PlantCeU Reports

Plant Cell Reports (1986) 5 : 150-154

© Springer-Verlag 1986

Plant regeneration by organogenesis in Glycine max M. S. Wright, S. M. Koehler, M. A. Hinchee, and M. G. Carnes Monsanto Agricultural Company, 700 Chesterfield Village Parkway, St. Louis, MO 63198, USA Received December 23, 1985 / Revised version received February 4, 1986 - Communicated by I. K. Vasil

MATERIALS AND METHODS

ABSTRACT A procedure for the regeneration of f e r t i l e plants by organogenesis from tissue cultures of soybeans, Glycine max is described. Seedswere germinated on reduced inorganic salt MS medium containing 5pM BA. Cotyledonary nodes were excised and cultured on the same medium. Presence of BA in the medium during seed germination and culture of nodal explants was required for multiple shoot and shoot-bud formation. Histological analyses established the de novo nature of shoot regeneration. Separatereduction of the concentration of inorganic salts or substitution of sucrose for fructose during culture had minimal effects on the regeneration response. Conversely, i f the BA was reduced, the inhibition response could not be overcome by increased salt concentration or altered carbon source.

The schematic outline of the protocol used is shown in Figure i. Surface s t e r i l i z e d seeds (3 seeds/lO0 x 25 mm Petri dish) of Glycine max (L.) Merrill cv. Wayne were germinated on MS (Murashige and Skoog, 1962) mediumcontaining half the recommended concentration of inorganic salts (1/2MS). Half the plates contained 5pM BA and the other half, no supplementary hormones. After 14 days, the cotyledonary nodes were excised from the seedlings. The epicotyl was removed within 2-3mm of the node and the hypocotyl and any a x i l l a r y shoots were cut flush with the node. The cotyledons were removed within 5mm of the node. The piece of nodal tissue cultured was 0.5-1.0 cm long and was placed basipetally on 1/2MS agar medium containing 5pM BA (1/2MS/SBA). Four nodal explants were placed in each I00 x 15 mm Petri dish. After three days half of the nodes from each germination

ABBREVIATIONS

Figure 1

Experimental Protocol for Establishing Conditions for Soybean Shoot Organogenesis

BA, benzyladenine; IAA, indoleacetic acid; SAS, secondary a x i l l a r y shoots; MS, Murashige and Skoog (1962) medium.

Germinate surface sterilized seed on I/2MS/5BA or I/2MS/0BA 14 days

INTRODUCTION Until recently, only regeneration of smallseeded legumes, i.e. a l f a l f a (Saunders and Bingham, 1982) and clover ( P e l l e t i e r and P e l l e t i e r , 1971), as opposed to large-seeded legumes, had been accomplished in v i t r o . Regeneration of plants from tissue cultures of soybean, commercially the most important species of legumes, has proven very difficult. There are reports of limited somatic embryogenesis in soybean tissue cultures (Christianson, et al. 1983 and Lippmann and Lippmann, 1984). Accounts of low frequency shoot morphogenesis (Cheng, et a l . , 1980) have also been published. This report describes conditions necessary for high frequency shoot production from soybean tissue culture. The conditions examinedwere (1) preconditioning the seedlings by germination on medium containing BA, (2) sequential reduction of inorganic salts, (3) reduction of BA during culture, and (4) use of fructose versus sucrose in the medium to stimulate shoot break.

Offprint requests to: M. S. Wright

Excise and explant to I/2MS/5EA 3 days

Subculture l I/4MS/5EA

[ I/2MS/5BA

4 days

[ 4 days

v (l)*

(2)

I/4MS IBA Fructose

1/4MS IBA Sucrose

v (3) 1/4MS 5BA Fructose

(4)

IS)

1/4MS 5BA Sucrose

(6)

1/2MS IBA Fructose

1/2MS IBA Sucrose

(7) 1/2MS 5BA Fructose

(8 ) 1/2MS 5BA Sucrose

14 days, ist transfer Count, Separate and Replate on same medium (2nd Subculture) I

21-30 Days, 2nd - 6th transfer

Count, Separate and Replate on same medium (3rd-7th Subculture)

*Experiment numbers refer only to treatments including BA throughout the total germination and culture period.

151 medium were transferred to fresh 1/2MS medium and h a l f to reduced s a l t medium, 1/4MS, both containing 5NM BA. Four days l a t e r , the nodes were again transferred. At t h i s point each node was plated separately in a 60 x 15 mm Petri dish. Salt concentration of the medium was not changed. Nodes cultured on 1/2MS were replated on 1/2MS and those that were on 1/4MS were replated on 1/4MS. At this subculture, INM BA was compared to 5NM BA as the hormone supplement and sucrose (30 g / l ) was compared to fructose (30 g / l ) as the carbohydrate source. There were ten nodes (plates) per treatment. After an a d d i t i o n a l 14 days, shoots and shoot-buds (shoots less than 0.5 cm t a l l ) were counted (Fig. 2a). Thereafter, the nodes were subdivided into

