Improvement of isolated microspore culture of ornamental kale (Brassica oleracea var. acephala): effects of sucrose concentration, medium replacement, and cold pre-treatment

Summary By evaluating the effects of the culture medium and cold pre-treatment on microspore embryogenesis, we have developed an efficient and reliable protocol for microspore culture and doubled-haploid plant regeneration of ornamental kale (Brassica oleracea var. acephala). Microspore embryogenesis at high frequency was obtained using a liquid NLN medium containing 16% (w/v) sucrose (NLN-16) without medium replacement. Of the genotypes tested, the cultivar ‘Peachy Dancing’ produced the highest yield of embryos (averaging 123.63 embryos per dish). Medium replacement was found to deplete embryo yield significantly in all three genotypes tested (namely, ‘Nagoya’, ‘Peachy Dancing’, and ‘P3’), when compared with continuous culture of microspores in the original incubation medium. A 48-h cold pre-treatment, delivered to the flower buds of ‘Nagoya’ and ‘P3’ before microspore isolation, significantly enhanced subsequent microspore embryogenesis; however, cold pre-treatment of flower buds had a negative effect on ‘Peachy Dancing’. Cotyledonary embryos were incubated on B5 basal medium and abnormal embryos on B5 medium containing 2% (w/v) sucrose, 1.5 mg l−1 6-benzylaminopurine, and 0.25 mg l−1 naphthaleneacetic acid. In each case, high quality embryos were regenerated that went on to produce healthy, vigorous plants. There were 38 – 50% spontaneous diploid, microspore-derived plants among the three genotypes tested.

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