Plant Cell Reports (1983) 2:119-121
Plant Cell Reports © Springer-Verlag 1983
MesophyU Protoplasts of Fenugreek (Trigonella foenumgraecum): Isolation, Culture and Shoot Regeneration N. S. Shekhawat ~ and A. W. Galston Department of Biology, Yale University, P.O. Box 6666, New Haven, CT 06511, USA Received February 8, 1983 / May 9, 1983 Communicated by J. Widholm
ABSTRACT H i g h y i e l d s of m e s o p h y l l p r o t o p l a s t s w e r e o b t a i n e d a f t e r t r e a t m e n t of l e a v e s of Trigonella foenumgraecum, a forage legume, with purified cellulase. Under appropriate c o n d i t i o n s of c u l t u r e up to 70% p l a t i n g e f f i c i e n c y c o u l d be o b t a i n e d . The p r o t o p l a s t - d e r i v e d c o l o n i e s d e v e l o p e d into r a p i d ly g r o w i n g g r e e n c a l l i and p r o d u c e d l e a f y s h o o t s on a m e d i u m c o n t a i n i n g 0.i m g / l each of b e n z y l a m i n o p u r i n e (BAP) and zeatin. A d d i t i o n of g l u t a m i n e and a s p a r a g i n e to the m e d i u m was f o u n d e s s e n t i a l for r a p i d c e l l d i v i s i o n , c a l l u s g r o w t h and d i f f e r e n t i a t i o n . ABBREVIATIONS BAP: 6 - b e n z y l a m i n o p u r i n e ; 2,4-D: 2 , 4 - d i chlorophenoxyacetic acid; GA3: g i b b e r e l l i c acid; NAA: ~ - n a p h t h a l e n e a c e t i c acid. INTRODUCTION In r e c e n t y e a r s , r e g e n e r a t i o n of p l a n t s f r o m i s o l a t e d p r o t o p l a s t s has b e e n an a c t i v e a r e a of r e s e a r c h , m a r r e d o n l y by the d i f f i culty experienced with such economically i m p o r t a n t g r o u p s as the c e r e a l s and l e g u m e s . This f a i l u r e has b e e n r e c o g n i z e d as a m a j o r d e t e r r e n t to the u t i l i z a t i o n of p r o t o p l a s t s in a p p l i e d p l a n t b r e e d i n g ( T h o m a s et al., 1979). T h e r e are a f e w r e p o r t s of the s u c c e s s f u l r e g e n e r a t i o n of c e r e a l p r o t o p l a s t s (see V a s i l , 1982) and the list of r e g e n e r a ting l e g u m e s p e c i e s has b e e n e x t e n d e d in the last t h r e e y e a r s ( G r e s s h o f f , 1980; Kao and M i c h a y l u k , 1980; S a n t o s et al., 1980; A r c i o n i et al., 1982; B h o j w a n i and W h i t e , 1982 and S h e k h a w a t and G a l s t o n , 1983). This l a b o r a tory has b e e n w o r k i n g on c e r e a l and l e g u m e p r o t o p l a s t s for s e v e r a l y e a r s ( B r e n n e m a n and G a l s t o n , 1975 and R e i d and G a l s t o n , 1975) and in the p r e s e n t c o m m u n i c a t i o n we r e p o r t on the i s o l a t i o n , c u l t u r e and r e g e n e r a t i o n of leaf p r o t o p l a s t s of T r i g o n e l l a f o e n u m g r a e c u m (fenugreek). This a n n u a l l e g u m e , a l r e a d y used as a v e g e t a b l e , f o d d e r and g r e e n m a n u r e , has the a d d i t i o n a l r e c o g n i z e d p o t e n t i a l of o l e o r e s i n and s t e r o i d p r o d u c t i o n for oral contraceptives (Lee, 1981). D i o s g e n i n and trigonelline production from tissue culture
of f e n u g r e e k h a v e also b e e n s t u d i e d ( R a d w a n and K o k a t e , 1980 and B r a i n and W i l l i a m s , 1983). Sen and G u p t a (1979) and M u l t a n i (1983) r e p o r t e d o r g a n o g e n e s i s f r o m the t i s s u e c u l t u r e s and Xu et al. (1982) c l a i m e d r o o t f o r m a t i o n f r o m the r o o t p r o t o p l a s t s of this p l a n t . MATERIALS
AND M E T H O D S
P l a n t m a t e r i a l and p r o t o p l a s t i s o l a t i o n : Seeds of T r i g o n e l l a f o e n u m g r a e c u m p u r c h a s e d f r o m K. K a l u s t y a m , 123 L e x i n g t o n A v e n u e , N e w York, w e r e p l a n t e d in v e r m i c u l i t e , s u b i r r i g a ted once a day w i t h 1.2 g/l H y p o n e x ( C o p l e y C h e m i c a l Co., C o p l e y , Ohio) in c o n t r o l l e d e n v i r o n m e n t g r o w t h c h a m b e r w i t h 260 ~E m - 2 s -I P h o t o n F l u x D e n s i t y (PFD) of c o m b i n e d f l u o rescent-incandescent l i g h t in a 1 6 - h r p h o t o p e r i o d at 24 ° C. The f i r s t l e a v e s of 15 d a y - o l d p l a n t s w e r e w a s h e d s e v e r a l t i m e s w i t h tap w a t e r . They w e r e then s u r f a c e s t e r i l i z e d w i t h 4 . 