PlantCell Reports

Plant Cell Reports (1995) 15:282-286

9 Springer-Verlag1995

Plant regeneration from protoplasts of immature Vigna sinensis cotyledons via somatic embryogenesis Xue-Bao Li 1, Zhi-Hong Xu 2, and Zhi-Ming Wei 2 t Department of Biology, Central China Normal University, Wuhan 430070, China 2 Shanghai Institute of Plant Physiology, Chinese Academy of Sciences, Shanghai 200032, China Received 13 September 1994/Revised version received 7 July 1995 - Communicated by J. Widholm

SUMMARY. Protoplasts were isolated from immature cotyledons of Vigna sinensis and cultured in a

INTRODUCTION Vigna sinensis (Vigna unguiculata, cowpea) is

modified MS Liquid medium containing 0. 2 m g / l 2, 4-dichlorophenoxyaeetie acid ( 2 , 4 - D ) , 1 rag/1 naphthaleneacetie acid ( N A A ) a n d 0. 5 rag/1 6-benzylaminopurine ( B A P ) i n the dark at a density of 1 )< 105/ml. T h e protoplasts began to divide in 3-5 days. Sustained cell division resulted in formation of cell clusters and small calli, with the cell division frequency and plating efficiency of cell colonies reaching 27. 7% and 1.70//00 respectively. When calli of 2 m m in size were transferred onto MSB medium ( M S salts and B5 vitamins) containing 500mg/l NaC1, 500 r a g / l casein hydrolysate ( C H ) , 2 mg/1 2 , 4 - D and 0. 5 rag/1 BAP for further g r o w t h , approximately 5 ~ of the calli developed embryogenically. T h e embryogenic calli were selected and subcultured on the same composition of MSB medium and were able to maintain somatic embryogenesis capacity in subculture for a long time. When the ealli were moved to MSB medium with 0. 1 rag/1 indole-3-acetic acid ( I A A ) , 0. 5mg/1 kinetin ( K T ) , 3-5 ~ mannitol and 2 ~ sucrose in the light, m a n y somatic embryos formed from the calli. Only part of the embryoids developed further to the cotyledonary stage, and the others died at the globular, heart-shaped or torpedo stages. Finally, some cotyledonary embryoids germinated and developed into plants or shoots. T h e shoots were readily rooted on 1/2 strength MS medium with 0. 1 - 0. 3 rag/1 indole-3-butyric acid ( I B A ) . T h e plants grew well in soil and were fertile.

one of the important vegetable crops in the world. Davey et al. (1974) cultured cowpea protoplasts isolated from leaves and obtained callus formation. Later, callus formation and root differentiation were also observed in cowpea protoplast culture (Jha and R o y , 1980 ; Steinbiss and Li, 1983 ;Gill et al. , 1987). In recent years, intensive studies on protoplast culture have led to some success in some grain legumes, including soybean (Glycine maze)(Wei and Xu, 1988~ Zhang et al. , 1 9 9 3 ) , pea (Pisum sativum) (Renate and Jacobsen, 1989), Adzuki bean (Phaseolus angularis) (Ge et al. , 1989), moth bean (Vigna aconitlfolia) (Shekhawat and Galston, 198 3 ~Gill and Eapen, 1986) and peanut (Arachis hypogaea) (Wei et al. , 1993). However, it has not been possible to regenerate shoots or plantlets from cowpea protoplasts. We describe here the conditions of somatic embryogenesis and plant regeneration from protoplasts of immature cotyledons of cowpea.

Key words: Vigna sinensisr protoplast culture~ somatic embryogenesis ~ plant regeneration.

Abbreviations = 2, 4-D, dichlorophenoxyacetic acid ~ NAA, naphthaleneacetic acid ~ BAP, 6-benzylaminopurine ~IAA, indole-3-acetic acid ~ KT, kinetin ~ IBA, indole-3-butyric acid CH, casein hydrolysate ~ CM, coconut milk ~ZT, zeatin. Correspondence to: Xue-Bao Li

MATERIALS AND METHODS Plant material Plants were cultivated in the phytotron of Shanghai Institute o:[ Plant Physiology (7500 lux, 12 hours/day, 25 ~ ) or in the fields from seeds of eowpea (eultivar Zhigang No. 28) for providing immature pods. Isolation and culture of protoplasts The immature pods, 10-15 days after anthesis, were collected and exposed to low temperature (4~ for 3-5 days. The pods were surface-sterilized with 70~(V/V)ethanol for 5 minutes, and then young seed were dissected from the pods under aseptic conditions. The cotyledons were cut transversely into approximately 1 mm thick slices after removing seed coats and plasmolizing for 2-3 hours in CPW-13M solution (CPW solution containing 13 ~ (W/V) mannitol, PHS. 6) (Frearson et aL , 1973). The cotyledon slices were then incubated in an enzyme solution containing 4% Onozuka R-10, 0. 3%

