Plant Cell Reports
Plant Cell Reports (1986) 5 : 85-88
© Springer-Verlag 1986
Plant regeneration from protoplast-derived callus of rice ( Oryza sativa L.) Yasuyuki Yamada, Yang Zhi-Qi, and Tang Ding-Tai Research Center for Cell and Tissue Culture, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606, Japan Received August 19, 1985 / Revised version received January 24, 1986 - Communicated by J. M. Widholm
ABSTRACT Protoplasts i s o l a t e d from cultured rice c e l l s of an A-58 cytoplasmic male s t e r i l e l i n e (A-58 MS)(Oryza s a t i v a L.) were used to i n v e s t i g a t e the regeneration of r i c e plants. A cultured c e l l l i n e (T3) of A-58 MS w i t h a high growth r a t e and dense cytoplasm was selected. About 10% of the protoplasts prepared from t h i s e s t a b l i s h e d c e l l l i n e p l a t e d in RY-2 (a new medium) formed c o l o n i e s . The c a l l i formed shoots and r o o t s in the r e g e n e r a t i o n medium and developed into whole plants. P r o t o p l a s t s a l s o were prepared from suspension cultures of 25 other v a r i e t i e s of r i c e using the same methods. The protoplasts i s o l a t e d from two of the 25 v a r i e t i e s , Fujiminori and Toyotama, had high rates of c e l l d i v i s i o n in RY-2 medium. Only p r o t o p l a s t derived c a l l i from Fujiminori, produced whole plants in the regeneration medium. ABBREVIATIONS LS : Linsmaier and Skoog (1965) 2,4-D : 2,4-dichlorophenoxyacetic acid BA : 6-benzyladenine MES : 2 - ( N - M o r p h o l i n o ) e t h a n e s u l f o n i c acid, monohydrate INTRODUCTION Pioneering work in methods to prepare protoplasts from monocotyledonous p l a n t s has developed and e s t a b l i s h e d techniques which produce c a l l u s from various cereals including maize (Potrykus et al 1979, Chourey & Zurawski 1981), pearl m i l l e t (Vasil & Vasil 1979), sorghum (Brar et al 1980), and r i c e (Deka and Sen 1976, Cai et al 1978, Wakasa 1984). Although many i n v e s t i g a t o r s have attempted to r e g e n e r a t e p l a n t l e t s from c e r e a l p r o t o p l a s t s , in o n l y a few cases, e.g. sugar cane ( S r i n i v a s a n & V a s i l 1985) and p e a r l m i l l e t ( V a s i l & V a s i l 1980), has success been reported. Our i n t e r e s t in the c u l t u r e of r i c e protoplasts and the regeneration of plants from these p r o t o p l a s t s is based on the o b j e c t i v e of forming hybrids by c e l l fusion with a cytoplasmic male s t e r i l e c e l l line. Formation of c a l l u s from protoplasts of rice has been reported by Deka and Sen (1976), Cai et al (1978) and Wakasa (1984). Although t h e i r reports have added to the i n f o r m a t i o n a v a i l a b l e on f o r m a t i o n from r i c e p r o t o p l a s t s , they did not succeed in r e g e n e r a t i n g p l a n t s from the c a l l i t h e y d e r i v e d from r i c e protoplasts. According to several reports on the regeneration of p l a n t s from r i c e c a l l i (Nishi et al 1968, Guha-
Offprint requests to: Y. Yamada
Mukherjee 1973, Wakasa 1981, Tamura 1981), the genotype or v a r i e t y of the o r i g i n a l plants is c r i t i c a l for the success of p l a n t regeneration. Therefore, we considered i t i m p o r t a n t in our experiments on p l a n t r e g e n e r a t i o n from r i c e p r o t o p l a s t s to screen f o r promising varieties from t h e l a r g e number in existence. The regeneration of plants from c a l l i of a number of v a r i e t i e s of rice has been reported (Wernicke et al 1981, Genovesi and M a g i l l 1982, Lai and Liu 1982, Heyser et al 1983, Fukui1983, Yamada and Lob 1984). The v a r i e t i e s of r i c e used in those r e g e n e r a t i o n e x p e r i m e n t s a l s o may be s u i t a b l e for plant regeneration from rice protoplasts. We here report the s e l e c t i o n of s u i t a b l e r i c e v a r i e t i e s f o r p l a n t r e g e n e r a t i o n , and the r e s u l t s of our experiments on the regeneration of plants from rice protoplasts, MATERIALS AND METHODS Callus formation : Seeds of the Oryza sativa A58 cytoplasmic male s t e r i l e l i n e (A-58 MS) were a g i f t from professor T, Kinoshita, Faculty of Agriculture, Hokkaido U n i v e r s i t y . The husked seeds were s t e r i l i z e d using 70% ( v / v ) ethanol for 30 sec then I% ( v / v ) sodium h y p o c h l o r i t e f o r 40 min, f o l l o w e d by 3 washings with s t e r i l i z e d d i s t i l l e d water. LS basal medium supplemented with 20 ~M 2,4-D, 3% (w/v) sucrose and I% (w/v) agar adjusted to pH 5.6 was used for c a l l u s formation. A-58 MS seeds were placed on the medium and incubated in the dark at 25°C, A f t e r 40 days, l g F.W. o f each primary c a l l u s was t r a n s f e r r e d to 300-ml f l a s k s c o n t a i n i n g 70 ml of LS l i q u i d medium and 20 ~M 2,4-D, and the c a l l u s cultured on a g y r a t o r y shaker at lO0 rpm in the dark, w i t h subculturing every 3 days, C a l l u s from the o t h e r 25 v a r i e t i e s of r i c e were produced by the same methods. Selection of the c e l l l i n e for p r o t o p l a s t c u l t u r e from A-58 MS: The T3 c e l l l i n e used f o r p r o t o p l a s t i s o l a t i o n and c u l t u r e was selected by the f o l l o w i n g methods from suspension cultures of 30 c e l l lines of A-58 MS because o f i t s f a s t g r o w t h and dense cytoplasm: l ) Every three days the t o t a l fresh weight of the c e l l s was determined under aseptic conditions. 2) At the time of s e l e c t i o n , the c e l l s chosen were viewed by microscopy in o r d e r to s e l e c t those c e l l lines with large numbers of clusters composed of lO-15 c e l l s . In a d d i t i o n , those c l u s t e r s in which c e l l s were round and rich in cytoplasm were selected, 3) To maintain the selected clusters, the medium c o n t a i n i n g f l o a t i n g c e l l s was decanted and the clusters transferred to fresh medium.