0 % of commercial bleach Clorox (containing 5.25% s o d i u m h y p o c h l o r i t e ) w i t h 0.2% v / v of surfactant Tween-80. A f t e r r i n s i n g the s t e r i lized leaves with sterile distilled water 6-7 times, we p e e l e d off the l o w e r e p i d e r m i s w i t h a fine f o r c e p s . The l e a v e s w e r e f l o a t ed w i t h the p e e l e d l o w e r s u r f a c e down, 8-10 l e a v e s per dish, on 4.0 ml of p r o t o p l a s t isolation medium. The y i e l d , q u a l i t y and p l a t i n g e f f i c i e n c y of the p r o t o p l a s t s c o u l d be i m p r o v e d s e v e r a l - f o l d by p r i o r d e s a l t i n g and p u r i f i c a t i o n of the e n z y m e on a S e p h a d e x G-50 c o l u m n , The f i n a l m e d i u m for p r o t o p l a s t i s o l a t i o n c o n t a i n e d 1.0 p e r c e n t d e s a l t e d Cellulase Onozuka R-10 (Gallard-Schlesinger C h e m i c a l Mfg. Corp., C a r l e P l a c e , N e w Y o r k ) ; 9.0% m a n n i t o l ; s a l t s (KNO 3, i01.0; K H 2 P O A , 27.2; C a C I 2 - 2 H 2 0 , 1 4 8 0 . 0 ; M g S O 4 " 7 H 2 0 , 2 4 0 . 0 and KI, 0.16 mg/l) and 3.1 m M M E S (2-{Nmorpholino}ethanesulphonic acid). The isol a t i o n m e d i u m was a d j u s t e d to pH 5.8 w i t h 0 . 1 N KOH, then f i l t e r - s t e r i l i z e d through a 0.2 p ~ p o r e size N a l g e n e f i l t e r . The l e a v e s w e r e i n c u b a t e d in the i s o l a tion m e d i u m at 30 ° C for 4 h. After digestion, the d e b r i s was r e m o v e d by f i l t e r i n g t h r o u g h a 55 ~m size N y l o n f i l t e r , and the
* On academic leave from the Department of Botany, University of Jodhpur, Jodhpur-342001, India, on the "Scheme of National Scholarship - 1980-81" awarded by the Ministry of Education and Culture, Government of India, New Delhi
120 protoplasts p e l l e t e d by c e n t r i f u g a t i o n at i00 X g for 5 min. The s u p e r n a t a n t fraction was d i s c a r d e d and the p r o t o p l a s t s resuspended and w a s h e d t w i c e w i t h c u l t u r e m e d i u m GS-I. The p r o t o p l a s t s w e r e t h e n p u r i f i e d by f l o a t ing o v e r 2 1 . 0 % W / V s u c r o s e s o l u t i o n w i t h C a C I 2 " 2 H 2 0 , 9 0 0 . 0 m g / l , o t h e r s a l t s of the i s o l a t i o n m e d i u m and 3.1 m M M E S b u f f e r ; pH 5.8. The p r o t o p l a s t pellet was resuspended in the s u c r o s e s o l u t i o n and c o v e r e d by a l a y e r of c u l t u r e m e d i u m GS-I. After centrif u g a t i o n at i00 X g for 5 min, the i n t a c t protoplasts f o r m e d a g r e e n r i n g in the c u l t u r e m e d i u m over the s u c r o s e . This culture m e d i u m l a y e r c o n t a i n i n g the p r o t o p l a s t s was r e m o v e d g e n t l y w i t h a P a s t e u r p i p e t t e , a f t e r w h i c h the p r o t o p l a s t s w e r e once a g a i n w a s h e d w i t h the c u l t u r e m e d i u m , and a f t e r repelleting, resus~ended in c u l t u r e m e d i u m at a d e n s i t y of i0 ~ p r o t o p l a s t s / m ! . C u l t u r e of P r o t o p l a s t s : The p r o t o p l a s t s w e r e c u l t u r e d in 25.0 ~i s i t t i n g d r o p s on the b o t t o m s of 60 x 15 m m p l a s t i c p e t r i d i s h e s (i0 d r o p s / d i s h ) . The c u l t u r e d i s h e s w e r e s e a l e d w i t h P a r a f i l m and k e p t in h u m i d p l a s t i c b o x e s (20 x 15 x i0 cm) to p r e v e n t desiccation at 26 ° C in d i f f u s e l i g h t (5 ~E m - 2 s -I PFD) for f i r s t two w e e k s and s u b s e q u e n t l y at 25 DE m - 2 s -I PFD (day l i g h t fluorescent tubes). The c u l t u r e m e d i a u s e d were: (i) GS'I, w h i c h c o n s i s t e d of MS m a c r o and m i c r o n u t r i e n t s ( M u r a s h i g e and Skoog, 1962); K M v i t a m i n s and o r g a n i c a c i d s (Kao and M i c h a y l u k , 1975); C a C I 2 " 2 H 2 0 , 900.0 mg/l; L - g l u t a m i n e and L - a s p a r a g i n e (2 m M each); a r g i n i n e i0 mg/l; m a n n i t o l 45 g/l; s u c r o s e 30 g/l; g l u c o s e 30 g/l; r i b o s e 500 m g / l and x y l o s e 300 mg/l. The h o r m o n e comb i n a t i o n of the c u l t u r e m e d i u m i n c l u d e d 0.5 m g / l e a c h of 2,4-D, NAA, BAP, z e a t i n and GA 3. The pH of the m e d i u m w a s a d j u s t e d to 5.8 before filter sterilization. (ii) GS-2, all the c o n s t i t u e n t s of G S - I m e d i u m but w i t h m a n n i t o l (20 g/l) and g l u c o s e (i0 g/l) and 1.0 m g / l e a c h of 2 , 4 - D and BAP. (iii) GS-3, as G S - 2 m e d i u m b u t w i t h o u t m a n n i t o l and w i t h 5.0 g/l g l u c o s e . (iv) GS-4, as G S - 3 m e d i u m but w i t h 0.i m g / l e a c h of BAP .and z e a t i n and no a u x i n . The p r o t o p l a s t s w e r e c u l t u r e d for the f i r s t 6 days in G S - I m e d i u m ; on the 6th day of c u l t u r e 25 ~I of GS-2 m e d i u m w a s a d d e d to e a c h c u l t u r e drop. A f t e r two w e e k s of c u l ture 25 ~i bf G S - 3 m e d i u m w a s a d d e d to the cultures. T h r e e to four w e e k old p r o t o p l a s t derived colonies were transferred to G S - 4 a g a r (0.8% p u r i f i e d agar) m e d i u m , and k e p t u n d e r 25 ~E m - 2 s -± PFD at 27 ° C. The c a l l i that d e v e l o p e d w e r e t r a n s f e r r e d to f r e s h m e d i u m a f t e r e a c h 15 days. The d i g e s t i o n of the c e l l w a l l w i t h e n z y m e and its r e g e n e r a t i o n in the c u l t u r e m e d i u m was m o n i t o r e d w i t h C a l c o f l u o r W h i t e staining, using a fluorescence microscope as d e s c r i b e d by N a g a t a a n d T a k e b e (1970). RESULTS
