Chu CC, Wang CC, Sun CS, Hsu C, Yin KC, Chu CY, Bio FY (1975) Sci Sin 18:659-668 Fukunaga Y, KingJ (1978) Plant Sci Lett 11:241-250 Fujimura T, Sakurai M, Akagi H, Negishi H, Hirose A (1985) Plant Tissue Culture Lett 2:74-75 Harms CT, Potrykus I (1978) Theor Appl Genet 53:57-63 Hayashi Y, KyozukaJ, Shimamoto K (1988) Mol Gen Genet 214: 610 Hayashimoto A, Li Z, Murai N (1990) Plant Physio193(in press) Kyozuka J, Hayashi Y, Shimamoto K (1987) Mol Gen Genet 206: 408-413 Muller AJ, Grafe R (1978) Mol Gen Genet 161:67-76 Murai, N, Sutton DW, Murray MJ, Slightom JL, Merlo DL, Reichert NA, Sengupta-Gopalan C, Stock CA, Baker RF, Kemp JD, Hall TC (1983) Science 222:476-482 Murashige T, Skoog F (1962) Physioi Plant 15:473-497 Ogura K, KyozukaJ, Hayashi Y, Koba T, Shimamoto K (1987) Thcor Appl Genet 74:670-676 Ohira K, Ojima K, Fujiwara A (1973) Plant Cell Physiol 14: 11131121 Shillito RD, Paszkowski J, Potrykus I (1983) Plant Cell Rep 2: 244247 Shimamoto K, Terada R, Izawa T, Fujimoto H (1989) Nature 338: 274-276 Toriyama K, Arimoto Y, Uchimiya H, Hinata K (1988) Bio/Technol 6:1072-1074 Toriyama K, Hinata K, Sasaki T (1986) Theor Appl Genet 73:16-19 Yamada Y, Yang ZQ, Tang DT (1986) Plant Cell Rep 5:85-88
Plant Cell Reports (1990) 9:216-220
9 Springer-Verlag 1990
Efficient plant regeneration from rice protoplasts in general medium Zhijian Li and Norimoto Murai Department of Plant Pathology and Crop Physiology, College of Agriculture, Louisiana State University, Baton Rouge, LA 70803-1720, USA Received March 9, 1990/Revised version received May 11, 1990
Communicated by G. C. Phillips
S u m m a r y . We e s t a b l i s h e d an efficient and reproducible procedure for protoplast propagation and fertile plant regeneration of rice (Oryza sativa L.) cultivars N i p p o n b a r e and Taipei 309. Selection of scutellum-derived secondary calli, the use of General medium and nurse culture were all found to be critical in the procedure. When 5 basal media (Murashige and Skoog, RY-2, modified R2, Amino Acid and General media) were compared, suspension callus growth rate, protoplast yield and plating efficiency were all about 30% higher in General medium than in the second-best R2 medium. Only one month was required to develop suspension cultures for protoplast isolation using General medium. A plating efficiency as high as 17% and a plant r e g e n e r a t i o n frequency of 67% were achieved by the improved procedure. Agronomic traits of protoplast- and seed-derived plants were found to be similar.
a small genome size (6 x 108 bp; n= 12) of which over 50% is single copy DNA. In addition, rice has a large germplasm collection, well-developed classical genetics, and restriction fragment length polymorphism maps. A n u m b e r of laboratories have reported plant regeneration from rice protoplasts (Fujimura et al. 1985, Coulibaly and Demarly 1986, Kyozuka et al. 1987, Hayashi et al. 1988). However, in most cases the procedures involved a long development period of suspension cell lines extending from six months to two years, and the quality of the resulting protoplasts was unpredictable (Abdullah et al. 1986, Toriyama et al. 1986, Yamada et al. 1986). Thus, the published procedures cannot readily be adopted for developing a reliable system for gene expression analysis in rice. As a first step in e s t a b l i s h i n g a reliable and easilyaccessible gene transfer system, we report here an efficient procedure for rice protoplast culture and fertile plant regeneration using the General medium of Chen (1986).
