Introduction of commercial protocol for in vitro proliferation of Brassica oleracea var. acephala

Document Type : Full Paper



In recent years, ornamental kale is commonly used in landscapes due to its colorful shoots and resistance to the diverce environmental conditions. Thus, its in vitro proliferation was studied in series of experiments. Different BA (0/10.1, 0.5 and 1 mg/L), 2,4-D (0.1, 0.5 mg/L) and silver nitrate (2, 4 mg/L) treatments were applied for the establishment stage. Treatments of the proliferation stage also included BA (0.1 and 0.5 mg/L) and IBA (0.1 and 0.5 mg/L). Acclimatization of in vitro plantlets was conducted using different concentrations of MS (complete and half strength) and vermicompost extract (direct and diluted). Results revealed that, using of 0.1 mg/L BA with one mg/L 2,4-D was able to provide regeneration in this ornamental plant, acceptably. Proliferation medium supplemented with 0.5 and 0.5 mg/L BA caused shoot development. Finally, in vitro plantlets were successfully adapted by transferring to media containing reduced MS medium without plant growth regulators. In conclusion, by using the relatively low cost, accessible media and the least concentrations of plant growth regulators, successful regeneration of kale ornamental were obtained.


Aslam, F. N., MacDonald, M. V., Loudon, P. & Ingram, D. S. (1990). Rapid-cycling Brassica species: inbreeding and selection of B. campestris for anther culture ability. Annal Botany, 65, 557-566.
Atiyeh, R., Lee, S., Edwards, C., Arancon, Q. & Metzger, J. (2002). The influence of humic acids drived from earthworm proessed organic wastes on plant growth. Bioresour Technology, 84(1), 7-14.
Baillie, A. M. R., D. Epp, J., Hucheson, D. & Keller, W. A. (1992). In vitro culture of isolated microspores and regeneration of plants in Brassica campestris. Plant Cell Report, 11, 234-237.
Biddington, N. L. (1992). The influence of ethylene in plant tissue culture. Plant Growth Regulations, 11, 173-187
Chamandoosti, F. & Afshari Azad, H. (2012). Somaclonal variation in resistance of canola (Brassica napus L.) to sclerotinai stem root. International journal of Agronomy and Plant Production, 3(12), 613-617.
Chaoui, H. I., Zibilske, L.M. & Ohno, T. (2003). Effects of earthworm casts and compost on soil microbial activity and plant nutrient availability. Journal Soil boil biochem, 35, 295-302.
Choi, P. S., Soh, W. Y. & Liu, J. R. (1996). Somatic embryogenesis and plant regeneration in cotyledonary explant cultures of Chinese cabbage. Plant Cell Tissue and Organ Culture, 44, 253-256.
Deane, C. R., Fuller, M. P. & Dix, P. J. (1997). Somatic embryogenesis in cauliflower (Brassica oleracea var. botrytis). Cruciferae Newsletter, 19, 43-44.
Gaspar, T., Kevers, C., Penel, C., Greppin, H., Reid, D. M. & Trevor, A. (1996). Plant hormones and plant growth regulators in plant tissue culture. In Vitro Cellular & Developmental Biology, 32, 272-289.
George, E. F., Hall, M. A. & De Klerk, G. J. (2008). Plant Propagation by Tissue Culture. 3rd Edition, Vol. 1. Springer, Dordrecht, the Netherlands. 501.
Khan, M. R., Rashid, H., Ansar, M. & Chaudry, Z. (2003). High frequency shoot regeneration and agrobacterium-mediated DNA transfer in canola (Brassica napus). Plant Cell Tissue and Organ Culture, 75, 223-231.
Kieffer, M., Fuller, M. P., Chauvin, J. E. & Schlesser, A. (1993). Anther culture of kale (Brassica oleracea L. convar, acephala (DC.) Alef.). Plant Cell, Tissue and Organ Culture, 33(3), 303-313.
Kim, J. H. & Botella, J. R. (2002). Callus Induction and Plant Regeneration from Broccoli (Brassica oleracea var. italica) for Transformation. Journal of Plant Biology, 45(3), 177-181.
Kohlenbach, H. W., Wenzel, G. & Hoffmann, F. (1982). Regeneration of Brassica napus plantlets in cultures from isolated protoplasts of haploid stem embryos as compared with leaf protoplasts. Z. Pflanzenphysiol, 105,131-142.
Kranz, E. (1988). Somatic embryogenesis in stationary phase suspension cultures derived from hypocotyl protoplasts of Brassica napus L. Plant Cell, Tissue and Organ Culture, 12, 141-146.
