Plant Cell Reports

Plant Cell Reports (1990) 9 : 4 2 7 - 4 3 0

9 Springer-Verlag 1990

Plant regeneration from callus and protoplasts of Brassica nigra (IC 257) through somatic embryogenesis Vibha Gupta, Abha Agnihotri, and V. Jagannathan Tata Energy Research Institute, 90 Jor Bagh, New Delhi 110003, India Received April 25, 1990/Revised version received September 10, 1990 - Communicated by E. D. Earle

Abstract

A m e t h o d to o b t a i n p l a n t s from e m b r y o g e n i c callus of Brassica nigra and protoplasts of hypocotyl explants is described. Callus was i n i t i a t e d on M u r a s h i g e and Skoog medium c o n t a i n i n g kinetin (kn) and 2,4-dichlorophenoxy acetic acid (2,4-D). Lowering of auxin induced embryo formation. Supplementation with gibberellic acid (GA 3 ) enhanced embryogenic response tenfold. P a s s a g e t h r o u g h l i q u i d medium d e v o i d of g r o w t h r e g u l a t o r s was e s s e n t i a l f o r t h e g r o w t h of e m b r y o s . S e c o n d a r y e m b r y o s w e r e p r o d u c e d on t r a n s f e r to solid basal medium. Embryogenic callus retained i t s m o r p h o g e n i c a b i l i t y e v e n a f t e r 12 s u b c u l t u r e s . Both primary and secondary embryos produced fertile plants. Hypocotyl-derived protoplasts were also regenerated to plants following the same protocol. The survival of plants on transfer to soil was about 80%. The seeds from plants derived from callus and protoplasts were viable. Key words:

Somatic embryogenesis, protoplasts, Brassica nigra, plant regeneration.

Abbreviations: 2,4-D: 2,4-dichlorophenoxy acetic acid; NAA: n a p h t h a l e n e a c e t i c a c i d ; IAA: i n d o l e a c e t i c a c i d ; k n : k i n e t i n ; GA3: g i b b e r e l l i c a c i d . Introduction

R e g e n e r a t i o n of p l a n t s b y p r o t o p l a s t c u l t u r e i s of v a l u e f o r g e n e t i c m a n i p u l a t i o n of p l a n t s p e c i e s . B r a s s i c a i s e c o n o m i c a l l y i m p o r t a n t as a s o u r c e of edible oil, condiments, vegetables and cattle fodder. E x t e n s i v e s t u d i e s h a v e b e e n made on c e l l a n d p r o t o p l a s t c u l t u r e a n d r e g e n e r a t i o n of s e v e r a l Brassica species. Relatively little work has, h o w e v e r , b e e n c a r r i e d out on B r a s s i c a n i g r a ( b l a c k mustard). It i s m a i n l y u s e d as a c o n d i m e n t in India, but is of importance as one of the components of agronomically important digenomic Brassicas, s u c h as B._t. j u n c e a a n d B. c a r i n a t a . P l a n t l e t f o r m a t i o n from c u l t u r e d a n t h e r s ( G o v i l et al. 1986) and shoot regeneration from callus ( D i e t e r t e t a l . 1982) o r d i r e c t l y from c o t y l e d o n s ( N a r a s i m h u l u & C h o p r a 1988) h a v e b e e n d e s c r i b e d w i t h B_z _. n i g r a i K l i m a s z e w s k a a n d K e l l e r (1986) o b t a i n e d s o m a t i c e m b r y o g e n e s i s in c e l l s u s p e n s i o n cultures, but the embryos gave rise to s t e r i l e plants. S c h e n c k a n d Hoffmann (1979) and G l i m e l i u s (1984) f a i l e d to o b t a i n p l a n t r e g e n e r a t i o n from B.

Offprint requests to." V. Jagannathan

nigra protoplasts. K l i m a s z e w s k a a n d K e l l e r (1986) reported callus production and somatic embryo f o r m a t i o n in p r o t o p l a s t culture, but the embryos d i d not grow i n t o p l a n t s . In a r e c e n t r e p o r t S a c r i s t a n et a l . (1989) d e s c r i b e d t r a n s f o r m a t i o n of protoplasts of B__. n i g r a b y c o c u l t i v a t i o n w i t h an Agrobacterium strain c o n t a i n i n g a gene e n c o d i n g resistance to h y g r o m y c i n and u s e d t h e r e s i s t a n t calli for further cell genetic studies. Only one of the many transformed cell lines produced transformed plants. In this paper we report the formation of primary embryos and secondary embryos from hypocotyl-derived callus as well as from protoplasts of B. ni r[zr_~ (var. IC 257) and r e g e n e r a t i o n of p l a n t s from t h e s e e m b r y o s . Materials and Methods

Plant Material S e e d s of Bc_. n i g r a ( v a t . from Dr. Shyam Prakash, R e s e a r c h I n s t i t u t e , New D e l h i .