Abbreviations. AA, Amino Acid medium (Muller and Grafe 1978);
Materials and Methods
MS, Murashige and Skoog (1962) medium.
Plant materials. Seeds of the rice (Oryza sativa L.) japonica cuitivar Nipponbare were generously provided by Dr. K. Okuno, Hokuriku
Introduction
We are interested in studying the regulation of gene expression in rice, a monocotyledonous plant. Our objective is to develop a homologous gene transfer system for rice to c i r c u m v e n t the difficulties encountered in heterologous dicot gene expression systems (Mural et al. 1983). Rice has a number of advantages for studying gene regulation and molecular genetics. Rice is an important cereal crop in which protoplast regeneration and transformation have been demonstrated (Toriyama et al. 1988, Shimamoto et al. 1989, Hayashimoto et al. 1990). Rice is diploid and has Offprint requests to: N. Murai
National Agricultural Experiment Station, Niigata, Japan. The cultivar Taipei 309 seeds were kindly supplied by Dr. M. C. Rush from the LSU Department of Plant Pathology and Crop Physiology who first introduced this cultivar into tissue culture. An Oc cell line was kindly provided by Dr. K. Syono, University of Tokyo. Another Oc cell line was provided by Dr. K. Shimamoto, Plantech Research Institute, Y o k o h a m a . All cell lines were c u l t u r e d in 125 ml E r l e n m e y e r flasks c o n t a i n i n g 25 ml of MS basal medium supplemented with 4.5/zM 2,4-D and 3% (w/v) sucrose. Cultures were gently rotated on a gyratorv shaker (80 rpm) under continuous fluorescent lighting (8.4 #tool m -'2 s "l) at 27~ in a growth chamber. Subcultures were repeated weekly. Ca//us bub~don. Dehulled mature seeds were sterilized in 70% (v/v) aqueous ethanol for 1.5 min then in 2.6% (w/v) sodium hypochlorite-
217 water for 45 rain with vigorous agitation, followed by 3 rinses with sterile distilled water. Sterilized seeds were plated on MS basal medium supplemented with 9 ~M 2,4-D, 3% (w/v) sucrose and 1% (w/v) Difco Bacto-agar, and cultured in the dark at 27~ for 14 d. After removal of e n d o s p e r m s and elongated shoots and roots, embryos were transferred to fresh callus induction medium and grown for another 14 d. Small friable secondary caUi developed from primary scuteUum-derived caUi were the starting material for cell suspension cultures.
Suspension culture. Five basal media tested for their effect on rapid initiation of cell suspension culture were MS, General (Chen 1986), RY-2 (Yamada eta/. 1986), modified R2 (Ohira eta/. 1973, Kyozuka et al. 1987), and AA media. The composition of General medium in mg p e r l i t e r m e d i u m is 3000 KNO3, 400 N H . H A P O 4 , 166 C a C I 2 " 2 H ~ O , 185 M g S O 4 - 7 H ~ O , 42.1 NabSeE/'DTA, 4.4 M n S O 4 - 4 I ~ O , 1.5 Z n S O 4 * 7 H _ t ~ , 1.6 H3BO3, 0.8 KI, 0.025 CuSO4"5HA~3, 0.25 NaMoO.-2~LLO, 0.025 COC12.6H20 , 1.0 thiamine.H~l, 0.5 pyridoxine./~CI, 0~ nicotinic acid, 2.0 glycine, 1.0 2,4-D and 30000 sucrose. Secondary scutellum callus (1 g) was transferred to 25 ml of suspension medium in a 125 ml Erienmeyer flask. Two-thirds of the original medium was replaced weekly with fresh medium for three weeks. Protoplast isolation. Suspension cells (3 g) were placed in a Petri dish (100 x 15 ram) with 20 ml of filter-sterilized enzyme solution containing 4% (w/v) Cellulase RS (Yakult), 1% (w/v) Macerozyme R10 and 0.4 M mannitol. After 3 h of incubation in the dark at 30~ without shaking, the enzymatic digests were passed through 40/zm and 20 t~m nylon meshes to remove undigested cell clumps and debris. Undigested cell clumps were washed twice with 20 ml of KMC solution (Harms and Potrykus 1978) to liberate protoplasts from cell dumps. The