AND
DISCUSSION
One g of l e a v e s r e l e a s e d a b o u t 3-5 x 106 protoplasts, r a n g i n g in size f r o m 25 to 40 ~m in d i a m e t e r , and c o n t a i n i n g b r i g h t g r e e n
chloroplasts a r r a n g e d on the p e r i p h e r y (Fig. i). The c u l t u r e d p r o t o p l a s t s increased in size and b e c a m e o b v i o u s l y v a c u o l a t e d a f t e r 24 h. The c h l o r o p l a s t s then d i s p e r s e d t h r o u g h o u t the p r o t o p l a s t s and b e c a m e l i g h t green. The p r o t o p l a s t s regenerated cell w a l l s w i t h i n 48 h of c u l t u r e and c h a n g e d in shape from sphericalto e l o n g a t e d or oval. Cell p l a t e f o r m a t i o n and the f i r s t cell d i v i s i o n w a s o b s e r v e d a f t e r 60 to 80 h (Fig. 2). The s e c o n d c e l l d i v i s i o n (Fig. 3) w a s o b s e r v e d on the 7th d a y of c u l t u r e b u t if G S - 2 m e d i u m w a s n o t a d d e d to t h e s e c u l t u r e s the s e c o n d cell d i v i s i o n o c c u r r e d a f t e r i0 days. F r o m 20 to 70 p e r c e n t of the c e l l s in different experiments produced microcolonies (Fig. 4) in two to t h r e e w e e k s . To a c c o m p l i s h this, it w a s e s s e n t i a l to k e e p the c u l t u r e s in d i f f u s e l i g h t for the i n i t i a l i0 days s i n c e e x p o s u r e of c u l t u r e s to h i g h intensity light caused brown precipitates to f o r m in the c u l t u r e d r o p s and p r o v e d d e l e t e r i o u s to the p r o t o p l a s t s . Likewise, s e r i a l and g r a d u a l a d d i t i o n of f r e s h m e d i u m wit~ r e d u c e d m a n n i t o l and g l u c o s e l e v e l s w a s found necessary for h i g h f r e q u e n c y d i v i s i o n and r a p i d and h e a l t h y g r o w t h ~ 0 f p r o t o p l a s t m d e r i v e d c e l l s and c o l o n i e s ~ The h i g h c o n c e n t r a t i o n of o s m o t i c u m c a u s e d o b v i o u s i n h i b i t i o n of c e l l d i v i s i o n . Jha and R o y (1980) and S h e k h a w a t and G a l s t o n (1983) f o u n d that with Visna species protoplasts gradual r e m o v a l of m a n n i t o l f r o m the c u l t u r e m e d i a i m p r o v e d the d i v i s i o n and g r o w t h of p r o t o plast-derived c e l l s and c o l o n i e s . On subc u l t u r e in G S - 3 m e d i u m , the c o l o n i e s r e a d i l y proliferated as l i g h t g r e e n c a l l i . On a g a r culture under high light intensity, the c a l l i b e c a m e hard, c o m p a c t and d e e p g r e e n ; globular, translucent nodul~r structures a p p e a r e d on the s u r f a c e s and p e r i p h e r i e s of t h e s e c a l l i (Fig. 5). When transferred to medium without auxin but containing 0.i m g / l e a c h of B A P and z e a t i n , t h e s e n o d u l a r b o d i e s f o r m e d g r e e n b u d s (Fig. 6), w h i c h in turn p r o d u c e d l e a f y s h o o t s (Fig. 7). Occurrence of i s o l a t e d l e a v e s w a s m o r e c o m m o n . This t y p e of r e s p o n s e was r e c o r d e d o n l y in 0.5 p e r c e n t of the c a l l i . T h e s e buds, s h o o t s and l e a v e s g r e w v e r y p o o r l y and s h o w e d a t e n d e n c y to d e d i f f e r e n t i a t e and d e f o r m . Xu et al. (1982) r e p o r t e d f a i l u r e to o b t a i n shoot differentiation from root protoplasts of this p l a n t w h i l e we f o u n d s u c h d i f f e r e n t i a t i o n to d e p e n d on g l u t a m i n e and a s p a r a gine. In m e d i a w i t h o u t t h e s e a m i n o a c i d s , c a l l u s g r o w t h w a s p o o r and the c a l l i s h o w e d b r o w n i n g of the m a r g i n s . A d d i t i o n of t h e s e a m i n o a c i d s to the c u l t u r e m e d i a e n h a n c e d both protoplast d i v i s i o n and s u b s e q u e n t g r o w t h of c o l o n i e s and c a l l i . These amino a c i d s , w h i c h are the d o m i n a n t ones in the p h l o e m sap o f m a n y l e g u m e s , h a v e a l s o b e e n u s e d for g e n e r a l c u l t u r e p u r p o s e s and for increasing the d i v i s i o n f r e q u e n c y of V i c i a narbonensis (Donn, 1 9 7 8 ) . T h e s e m e t h o d s , w h i c h we h a v e f o u n d h i g h l y efficient, are n o w b e i n g u s e d r o u t i n e l y in our l a b o r a t o r y for c u l t u r e and r e g e n e r a t i o n of p r o t o p l a s t s of V i g n a a c o n i t i f o l i a , and some o t h e r l e g u m e s .