two to four pieces each and shoot and shoot-bud counts taken every 21-30 days p r i o r to t r a n s f e r to fresh media through six a d d i t i o n a l transfers. At each t r a n s f e r , shoots were removed to a rooting medium for f u r t h e r maturation. Shoots were rooted in 100 x 15 mm Petri dishes on B5 medium (Gamborg et a l . , 1968) containing no exogenous hormones (MB5-O). After about one month, the rooted shoots were transferred to fresh MB5-O in Plant-Cons (Flow Labs) f o r three to four weeks. The p l a n t l e t s with two to three t r i f o l i a t e leaves were then planted in gravel contained in p l a s t i c boxes f i t t e d with covers. The plants were watered d a i l y with 1/2 strength modified Hoagland's n u t r i e n t s o l u t i o n ( L i p s t e i n , 1972). High humidity was maintained. As the plants adjusted and grew, the humidity was gradually decreased u n t i l the plants were able to be placed in pots and subsequently grown to maturity in the greenhouse under standard conditions. Seeds were collected from f u l l y mature tissue culture-derived plants. All manipulations p r i o r to the gravel culture were a s e p t i c a l l y performed. All cultures were incubated in 12 hours of l i g h t , ~ 39NE/sec, and 12 hours of darkness at 21-25°C. Cotyledonary node tissue at d i f f e r e n t stages of culture was fixed in formalin:lO% acetic acid:l% chromic acid:water ( 1 : 2 : 3 : 4 ) , dehydrated in the ethyl a l c o h o l - t e r t i a r y butyl alcohol series and embedded in Paraplast. Paraplast-embedded tissue was soaked in g l a c i a l acetic acid:70% ethanol (1:4)

Figure 2a A representative cultured node at the f i r s t count 21 days from explant, t r e a t ment 7. Seed was germinated on 1/2MS/ 5BA, 14 days, excised and explanted onto 1/2MS/SBA, 3 days, subcultured on 1/2MS/5BA, 4 days, and cultured on 1/2MSf/5BA.

for 1-3 days to soften the tissue p r i o r to sectioning. Longitudinal sections (10 Nm) were cut and stained in safranin and fast green for h i s t o l o g i c a l examination.

RESULTS & DISCUSSION Our results c l e a r l y demonstrate that m u l t i p l e plant regeneration can be r e l i a b l y obtained from tissue cultures of soybean. As shown in Table 1, germination of the seeds on media containing BA, a synthetic c y t o k i n i n , was found to be useful for m u l t i p l e bud production. In subsequent culture of the excised nodes, only those germinated on BA produced m u l t i p l e buds. In most of the treatments, bud production peaked in the t h i r d t r a n s f e r a f t e r three months of culture with approximately 20 buds per node. As expected, shoot production peaked in the fourth t r a n s f e r with 20 shoots per node a f t e r four months. Treatments 3 and 8 are exceptions. Treatment 3 consistently produced a large number of buds; however, many did not mature. Treatment 8 peaked in shoot production at the fourth t r a n s f e r , but the number of shoots showed an unexpected increase at the sixth transfer. Unfortunately, the experiment was terminated before the seventh t r a n s f e r and i t is not known how long these two treatments would have continued increased bud and shoot production.

Figure 2b

Wayne soybean seedling germinated 14 days on 1/2MS/5BA. Note the thickened nodal area which w i l l give r i s e to the m u l t i p l e shoots and buds.

Tran Thanh Van (1981) has stated that "the 'inherent c e l l u l a r state' which the explant has brought along into the culture medium a f t e r i t s excision, as well as the 'past of the mother p l a n t , ' are as important components [to regenerat i o n ] as the n u t r i t i o n a l , phytohormonal, or environmental factors being applied to the explant." Thus, even with i d e n t i c a l culture conditions, but without germination on BA medium, no m u l t i p l e bud formation was observed in the present experiments.

152 T a b l e i.

Shoots

(SHT), b u d s

(B) and s e c o n d a r y a x i l l a r y shoots

t h r o u g h six s u c c e s s i v e t r a n s f e r s .

days on I / 2 M S / 5 B A and four days on 1/2 or I/4MS/5BA, All c o u n t s w e r e b a s e d on i0 n o d e s / t r e a t m e n t

Germination

Treatment

SHT

*BA -BA

i) I/4MSf/IBA

2±i

+BA -BA

Ist Transfer B

(SAS) o b s e r v e d f r o m c o t y l e d o n a r y nodes of v a r i e t y W a y n e

All seeds w e r e g e r m i n a t e d 14 days on 1/2MS m e d i a ± 5 pM BA, c u l t u r e d t h r e e t h e n t r a n s f e r r e d to one of the e i g h t t r e a t m e n t s

listed.

and are r e p o r t e d ± STD error.

SAS

2nd Transfer SHT B

3rd Transfer SHT B

4th Transfer SHT B

5th Transfer SHT B

6th Transfer SHT B

6±]

3±]

3±I

4±I

4±1

3±I

I±I

2±1

0±0

I±I

6±I 0

2±0

0

2) I/4MSs/IBA

2±I 0

4i2 0

2±0