combined filtrates were centrifuged at 130 x g for 8 min. The protoplast pellets were washed 3 times by suspending in KMC solution and centrifuging at 130 x g for 4 rain. Purified protoplasts were suspended in a protoplast medium consisting of General or one of the other four basal media supplemented with 4.5 btM 2,4-D and 0.4 M sucrose. Protoplast density was determined using a . h e m a c y t o m e t e r u n d e r an inverted microscope at 200 x magnification. Protoplast culture. Protoplasts were cultured using the agarose bead method of Shillito eta/. (1983) and the mixed nurse culture method of Kyozuka et al. (1987). One ml of 2 x 106 protoplasts in suspension was mixed gently with a n equal volume of prewarmed protoplast medium containing 2.5% (w/v) SeaPlaque agarose (FMC). The protoplast mixture was then poured into a Petri dish (60 x 15 ram). The solidified agarose sheet was cut into 10 mm square blocks and was suspended in 5 ml of protoplast medium. About 100 mg of vigorously-growing nurse cells were added to each plate. The plates were rotated gently on a gyratory shaker (30 rpm) in the dark at 25~ for 10 d. At the end of the nurse culture period, agarose blocks were washed twice in protoplast medium to remove nurse cells, transferred to fresh protoplast medium and maintained for an additional 4 d. Agarose blocks were transferred to fresh protoplast medium and maintained for another 14 d, at which time the plating efficiency was determined. The plating efficiency of protoplasts was defined as a percentage of the number of dividing ~olonies per total number of protoplasts originally plated in a 1 mm agarose block. The average numl~r of dividing protoplasts was obtained by counting 10 blocks of 1 mm size. F o r plant regeneration, agarose blocks containing 14-d old protoplast colonies were t r a n s f e r r e d to a soft agarose medium c o n t a i n i n g G e n e r a l o r o n e of t h e o t h e r four basal media supplemented with 4.5 tzM 2,4-D, 3% (w/v) sucrose and 0.25% (w/v)
Sigma type I agarose. Protoplast colonies were propagated on a sof~ a~arose medium under continuous fluorescent lighting (56/zmol m s ) at 27~ for 2 to 4 weeks or until the colony size reached 2 mm in diameter.
Plant regeneration. Protoplast-derived calli were transferred onto a plant regeneration medium containing N6 basal medium (Chu et aL 1975), 2% (w/v) sucrose, 3% (w/v) sorbitol, and 0.7% (w/v) Sigma type I agarose. CaUi were maintained under continuous fluorescent fighting at 27~ and subcultured every 21 d. The number of calli producing regenerated plants and the total number of regenerated plants were counted after 50 d in N6 medium. Regenerated plants were transferred to 20era plastic pots containing rice field soil (Crowley silt loam). The pots were placed in the greenhouse bench flooded with tap water 5 cm above the soil surface.
Results and Discussion Five basal media were selected for comparison. MS, RY-2 (Yamada et al. 1986), modified R2 (Fujimura et al. 1985, Kyozuka et al. 1987) and AA media (Abdullah et al. 1986, Toriyama et al. 1986) have been used successfully for rice protoplast culture. General medium (Chen 1986) was derived from N6 medium (Chu et al. 1975) for improvement of callus induction from anther culture of both japonica and indica rice cultivars, but had not been tested previously for protoplast culture. Explant source for suspension culture.
The scutellum-derived primary calli of Nipponbare formed small friable secondary calli upon one subculture in callus induction medium. Careful selection of these friable secondary calli was found essential in establishing rapidly-growing suspension cultures. Suspension callus growth rate.