121
Figs.
1-6.
Regeneration
of m e s o p h y l l
protoplasts
of
Trigonella
foenumgraecum.
Fig. i. Freshly isolated protoplasts. (Bar = 25 ~m). Figs. 2 a n d 3. F i r s t and s e c o n d c e l l d i v i s i o n s r e s p e c t i v e l y . (Bar = 25 Fig. 4. Protoplast-derived colony. (Bar = 25 ~m). Fig. 5. Protoplast-derived calli. (Bar = 1 cm)° Figs. 6 and 7. Bud and l e a f y s h o o t f o r m e d on p r o t o p l a s t - d e r i v e d calli. 1 cm r e s p e c t i v e l y ) . ACKNOWLEDGEMENTS: We a r e t h a n k f u l to A n n a F r a n c e s c o n i for her e x p e r t s e c r e t a r i a l assistance. NSS is g r a t e f u l to the M i n i s t r y of E d u c a t i o n and Culture, Government of India, for g r a n t i n g National scholarship, 1980-~i.
REFERENCES A r c i o n i S, D a v e y MR, S a n t o s A V P Dos, C o c k i n g EC (1982) Z. P f l a n z e n p h y s i o l . 106:105-110 B h o j w a n i SS, W h i t e D W R (1982) P l a n t Sci. Lett. 26: 2 6 5 - 2 7 1 B r a i n KR, W i l l i a m s M H (1983) P l a n t C e l l R e p o r t s 2: 7-10 B r e n n e m a n FN, G a l s t o n A W (1975) B i o c h e m . Physiol. Pflanzen 168: 4 5 3 - 4 7 1 D o n n G (1978) Z. P f l a n z e n p h y s i o l . 86: 6 5 - 7 5 G r e s s h o f f PM (1980) Bot. Gaz. 141: 1 5 7 - 1 6 4 Jha TB, R o y SC (1980) I n d i a n J. Exp. Biol. 18: 8 7 - 8 9 Kao KN, M i c h a y l u k M R (1975) P l a n t a 126: 1 0 5 ii0
]Jm) ~
(Bar
= 500
~m
and
K a o KN, M i c h a y l u k M R (1980) Z. P f l a n z e n physiol. 96: 1 3 5 - 1 4 1 L e e P (1981) W o r l d F a r m i n g 23: 1 4 - 1 8 M u l t a n i DS (1983) In: Sen SK, G i l e s KL (eds) P l a n t C e l l C u l t u r e in C r o p I m p r o v e m e n t , P l e n u m Press, N e w York, pp 4 3 5 - 4 3 9 Murashige T, S k o o g F (1962) P h y s i o l . P l a n t . 15: 4 7 3 - 4 9 7 N a g a t a T, T a k e b e I (1970) P l a n t a 92: 3 0 1 - 3 0 8 R a d w a n SS, K o k a t e CK (1980) P l a n t a 147: 340344 R e i d RK, G a l s t o n A W (1975) B i o c h e m . P h y s i o l . P f l a n z e n 168: 4 7 3 - 4 8 2 S a n t o s A V P Dos, O u t k a DE, C o c k i n g EC, D a v e y M R (1980) Z. P f l a n z e n p h y s i o l . 99: 2 6 1 - 2 7 0 Sen B, G u p t a S (1979) P h y s i o l . P l a n t . 45: 425-428 S h e k h a w a t NS, G a l s t o n A W (1983) P l a n t Sci. Lett. (in p r e s s ) T h o m a s E, K i n g PJ, P o t r y k u s I (1979) Z. Pflanzenzuchtg. 82: 1-30 V a s i l IK (1982) In: V a s i l IK, S c o w c r o f t WR, F r e y KJ (eds) P l a n t I m p r o v e m e n t and Somatic Cell Genetics, A c a d e m i c Press, N e w Y o r k , pp 1 7 9 - 2 0 3 Xu ZH, D a v e y MR, C o c k i n g EC (1982) Z. Pflanzenphysiol. 107: 2 3 1 - 2 3 5
Plant Cell Reports © Springer-Verlag 1983
MesophyU Protoplasts of Fenugreek (Trigonella foenumgraecum): Isolation, Culture and Shoot Regeneration N. S. Shekhawat ~ and A. W. Galston Department of Biology, Yale University, P.O. Box 6666, New Haven, CT 06511, USA Received February 8, 1983 / May 9, 1983 Communicated by J. Widholm
ABSTRACT H i g h y i e l d s of m e s o p h y l l p r o t o p l a s t s w e r e o b t a i n e d a f t e r t r e a t m e n t of l e a v e s of Trigonella foenumgraecum, a forage legume, with purified cellulase. Under appropriate c o n d i t i o n s of c u l t u r e up to 70% p l a t i n g e f f i c i e n c y c o u l d be o b t a i n e d . The p r o t o p l a s t - d e r i v e d c o l o n i e s d e v e l o p e d into r a p i d ly g r o w i n g g r e e n c a l l i and p r o d u c e d l e a f y s h o o t s on a m e d i u m c o n t a i n i n g 0.i m g / l each of b e n z y l a m i n o p u r i n e (BAP) and zeatin. A d d i t i o n of g l u t a m i n e and a s p a r a g i n e to the m e d i u m was f o u n d e s s e n t i a l for r a p i d c e l l d i v i s i o n , c a l l u s g r o w t h and d i f f e r e n t i a t i o n . ABBREVIATIONS BAP: 6 - b e n z y l a m i n o p u r i n e ; 2,4-D: 2 , 4 - d i chlorophenoxyacetic acid; GA3: g i b b e r e l l i c acid; NAA: ~ - n a p h t h a l e n e a c e t i c acid. INTRODUCTION In r e c e n t y e a r s , r e g e n e r a t i o n of p l a n t s f r o m i s o l a t e d p r o t o p l a s t s has b e e n an a c t i v e a r e a of r e s e a r c h , m a r r e d o n l y by the d i f f i culty experienced with such economically i m p o r