Krzy┼╝anowska, D., Górecka, K., Kiszczak, W. & Kowalska, U. (2006). The effect of genotype and medium on plant regeneration from androgenic embryos. Journal of Fruit and Ornamental Plant Research, 14(1), 121-127.
Lelu, M. A. & Bollon, H. (1990). Cabbage Brassica oleracea var. capitata and Brussels Sprout Brassica oleracea var. gemmifera: In vitro Production of Haploids. Biotechnology in Agriculture and Forestry, 12, 358-373.
Lichter, R. (1989). Efficient yield of embryo by culture of isolated microspores of different Brassicaceae species. Plant Breed, 103, 119-123.
Muscolo, A., Bovalo, F. & Nardi, S. (1999). Earthworm humic matter produces auxin-like effects on Daucus Carota cell growth and nitrate metabolism. Soil Biology and Biochemistry, 31, 1303-1311.
Ono, Y., Takahata, Y. & Kaizuma, N. (1994). Effect of genotype on shoot regeneration from cotyledonary explants of rapeseed(Brassica napus L.). Plant Cell Report, 14, 13-17.
Palmer, C. E. (1992). Enhanced shoot regeneration from Brassica campestris by silver nitrate. Plant Cell Report, 11, 541-545.
Pental, D., Pradhan, A. K., Sodhi, Y. S. & Mukhopadhyay, A. (1990). Variation amongst Brassica juncea cultivars for regeneration from hypocotyl explants and optimization of conditions for Agrobacterium-mediatedgenetic transformation. Plant Cell Report, 12, 462-467.
Phogat, S. K., Burma, P. K. & Pental, D. (2000). High frequency regeneration of Brassica napus varieties and genetic transformation of stocks containing fertility restorer genes of two cytoplasmic male sterility systems. Journal Plant Biochemestry and Biotechnology, 9, 73-79.
Pua, E. C. & Chi, G. L. (1993). De novo shoot morphogenesis and plant growth of mustard (Brassica juncea) in vitro in relation to ethylene. Physiology Plant, 88, 467-474.
Rajagopalan, C. (2000). Export potential of Indian floriculture and need of policy environment. Floric Today, 9, 29-33.
Sato, T., Nishio, T. & Hirai, M. (1989). Plant regeneration from isolated microspore cultures of Chinese cabbage(Brassica campestris ssp. Pekinensis). Plant Cell Report, 8, 486-488.
Skoog, F. & Miller, C. (1957). Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symposia of the Society for Experimental Biology, 11, 118-131.
Tavakkol Afshari, R., Angoshtari, R. & Kalantari, S. (2011). Effects of light and different plant growth regulators on induction of callus growth in rapeseed (Brassica napus L.)genotypes. Plant Omics Journal, 4(2), 60-67.
Tomati, U., Grappelli, A. & Galli, E. (1987). The presence of growth regulators in earthworm-worked wastes. In: Bonvicini Paglioi A.M., Omodeo P. (Eds.), On Earthworms. Proceeding of International Symposium on Earthworms. Selected Symposia and Monographs. Unione Zoologica Italiana, 2, Mucchi, Modena, pp. 423-435.
Tuncer, B. & Yanmaz, R. (2011). Effects of Colchicine and High Temperature Treatments on Isolated Microspore Culture in Various Cabbage(Brassica oleraceae)Types. Journal of Agriculture & Biology, 13(5), 819-822.
Turgut, K., Barghchi, M. & Scott, R. (1998). Efficient shoot regeneration and somatic embryogenesis from immature cotyledons of Brassica napus L. Plant Breeding, 117, 503-504.
Wang, Y., Tong, Y., Li, Y., Zhang, Y., Zhang, J., Feng, J. & Feng, H. (2011). High frequency plant regeneration from microspore-derived embryos of ornamental kale (Brassica oleracea L. var. acephala). Scientia Horticulturae, 130, 296-302.
Warwick, S. I. (2011). Brassicaceae in Agriculture. In: R. Schmidt & I. Bancroft (Eds). Genetics and Genomics of the Brassicaceae. (9, 33-66). Springer Verlag, New York.
Wei, Z., Qiang, F., Xigang D. & Manzhu, B. (2008). The culture of isolated microspores of ornamental kale (Brassica oleracea var.acephala) and the importance of genotype to embryo regeneration. Scientia Horticulturae,117, 69-72.
Zhang, F. L., Takahata, Y. & Xu, J. B. (1998). Medium and genotype factors influencing shoot regeneration from cotyledonary explants of Chinese cabbage(Brassica campestris L. ssp. pekinensis). Plant Cell Report, 17, 780-786.
Zhang, W., Fu, Q., Dai, X.G. & Bao, M.Z. (2008). The culture isolated microspores of ornamental kale (Brassica oleracea var. acephala) and the importance of genotype to embryo regeneration. Scientia Horticulturae, 117, 69-72.