86 Protop~ast isolation: One gram (F.W.) o f a 3d a y - o l d suspension of c u l t u r e d c e l l s was added t o 20 ml of f i l t e r - s t e r i l i z e d enzyme s o l u t i o n t h a t consisted o f 2% C e l l u l a s e Onozuka RS ( Y a k u l t P h a r m a c e u t i c a l Industry Co..Ltd.). 2% M a c e r o z y m e R-IO ( Y a k u l t Pharmaceutical I n d u s t r y Co.. L t d . ) , 0.1% P e c t o l y a s e Y-23 (Seishin Pharmaceutical Co., Ltd.). I% D r i s e l a s e ( K y o w a H a k k o K o g y o Co., Ltd.), 0.8 % calf s e r u m ( N a k a r a i Chem. Co., L t d . ) , 80 mM CaCI2.2H20, 0.125 mM MgCl 2 and 0.5 mM MES, in hormone-Tree LS medium c o n t a i n i n g 0.3 M g l u c o s e . The pH o f t h e s o l u t i o n was a d j u s t e d t o 5.6 b e f o r e i t was f i l t e r sterilized. A f t e r ca. 9h o f i n c u b a t i o n in t h e d a r k w i t h o u t shaking, protoplasts were separated from the undigested c e l l s by f i l t r a t i o n through a 40 ~m nylon filter then c e n t r i f u g a t e d a t 250 x g f o r 4 min. The p r o t o p l a s t s were c o l l e c t e d and washed 3 t i m e s w i t h washing medium (0.8 % c a l f serum ( N a k a r a i Chem. Co., L t d . ) , 80 mM CaCI2.2H20. 0.125 mM MgCI 2 and 0.5 mM MES. in h o r m o n e - T r e e LS medium c o n t a l n i n g 0.3 M g l u c o s e , pH 5.6). then s i e v e d a g a i n t h r o u g h a 40 ~M nylon filter. All protoplasts passing through the filter were c o l l e c t e d . Y i e l d s o f p r o t o p l a s t s were c a l c u l a t e d w i t h a hemocytometer before p l a t i n g . P r o t o p l a s t c u l t u r e : The A-58 MS and 25 v a r i e t i e s of r i c e p r o t o p l a s t s were c u l t u r e d in m o d i f i e d medium in which the i n o r g a n i c elements were the same as in LS medium and the v i t a m i n s and organic elements the same as i n K a o ' s meduim. M o d i f i c a t i o n s w e r e made by checking the effects on p r o t o p l a s t division c o n c e n t r a t i o n s of ammonium and f e r r i c ions and o f the a d d i t i o n of c a l f serum. The i s o l a t e d p r o t o p l a s t s a t a fina~n l a t i n g d e n s i t y o f 2.5 x 105/ml were c u l t u r e d in ~ u ~ 15 mm p l a s t i c p e t r i - d i s h e s c o n t a i n i n g 2 ml of newly prepared l i q u i d medium. These dishes were sealed w i t h P a r a f i l m M and kept at 25°C in the dark w i t h o u t shaking. Plant regeneration: A f t e r 40-50 days of c u l t u r e in l i q u i d RY-2 medium ( T a b l e I ) , 0.5 g (F.W.) o f t h e p r o t o p l a s t - d e r i v e d c a l l u s formed was t r a n s f e r r e d t o lO0-ml f l a s k s c o n t a i n i n g 15 ml o f l i q u i d N6 medium (Chu e t a l 1975) ( s u p p l e m e n t e d w i t h 8% s u c r o s e , b u t w i t h o u t hormones), and s u b c u l t u r e d on a g y r a t o r y Table 1. Composition o f RY-2 medium f o r r i c e protoplasts. a) ~vlineral Salt (mgll) (NH 4)2sO4 67 KI 0.83 KNO3 1900 H3BO3 6.20 KH2PO4 170 ZnSO4. LIH20 8.60 CaCI2.2H20 440 Na2MoOLI.2H20 0.25 MgSOLI.7H20 370 CuSO4. 5H20 0.025 MnSO4, 4H20 22.3 CoCl2. 6H20 0.025 EDTA-Na-Fe Sali" 19.25 b) Organic acid (mg/l)(adjusted to pH 5.5 with NH4OH) Sodium pyruvate 5 Malic acid 10 Citric acid 10 Fumaric acid 10 c) Vitamins (mgll) Nicotinamide I Biotin 0.005 Pyridoxine HCI I Choline chloride 0.5 Thiamine HCl 10 Riboflavin 0.I D Calcium pantothenate 0.5 Ascorbic acid I Folio acid 0.2 Vitamin A 0.005 ~-Aminobenzoic acid 0.01 Vitamin D3 0.005 Vitamin B12 0.005 d) Inositol (mgll)~ 100 e) Hormone (x10-~M) 2,4 D 2 f) MES (mM} I g) Calf serum (Nakarai Chem. Co.) (ml) 8 h) Glucose (M) 0.5 i) pH 5.6 (NaOH)Fllter Sterilized. a): Linsmaier-Skoog medium (1965) w i t h m o d i f i c a t i o n s . b . c . d ) : According t o Kao (1977).
sha~er a t I00 rpm in t h e d a r k f o r 17 days. The c e l l aggregates that formed were transplanted to a r e g e n e r a t i o n agar medium (LS medium supplemented w i t h 8% ( w / v ) s u c r o s e . 0.8% ( w / v ) a g a r and 4 x IO-~M BA, pH 5.6). These c u l t u r e s f o r p l a n t r e g e n e r a t i o n were maintained a t 25% under continuous f l u o r e s c e n t l i g h t o f 3500 l u x . The e x t e n t o f p l a n t r e g e n e r a t i o n was assessed v i s u a l l y . A1 1 t h e p l a n t s t h a t regenerated were potted in v e r m i c u l i t e and placed in a t r a n s p a r e n t p l a s t i c c a b i n e t t o a c c l i m a t i z e them, a f t e r which they were t r a n s f e r r e d to a greenhouse. RESULTS Selection of the cell line for the source of ~to~a~ts: A cell line, T 3, s e l e c t e d from s u s p e n s i o n - c u l t u r e d A-58 MS c e l l s - a f t e r I0 months had a h i g h g r o w t h r a t e and opaque, s l i g h t l y yellow cytoplasms w i t h s m a l l vacuoles. These T3 c e l l s were a l m o s t round. In c o n t r a s t , t h e u n s e l e c t e d A-58 MS c e l l s had v a r i o u s shapes and c o l o r l e s s , t r a n s p a r e n t cytoplasms w i t h l a r g e vacuoles. P r o t o p l a s t s were i s o l a t e d from suspension c e l l s o f both t h e T 3 and o r i g i n a l A-58 MS c e l l l i n e s . The p r o t o p l a s t y i e l d from T 3 was 6 t i m e s t h a t from t h e o r i g i n a l A-58 MS ( T a b l e ~ ) . After purification, by two f i l t r a t i o n s through a 40 um f i l t e r , we confirmed by microscopy t h a t o n l y p r o t o p l a s t s were present. ~mprovement o f t h e LS medium f o r p r o t o p l a s t culture; E f f e c t s of v a r i o u s f a c t o r s on T~ p r o t o p l a s t ~ o n were examined. Sugars, s u g a ~ a T c o h o l s and the concentrations o f NH4+ and Fe j + w e r e v e r y i m p o r t a n t f a c t o r s f o r the c u l t u r e of r i c e p r o t o p l a s t s . No p r o t o p l a s t s s u r v i v e d a f t e r 4 weeks in media in which t h e o s m o t i c p r e s s u r e s o f 0.2, 0.3, 0.4, 0.5, and 0.6 M had been a d j u s t e d w i t h m a n n i t o l o r s o r b i t o l . O n l y a t t h e o s m o t i c p r e s s u r e s 0.4 and 0.5 M p r o d u c e d by glucose d i d p r o t o p l a s t s d i v i d e w e l l . The r a t e of c o l o n y f o r m a t i o n was 9.6% a t 0.5 M o s m o t i c p r e s s u r e b u t d e c r e a s e d t o 4% a t 0.4 M ( w i t h c a l f serum, see below). At the 0.3 M o s m o t i c p r e s s u r e produced by sucrose, c o l o n i e s formed w i t h o n l y 0.4% e f f i c i e n c y . Ammonium ion was an i m p o r t a n t f a c t o r in o b t a i n i n g a high r a t e of p r o t o p l a s t d i v i s i o n . The o r i g i n a l LS medium (1965) c o n t a i n s 1650 m g / l o f NH4+ ion. but a t t h i s c o n c e n t r a t i o n , no c o l o n i e s formed. We t h e r e f o r e d e t e r m i n e d t h e optimum c o n c e n t r a t i o n o f NH~+ f o r p r o t o p l a s t d i v i s i o n by t r i a l and e r r o r and found t h a t a c o n c e n t r a t i o n o f 67 m g / l o f NH4+ gave t h e b e s t colony formation. F e r r i c i o n a l s o was an i m p o r t a n t factor for rice protoplast culture. But a t t h e c o n c e n t r a t i o n o f 38 m g / l used in s t a n d a r d LS medium (1965). no c o l o n y f o r m a t i o n t o o k p