t a n t g r o u p s as the c e r e a l s and l e g u m e s . This f a i l u r e has b e e n r e c o g n i z e d as a m a j o r d e t e r r e n t to the u t i l i z a t i o n of p r o t o p l a s t s in a p p l i e d p l a n t b r e e d i n g ( T h o m a s et al., 1979). T h e r e are a f e w r e p o r t s of the s u c c e s s f u l r e g e n e r a t i o n of c e r e a l p r o t o p l a s t s (see V a s i l , 1982) and the list of r e g e n e r a ting l e g u m e s p e c i e s has b e e n e x t e n d e d in the last t h r e e y e a r s ( G r e s s h o f f , 1980; Kao and M i c h a y l u k , 1980; S a n t o s et al., 1980; A r c i o n i et al., 1982; B h o j w a n i and W h i t e , 1982 and S h e k h a w a t and G a l s t o n , 1983). This l a b o r a tory has b e e n w o r k i n g on c e r e a l and l e g u m e p r o t o p l a s t s for s e v e r a l y e a r s ( B r e n n e m a n and G a l s t o n , 1975 and R e i d and G a l s t o n , 1975) and in the p r e s e n t c o m m u n i c a t i o n we r e p o r t on the i s o l a t i o n , c u l t u r e and r e g e n e r a t i o n of leaf p r o t o p l a s t s of T r i g o n e l l a f o e n u m g r a e c u m (fenugreek). This a n n u a l l e g u m e , a l r e a d y used as a v e g e t a b l e , f o d d e r and g r e e n m a n u r e , has the a d d i t i o n a l r e c o g n i z e d p o t e n t i a l of o l e o r e s i n and s t e r o i d p r o d u c t i o n for oral contraceptives (Lee, 1981). D i o s g e n i n and trigonelline production from tissue culture
of f e n u g r e e k h a v e also b e e n s t u d i e d ( R a d w a n and K o k a t e , 1980 and B r a i n and W i l l i a m s , 1983). Sen and G u p t a (1979) and M u l t a n i (1983) r e p o r t e d o r g a n o g e n e s i s f r o m the t i s s u e c u l t u r e s and Xu et al. (1982) c l a i m e d r o o t f o r m a t i o n f r o m the r o o t p r o t o p l a s t s of this p l a n t . MATERIALS
AND M E T H O D S
P l a n t m a t e r i a l and p r o t o p l a s t i s o l a t i o n : Seeds of T r i g o n e l l a f o e n u m g r a e c u m p u r c h a s e d f r o m K. K a l u s t y a m , 123 L e x i n g t o n A v e n u e , N e w York, w e r e p l a n t e d in v e r m i c u l i t e , s u b i r r i g a ted once a day w i t h 1.2 g/l H y p o n e x ( C o p l e y C h e m i c a l Co., C o p l e y , Ohio) in c o n t r o l l e d e n v i r o n m e n t g r o w t h c h a m b e r w i t h 260 ~E m - 2 s -I P h o t o n F l u x D e n s i t y (PFD) of c o m b i n e d f l u o rescent-incandescent l i g h t in a 1 6 - h r p h o t o p e r i o d at 24 ° C. The f i r s t l e a v e s of 15 d a y - o l d p l a n t s w e r e w a s h e d s e v e r a l t i m e s w i t h tap w a t e r . They w e r e then s u r f a c e s t e r i l i z e d w i t h 4 . 0 % of commercial bleach Clorox (containing 5.25% s o d i u m h y p o c h l o r i t e ) w i t h 0.2% v / v of surfactant Tween-80. A f t e r r i n s i n g the s t e r i lized leaves with sterile distilled water 6-7 times, we p e e l e d off the l o w e r e p i d e r m i s w i t h a fine f o r c e p s . The l e a v e s w e r e f l o a t ed w i t h the p e e l e d l o w e r s u r f a c e down, 8-10 l e a v e s per dish, on 4.0 ml of p r o t o p l a s t isolation medium. The y i e l d , q u a l i t y and p l a t i n g e f f i c i e n c y of the p r o t o p l a s t s c o u l d be i m p r o v e d s e v e r a l - f o l d by p r i o r d e s a l t i n g and p u r i f i c a t i o n of the e n z y m e on a S e p h a d e x G-50 c o l u m n , The f i n a l m e d i u m for p r o t o p l a s t i s o l a t i o n c o n t a i n e d 1.0 p e r c e n t d e s a l t e d Cellulase Onozuka R-10 (Gallard-Schlesinger C h e m i c a l Mfg. Corp., C a r l e P l a c e , N e w Y o r k ) ; 9.0% m a n n i t o l ; s a l t s (KNO 3, i01.0; K H 2 P O A , 27.2; C a C I 2 - 2 H 2 0 , 1 4 8 0 . 0 ; M g S O 4 " 7 H 2 0 , 2 4 0 . 0 and KI, 0.16 mg/l) and 3.1 m M M E S (2-{Nmorpholino}ethanesulphonic acid). The isol a t i o n m e d i u m was a d j u s t e d to pH 5.8 w i t h 0 . 1 N KOH, then f i l t e r - s t e r i l i z e d through a 0.2 p ~ p o r e size N a l g e n e f i l t e r . The l e a v e s w e r e i n c u b a t e d in the i s o l a tion m e d i u m at 30 ° C for 4 h. After digestion, the d e b r i s was r e m o v e d by f i l t e r i n g t h r o u g h a 55 ~m size N y l o n f i l t e r , and the