A significant difference among 5 basal media in the growth rate of suspension cultures was observed after weekly subculture for 3 successive passages. General medium supported the highest cell growth rate (2.87 g/flask), almost tripling the initial fresh weight during the 3 week culture period. The value represents the average fresh weights of 3 flasks in 3 independent experiments. Modified R2 medium ranked second in promoting cell growth (2.13 g/flask), while callus weight increases (1.71, 1.65 and 1.51 g/flask) were less pronounced in RY, MS and AA suspension media, respectively. The appearances of cell aggregate calli in the 5 media were distinct from each other. Suspensions in General medium readily produced light yellow and finely-dispersed cell aggregates. A similar appearance of the cell aggregates was observed in suspension cultures in modified R2 medium. However, calli inoculated in RY and MS suspension media did not
218
Fig. 1. Plant regeneration from rice protoplasts cultured in General medium. (a) Protoplasts isolated from Nipponbaresuspension calli in General medium. Bar represents 10/,m. (b and c) Aggregates of 4 to 20 cells at the end of 10-d nurse culture. Bar represents 10/,m. (d) Protoplastderived colonies in agarose beads after 14 d in soft agarose medium. Bar represents 1 era. (e) Fertile protoplast-derived plants in the greenhouse.
grow noticeably during the first week and instead turned slightly brown. A f t e r subculturing these aggregates produced new tissue very slowly. Calli in AA suspension medium soon became white, formed small callus clumps and grew very slowly.
Protoplast yield. The basal media employed in suspension cultures also significantly influenced the protoplast yield and plating efficiency. To compare the medium effect, suspensions were subcultured weekly for 4 weeks in the 5 separate liquid m e d i a and h a r v e s t e d cells w e r e d i g e s t e d enzymatically to release protoplasts. Cells cultured in General m e d i u m released the largest n u m b e r of protoplasts, p r o d u c i n g an average of 8.0 million protoplasts per g fresh weight of suspension in 4 independent experiments. Cultures in modified R2 medium produced about 40% fewer protoplasts (5.1 x 106/g) than those in General medium. Consistent p r o t o p l a s t yields w e r e d i f f i c u l t to o b t a i n from suspension cultures in RY, AA and MS media (3.1, 3.0 and 2.3 x 106/g, respectively) for the short culture
period. The low protoplast yield of these cultures is probably a reflection of the slow growth rate of these suspensions. The average protoplast yields obtained from R2 and AA cultures in this study were similar to the protoplast yields in previous reports (Kyozuka et al. 1987, Abdullah et al. 1986). M o r e than 95% of protoplasts isolated from suspension cultures in General medium exhibited a typical spherical shape with dense cytoplasm (Fig. 1, a). Little contamination by intact single cells and fewer than 5% s p o n t a n e o u s l y - f u s e d p r o t o p l a s t s were observed in these protoplasts. In contrast, protoplasts isolated from suspension cultures in the other four media contained single elongated cells and small cell clumps to various extents.