283 Macerozyme R-10 and 2% Hemicellulase dissolved in CPW9M solution (CPW solution with 9~ mannitol, PHS. 6) on a shaker (50 rev/min) at 25~ for 16-20 hours. The enzyme mixture was filtered through a 45 /*m nylon sieve to remove debris, and the protoplasts were harvested by centrifugation (80 X g) for 5 minutes. Pelleted protoplasts were resuspended in CPW-21S (the same salts solution as CPW-13M, but with 21% sucrose replacing mannitol) and centrifuged again. The floating protoplasts were removed, washed three times with CPW-gM solution by resuspending and centrifuging. Protoplasts were resuspended at a density of about 1 X 105/ml in modified MS liquid medium (Murashige and Skoog, 1962) supplemented with 0. 2rag/1 2, 4-D, 1 rag/1 NAA, 0.5rag/1 BAP,CH250 rag/l, CM 20 ml/1 and 7% glucose. Two ml of protoplast suspension solution was put into a 60 • 14mm Petri dish sealed with parafilm and cultured in the dark (25+ 1 ~ ). The cell division frequency was calculated as the percentage of counted cells that divided after 10 days. The cultures were moved under diffuse light of 500-800 lux after 15 days, and the media were diluted at 7-10 day intervals with 0. 5ml MS liquid medium containing 3% sucrose and the same growth regulators as above. All experiments were replicated 3-

5times.

Plant regeneration When protoplast-derived calli grew to about 2 mm in size, they were transferred onto MSB agar medium, containing MS minerals and B5 organic components (Gamborg et al. , 1968), supplemented with 500 mg/l NaCI, 500 rag/1 CH, 2 rag/1 2,4-D and 0.5rag/1 BAP for further growth. Embryogenic catli developing on this medium were suhcultured on MSB medium with the same composition. The embryogenic calli were transferred onto MSB medium with 0.1 mg/l IAA, 0.5rag/1 KT, 2% sucrose and 3-5% mannitol in the light ( 2000 lux, providing by daylight fluorescent tubes, 12 hours / day , 25 fl: 1 ~ for somatic embryo formation and development, then they were transfered to MSB medium without mannitol for somatic embryo germination. Shoots 3-4 cm in height were cut and rooted on 1/2 strength MS medium with 0. 1-0. 3rag/1 indole-3-butyric acid (IBA). The regenerated plants were transplanted to soil in pots and grew in the field or greenhouse. All of experiments were replicated 3-5times. The composition of culture media used are shown in Table 1.

Table 1. Components of different media in protoplast culture and callus differentiation of cowpea immature cotyledons (rag/

1) medium

Pl

P2

Pa

P4

Ps

basic constituents

MS

MSB

MSB

MSB

1/2 MS

CH250

CH500

CH500

CH500

CH500

CM20ml glucose 7~

NaCI500 sucrose 3% agar O. 7 0/~

mannitol 3-5% sucrose 2 % agar O. 7%

sucrose 2 % agar O. 7%

sucrose 2 % agar O. 7%

2,4-D 0. 2 NAA 1.0 BAP 0.5

2,4-D 2.0 BAP 0.5

IAA 0. 1 KT 0.5

IAA 0.1 KT 0.5

IBA o. 1-0. 3

growth regulators pH

5.6

5.8

5.8

5.8

5.8

complementary constituents

RESULTS AND DISCUSSION Isolation o f protoplasts One of the important factors in protoplast isolation is the age of immature cotyledons used. Our experimental results indicated that the cowpea immature cotyledons less than 3-5 m m in length could not be used to isolate protoplasts, and the protoplasts from the immature cotyledons more than 10-12 m m in length, in which ceils contained a large amount of starch grains , disrupted within 24-48 hours of culture. T h e r e f o r e , the immature cotyledons 5-10 m m in length at 10-15 days-old were used for protoplast isolation. Viable protoplasts could be isolated from the cotyledons (Fig. 1) with a yield of 1. 5-2 X 106 protoplasts/g fresh weight.