l a c e . At t h e Fe j + c o n c e n t r a t i o n o f 19 m g / l , marked c o l o n y f o r m a t i o n o c c u r r e d . A n o t h e r i m p o r t a n t i m p r o v e m e n t was made by t h e a d d i t i o n o f c a l f serum t o t h e c u l t u r e medium. W i t h o u t c a l f serum, c o l o n y f o r m a t i o n a t o s m o t i c p r e s s u r e s o f 0.4 and 0.5 M g l u c o s e were 2% and 5%. An a d d i t i o n o f 8% c a l f serum t o 0.4 and 0.5 M g l u c o s e increased colony f o r m a t i o n t o 4% and 9.6%. Eventually. a suitable medium (RY-2) f o r T p r o t o p l a s t c u l t u r e was o b t a i n e d , t h e c o m p o s i t i o n o~ which is l i s t e d in Table 1 A p p r o x i m a t e l y 10% of the p l a t e d p r o t o p l a s t s p r o d u c e d c e l l c o l o n i e s in t h i s medium a f t e r 4 weeks of c u l t u r e . Isolation and c u l t u r e o f p r o t o p ] a s t s from 25 Table 2. P r o p e r t i e s of the T3 c e l l from A-58 MS c e i l s . T3 Growth rate (% increase in fresh I 03 weight in 3 d) Yield of protoplasts {I061g fresh weight) 8. I Cytoplasm dense Vacuole small
line selected Original A-58 MS 37
I. 3 clear large
87 v a r i e t i e s of r i c e : P r o t o p l a s t s were prepared from suspension c u l t u r e s of 25 v a r i e t i e s of r i c e by the methods used to get A-58 MS protoplasts (Fig I-A), but no c e l l s e l e c t i o n was done. Suspension-cultured c e l l s of Fujiminori and Toyotama, however, grew very r a p i d l y and had dense cytoplasms l i k e the A-58 MS (T3) c e l l l i n e , and even though t h e r e was no s p e c i a l s e l e c t i o n of these c e l l s , s i g n i f i c a n t l y d i f f e r e n t y i e l d s were obtained (Fig 2). RY-2 medium, which was s u i t a b l e for the growth of protoplasts i s o l a t e d from A-58 MS (T3) c e l l s , was used t o c u l t u r e t h e s e protoplasts. 0 n l y two v a r i e t i e s , F u j i m i n o r i and Toyotama, showed a high frequency of p r o t o p l a s t d i v i s i o n and colony formation ( r e s p e c t i v e l y 8.4% and 5,6% of the plated p r o t o p l a s t s formed c o l o n i e s , ) ( F i g I-B,C,D), Norin-l, Norin-22 and Akage had low rates of c e l l d i v i s i o n , and no colonies formed. None of the other v a r i e t i e s showed p r o t o p l a s t d i v i s i o n or colony formation (Table 3). Regeneration of plants from A-58 MS (T3) an__ddFujiminori c e l l s : After colonies That had formed from protoplasts were cultured in N6 l i q u i d medium for 17 days, the c e l l s ( F u j i m i n o r i , Toyotama and A-58 MS (T3)) were placed in r e g e n e r a t i o n medium. A f t e r 30 days of subculture, green spots appeared on some c e l l s in the Fujminori and A-58 MS (T3) c a l l i (Shimada & Yamada 1979), No spots were present on c e l l s in Toyotama c a l l u s . Some green spots grew, e v e n t u a l l y forming shoots. A f t e r 40 days, a p p r o x i m a t e l y l-cm long c o l e o p t i l e s and f i r s t leaves emerged from these shoots (Fig 3-A), A f t e r 50 days, second and t h i r d leaves had developed and the rooted p l a n t l e t s grew to about 5 cm (Fig 3-B). A f t e r 60 days, the l e a v e s of the regenerated p l a n t s had reached the tops of the flasks and roots had elongated and expanded throughout the agar. These plants were potted and transferred to a greenhouse where they continued to grow (Fig. 3-C). One r i c e p l a n t was regenerated from the p r o t o p l a s t d e r i v e d A-58 MS (T3) c e l l s and e i g h t from the Fujiminori c e l l s ,
and c o l o n y Fig I. P r o t o p l a s t s , cell division f o r m a t i o n by F u j i m i n o r i , (A) I s o l a t e d p r o t o p l a s t s from s u s p e n s i o n - c u l t u r e d c e l l s of F u j i m i n o r i . (B) C e l l d i v i s i o n a f t e r 12 days of c u l t u r e , (C) C e l l c l u s t e r s formed a f t e r 25 days o f c u l t u r e . (D) Colonies derived from Fujiminori protoplasts a f t e r 30 days. Bar: I0 ~m.
~
IYOKAZE IR36 YAMABOHSHI SASASHIGURE NORIN-1
K~MENO0 SHIBATA
KOSHIHIKARI I SHIOKARI NORIN-22 IASAHI REISUI
DISCUSSION Rice p l a n t s were s u c c e s s f u l l y regenerated from p r o t o p l a s t s of A-58 MS (T3) c e l l s and F u j i m i n o r i cells, This is the f i r s t r e p o r t e d case of p l a n t regeneration from protoplasts of rice (0. s a t i v a L.). Our success is due to the f a c t t h a t (1) a c e l l l i n e w i t h a good growth r a t e and dense cytoplasm was s e l e c t e d , (2) t h a t RY-2, a medium s u i t a b l e f o r p r o t o p l a s t c u l t u r e , was d e v e l o p e d and (3) t h a t a v a r i e t y of rice genotypes were used. The c e l l l i n e (T3) s e l e c t e d from A-58 MS c e l l s showed a very good growth r a t e and i t s c e l l s had opaque, y e l l o w cytoplasms with small vacuoles and a high frequency of p r o t o p l a s t d i v i s i o n . The Fujiminori c e l l s , which we did not put through the s e l e c t i o n procedure, had c h a r a c t e r i s t i c s s i m i l a r to the selected T~ c e l l s , Under microscopy these c h a r a c t e r i s t i c s are useful markers for the s e l e c t i o n of c e l l s capable of regenerating plantlets, By s e l e c t i n g a c e l l l i n e that has a dense cytoplasm, one may obtain a l i n e that can d i f f e r e n t i a t e plants. Of the o t h e r 25 v a r i e t i e s , about I0 ( i n c l u d i n g Fujiminori and Toyotama) showed h i g h , or and r e l a t i v e l y high, c e l l growth rates. But as stated, only protoplasts of Toyotama and F u j i m i n o r i d i v i d e d w e l l ; therefore, the s e l e c t i o n of a c e l l l i n e that has a good growth r a t e is necessary, but not s u f f i c i e n t , condition for the regeneration of rice p l a n t l e t s . Protoplast d i v i s i o n was s t r o n g l y affected by the c u l t u r e medium used. By improving standard LS medium we developed RY-2 medium which is e f f e c t i v e f o r p r o t o p l a s t d i v i s i o n . As shown in Table 2, c a l f serum was added and glucose used as the osmoticum, the amounts of ammonium and f e r r i c ions decreased. These changes together with the other modifications l i s t e d
IPOKKALI IYAMABI
KO
NIHONBARE FUJIMINORI
t
A-58(fertile)
IIOMACHI KUSABUE
ITOYOTAMA AIKOKU IZAIHO I NAKATESHINSENBON I DOKAI I AKAGE
0
I
I
t
I
I
1
2
3
4
5
Number of protoplasts (xl06/g fresh weight) Fig, 2, Yields of protoplasts i s o l a t e d from cultured c e l l s of 25 rice v a r i e t i e s . Table 3. Rice v a r i e t i e s forming colonies or showing c e l l d i v i s i o n of protoplasts. Colony formation
A - 5 8 MS ( T 3 )
(9.6%) I )
Fujiminori
(8.4%) I) (5.6%) I)
Toyotama Cell d i v i s i o n a)
Norin- 1
Norin-22 Akage
(0.01%) 2) (0.01%) 2) (0.01%) 2)
I) (No, of coloniesformed)/(No, of inoculated protoplasts) x lO0 2) (No. of divided p r o t o p l a s t s ) / (No. of inoculated protoplasts) x lO0 3) No c e l l d i v i s i o n took place in the unlisted v a r i e t i e s ,
88
Fig, 3, Rice p l a n t regeneration from Fujiminori p r o t o p l a s t - d e r i v e d c e l l s , ( B ) Developing rice plants plants growing in a greenhouse.