Protoplast plating efficiency. To evaluate the medium effect, protoplasts isolated from suspensions in a given basal m e d i u m were propagated in the same basal m e d i u m plus 0.4 M sucrose and 4.5 tzM 2,4-D. Cell wall regeneration and the first cell divisions were observed after 3 d of nurse
219 culture in all cases. However, subsequent active cell divisions were more evident in protoplast cultures in General and modified R2 media than in other media. Compact colonies of protoplasts (visible to the naked eye) in General and modified R2 media were observed at the 16-cell stage within. 10 d (Fig. 1, b and c). These colonies grew vigorously and developed calli of about 1 mm in diameter after 4 weeks of protoplast culture (Fig. 1, d). Smaller, slowly-growing colonies were obtained from protoplast cultures in other media. The protoplast plating efficiency in the 5 basal media essentially correlated with the suspension growth rate and protoplast yield. The highest plating efficiency was obtained in protoplasts maintained in General medium (7.3%), the second highest in R2 medium (4.6%), then followed by RY (2.1%), MS (2.0%) and AA (1.9%). The protoplast plating efficiency represents an average of 2 independent experiments. The use of General medium in protoplast cultures appeared to promote the highest plating efficiency of protoplasts isolated from suspension culture in General medium. When protoplasts derived from suspension cells in General medium were cultured in the 5 separate protoplast media, the plating efficiency was 10.4 % in General medium, 7.4 % in R2 medium, 2.2 % in MS medium, 1.2 % in R Y m e d i u m and 0.5 % in A A medium. Thus, the high plating efficiency in G e n e r a l m e d i u m a p p e a r e d to be due to the combination of the superior quality of protoplasts isolated from suspension culture in General medium, as well as the growth promotion effect of General medium in protoplast culture. The superior effect of General medium may in part be due to the use of (NH4)H2PO 4 as the sole source of ammonium and phosphorus elements. Effects of nitrogen sources on rice suspension cell growth and p r o t o p l a s t yield have b e e n p r e v i o u s l y s t u d i e d (Fukunaga and King, 1978). A high concentration of ammonium ion appeared to inhibit rice protoplast growth (Yamada et al. 1986). General m e d i u m contains a low concentration of ammonium ion (3.5 raM) compared to R2 and MS media.
Nurse cell quantity and culture duration. The addition of nurse cells in protoplast culture has been shown to be critical in p r o m o t i n g vigorous p r o t o p l a s t g r o w t h ( K y o z u k a et al. 1987). We determined the nurse cell quantity and nurse culture duration that produced the highest protoplast division f r e q u e n c y in G e n e r a l m e d i u m . The m a x i m u m protoplast plating efficiency was found at nurse cell concentrations between 100 and 200 mg per 5 ml of protoplast medium (Fig. 2). Less than 100 mg of nurse cells may not supply e n o u g h p u t a t i v e growthstimulating substances, whereas 200 mg or more of nurse cells may compete for nutrients with protoplasts.
20
r
"2 r
10
5-
0
it 0
200
t 400
i 600
t 800
Milligram nurse cells per 5 ml Fig. 2. Protoplast plating efficiency dependent on nurse cell quantity.
Nipponbare protoplasts isolated from suspension cells in General medium were cultured in the presence of the specified amount of Oc cells for 10 days. The values represent an average of 3 independent experiments except those for 25 and 50 mg which were obtained from a single experiment. The bar represents a standard error.
Nurse culture for 8 d supported the highest plating efficiency of b o t h Nipponbare and Taipei 309 protoplasts, 13.7 and 17.3% respectively, in the presence of 100 mg of Oc cells per 5 ml medium. The plating efficiencies of Nipponbare and Taipei 309 protoplasts were 0 and 0.7% without nurse culture, 4.3 and 3.5% after 4 d of nurse culture, 13.8 and 17.3% after 8 d, 12.8 and 13.4% after 12 d, and 6.5 and 14.4% after 16 d of nurse culture, respectively. Two successive 4- or 8-d cultures had no greater protoplast plating efficiency, 4.2 and 9.3% for Nipponbare, and 6.8 and 15.5% for Taipei 309, respectively, than single 4- or 8-d cultures. The plating efficiency of Nipponbare p r o t o p l a s t s was an a v e r a g e of 2 i n d e p e n d e n t experiments while that of Taipei 309 was from a single experiment.
Plant regeneration. After transfer to hormone-free N6 medium, protoplastderived calli initially grew slowly with browning in older callus tissue. Subcultures at shorter intervals, such as 2 to 3 weeks, ensured consistent growth and subsequent differentiation of cultured callus. The formation of roots and shoots occurred within 20 d after transfer. Complete plants were regenerated after 50 d of culture (Fig. 1, e). General medium was not effective in promoting plant regeneration. A total of 141 regenerated plants (plant regeneration frequency = 67%) on N6 medium were obtained from 30 calli derived from protoplasts cultured in General medium. The a v e r a g e n u m b e r of plants per regenerating callus was 7.0, with some calli producing more than 15 plants. One advantage of the improved procedure is that it shortens to five months the time required from the
220
initiation of callus induction to c o m p l e t e plant regeneration. Only one month was required to develop suspension cultures from which protoplasts were isolated. In addition, one month was required for callus induction, one month for protoplast propagation and two months for plant regeneration. Thus, gene expression in transgenic rice plants may be analyzed three months after protoplast transformation, when suspension cells are available.