* On academic leave from the Department of Botany, University of Jodhpur, Jodhpur-342001, India, on the "Scheme of National Scholarship - 1980-81" awarded by the Ministry of Education and Culture, Government of India, New Delhi
120 protoplasts p e l l e t e d by c e n t r i f u g a t i o n at i00 X g for 5 min. The s u p e r n a t a n t fraction was d i s c a r d e d and the p r o t o p l a s t s resuspended and w a s h e d t w i c e w i t h c u l t u r e m e d i u m GS-I. The p r o t o p l a s t s w e r e t h e n p u r i f i e d by f l o a t ing o v e r 2 1 . 0 % W / V s u c r o s e s o l u t i o n w i t h C a C I 2 " 2 H 2 0 , 9 0 0 . 0 m g / l , o t h e r s a l t s of the i s o l a t i o n m e d i u m and 3.1 m M M E S b u f f e r ; pH 5.8. The p r o t o p l a s t pellet was resuspended in the s u c r o s e s o l u t i o n and c o v e r e d by a l a y e r of c u l t u r e m e d i u m GS-I. After centrif u g a t i o n at i00 X g for 5 min, the i n t a c t protoplasts f o r m e d a g r e e n r i n g in the c u l t u r e m e d i u m over the s u c r o s e . This culture m e d i u m l a y e r c o n t a i n i n g the p r o t o p l a s t s was r e m o v e d g e n t l y w i t h a P a s t e u r p i p e t t e , a f t e r w h i c h the p r o t o p l a s t s w e r e once a g a i n w a s h e d w i t h the c u l t u r e m e d i u m , and a f t e r repelleting, resus~ended in c u l t u r e m e d i u m at a d e n s i t y of i0 ~ p r o t o p l a s t s / m ! . C u l t u r e of P r o t o p l a s t s : The p r o t o p l a s t s w e r e c u l t u r e d in 25.0 ~i s i t t i n g d r o p s on the b o t t o m s of 60 x 15 m m p l a s t i c p e t r i d i s h e s (i0 d r o p s / d i s h ) . The c u l t u r e d i s h e s w e r e s e a l e d w i t h P a r a f i l m and k e p t in h u m i d p l a s t i c b o x e s (20 x 15 x i0 cm) to p r e v e n t desiccation at 26 ° C in d i f f u s e l i g h t (5 ~E m - 2 s -I PFD) for f i r s t two w e e k s and s u b s e q u e n t l y at 25 DE m - 2 s -I PFD (day l i g h t fluorescent tubes). The c u l t u r e m e d i a u s e d were: (i) GS'I, w h i c h c o n s i s t e d of MS m a c r o and m i c r o n u t r i e n t s ( M u r a s h i g e and Skoog, 1962); K M v i t a m i n s and o r g a n i c a c i d s (Kao and M i c h a y l u k , 1975); C a C I 2 " 2 H 2 0 , 900.0 mg/l; L - g l u t a m i n e and L - a s p a r a g i n e (2 m M each); a r g i n i n e i0 mg/l; m a n n i t o l 45 g/l; s u c r o s e 30 g/l; g l u c o s e 30 g/l; r i b o s e 500 m g / l and x y l o s e 300 mg/l. The h o r m o n e comb i n a t i o n of the c u l t u r e m e d i u m i n c l u d e d 0.5 m g / l e a c h of 2,4-D, NAA, BAP, z e a t i n and GA 3. The pH of the m e d i u m w a s a d j u s t e d to 5.8 before filter sterilization. (ii) GS-2, all the c o n s t i t u e n t s of G S - I m e d i u m but w i t h m a n n i t o l (20 g/l) and g l u c o s e (i0 g/l) and 1.0 m g / l e a c h of 2 , 4 - D and BAP. (iii) GS-3, as G S - 2 m e d i u m b u t w i t h o u t m a n n i t o l and w i t h 5.0 g/l g l u c o s e . (iv) GS-4, as G S - 3 m e d i u m but w i t h 0.i m g / l e a c h of BAP .and z e a t i n and no a u x i n . The p r o t o p l a s t s w e r e c u l t u r e d for the f i r s t 6 days in G S - I m e d i u m ; on the 6th day of c u l t u r e 25 ~I of GS-2 m e d i u m w a s a d d e d to e a c h c u l t u r e drop. A f t e r two w e e k s of c u l ture 25 ~i bf G S - 3 m e d i u m w a s a d d e d to the cultures. T h r e e to four w e e k old p r o t o p l a s t derived colonies were transferred to G S - 4 a g a r (0.8% p u r i f i e d agar) m e d i u m , and k e p t u n d e r 25 ~E m - 2 s -± PFD at 27 ° C. The c a l l i that d e v e l o p e d w e r e t r a n s f e r r e d to f r e s h m e d i u m a f t e r e a c h 15 days. The d i g e s t i o n of the c e l l w a l l w i t h e n z y m e and its r e g e n e r a t i o n in the c u l t u r e m e d i u m was m o n i t o r e d w i t h C a l c o f l u o r W h i t e staining, using a fluorescence microscope as d e s c r i b e d by N a g a t a a n d T a k e b e (1970). RESULTS
AND
DISCUSSION
One g of l e a v e s r e l e a s e d a b o u t 3-5 x 106 protoplasts, r a n g i n g in size f r o m 25 to 40 ~m in d i a m e t e r , and c o n t a i n i n g b r i g h t g r e e n