protoplasts, (A) Shoot and root regeneration from the regenerated from the p r o t o p l a s t s , (C) Regenerated
were needed to obtain a high frequency of protoplast d i v i s i o n in A-58 MS (T3), F u j i m i n o r i and Toyotama cel Is. We were a b l e to regenerate nine p l a n t s from the p r o t o p l a s t - d e r i v e d cel Is of two of these three v a r i e t i e s , (8 plants from Fujiminori and one from A-58 MS (T3)), A critical p o i n t is whether there were cel Is mixed with our protoplasts, I t is almost impossible to c o n f i r m whether al 1 the c o l o n i e s o r i g i n a t e d from s i n g l e protoplasts, But, we isolated both the T3 and Fujiminori protoplasts from suspension-cultured c e i l s then f i l t e r e d them twice through a 40 ~m nylon f i l t e r to p u r i f y them as much as p o s s i b l e , The F u j i m i n o r i protoplast preparation p u r i f i e d in Lbhis way contained about 8 s i n g l e cel Is~ per I x I0 ~ p r o t o p l a s t s , We p l a t e d samples 5 x I0 J p r o t o p l a s t s in p e t r i dishes, and the average colony formation was about 10%, Even assuming t h a t al 1 s i n g l e cel Is present in the dish formed c o l o n i e s , at most t h e r e would be o n l y 4~ single-cell d e r i v e d c o l o n i e s among the 5 x I0 c o l o n i e s in the dish; the r a t i o of the former to the l a t t e r being less than 0,1%, In our r e g e n e r a t i o n e x p e r i m e n t ~ w i t h Fujiminori p r o t o p l a s t , 396 c l o n e s from 5 x I0 ~ c o l o n i e s were used, Four c l o n e s r e g e n e r a t e d r i c e p l a n t s ; a r e g e n e r a t i o n r a t i o of about I%. Therefore, the possibility t h a t a l l the regenerated p l a n t s were derived from c o n t a m i n a t i n g single cel Is is statistically negligible, The problem addressed in t h i s study is how to increase the p l a n t r e g e n e r a t i o n r a t i o from r i c e protoplasts, A l t h o u g h d i f f e r e n t i a t i o n of shoots and r e g e n e r a t i o n of p l a n t l e t s from c a l l u s has been achieved with several v a r i e t i e s of rice, the c a l l i of many other v a r i e t i e s could not i n i t i a t e shoots (Kawata 1968, Daroyan & Smetamin 1979, Bhattachary & Sen 1980) or the a b i l i t y to i n i t i a t e shoots was l o s t a f t e r protracted subculturing (Nakano et al 1975, Daroyan & Smetamin 1979, Inoue & Maeda 1980), The c a l l i (Fujiminori and A-58 MS) we used t o i s o l a t e protoplasts had been cultured f o r more than two years. Therefore, the r e g e n e r a t i o n t o t i p o t e n c y should have been lower than that of the o r i g i n a l c a l l u s , I t may be p o s s i b l e t o i n c r e a s e the p l a n t r e g e n e r a t i o n r a t i o from p r o t o p l a s t s by using newly induced cal l i , Improvement of the regeneration r a t i o has been reported by Heyser and his associates (1983) who found t h a t about 20% of t h e i r r o o t - d e r i v e d embryogenic c a l l u s produced shoots a f t e r 50 weeks of culture, whereas o n l y 6% o f r o o t - d e r i v e d nonembryogenic c a l l u s did so. The regeneration of pearl m i l l e t plants also has been reported from embryogenic c a l l u s (Vasil & Vasil 1980), We a l s o found a few embryogenic c a l l i d u r i n g protoplast c u l t u r e in N6 medium, I f the regeneration of rice plants from protoplasts requires the formation of embryogenic cel Is, we must f i n d a way to increase the embryogenic cal l i of r i c e in order to increase r i c e p l a n t regeneration. Several p o s s i b i l i t i e s that might operate in a h i g h e r r e g e n e r a t i o n frequency of
p l a n t s from p r o t o p l a s t s of r i c e are c u r r e n t l y investigated in our laboratory.
being
ACKNOWLEDGMENTS We are d e e p l y g r a t e f u l to Professor A. Gibor of the U n i v e r s i t y of C a l i f o r n i a , Santa Barbara, Dr, H, Hori of our I n s t i t u t e and Mrs P, Yamada for t h e i r help in the preparation of t h i s manuscript, REFERENCES Bhattacharya P, Sen SK (1980) Theor. Appl, Genet, 58: 87-90. Brar DS, Rambold S, Constabel F, Gamborg OL (1980) Z. Pflanzenphysiol. 96: 269-275, Cai QG, Qian YQ, Zhou YL, Wu SX (1978) Acta, Bot. S i n i c a . 20 (2): 97-I02. Chourey PS, Zurawski DB (1981) Theor, appl. Genet, 59: 341-344, Deka PC, Sen SK (1976) Molec. Gen. Genet, 145: 239244. Daroyan El, Smetanin AP (1979) Sobiet P l a n t P h y s i o l , 26: 261-266. Fukui K (1983) Theo. Appl. Genet. 65: 225-230. Genovesi AD, M a g i l l CW (1982) Plant c e l l Reports. l: 257-260. Guha-Mukherjee S (1973) J. Exp. Bot. 24: 139-144. Heyser JW, Dykes TA, Demott KJ, Nobors NW (1983) P l a n t Sci, Lett. 29: 175-181, Inoue M, Maeda E (1980) Japan, J Crop Sci, 49: 167174. Kao KN (1977) Molee. Gen, Genet. 150: 225-230. Kawata S, Ishihara A (1968) Proc. Japan Acad. 44: 549553. Lai KL, Liu LF (1982) Crop Sci. 51:70-74, Limsmaier EF, Skoog F (1965) P h y s i o l . P l a n t , 18: I 0 0 127. Nakano H, Tashiro T, Maeda E ( 1 9 6 5 ) Z. Pflanzenphysiol. 76: 444-449, Nishi T, Yamada Y, Takahashi E (1968) Nature 219: 508-509. Potrykus I, Harms CT, Lorz H (1979) Theor, Appl, Ginet. 54: 209-214, Sarinivasan, Vasil IK (1985) Amer, J, Bot, in press. Shimada T, Yamada Y (1979) Japan, J, Genetics. 54: 379-385. Tamura S (1981) Mem.Fac, A g r i . N i i g a t a Univ. 18:1-52, Vasil V, Vasil IK (1979) Z, Pflanzenphysiol, 92: 379383, Vasil V, Vasil IK (1980) Theor, Appl, Genet, 56: 97-99, Wakasa k (1981) Nat. Inst, Agric, Sci,D 33:122-200. Wakasa K (1984) J. Plant, Physiol. 117: 223-231. Wernicke W, Brettel R, Wakizaka T, Potrykus I (1981) Z. Pflanzenphysiol 103: 361-365, Yamada Y, Loh WH (1984) In: Ammirato PV, Evans DA, Sharp WR, Yamada Y (eds) Handbook of P l a n t C e l l Culture, Vol 3, Macmillan, N.Y., 151-170,
Plant Cell Reports (1986) 5 : 85-88
© Springer-Verlag 1986
