L-serine, 0.25 mg/liter 6-benzylaminopurine, 0.5 rag/liter 2,4-dichlorophenoxyacetic acid, ... protoplasts developed into plantlets within 4 wk after transfer onto 2N ...
IN VITROCELLULAR~t DEVELOPMENTALBIOLOGY Volume 23, Number 6, June 1987 9 1987 Tissue Culture Association,Ipc.
PLANT REGENERATION
F R O M B R A S S I C A N I G R A IL.D K O C H S T E M P R O T O P L A S T S
PHAN V. CHUONG', K. P. PAULS. ANDW. D. BEVERSDORF
Crop Science Department. Universityof Guelph. Guelph. Ontario. Canada N1G 2W1 (Received 11 December 1986; accepted 11 December 1986)
SUMMARY Protoplasts of Brassica nigra (L.) Koch were isolated from stem peels of bolting racemes and cultured in 1.5 ml of VN1 liquid medium. The protoplasts in the liquid medium were plated on top of half strength MS medium supplemented with 400 mg/liter glutamine, 15 rag/liter glutathione, 50 mg/liter L-serine, 0.25 mg/liter 6-benzylaminopurine, 0.5 rag/liter 2,4-dichlorophenoxyacetic acid, 1.5% sucrose, and 5% mannitol, pH 5.7, solidified with 0.3% agarose. Ten percent of calli obtained from the protoplasts developed into plantlets within 4 wk after transfer onto 2N regeneration medium which contains MS salts plus 200 rag/liter casein hydrolysate, 0.625 mg/liter 6-benzylaminopurine, 0.625 mg/liter kinetin, 0.625 rag/liter 6-(y,y-dimethylallylamino)-purine, 0.625 mg/liter zeatin, 0.5 mg/liter 1-naphthaleneacetic acid, 1.5% sucrose, and 0.4% agarose. This is the first report of plant regeneration from B. nigra protoplasts.
Key words: Brassica nigra; protoplast culture; regeneration. mannitol, pH 5.7 {filter sterilized). The preparations were agitated for 16 h with a rotary shaker reciprocating at 80 rpm. The protoplasts released by this treatment were filtered through two layers of nylon screen and washed as described in a previous paper ~5). After the last wash, protoplasm were resuspended in filter-sterilized VN1 medium consisting of onethird strength CPW, 13% mannitol, and two-third strength modified Nitsch and-Nitsch medium t5) without antibiotics; this mixture was supplemented with 0.25 rag/liter 6benzylaminopurine and 0.5 mg/liter 2,4-dichlorophenoxyacetic acid. One and a hal/ milliliter of the protoplast suspension containing l0 s cells/ml were plated on top of a thin layer of 0.3% agarose containing hal/ strength Murashige and Skoog medium salts I13) supplemented with 400 rag/liter glutamine, 15 rag/liter glutathione, 50 rag/liter L-serine, 0.25 mg/liter 6-benzylaminopurine, 0.5 mg/liter 2,4dichlorophenoxyacetic acid, 1.5% sucrose, and 5% mannitol, pH 5.7, in 15 X 60-mm Falcon plates. The protoplast cultures were incubated at 25 ~ C in the dark for 10 to 15 d and then gradually acclimated to the light. After 4 wk in culture, the microcalli that developed from protoplasts were subcultured onto 2N regeneration medium containing MS medium ingredients plus 200 mg/liter casein hydrolysate, 0.625 rag/liter 6benzylaminopurine, 0.625 mg/liter kinetin, 0.625 mg/ liter zeatin, 0.625 mg/liter 6-{r,r-dimethylallyamino~purine, 0.5 mg/liter 1-napthaleneacetic acid, 1.5% sucrose, and 0.4% agarose, pH 5.7 iadjusted before adding agaroseL Shoots that developed from the callus were excised and then induced to root by subculturing onto H3 medium containing MS salts supplemented with 1 rag/liter 1-napthaleneacetic acid, 3% sucrose, 0.6% agar, pH 5.7.