IC 257) Indian

were a gift Agricultural

Culture Conditions Seeds were surface sterilized with 0.02% m e r c u r i c c h l o r i d e and grown a s e p t i c a l l y on 0.7% a g a r in 16 h l i g h t (2000 l u x ) a n d 8 h d a r k periods. All o p e r a t i o n s w e r e c a r r i e d out a t 25+20C and i n c u b a t i o n of c u l t u r e s in l i g h t in a l l t h e f o l l o w i n g p r o c e d u r e s was in 16 h l i g h t (2000 l u x ) and 8 h clark p e r i o d s u n l e s s o t h e r w i s e s t a t e d . A four step procedure was found to be essential for obtaining plants. (I) Hypocotyl segments (0.5-1.0 era) from I0 day old seedlings w e r e c u l t u r e d on MS medium ( M u r a s h i g e a n d Skoog 1962) s u p p l e m e n t e d w i t h g r o w t h r e g u l a t o r s ( k n 1 ppm and 2,4-D 1 ppm ) for callus formation. S u b c u l t u r i n g of c a l l i was done e v e r y 3-4 w e e k s . The cultures were maintained under light. (2) Both s p h e r i c a l and friable callus were obtained from e a c h e x p l a n t , a n d o n l y t h e s p h e r i c a l p o r t i o n s were transferred to MS medium c o n t a i n i n g kn (1 p p m ) , NAA (0.1 p p m ) a n d GA3 (1 p p m ) m a i n t a i n i n g t h e same c u l t u r e c o n d i t i o n s . (3) Somatic ~ m b r y o s were produced in all cultures. They were t r a n s f e r r e d to l i q u i d MS b a s a l medium c o n t a i n i n g 3% s u c r o s e b u t no g r o w t h r e g u l a t o r s (A3) a n d k e p t on

428 a shaker (60 rpm) for a week. (4) Embryos with proper roots and cotyledons were subcultured on MS medium containing 1% sucrose (AI) and no growth regulators to obtain plants.

Table 1 E f f e c t of s u p p l e m e n t s on g r o w t h a n d o r g a n o g e n e s i s from h y p o c o t y l - d e r i v e d callus

Protoplast isolation Supplements Protoplast isolation and culture conditions were according to the procedure of Glimelius et al. (1984) with slight modifications. Seeds were germinated on B~ (Gamborg et al. 1968) medium with calcium nitr~ate (I00 mg/l) in the dark for 2 days and then kept under light for 4 more days. Hypocotyls were cut into 2-3 m m pieces while immersed in C P W 9 M medium (CPW salts, Frearson et al. 1973 containing 9% mannitol). They were transferred to the C P W 9 M medium supplemented with kn 1 p p m and 2,4-D I p p m and 0.5% (w/v) each of cellulase and macerozyme R-10 (Onozuka, Japan) and incubated overnight at 25~ in the dark. Protoplasts were sieved through stainless steel mesh (80 u), washed twice in C P W 9 M medium and layered gently on G P W 18S (CPW containing 18% sucrose). After centrifugation for 5 rain at I000 g the protoplasts formed a band at the interface. They were collected, washed with and suspended in modified Kao's medium containing 0.4 M gl~cose (Glimelius 1984) at a cell density of 2-Sxl0~/ml. Protoplast culture Protoplasts were cultured in the dark in 60x15 mm plastic petri dishes (Costar, 205 Broadway, Cambridge, M A 02139, USA), each dish containing 4 ml of modified Kao's medium with 0.4 M glucose and 1 p p m each of kn and 2,4-D. The division frequency (ratio of the number of dividing protoplasts to the total intact protoplast number) was determined after 7-8 days of culture. Osmotic concentration of the culture medium was lowered progressively at intervals of 3 days by the addition of 0.5-0.7 ml of Kao and Michayluk's (1974) medium with 0.1 M sucrose instead of 0.4 M glucose but with the same growth regulator composition. When small aggregates were obtained, the calli were plated on K3 (Nagy & Maliga 1976) medium with 0.25% agarose, and the same growth regulator supplements. These cultures were kept under light for about a m o n t h . Plant regeneration Plants were successfully raised from the protoplast derived colonies, when 2-3 m m in diameter, using the same procedure as described earlier for plant regeneration from callus.