chloroplasts a r r a n g e d on the p e r i p h e r y (Fig. i). The c u l t u r e d p r o t o p l a s t s increased in size and b e c a m e o b v i o u s l y v a c u o l a t e d a f t e r 24 h. The c h l o r o p l a s t s then d i s p e r s e d t h r o u g h o u t the p r o t o p l a s t s and b e c a m e l i g h t green. The p r o t o p l a s t s regenerated cell w a l l s w i t h i n 48 h of c u l t u r e and c h a n g e d in shape from sphericalto e l o n g a t e d or oval. Cell p l a t e f o r m a t i o n and the f i r s t cell d i v i s i o n w a s o b s e r v e d a f t e r 60 to 80 h (Fig. 2). The s e c o n d c e l l d i v i s i o n (Fig. 3) w a s o b s e r v e d on the 7th d a y of c u l t u r e b u t if G S - 2 m e d i u m w a s n o t a d d e d to t h e s e c u l t u r e s the s e c o n d cell d i v i s i o n o c c u r r e d a f t e r i0 days. F r o m 20 to 70 p e r c e n t of the c e l l s in different experiments produced microcolonies (Fig. 4) in two to t h r e e w e e k s . To a c c o m p l i s h this, it w a s e s s e n t i a l to k e e p the c u l t u r e s in d i f f u s e l i g h t for the i n i t i a l i0 days s i n c e e x p o s u r e of c u l t u r e s to h i g h intensity light caused brown precipitates to f o r m in the c u l t u r e d r o p s and p r o v e d d e l e t e r i o u s to the p r o t o p l a s t s . Likewise, s e r i a l and g r a d u a l a d d i t i o n of f r e s h m e d i u m wit~ r e d u c e d m a n n i t o l and g l u c o s e l e v e l s w a s found necessary for h i g h f r e q u e n c y d i v i s i o n and r a p i d and h e a l t h y g r o w t h ~ 0 f p r o t o p l a s t m d e r i v e d c e l l s and c o l o n i e s ~ The h i g h c o n c e n t r a t i o n of o s m o t i c u m c a u s e d o b v i o u s i n h i b i t i o n of c e l l d i v i s i o n . Jha and R o y (1980) and S h e k h a w a t and G a l s t o n (1983) f o u n d that with Visna species protoplasts gradual r e m o v a l of m a n n i t o l f r o m the c u l t u r e m e d i a i m p r o v e d the d i v i s i o n and g r o w t h of p r o t o plast-derived c e l l s and c o l o n i e s . On subc u l t u r e in G S - 3 m e d i u m , the c o l o n i e s r e a d i l y proliferated as l i g h t g r e e n c a l l i . On a g a r culture under high light intensity, the c a l l i b e c a m e hard, c o m p a c t and d e e p g r e e n ; globular, translucent nodul~r structures a p p e a r e d on the s u r f a c e s and p e r i p h e r i e s of t h e s e c a l l i (Fig. 5). When transferred to medium without auxin but containing 0.i m g / l e a c h of B A P and z e a t i n , t h e s e n o d u l a r b o d i e s f o r m e d g r e e n b u d s (Fig. 6), w h i c h in turn p r o d u c e d l e a f y s h o o t s (Fig. 7). Occurrence of i s o l a t e d l e a v e s w a s m o r e c o m m o n . This t y p e of r e s p o n s e was r e c o r d e d o n l y in 0.5 p e r c e n t of the c a l l i . T h e s e buds, s h o o t s and l e a v e s g r e w v e r y p o o r l y and s h o w e d a t e n d e n c y to d e d i f f e r e n t i a t e and d e f o r m . Xu et al. (1982) r e p o r t e d f a i l u r e to o b t a i n shoot differentiation from root protoplasts of this p l a n t w h i l e we f o u n d s u c h d i f f e r e n t i a t i o n to d e p e n d on g l u t a m i n e and a s p a r a gine. In m e d i a w i t h o u t t h e s e a m i n o a c i d s , c a l l u s g r o w t h w a s p o o r and the c a l l i s h o w e d b r o w n i n g of the m a r g i n s . A d d i t i o n of t h e s e a m i n o a c i d s to the c u l t u r e m e d i a e n h a n c e d both protoplast d i v i s i o n and s u b s e q u e n t g r o w t h of c o l o n i e s and c a l l i . These amino a c i d s , w h i c h are the d o m i n a n t ones in the p h l o e m sap o f m a n y l e g u m e s , h a v e a l s o b e e n u s e d for g e n e r a l c u l t u r e p u r p o s e s and for increasing the d i v i s i o n f r e q u e n c y of V i c i a narbonensis (Donn, 1 9 7 8 ) . T h e s e m e t h o d s , w h i c h we h a v e f o u n d h i g h l y efficient, are n o w b e i n g u s e d r o u t i n e l y in our l a b o r a t o r y for c u l t u r e and r e g e n e r a t i o n of p r o t o p l a s t s of V i g n a a c o n i t i f o l i a , and some o t h e r l e g u m e s .
