Plant Tissue Cult. 4(2) : 77-84, 1994

High Planting Efficiency and Plant Regeneration from Hypocotyl Protoplast of Brassica oleracea var. italica cv. Shanghai No. 1

Zhongxin Zhong and Xian Li

Horticultural Research Institute, Shanghai Academy of Agricultural Science, Shanghai 201 106, P.R. China

Key words: Broccoli, Hypocotyl, Protoplasts, Regenerated plants

Abastract

A procedure is described for regeneration of plants from hypocotyl protoplast of the hybrid cultivar, Shanghai No. 1 (Brassicaca oleracea var. italica). Hypocotyl protoplasts were isolated from in vitro grown seedlings cultured at room temperature (20-25⁰C) under natural illumination. After purification of the protoplasts, higher protoplast yield (1 ? 10⁶/g, fresh weight) was obtained. Using agarose bead culture method, the purified protoplasts were cultured in K8P medium supplemented with 1.0 mg/1 NAA, 0.1 mg/1 2,4-D, 1.0 mg/1 zeatin and 0.6% agarose. The continued division of regenerated cells and formation of micro calli were observed; and the division frequency and planting efficiency were 35 and 5% respectively, which was more than what was observed in suspension and two-layer cultures. It indicated that the conditions of in vitro grown seedlings and agarose bead culture method are suitable for isolation and culture of hypocotyl protoplasts of broccoli. Multiple shoots were induced on differentiation medium of MS supplemented with 0.1 mg/1 IAA, 2.0 mg/1 zeatin, 40 mg/1 adenine and 100 mg/1 glutamine in which the differentiation rate reached 20%. After transfer of differentiated shoots to the rooting medium consisting of half strength MS medium supplemented with 0.1 mg/1 IBA, shoots rooted and formed intact plantlets. The latter established themselves following their transplantation to potted soil.

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