Results Callus initiation and somatic embryogenesis All the hypocotyl explants produced spherical callus as well as friable callus, both of which were pale yellow, after 3-4 weeks of culture on MS medium supplemented with kn (I.0 ppm) and 2,4-D (I. 0 ppm). The spherical callus was subcultured in 12 tubes each of different media (Table 1) for further embryogenic /organogenic response. 10-15 torpedo-shaped somatic embryos (fig. Ic) were obtained from one gram of spherical callus on reducing the concentration of 2,4-D in the callusing medium to 0.05 ppm.

I. 2. 3. 4. 5. 6. 7. 8. 9. I0. II

Calinsing

kn(1) + kn(1)+2,4-D(1) ++ kn(1)+IAA(1) + kn(1)+NAA(1) + kn(0.5)+2,4-D(0.05) + kn(0.5)+IAA(0.1) + kn(0.5)+NAA(0.1) + kn(1)+NAA(0.1) + kn(1)+2,4-D(0.05) + kn(1)+NAA(0.1)+GA.(1) + kn(I.5)+NAA(0.1)+~A3(I ) +

Organogenesis

+R +R +R +* +R +R +R +* +R +R

Embryogenesis

+ + + + +++

MS medium Was supplemented with growth regulators at concentrations shown in parenthesis (in ppm) * Abnormal growth of shoots R Roots +,++ Relative rate of callus growth or extent of organogenesis

Replacement of 2,4-D with 0.I p p m of NAA gave nearly double the number of embryos, while complete removal of auxin failed to induce embryogenesis. These experiments were repeated thrice with similar results. Several growth regulating substances such as gibberellic acid, phenyl acetic acid, abscisic acid, adenine and methionine etc. were tested for their effect on embryogenic response on kn, NAA medium. A tenfold increase was observed with 1 p p m of gibb erellic acid (GA 3 ). None of the other substances had any effect. Increase or decrease of kinetin from 1 p p m in the callusing medium was found to be detrimental for embryogenesis. After 3 to 4 weeks of culture on embryo differentiation medium, torpedo shaped embryos developed further into bipolar structures and after 3-7 more days they were ready to be transferred to a medium devoid of growth regulators. The callus continued to produce embryos even after I012 subcultures on this medium. Secondary

embryogenesis

and plant formation

The embryos produced on embryo differentiation medium gave rise to secondary embryos when subcultured on solidified A~ medium 5 devoid of growth regulators. Each embryo gave rise to 6-7 secondary embryos from the hypocotyl and cotyledonary region after enlargement (fig. le). These secondary embryos developed in the same manner as the primary embryos during subsequent culture. Both primary and secondary embryos readily developed distinct roots and shoots (fig. Id) on transfer to liquid A 3 medium. After a week these plants were transferred to solidified AI medium. If left for more than a week in the liquid medium, the embryos became hard and failed to grow further. For a week the plants were grown on

429

Fig. I: Development of plants from Brassica ni~ra hypocotyls and hypocotyl protoplasts (a) Freshly isolated hypocotyl protoplasts (X200). (b)-Dividing protoplasts forming colonies (X200) 8 days after culture. (c) Somatic embryos from hypocotyl-derived callus at various stages of development (X5.3). Similar somatic embryos were also formed from protoplast-derived callus (Photo not shown). (d) Plantlet obtained from hypocotyl-derived embryo (X5). (e) Primary embryo from hypocotyl showing secondary embryos. (f) Protoplast-derived plant growing in a pot. moist sand under aseptic conditions for hardening. Subsequently they were transplanted into peatmosssoil (1:3) in p o t s a n d a l l o w e d to d e v e l o p to maturity under natural conditions. About 90% of the embryos, both primary and secondary, formed plants. Out of 169 p l a n t s t r a n s f e r r e d to s o i l , 134 survived, flowered and set seeds. Plants were not hand-pollinated but those derived from d i f f e r e n t p a r e n t s w e r e grown t o g e t h e r to p e r m i t natural crossing. B_.:. n i g r a i s r e p o r t e d to b e s e l f incompatible but no direct tests were made r e g a r d i n g c o m p a t i b i l i t y of p l a n t s d e r i v e d from t h e same p a r e n t . 89% of t h e p o d s c o n t a i n e d s e e d s a n d on sowing 81% of t h e seeds produced healthy plants. Protoplast

culture and embryo formation

P r o t o p l a s t s i s o l a t e d from h y p o c o t y l s were a m i x t u r e of l a r g e h i g h l y v a c u o l a t e d p r o t o p l a s t s a n d small protoplasts with dense cytoplasm (fig. la).