121
Figs.
1-6.
Regeneration
of m e s o p h y l l
protoplasts
of
Trigonella
foenumgraecum.
Fig. i. Freshly isolated protoplasts. (Bar = 25 ~m). Figs. 2 a n d 3. F i r s t and s e c o n d c e l l d i v i s i o n s r e s p e c t i v e l y . (Bar = 25 Fig. 4. Protoplast-derived colony. (Bar = 25 ~m). Fig. 5. Protoplast-derived calli. (Bar = 1 cm)° Figs. 6 and 7. Bud and l e a f y s h o o t f o r m e d on p r o t o p l a s t - d e r i v e d calli. 1 cm r e s p e c t i v e l y ) . ACKNOWLEDGEMENTS: We a r e t h a n k f u l to A n n a F r a n c e s c o n i for her e x p e r t s e c r e t a r i a l assistance. NSS is g r a t e f u l to the M i n i s t r y of E d u c a t i o n and Culture, Government of India, for g r a n t i n g National scholarship, 1980-~i.
REFERENCES A r c i o n i S, D a v e y MR, S a n t o s A V P Dos, C o c k i n g EC (1982) Z. P f l a n z e n p h y s i o l . 106:105-110 B h o j w a n i SS, W h i t e D W R (1982) P l a n t Sci. Lett. 26: 2 6 5 - 2 7 1 B r a i n KR, W i l l i a m s M H (1983) P l a n t C e l l R e p o r t s 2: 7-10 B r e n n e m a n FN, G a l s t o n A W (1975) B i o c h e m . Physiol. Pflanzen 168: 4 5 3 - 4 7 1 D o n n G (1978) Z. P f l a n z e n p h y s i o l . 86: 6 5 - 7 5 G r e s s h o f f PM (1980) Bot. Gaz. 141: 1 5 7 - 1 6 4 Jha TB, R o y SC (1980) I n d i a n J. Exp. Biol. 18: 8 7 - 8 9 Kao KN, M i c h a y l u k M R (1975) P l a n t a 126: 1 0 5 ii0
]Jm) ~
(Bar
= 500
~m
and
K a o KN, M i c h a y l u k M R (1980) Z. P f l a n z e n physiol. 96: 1 3 5 - 1 4 1 L e e P (1981) W o r l d F a r m i n g 23: 1 4 - 1 8 M u l t a n i DS (1983) In: Sen SK, G i l e s KL (eds) P l a n t C e l l C u l t u r e in C r o p I m p r o v e m e n t , P l e n u m Press, N e w York, pp 4 3 5 - 4 3 9 Murashige T, S k o o g F (1962) P h y s i o l . P l a n t . 15: 4 7 3 - 4 9 7 N a g a t a T, T a k e b e I (1970) P l a n t a 92: 3 0 1 - 3 0 8 R a d w a n SS, K o k a t e CK (1980) P l a n t a 147: 340344 R e i d RK, G a l s t o n A W (1975) B i o c h e m . P h y s i o l . P f l a n z e n 168: 4 7 3 - 4 8 2 S a n t o s A V P Dos, O u t k a DE, C o c k i n g EC, D a v e y M R (1980) Z. P f l a n z e n p h y s i o l . 99: 2 6 1 - 2 7 0 Sen B, G u p t a S (1979) P h y s i o l . P l a n t . 45: 425-428 S h e k h a w a t NS, G a l s t o n A W (1983) P l a n t Sci. Lett. (in p r e s s ) T h o m a s E, K i n g PJ, P o t r y k u s I (1979) Z. Pflanzenzuchtg. 82: 1-30 V a s i l IK (1982) In: V a s i l IK, S c o w c r o f t WR, F r e y KJ (eds) P l a n t I m p r o v e m e n t and Somatic Cell Genetics, A c a d e m i c Press, N e w Y o r k , pp 1 7 9 - 2 0 3 Xu ZH, D a v e y MR, C o c k i n g EC (1982) Z. Pflanzenphysiol. 107: 2 3 1 - 2 3 5