Plant regeneration from protoplast-derived callus of rice ( Oryza sativa L.) Yasuyuki Yamada, Yang Zhi-Qi, and Tang Ding-Tai Research Center for Cell and Tissue Culture, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606, Japan Received August 19, 1985 / Revised version received January 24, 1986 - Communicated by J. M. Widholm
ABSTRACT Protoplasts i s o l a t e d from cultured rice c e l l s of an A-58 cytoplasmic male s t e r i l e l i n e (A-58 MS)(Oryza s a t i v a L.) were used to i n v e s t i g a t e the regeneration of r i c e plants. A cultured c e l l l i n e (T3) of A-58 MS w i t h a high growth r a t e and dense cytoplasm was selected. About 10% of the protoplasts prepared from t h i s e s t a b l i s h e d c e l l l i n e p l a t e d in RY-2 (a new medium) formed c o l o n i e s . The c a l l i formed shoots and r o o t s in the r e g e n e r a t i o n medium and developed into whole plants. P r o t o p l a s t s a l s o were prepared from suspension cultures of 25 other v a r i e t i e s of r i c e using the same methods. The protoplasts i s o l a t e d from two of the 25 v a r i e t i e s , Fujiminori and Toyotama, had high rates of c e l l d i v i s i o n in RY-2 medium. Only p r o t o p l a s t derived c a l l i from Fujiminori, produced whole plants in the regeneration medium. ABBREVIATIONS LS : Linsmaier and Skoog (1965) 2,4-D : 2,4-dichlorophenoxyacetic acid BA : 6-benzyladenine MES : 2 - ( N - M o r p h o l i n o ) e t h a n e s u l f o n i c acid, monohydrate INTRODUCTION Pioneering work in methods to prepare protoplasts from monocotyledonous p l a n t s has developed and e s t a b l i s h e d techniques which produce c a l l u s from various cereals including maize (Potrykus et al 1979, Chourey & Zurawski 1981), pearl m i l l e t (Vasil & Vasil 1979), sorghum (Brar et al 1980), and r i c e (Deka and Sen 1976, Cai et al 1978, Wakasa 1984). Although many i n v e s t i g a t o r s have attempted to r e g e n e r a t e p l a n t l e t s from c e r e a l p r o t o p l a s t s , in o n l y a few cases, e.g. sugar cane ( S r i n i v a s a n & V a s i l 1985) and p e a r l m i l l e t ( V a s i l & V a s i l 1980), has success been reported. Our i n t e r e s t in the c u l t u r e of r i c e protoplasts and the regeneration of plants from these p r o t o p l a s t s is based on the o b j e c t i v e of forming hybrids by c e l l fusion with a cytoplasmic male s t e r i l e c e l l line. Formation of c a l l u s from protoplasts of rice has been reported by Deka and Sen (1976), Cai et al (1978) and Wakasa (1984). Although t h e i r reports have added to the i n f o r m a t i o n a v a i l a b l e on f o r m a t i o n from r i c e p r o t o p l a s t s , they did not succeed in r e g e n e r a t i n g p l a n t s from the c a l l i t h e y d e r i v e d from r i c e protoplasts. According to several reports on the regeneration of p l a n t s from r i c e c a l l i (Nishi et al 1968, Guha-
Offprint requests to: Y. Yamada
Mukherjee 1973, Wakasa 1981, Tamura 1981), the genotype or v a r i e t y of the o r i g i n a l plants is c r i t i c a l for the success of p l a n t regeneration. Therefore, we considered i t i m p o r t a n t in our experiments on p l a n t r e g e n e r a t i o n from r i c e p r o t o p l a s t s to screen f o r promising varieties from t h e l a r g e number in existence. The regeneration of plants from c a l l i of a number of v a r i e t i e s of rice has been reported (Wernicke et al 1981, Genovesi and M a g i l l 1982, Lai and Liu 1982, Heyser et al 1983, Fukui1983, Yamada and Lob 1984). The v a r i e t i e s of r i c e used in those r e g e n e r a t i o n e x p e r i m e n t s a l s o may be s u i t a b l e for plant regeneration from rice protoplasts. We here report the s e l e c t i o n of s u i t a b l e r i c e v a r i e t i e s f o r p l a n t r e g e n e r a t i o n , and the r e s u l t s of our experiments on the regeneration of plants from rice protoplasts, MATERIALS AND METHODS Callus formation : Seeds of the Oryza sativa A58 cytoplasmic male s t e r i l e l i n e (A-58 MS) were a g i f t from professor T, Kinoshita, Faculty of Agriculture, Hokkaido U n i v e r s i t y . The husked seeds were s t e r i l i z e d using 70% ( v / v ) ethanol for 30 sec then I% ( v / v ) sodium h y p o c h l o r i t e f o r 40 min, f o l l o w e d by 3 washings with s t e r i l i z e d d i s t i l l e d water. LS basal medium supplemented with 20 ~M 2,4-D, 3% (w/v) sucrose and I% (w/v) agar adjusted to pH 5.6 was used for c a l l u s formation. A-58 MS seeds were placed on the medium and incubated in the dark at 25°C, A f t e r 40 days, l g F.W. o f each primary c a l l u s was t r a n s f e r r e d to 300-ml f l a s k s c o n t a i n i n g 70 ml of LS l i q u i d medium and 20 ~M 2,4-D, and the c a l l u s cultured on a g y r a t o r y shaker at lO0 rpm in the dark, w i t h subculturing every 3 days, C a l l u s from the o t h e r 25 v a r i e t i e s of r i c e were produced by the same methods. Selection of the c e l l l i n e for p r o t o p l a s t c u l t u r e from A-58 MS: The T3 c e l l l i n e used f o r p r o t o p l a s t i s o l a t i o n and c u l t u r e was selected by the f o l l o w i n g methods from suspension cultures of 30 c e l l lines of A-58 MS because o f i t s f a s t g r o w t h and dense cytoplasm: l ) Every three days the t o t a l fresh weight of the c e l l s was determined under aseptic conditions. 2) At the time of s e l e c t i o n , the c e l l s chosen were viewed by microscopy in o r d e r to s e l e c t those c e l l lines with large numbers of clusters composed of lO-15 c e l l s . In a d d i t i o n , those c l u s t e r s in which c e l l s were round and rich in cytoplasm were selected, 3) To maintain the selected clusters, the medium c o n t a i n i n g f l o a t i n g c e l l s was decanted and the clusters transferred to fresh medium.