The effects o f basic media and growth regulators an division o f protoplasts It was noticed that during protoplast c u l t u r e , t h e

g r o w t h state and division frequency of regenerated cells varied significantly with different basic media containing different g r o w t h regulators ( Table 2 ). Comparing the effects of M S , B 5 and K 8 P ( K a o and Michayluk, 1975) media on protoplast culture , M S medium was the most suitable for the g r o w t h and division of cowpea protoplasts. Modified M S medium supplemented with 0. 2 rag/1 2, 4-D, 1 rag/1 N A A and 0. 5 m g / l B A P (P1 m e d i u m ) gave the best result. A b o u t 8 7 . 4 ~ of the protoplasts survived at 2 days of culture in Pt medium ( T a b l e 2 ). T h e protoplasts started to divide after 3-5 days of culture (Fig. 2) , and cell division occurred at an average frequency of 2 7 . 7 % after 10 days of culture in P1 m e d i u m ( T a b l e 2 ) . T h e cultures were moved to diffuse light (500800 lux) to promote cell colony formation (Fig. 3). Protoplast-derived caUi could be observed with the naked eye after 4-6 weeks of culture ( Fig. 4) . T h e average plating efficiency of cell colonies reached about 1 . 7 ~ w h e n the protoplasts were cultured in P1

284 medium (Table 2) . Furthermore , our experimental results showed that glucose as Single carbon source and osmotic stablizer was more suitable for immature cotyledon protoplast culture than sucrose and mannitol. Only 10. 5 - - 1 4 . 1% cell division frequency and 0. 2 - 0. 5% cell plating efficiency were obtained

when the protoplasts were cultured in MS medium with 3~ glucose (or sucrose) and 7% mannitol, but the cell division frequency and plating efficiency were raised to 2 5 . 1 - - 3 0 . 9% and 1 . 6 - - 1 . 9 ~ ,respectively, when 7% glucose, instead of sucrose and mannitol, was added in the medium.

Table 2 Effects of basic MS, B5 and KSp media with different growth regulator compositions on cukure of immature cotyledon protop/asts of cowpea Cell Plating Growth regulator Protoplast division efficiency of Media compositions viability frequency cell colonies (rag/l) (%) (~) (%)

MS

2,4-D0.2,NAA1,BAP0.5 2,4-D0.2, NAA1 ,ZT0.5 2,4-D1 ,BAP1 2,4-D1,BAP0. 5

87.4 80.2 85.3 73.1

27.7 15.3 21.5 10.3

1.7 0.8 1.1 0.2

B5

2,4-D0.2,NAA1,BAP0.5 2,4-I)0. 2, NAA1,ZT0. 5 2,4-DI,BAP1 2,4-DI ,BAP0.5

70. 2 71.3 76.5 62.4

20. 5 19.6 21.5 I0.3

1.3 0. 9 I. 0 0.2

K8P

2,4-D0.2,NAAI ,BAP0.5 2,4-D0.2, NAAI,ZT0.5 2,4-DI,BAPI 2,4-D1,BAP0.5

61.0 57.8 60. 2 49.9

15.2 16. i 13.6 8.2

I. 1 0.5 0. 7 0.1

Mean in three replicate experiments.

Embryogenic callus formation and embryoid dif fe.rentiation There is a close relationship between the formation of embryogenic calli and growth regulator compositions in the medium. When calli of approximately 2 mm in size formed in Pt medium were transferred onto MSB agar medium with 0. 1-0. 5 rag/1 IAA and 1-5 rag/1 BAP ( K T or Z T ) , they grew and became dark-green, compact calli in the light (daylight fluorescent tubes). No shoots, but only a few roots formed from the calli in subsequent culture on this medium. When calli formed in P1 medium were transferred onto MSB medium containing 1-10 mg/l 2 , 4 - D or 1-30 rag/1 N A A alone, they grew rapidly, but became soft. No embryogenic calli were observed in subculture on the same medium as above. The calli failed to form shoots or embryoids when they were transferred to differentiation medium. However, the use of 1-2 rag/1 2 , 4 - D in combination with 0. 5 mg/l BAP stimulated the formation of embryogenic calli. After calli formed in P1 medium were transferred onto MSB medium with 2 m g / l 2, 4-D, 0. 5 rag/1 B A P , 500 rag/1 NaCI and 500 rag/1 CH (P2 medium), approximately 5o/oo of them developed into rapidly growing, yellow ( or yellowish) , compact and nodular embryogenic calli. T h e embryogenic calli