INTRODUCTION Protoplasts of five Brassica species have been demonstrated to be totipotent I1,3,4,6,9-11,16,19). In this list only B. nigra is absent from the six genotypes that are related in the Brassica genus as described by U [18L This report describes a procedure for plant regeneration from protoplasts isolated from stem peels of B. nigra. Currently interest in this area is being stimulated by the possible applications of protoplast fusion in Brassica species for crop improvement {2,7,12,14,15,17). MATERIALS AND METHODS Plants of B. nigra cv R-3099 were grown at 25~ C in pots containing Metro-mix 245 tGrace & Co of Canada, OntarioL Light was supplied for 16 h daily by fluorescent tubes to give approximately 200 ~mol per m 2 per s of photosynthetic radiation at the bench level. Plants were fertilized daily to induce optimal growth. The main racemes of the plants were sampled when the internodes were about 2 to 3 cm long. Two or three of the uppermost internode segments were immersed in 70% ethanol for several seconds and then placed in 5% of commercial bleach {5.25% sodium hyprochlorite} for 10 to 15 rain. After rinsing several times with sterile deionized water, the stems were peeled with forceps and the peelings were placed into an enzyme mixture consisting of 1.5% Cellulase Onozuka R-10 ~Yakult Pharmaceutical Industries, Nishinomiya, Japan), 0.3% Macerase [Calbiochem, La Jolla, CA) in CPW salts ~19), and 10% ' To whom correspondence should be addressed at Plant Research Centre, Central Experiment Farm, Building 21, Ottawa, Ontario, Canada K1A 0C6. 449
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FIG. 1. Freshly isolated protoplasts from stem peels of B. nigra cv R-3099. Arrow indicates type C protoplast. Bar = 50 ~m. FIG. 2. First division of stem peel protoplasts after 5 d in culture. Bar = 5 0 ~ m . FIG. 3. Microcalli derived from stem peel protoplasts settling on the agarose layer after 3 wk in culture. Bar -= 1000 ~m. FI6. 4. Plate of microcalli developed from protoplasts after 4 wk in culture. Bar = 10 m m . FIG. 5. Shoot initiation on callus derived from stem peel protoplasts 4 wk after transfer of the microcalli onto 2N medium. Bar = 10 m m . FIG. 6. Mature plants recovered from stem peel protoplasts of B. nigra cv R-3099.
BRASSICA NIGRA PROTOPLAST CULTURE
RESULTS AND DISCUSSION The stem peels, which were approximately 0.3 to 0.5-mm thick, were microscopically observed to consist of the epidermis, cortex, and vascular bundles of the stem. This was also evident from the fact that the crude protoplast preparations contained xylem elements. Protoplasts isolated from the stem peels could be classified into three types: ta) type A, small, nonpigmented, vacuolated, chloroplast/tee protoplasts; (b) type B, large protoplasts containing many chloroplasts; and (c) type C, nonpigmented, densely cytoplasmic protoplasts (Fig. 1 ). After plating on the thin agarose medium the protoplasts drifted freely in the liquid phase of the medium and did not adhere to the agarose surface. The first cell divisions were observed 3 to 4 d after initiating the cultures. Second and third divisions occurred after 7 d (Fig. 2). Cell division frequencies were 5% at Day 5 and 15% by D a y 10. Cell browning and lysis occurred at Day 10 but not more than 5% of microcolonies were affected. Microcolonies ranging in size from 300 to 500 /~m were observable with unaided eyes after 3 wk of culture (Fig. 3). Usually by this time the volume of the liquid media had decreased enough to leave the microcolonies exposed on the surface of the agarose layer. Exposed microcolonies tended to develop faster than those that remained submerged in the liquid medium. By the 4th wk microcalli about 500 to 700 #m were observed IFig. 4). Typically, 600 to 1000 microcalli were produced per l0 s cells plated. The microcalli were transferred to 2N medium to induce cell proliferation and shoot formation. One month after transfer approximately 10% of the calli which had developed from microcalli produced shoots iFig. 5). Subculture of excised shoots onto H3 medium induced rooting within 2 to 3 wk. Rooted shoots were transplanted to pots containing Metro-mix and grown to maturity (Fig. 6). Protoplasts, which were highly cytoplasmic and showed active streaming (type C), seemed to have the highest cell division frequency of the three types of protoplasts described earlier. Protoplasts without chloroplasts accounted for 90% of the dividing cells seen in the cultures. Similar results were observed for the stem protoplasts isolated from haploid lines of B. napus tunpublished data). The agarose layer served primarily to prevent the protoplasts from adhering to the bottom of the plate, thereby minimizing the damage to microcalli during their transfer to regeneration medium. In addition, several other beneficial effects were noted. The agarose layer served as a supplementary source of nutrients, and provided a support medium for the microcolonies that became exposed during their growth, thus facilitating gas exchange. Moreover, it seemed from comparisons of cultures with and without the agarose layer that cell browning and lysis was minimized in those in which the layer was present. The results of this study indicate that stem peels are a good source of protoplasts for plant regeneration in B. nigra. Large numbers of protoplasts can easily be obtained after enzymatic digestion of the cell walls in this
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tissue. The protoplasts seem to be stable during the isolation procedures and show little tendency to lyse and release brown particles into the culture medium. Because stem sections can be subjected to rigorous surface sterilization procedures, contamination of the protoplast cultures is not a problem if the original plant is disease-free and has not suffered insect damage. The amount of stem material that can be obtained from one plant can be increased by trimming the old raceme to induce new ones to develop. Plant regeneration frequencies from calli derived from Brassica protoplasts have been reported to range from 1 to 70%. Specifically, 70% regeneration from B. napus hypocotyl protoplasts (1,9), 20% from B. carinata hypocotyl protoplasts ~6), 5 to 20% from B. juncea mesophyll protoplasts (10), 20% from B. oleracea mesophyll protoplasts (8), and 1% from B. campestris hypocotyl protoplasts (9) have been observed. Compared to these results a regeneration frequency of 10% for B. nigra protoplast-derived calli is on the low side but is still usable for protoplast fusion experiments.