86 Protop~ast isolation: One gram (F.W.) o f a 3d a y - o l d suspension of c u l t u r e d c e l l s was added t o 20 ml of f i l t e r - s t e r i l i z e d enzyme s o l u t i o n t h a t consisted o f 2% C e l l u l a s e Onozuka RS ( Y a k u l t P h a r m a c e u t i c a l Industry Co..Ltd.). 2% M a c e r o z y m e R-IO ( Y a k u l t Pharmaceutical I n d u s t r y Co.. L t d . ) , 0.1% P e c t o l y a s e Y-23 (Seishin Pharmaceutical Co., Ltd.). I% D r i s e l a s e ( K y o w a H a k k o K o g y o Co., Ltd.), 0.8 % calf s e r u m ( N a k a r a i Chem. Co., L t d . ) , 80 mM CaCI2.2H20, 0.125 mM MgCl 2 and 0.5 mM MES, in hormone-Tree LS medium c o n t a i n i n g 0.3 M g l u c o s e . The pH o f t h e s o l u t i o n was a d j u s t e d t o 5.6 b e f o r e i t was f i l t e r sterilized. A f t e r ca. 9h o f i n c u b a t i o n in t h e d a r k w i t h o u t shaking, protoplasts were separated from the undigested c e l l s by f i l t r a t i o n through a 40 ~m nylon filter then c e n t r i f u g a t e d a t 250 x g f o r 4 min. The p r o t o p l a s t s were c o l l e c t e d and washed 3 t i m e s w i t h washing medium (0.8 % c a l f serum ( N a k a r a i Chem. Co., L t d . ) , 80 mM CaCI2.2H20. 0.125 mM MgCI 2 and 0.5 mM MES. in h o r m o n e - T r e e LS medium c o n t a l n i n g 0.3 M g l u c o s e , pH 5.6). then s i e v e d a g a i n t h r o u g h a 40 ~M nylon filter. All protoplasts passing through the filter were c o l l e c t e d . Y i e l d s o f p r o t o p l a s t s were c a l c u l a t e d w i t h a hemocytometer before p l a t i n g . P r o t o p l a s t c u l t u r e : The A-58 MS and 25 v a r i e t i e s of r i c e p r o t o p l a s t s were c u l t u r e d in m o d i f i e d medium in which the i n o r g a n i c elements were the same as in LS medium and the v i t a m i n s and organic elements the same as i n K a o ' s meduim. M o d i f i c a t i o n s w e r e made by checking the effects on p r o t o p l a s t division c o n c e n t r a t i o n s of ammonium and f e r r i c ions and o f the a d d i t i o n of c a l f serum. The i s o l a t e d p r o t o p l a s t s a t a fina~n l a t i n g d e n s i t y o f 2.5 x 105/ml were c u l t u r e d in ~ u ~ 15 mm p l a s t i c p e t r i - d i s h e s c o n t a i n i n g 2 ml of newly prepared l i q u i d medium. These dishes were sealed w i t h P a r a f i l m M and kept at 25°C in the dark w i t h o u t shaking. Plant regeneration: A f t e r 40-50 days of c u l t u r e in l i q u i d RY-2 medium ( T a b l e I ) , 0.5 g (F.W.) o f t h e p r o t o p l a s t - d e r i v e d c a l l u s formed was t r a n s f e r r e d t o lO0-ml f l a s k s c o n t a i n i n g 15 ml o f l i q u i d N6 medium (Chu e t a l 1975) ( s u p p l e m e n t e d w i t h 8% s u c r o s e , b u t w i t h o u t hormones), and s u b c u l t u r e d on a g y r a t o r y Table 1. Composition o f RY-2 medium f o r r i c e protoplasts. a) ~vlineral Salt (mgll) (NH 4)2sO4 67 KI 0.83 KNO3 1900 H3BO3 6.20 KH2PO4 170 ZnSO4. LIH20 8.60 CaCI2.2H20 440 Na2MoOLI.2H20 0.25 MgSOLI.7H20 370 CuSO4. 5H20 0.025 MnSO4, 4H20 22.3 CoCl2. 6H20 0.025 EDTA-Na-Fe Sali" 19.25 b) Organic acid (mg/l)(adjusted to pH 5.5 with NH4OH) Sodium pyruvate 5 Malic acid 10 Citric acid 10 Fumaric acid 10 c) Vitamins (mgll) Nicotinamide I Biotin 0.005 Pyridoxine HCI I Choline chloride 0.5 Thiamine HCl 10 Riboflavin 0.I D Calcium pantothenate 0.5 Ascorbic acid I Folio acid 0.2 Vitamin A 0.005 ~-Aminobenzoic acid 0.01 Vitamin D3 0.005 Vitamin B12 0.005 d) Inositol (mgll)~ 100 e) Hormone (x10-~M) 2,4 D 2 f) MES (mM} I g) Calf serum (Nakarai Chem. Co.) (ml) 8 h) Glucose (M) 0.5 i) pH 5.6 (NaOH)Fllter Sterilized. a): Linsmaier-Skoog medium (1965) w i t h m o d i f i c a t i o n s . b . c . d ) : According t o Kao (1977).
sha~er a t I00 rpm in t h e d a r k f o r 17 days. The c e l l aggregates that formed were transplanted to a r e g e n e r a t i o n agar medium (LS medium supplemented w i t h 8% ( w / v ) s u c r o s e . 0.8% ( w / v ) a g a r and 4 x IO-~M BA, pH 5.6). These c u l t u r e s f o r p l a n t r e g e n e r a t i o n were maintained a t 25% under continuous f l u o r e s c e n t l i g h t o f 3500 l u x . The e x t e n t o f p l a n t r e g e n e r a t i o n was assessed v i s u a l l y . A1 1 t h e p l a n t s t h a t regenerated were potted in v e r m i c u l i t e and placed in a t r a n s p a r e n t p l a s t i c c a b i n e t t o a c c l i m a t i z e them, a f t e r which they were t r a n s f e r r e d to a greenhouse. RESULTS Selection of the cell line for the source of ~to~a~ts: A cell line, T 3, s e l e c t e d from s u s p e n s i o n - c u l t u r e d A-58 MS c e l l s - a f t e r I0 months had a h i g h g r o w t h r a t e and opaque, s l i g h t l y yellow cytoplasms w i t h s m a l l vacuoles. These T3 c e l l s were a l m o s t round. In c o n t r a s t , t h e u n s e l e c t e d A-58 MS c e l l s had v a r i o u s shapes and c o l o r l e s s , t r a n s p a r e n t cytoplasms w i t h l a r g e vacuoles. P r o t o p l a s t s were i s o l a t e d from suspension c e l l s o f both t h e T 3 and o r i g i n a l A-58 MS c e l l l i n e s . The p r o t o p l a s t y i e l d from T 3 was 6 t i m e s t h a t from t h e o r i g i n a l A-58 MS ( T a b l e ~ ) . After purification, by two f i l t r a t i o n s through a 40 um f i l t e r , we confirmed by microscopy t h a t o n l y p r o t o p l a s t s were present. ~mprovement o f t h e LS medium f o r p r o t o p l a s t culture; E f f e c t s of v a r i o u s f a c t o r s on T~ p r o t o p l a s t ~ o n were examined. Sugars, s u g a ~ a T c o h o l s and the concentrations o f NH4+ and Fe j + w e r e v e r y i m p o r t a n t f a c t o r s f o r the c u l t u r e of r i c e p r o t o p l a s t s . No p r o t o p l a s t s s u r v i v e d a f t e r 4 weeks in media in which t h e o s m o t i c p r e s s u r e s o f 0.2, 0.3, 0.4, 0.5, and 0.6 M had been a d j u s t e d w i t h m a n n i t o l o r s o r b i t o l . O n l y a t t h e o s m o t i c p r e s s u r e s 0.4 and 0.5 M p r o d u c e d by glucose d i d p r o t o p l a s t s d i v i d e w e l l . The r a t e of c o l o n y f o r m a t i o n was 9.6% a t 0.5 M o s m o t i c p r e s s u r e b u t d e c r e a s e d t o 4% a t 0.4 M ( w i t h c a l f serum, see below). At the 0.3 M o s m o t i c p r e s s u r e produced by sucrose, c o l o n i e s formed w i t h o n l y 0.4% e f f i c i e n c y . Ammonium ion was an i m p o r t a n t f a c t o r in o b t a i n i n g a high r a t e of p r o t o p l a s t d i v i s i o n . The o r i g i n a l LS medium (1965) c o n t a i n s 1650 m g / l o f NH4+ ion. but a t t h i s c o n c e n t r a t i o n , no c o l o n i e s formed. We t h e r e f o r e d e t e r m i n e d t h e optimum c o n c e n t r a t i o n o f NH~+ f o r p r o t o p l a s t d i v i s i o n by t r i a l and e r r o r and found t h a t a c o n c e n t r a t i o n o f 67 m g / l o f NH4+ gave t h e b e s t colony formation. F e r r i c i o n a l s o was an i m p o r t a n t factor for rice protoplast culture. But a t t h e c o n c e n t r a t i o n o f 38 m g / l used in s t a n d a r d LS medium (1965). no c o l o n y f o r m a t i o n t o o k p