Agronomic traits. Protoplast-derived plants grown in the greenhouse exhibited no apparent phenotypic variation when compared to seed-derived plants of Nipponbare. The mean plant height and panicle length of 17 protoplastderived plants were 74.5 ___ 1.9 and 15.8 __. 0.4 cm, respectively ( m e a n __. SE). The average number of tillers per plant and spikelets per panicle were 8.7 _ 0.5 and 64.3 _ 3.3, respectively. Seed fertility of these plants ranged from 3.4 to 93.7%, with an average of 65.4 %. A low frequency of somaclonal variation among protoplast-derived rice plants in the field has been reported previously (Ogura et aL 1987). Acknowledgmnent~ We wish to thank Andreana Lisca and Christian Knaak for excellent technical assistance; Dr. Ko Shimamoto for advice in protoplast culture; Drs. David J. Longstreth, Mary E. Musgrave, Milton C. Rush and Mark D. Burow for critical reading of the manuscript; and all members of the Plant Molecular Biology Laboratory for discussion and encouragement. This work was supported in part by grants from the Louisiana Education Quality Support Fund (1987-90)-RD-A-6 and from the Louisiana State University System BiotechnologyProgram to N. M.
References Abdullah R, Cocking EC, Thompson JA (1986) Bio/Technol 4: 10871090 Chen Y (1986) In: Hu H, Yang HY (eds) Haploids of Higher Plants in vitro, China Academic Publishers and Springer-Verlag, Germany, pp 1-25 CoulibalyMY, Demarly Y (1986) Z Pflanzenzuchtg96: 79-81. Chu CC, Wang CC, Sun CS, Hsu C, Yin KC, Chu CY, Bio FY (1975) Sci Sin 18:659-668 Fukunaga Y, KingJ (1978) Plant Sci Lett 11:241-250 Fujimura T, Sakurai M, Akagi H, Negishi H, Hirose A (1985) Plant Tissue Culture Lett 2:74-75 Harms CT, Potrykus I (1978) Theor Appl Genet 53:57-63 Hayashi Y, KyozukaJ, Shimamoto K (1988) Mol Gen Genet 214: 610 Hayashimoto A, Li Z, Murai N (1990) Plant Physio193(in press) Kyozuka J, Hayashi Y, Shimamoto K (1987) Mol Gen Genet 206: 408-413 Muller AJ, Grafe R (1978) Mol Gen Genet 161:67-76 Murai, N, Sutton DW, Murray MJ, Slightom JL, Merlo DL, Reichert NA, Sengupta-Gopalan C, Stock CA, Baker RF, Kemp JD, Hall TC (1983) Science 222:476-482 Murashige T, Skoog F (1962) Physioi Plant 15:473-497 Ogura K, KyozukaJ, Hayashi Y, Koba T, Shimamoto K (1987) Thcor Appl Genet 74:670-676 Ohira K, Ojima K, Fujiwara A (1973) Plant Cell Physiol 14: 11131121 Shillito RD, Paszkowski J, Potrykus I (1983) Plant Cell Rep 2: 244247 Shimamoto K, Terada R, Izawa T, Fujimoto H (1989) Nature 338: 274-276 Toriyama K, Arimoto Y, Uchimiya H, Hinata K (1988) Bio/Technol 6:1072-1074 Toriyama K, Hinata K, Sasaki T (1986) Theor Appl Genet 73:16-19 Yamada Y, Yang ZQ, Tang DT (1986) Plant Cell Rep 5:85-88