REFERENCES 1. Barsby. T. L.; Yarrow. S. A.: Shepard, J. K. A rapid and efficient alternative procedure for the regeneration of plants from hypocotyl protoplasts of Brassica napus. Plant Cell Rep. 5:101-103: 1986. 2. Barsby. T. L.; Chuong, P. V.; Yarrow, S. A.. et al. Somatic transfer of cytoplasmic traits in Brassica. Theor. Appl. Genet. (in press!. 3. Bidney. D. L.; Shepard, J. F.; Kaleikan, E. Regeneration of plants from mesophyll protoplasts of Brassica oleracea. Protoplasma 117:89-92; 1983. 4. Chatterjee, G.; Sikdar, S. R.: Das, S., et al. Regeneration of plantlets from mesophyU protoplasts of Brassica juncea (L.) Czern. Plant Cell Rep. 4:245-247; 1985. 5. Chuong, P. V.: Pauls. K. P.; Beversdorf, W. D. A simple culture method for Brassica hypocotyl protoplasts. Plant Cell Rep. 4:4-6: 1985. 6. Chuong, P. V.; Pauls, K. P.; Beversdorf, W. D. Protoplast culture and plant regeneration from Brassica carinata Braun. Plant Cell Rep. 6:67-69; 1987. 7. Chuong, P. V.; Powell, A. D.; Pauls, K. P., et al. Somatic transfer of cytoplasmic traits in Brassica napus by haploid protoplast fusion. Proc. Cruci/er Genet. Workshop III: Univ. of Guelth, Guelth, Ontario, Canada; 1986: 62. 8. Fu, Y.; Jia, S.; Lin, Y. Plant regeneration from mesophyll protoplast culture of cabbage (Brassica oleracea var capitata). Theor. Appl. Genet. 71:495-499; 1985. 9. Glimelius. K. High growth rate and regeneration capacity of hypocotyl protoplasts in some Brassicaceae. Physiol. Plant. 61:38-44: 1984. 10. Kao. H. M.; Seguin-Swartz. G. Culture and regeneration of plants from mesophyll protoplasts of Brassica napus and B. juncea. Proc. Crucifer Genet. Workshop IlI: Univ. of Guelth, Guelth. Ontario. Canada; 1986: 59. 11. Kartha, K. K.; Michayuk, M. R.; Kao. K. H., et al. Callus formation and plant regeneration from mesophyll protoplasts of rape plant. Plant Sci. Lett. 3:265-271; 1974. 12. Klimaszewska, K. A.; Keller, W. A. Protoplast fusion of Brassica napus with other Cruci/erous species. Proc. Cruclfer Genet. Workshop III: Univ. of Guelth. Guelth. Ontario, Canada; 1986: 59.
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13. Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497; 1962. 14. Pelletier, G.; Primard, C.; Vedel, F., et al. Intergeneric cytoplasmic hybridization in Cruciferae by protoplast fusion. Mol. Gen. Genet. 191:244-250; 1983. 15. Schenck, H. R. A.; Robbelen, G. Somatic hybrids by fusion of protoplasts from Brassica olerace and B. campestris. Z. Pflanzenzucht. 89:278-288; 1982. 16. Sproule, A.; Keller, W. A.; Webb, J. A. Plant regeneration from stem cortex protoplast of Brassica ]uncea. Proc. Crucifer
Genet. Workshop III: Univ. of Guehh, Guelth, Ontario, Canada; 1986: 60. 17. Taguehi, T.; Kameya, T. Production of somatic hybrid plants between cabbage and Chinese cabbage through protopiast fusion. Jpn. J. Breed. 36:185-189; 1986. 18. U, N. Genomic analysis in Brassica with special reference to the experimental formation of Brassica napus and peculiar mode of fertilization. Jpn. J. Bot. 7:389-543; 1935. 19. Xu, Z. H.; Davey, M. R.; Cocking, E. C. Plant regeneration from root protoplasts of Brassica. Plant Sci. Lett. 24:117-121; 1982.