l a c e . At t h e Fe j + c o n c e n t r a t i o n o f 19 m g / l , marked c o l o n y f o r m a t i o n o c c u r r e d . A n o t h e r i m p o r t a n t i m p r o v e m e n t was made by t h e a d d i t i o n o f c a l f serum t o t h e c u l t u r e medium. W i t h o u t c a l f serum, c o l o n y f o r m a t i o n a t o s m o t i c p r e s s u r e s o f 0.4 and 0.5 M g l u c o s e were 2% and 5%. An a d d i t i o n o f 8% c a l f serum t o 0.4 and 0.5 M g l u c o s e increased colony f o r m a t i o n t o 4% and 9.6%. Eventually. a suitable medium (RY-2) f o r T p r o t o p l a s t c u l t u r e was o b t a i n e d , t h e c o m p o s i t i o n o~ which is l i s t e d in Table 1 A p p r o x i m a t e l y 10% of the p l a t e d p r o t o p l a s t s p r o d u c e d c e l l c o l o n i e s in t h i s medium a f t e r 4 weeks of c u l t u r e . Isolation and c u l t u r e o f p r o t o p ] a s t s from 25 Table 2. P r o p e r t i e s of the T3 c e l l from A-58 MS c e i l s . T3 Growth rate (% increase in fresh I 03 weight in 3 d) Yield of protoplasts {I061g fresh weight) 8. I Cytoplasm dense Vacuole small
line selected Original A-58 MS 37
I. 3 clear large
87 v a r i e t i e s of r i c e : P r o t o p l a s t s were prepared from suspension c u l t u r e s of 25 v a r i e t i e s of r i c e by the methods used to get A-58 MS protoplasts (Fig I-A), but no c e l l s e l e c t i o n was done. Suspension-cultured c e l l s of Fujiminori and Toyotama, however, grew very r a p i d l y and had dense cytoplasms l i k e the A-58 MS (T3) c e l l l i n e , and even though t h e r e was no s p e c i a l s e l e c t i o n of these c e l l s , s i g n i f i c a n t l y d i f f e r e n t y i e l d s were obtained (Fig 2). RY-2 medium, which was s u i t a b l e for the growth of protoplasts i s o l a t e d from A-58 MS (T3) c e l l s , was used t o c u l t u r e t h e s e protoplasts. 0 n l y two v a r i e t i e s , F u j i m i n o r i and Toyotama, showed a high frequency of p r o t o p l a s t d i v i s i o n and colony formation ( r e s p e c t i v e l y 8.4% and 5,6% of the plated p r o t o p l a s t s formed c o l o n i e s , ) ( F i g I-B,C,D), Norin-l, Norin-22 and Akage had low rates of c e l l d i v i s i o n , and no colonies formed. None of the other v a r i e t i e s showed p r o t o p l a s t d i v i s i o n or colony formation (Table 3). Regeneration of plants from A-58 MS (T3) an__ddFujiminori c e l l s : After colonies That had formed from protoplasts were cultured in N6 l i q u i d medium for 17 days, the c e l l s ( F u j i m i n o r i , Toyotama and A-58 MS (T3)) were placed in r e g e n e r a t i o n medium. A f t e r 30 days of subculture, green spots appeared on some c e l l s in the Fujminori and A-58 MS (T3) c a l l i (Shimada & Yamada 1979), No spots were present on c e l l s in Toyotama c a l l u s . Some green spots grew, e v e n t u a l l y forming shoots. A f t e r 40 days, a p p r o x i m a t e l y l-cm long c o l e o p t i l e s and f i r s t leaves emerged from these shoots (Fig 3-A), A f t e r 50 days, second and t h i r d leaves had developed and the rooted p l a n t l e t s grew to about 5 cm (Fig 3-B). A f t e r 60 days, the l e a v e s of the regenerated p l a n t s had reached the tops of the flasks and roots had elongated and expanded throughout the agar. These plants were potted and transferred to a greenhouse where they continued to grow (Fig. 3-C). One r i c e p l a n t was regenerated from the p r o t o p l a s t d e r i v e d A-58 MS (T3) c e l l s and e i g h t from the Fujiminori c e l l s ,
and c o l o n y Fig I. P r o t o p l a s t s , cell division f o r m a t i o n by F u j i m i n o r i , (A) I s o l a t e d p r o t o p l a s t s from s u s p e n s i o n - c u l t u r e d c e l l s of F u j i m i n o r i . (B) C e l l d i v i s i o n a f t e r 12 days of c u l t u r e , (C) C e l l c l u s t e r s formed a f t e r 25 days o f c u l t u r e . (D) Colonies derived from Fujiminori protoplasts a f t e r 30 days. Bar: I0 ~m.
~
IYOKAZE IR36 YAMABOHSHI SASASHIGURE NORIN-1
K~MENO0 SHIBATA
KOSHIHIKARI I SHIOKARI NORIN-22 IASAHI REISUI
DISCUSSION Rice p l a n t s were s u c c e s s f u l l y regenerated from p r o t o p l a s t s of A-58 MS (T3) c e l l s and F u j i m i n o r i cells, This is the f i r s t r e p o r t e d case of p l a n t regeneration from protoplasts of rice (0. s a t i v a L.). Our success is due to the f a c t t h a t (1) a c e l l l i n e w i t h a good growth r a t e and dense cytoplasm was s e l e c t e d , (2) t h a t RY-2, a medium s u i t a b l e f o r p r o t o p l a s t c u l t u r e , was d e v e l o p e d and (3) t h a t a v a r i e t y of rice genotypes were used. The c e l l l i n e (T3) s e l e c t e d from A-58 MS c e l l s showed a very good growth r a t e and i t s c e l l s had opaque, y e l l o w cytoplasms with small vacuoles and a high frequency of p r o t o p l a s t d i v i s i o n . The Fujiminori c e l l s , which we did not put through the s e l e c t i o n procedure, had c h a r a c t e r i s t i c s s i m i l a r to the selected T~ c e l l s , Under microscopy these c h a r a c t e r i s t i c s are useful markers for the s e l e c t i o n of c e l l s capable of regenerating plantlets, By s e l e c t i n g a c e l l l i n e that has a dense cytoplasm, one may obtain a l i n e that can d i f f e r e n t i a t e plants. Of the o t h e r 25 v a r i e t i e s , about I0 ( i n c l u d i n g Fujiminori and Toyotama) showed h i g h , or and r e l a t i v e l y high, c e l l growth rates. But as stated, only protoplasts of Toyotama and F u j i m i n o r i d i v i d e d w e l l ; therefore, the s e l e c t i o n of a c e l l l i n e that has a good growth r a t e is necessary, but not s u f f i c i e n t , condition for the regeneration of rice p l a n t l e t s . Protoplast d i v i s i o n was s t r o n g l y affected by the c u l t u r e medium used. By improving standard LS medium we developed RY-2 medium which is e f f e c t i v e f o r p r o t o p l a s t d i v i s i o n . As shown in Table 2, c a l f serum was added and glucose used as the osmoticum, the amounts of ammonium and f e r r i c ions decreased. These changes together with the other modifications l i s t e d
IPOKKALI IYAMABI
KO
NIHONBARE FUJIMINORI
t
A-58(fertile)
IIOMACHI KUSABUE
ITOYOTAMA AIKOKU IZAIHO I NAKATESHINSENBON I DOKAI I AKAGE
0
I
I
t
I
I
1
2
3
4
5
Number of protoplasts (xl06/g fresh weight) Fig, 2, Yields of protoplasts i s o l a t e d from cultured c e l l s of 25 rice v a r i e t i e s . Table 3. Rice v a r i e t i e s forming colonies or showing c e l l d i v i s i o n of protoplasts. Colony formation
A - 5 8 MS ( T 3 )
(9.6%) I )
Fujiminori
(8.4%) I) (5.6%) I)
Toyotama Cell d i v i s i o n a)
Norin- 1
Norin-22 Akage
(0.01%) 2) (0.01%) 2) (0.01%) 2)
I) (No, of coloniesformed)/(No, of inoculated protoplasts) x lO0 2) (No. of divided p r o t o p l a s t s ) / (No. of inoculated protoplasts) x lO0 3) No c e l l d i v i s i o n took place in the unlisted v a r i e t i e s ,
88
Fig, 3, Rice p l a n t regeneration from Fujiminori p r o t o p l a s t - d e r i v e d c e l l s , ( B ) Developing rice plants plants growing in a greenhouse.