H y p o c o t y l s from 6 d a y o l d s e e d l i n g s g a v e maximum y i e l d of p r o t o p l a s t s (1.6 x 10~ per ml). The f i r s t d i v i s i o n was o b s e r v e d on t h e 2nd o r 3 r d d a y of c u l t u r e b u t i f g r o w t h r e g u l a t o r s w e r e a d d e d to the enzyme solution, the first division occurred w i t h i n one d a y . The average division frequency in 15 s e t s of e x p e r i m e n t s was 33% a f t e r 7-8 d a y s of c u l t u r e ( f i g . l b ) . The calli obtained on t h e a g a r o s e medium w e r e t r a n s f e r r e d to MS medium w i t h 1 p p m e a c h of kn and 2,4-D for proliferation and then s u b c u l t u r e d on t h e e m b r y o d i f f e r e n t i a t i o n m e d i u m . 52% of t h e p r o t o p l a s t derived calli produced a large number of embryos within 30 d a y s of subculture. In c o n t r a s t t o t h e e x p l a n t d e r i v e d callus, the protoplast derived callus produced both embryos and roots. The p r i m a r y e m b r y o s also formed secondary embryos. The development of e m b r y o s to p l a n t s was t h e same as w i t h t h e hypocotyl derived embryos, i.e. like that shown

430 in f i g s . l c - e . Out of 32 p l a n t s t r a n s f e r r e d to s o i l , 25 w e r e grown to m a t u r i t y ( f i g . l f ) . The s e e d numbers of two p l a n t s w h i c h w e r e counted w e r e 644 and 1032. About 75% of t h e s e e d s germinated.

Discussion Plant regeneration from protoplasts of i m p o r t a n t c r o p s p e c i e s i s d i f f i c u l t and i s a major o b s t a c l e to t h e a p p l i c a t i o n of m e t h o d s of g e n e t i c e n g i n e e r i n g to p l a n t s . The p r e s e n t s t u d y o u t l i n e s an e f f i c i e n t and r e p r o d u c i b l e method for the f o r m a t i o n of f e r t i l e p l a n t s in l a r g e numbers from somatic t i s s u e s and p r o t o p l a s t s of B__:_.n i 6 r a . The induction of embryogenesis by lowering the auxin concentration in the medium is similar to that described for other plant species such as carrot (Halperin and Wetherell 1964) and Brassicas (Glimelius 1984, Pareek and Chandra 1988). Embryo differentiation started within four weeks of subculture of B__t. nigra callus and embryo production continued even after 10-12 months of subculturing. The formation of secondary embryos in large number from embryos derived either from callus or protoplasts greatly enhances the e f f i c i e n c y of t h e r e g e n e r a t i o n s y s t e m . It i s n o t e w o r t h y that successive transfers t h r o u g h four d i f f e r e n t media w e r e n e c e s s a r y for plant production. For normal growth and d e v e l o p m e n t of e m b r y o s , a s h o r t p a s s a g e in l i q u i d basal medium without growth regulators was essential. E m b r y o g r o w t h on media w i t h o u t growth regulators for plant formation has also been d e s c r i b e d for B_a_. c a m p e s t r i s ( B h a t t a c h a r y a and Sen 1980) and B__t juncea ( K i r t i and C h o p r a 1989). With B__u n i ~ r a , however, only passage through l i q u i d medium w i t h o u t g r o w t h r e g u l a t o r s gave r i s e to p l a n t s w h e r e a s growth on s o l i d medium of t h e same c o m p o s i t i o n gave r i s e only to s e c o n d a r y embryos. GA3 was found to be m a r k e d l y s t i m u l a t o r y for e m b r y o g e n e s i s in Bz_. n i g r a as was a l s o o b s e r v e d with leaf callus (Sacristan 198t ) and callus d e r i v e d from m e s o p h y l l p r o t o p l a s t s of r a p e s e e d (Kartha et a l . 1974, P e l l e t i e r et a l . 1983). The protoplast regeneration procedure d e s c r i b e d in t h i s p a p e r i s being u s e d for t h e i n t r o d u c t i o n of kanamycin m a r k e r gene along w i t h GUS r e p o r t e r gene into B__u. n i g r a by d i r e c t DNA u p t a k e and t h e p r o d u c t i o n of s y n t h e t i c B__u. juncea from B__t camloestris and t r a n s f o r m e d B t. n i ~ r a . Acknowledgements We thank Ms. Rina Aggarwal for technical assistance and Dr. Arundhati Mukhopadhyay and Dr. Deepak Pental for providing useful suggestions during the protoplast work.

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