were selected and subcultured on the same composition of P2 medium every 2 weeks (Fig. 5). In order to support initiation and differentiation of somatic embryos to cotyledonary stage, the embryogenic calli were transferred from P2 medium to MSB medium with a lower concentration of growth regulators or without growth regulators. By comparing different growth regulator combinations, we found that using 0. 1 rag/1 IAA in combination with 0. 5 mg/l K T gave a better effect on formation of cowpea somatic embryos. For pea somatic embryo differentiation, enriching MSB medium with 3-5% mannitol to increase osmolarity and lowering the sucrose concentration to 2 % in the medium were necessary (Renate and Jacobson, 1989). In our experiment of cowpea embryoid differentiation, a similar result was also obtained. The embryogenic calli were transferred to MSB medium containing 0. 1 rag/1 I A A , 0 . 5 mg/1 K T , 3-5% mannitol and 2% sucrose (P3 medium) in the light (daylight fluorescent tubes). After a period of 10-15 days on this medium, many somatic embryos were induced to form in all the calli (Fig. 6). Only a portion of the somatic embryos developed further to the cotyledonary stage (Fig. 7 ) , and the others died at the globular, heartshaped or torpedo stages.

285

Fig. 1. Freshly isolated protoplasts of cowpea. 400 X Fig. 2. First and second divisions of protoplast-derived cells [n MS liquid medium after 5 days o~ culture. 4 0 0 X Fig. 3. Cell cluster from protoplast in MS liquid medium after 14 days of culture. 100X, Fig. 4. Calli formed in MS liquid medium after 30 days of protoplast culture in 60• 14ram Petri dish. Fig. 5. Embryogenie calli subcultured on MSB agar medium (P2 medium,flask of 60mm diameter). Fig. 6. Somatic embryos observed on the callus on MSB medium (P~ medium ,~lask of 80mm diameter). Fig. 7. An embryoid at the cotyledonary stage. 5 X Fig. 8. Plantlets developing ~rom embryoids on P4 medium(flask of 80ram diameter). Fig. 9. Shoots developing from embryoids on P, medium(tube of 50 mm diameter). Fig. 10. Plants regenerated from eowpea protoplasts in pot(30cm diameter).

286

Germination o f somatic embryos The cotyledonary embryoids did not develop further if they were not moved to fresh medium. In order to promote germination of the embryoids, the effects of some different growth regulator combinations were studied. The embryoids failed to germinate when they were transferred to growth regulator-free MSB medium. The embryoids germinated rapidly after they were transferred to a fresh MSB medium with lower concentration of growth regulators (P4 medium). However, only about 10-15% of the germinated embryoids developed further to form plantlets (Fig. 8 ) , and many of them recallussed, or only shoots developed and the root meristem recallussed at opposite poles (Fig. 9 ). The same phenomenon of embryoid recallussing (dedifferentiation) was also observed in Vigna aconitifolia (Eapen and George, 1990). All the shoots of 3-4 cm in height were readily rooted on half-strength MS medium with 0.1-0. 3 rag/1 IBA (Pc m e d i u m ) , resulting in the regeneration of whole plantlets. A total of 21 regenerated plantlets were obtined from the protoplasts in the three experiments. The plants were successfully transplanted to soil ,and grew normally and were fertile (Fig. 10). Seeds were obtained from the plants. CONCLUSIONS In protoplast culture of grain legumes, immature cotyledon protoplasts have been employed in protoplast culture studies (Wei and Xu, 1988~ Zhang, 1993;Wei et al. , 1993),which led to success of plant regeneration from protoplasts. Our experimental results also show that immature cotyledons, which are obtained and surface sterilized readily, are excellent material for protoplast culture of cowpea. Plant protoplast culture is a lengthy process. It can readily be seen that growth regulators in the subculture medium has a critical effect on somatic embryogenesis and plant regeneration. Ge et al. (1989) found that using relatively higher concentration of 2, 4-D in combination with lower concentration of BAP could effectively promote the formation of embryo-like structure of calli for protoplast culture of Adzuki bean. The effect of NaC1 and KC1 on increasing somatic embryogenesis frequency was studied (Galiba and Yanmada,

1988). Renate and Jacobsen(1989) indicated that using 3-5~ mannitol to increase osmolarity of medium was imperative for the formation and development of pea embryoids . In our expeximent, we combined 2, 4-D with BAP for the amplification of calli which could develop into embryogenic calli. Meanwhile, 500 mg/1 NaC1 was supplemented in the medium. The embryogenic calli were able to maintain their somatic embryogenesis capacity in subculture for a long time. When healthy embryogenic calli with frequent subculturing were transferred to MSB medium containing 0. 1 mg/1 1AA, 0. 5mg/1 K T , 3-5% mannitol and 2 ~ sucrose, somatic embryos quickly developed. T o date, there were no reports of plant regeneration for protoplasts of cowpea. Thus this report is the first to describe the production of plants from immature cotyledon protoplasts of cowpea by somatic embryogenesis.

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