protoplasts, (A) Shoot and root regeneration from the regenerated from the p r o t o p l a s t s , (C) Regenerated
were needed to obtain a high frequency of protoplast d i v i s i o n in A-58 MS (T3), F u j i m i n o r i and Toyotama cel Is. We were a b l e to regenerate nine p l a n t s from the p r o t o p l a s t - d e r i v e d cel Is of two of these three v a r i e t i e s , (8 plants from Fujiminori and one from A-58 MS (T3)), A critical p o i n t is whether there were cel Is mixed with our protoplasts, I t is almost impossible to c o n f i r m whether al 1 the c o l o n i e s o r i g i n a t e d from s i n g l e protoplasts, But, we isolated both the T3 and Fujiminori protoplasts from suspension-cultured c e i l s then f i l t e r e d them twice through a 40 ~m nylon f i l t e r to p u r i f y them as much as p o s s i b l e , The F u j i m i n o r i protoplast preparation p u r i f i e d in Lbhis way contained about 8 s i n g l e cel Is~ per I x I0 ~ p r o t o p l a s t s , We p l a t e d samples 5 x I0 J p r o t o p l a s t s in p e t r i dishes, and the average colony formation was about 10%, Even assuming t h a t al 1 s i n g l e cel Is present in the dish formed c o l o n i e s , at most t h e r e would be o n l y 4~ single-cell d e r i v e d c o l o n i e s among the 5 x I0 c o l o n i e s in the dish; the r a t i o of the former to the l a t t e r being less than 0,1%, In our r e g e n e r a t i o n e x p e r i m e n t ~ w i t h Fujiminori p r o t o p l a s t , 396 c l o n e s from 5 x I0 ~ c o l o n i e s were used, Four c l o n e s r e g e n e r a t e d r i c e p l a n t s ; a r e g e n e r a t i o n r a t i o of about I%. Therefore, the possibility t h a t a l l the regenerated p l a n t s were derived from c o n t a m i n a t i n g single cel Is is statistically negligible, The problem addressed in t h i s study is how to increase the p l a n t r e g e n e r a t i o n r a t i o from r i c e protoplasts, A l t h o u g h d i f f e r e n t i a t i o n of shoots and r e g e n e r a t i o n of p l a n t l e t s from c a l l u s has been achieved with several v a r i e t i e s of rice, the c a l l i of many other v a r i e t i e s could not i n i t i a t e shoots (Kawata 1968, Daroyan & Smetamin 1979, Bhattachary & Sen 1980) or the a b i l i t y to i n i t i a t e shoots was l o s t a f t e r protracted subculturing (Nakano et al 1975, Daroyan & Smetamin 1979, Inoue & Maeda 1980), The c a l l i (Fujiminori and A-58 MS) we used t o i s o l a t e protoplasts had been cultured f o r more than two years. Therefore, the r e g e n e r a t i o n t o t i p o t e n c y should have been lower than that of the o r i g i n a l c a l l u s , I t may be p o s s i b l e t o i n c r e a s e the p l a n t r e g e n e r a t i o n r a t i o from p r o t o p l a s t s by using newly induced cal l i , Improvement of the regeneration r a t i o has been reported by Heyser and his associates (1983) who found t h a t about 20% of t h e i r r o o t - d e r i v e d embryogenic c a l l u s produced shoots a f t e r 50 weeks of culture, whereas o n l y 6% o f r o o t - d e r i v e d nonembryogenic c a l l u s did so. The regeneration of pearl m i l l e t plants also has been reported from embryogenic c a l l u s (Vasil & Vasil 1980), We a l s o found a few embryogenic c a l l i d u r i n g protoplast c u l t u r e in N6 medium, I f the regeneration of rice plants from protoplasts requires the formation of embryogenic cel Is, we must f i n d a way to increase the embryogenic cal l i of r i c e in order to increase r i c e p l a n t regeneration. Several p o s s i b i l i t i e s that might operate in a h i g h e r r e g e n e r a t i o n frequency of
p l a n t s from p r o t o p l a s t s of r i c e are c u r r e n t l y investigated in our laboratory.
being
ACKNOWLEDGMENTS We are d e e p l y g r a t e f u l to Professor A. Gibor of the U n i v e r s i t y of C a l i f o r n i a , Santa Barbara, Dr, H, Hori of our I n s t i t u t e and Mrs P, Yamada for t h e i r help in the preparation of t h i s manuscript, REFERENCES Bhattacharya P, Sen SK (1980) Theor. Appl, Genet, 58: 87-90. Brar DS, Rambold S, Constabel F, Gamborg OL (1980) Z. Pflanzenphysiol. 96: 269-275, Cai QG, Qian YQ, Zhou YL, Wu SX (1978) Acta, Bot. S i n i c a . 20 (2): 97-I02. Chourey PS, Zurawski DB (1981) Theor, appl. Genet, 59: 341-344, Deka PC, Sen SK (1976) Molec. Gen. Genet, 145: 239244. Daroyan El, Smetanin AP (1979) Sobiet P l a n t P h y s i o l , 26: 261-266. Fukui K (1983) Theo. Appl. Genet. 65: 225-230. Genovesi AD, M a g i l l CW (1982) Plant c e l l Reports. l: 257-260. Guha-Mukherjee S (1973) J. Exp. Bot. 24: 139-144. Heyser JW, Dykes TA, Demott KJ, Nobors NW (1983) P l a n t Sci, Lett. 29: 175-181, Inoue M, Maeda E (1980) Japan, J Crop Sci, 49: 167174. Kao KN (1977) Molee. Gen, Genet. 150: 225-230. Kawata S, Ishihara A (1968) Proc. Japan Acad. 44: 549553. Lai KL, Liu LF (1982) Crop Sci. 51:70-74, Limsmaier EF, Skoog F (1965) P h y s i o l . P l a n t , 18: I 0 0 127. Nakano H, Tashiro T, Maeda E ( 1 9 6 5 ) Z. Pflanzenphysiol. 76: 444-449, Nishi T, Yamada Y, Takahashi E (1968) Nature 219: 508-509. Potrykus I, Harms CT, Lorz H (1979) Theor, Appl, Ginet. 54: 209-214, Sarinivasan, Vasil IK (1985) Amer, J, Bot, in press. Shimada T, Yamada Y (1979) Japan, J, Genetics. 54: 379-385. Tamura S (1981) Mem.Fac, A g r i . N i i g a t a Univ. 18:1-52, Vasil V, Vasil IK (1979) Z, Pflanzenphysiol, 92: 379383, Vasil V, Vasil IK (1980) Theor, Appl, Genet, 56: 97-99, Wakasa k (1981) Nat. Inst, Agric, Sci,D 33:122-200. Wakasa K (1984) J. Plant, Physiol. 117: 223-231. Wernicke W, Brettel R, Wakizaka T, Potrykus I (1981) Z. Pflanzenphysiol 103: 361-365, Yamada Y, Loh WH (1984) In: Ammirato PV, Evans DA, Sharp WR, Yamada Y (eds) Handbook of P l a n t C e l l Culture, Vol 3, Macmillan, N.Y., 151-170,
