engleski

Formation of embryogenic callus in hairy roots of pumpkin(Cucurbita pepo L.)

Excised cotyledons from 8-d-old pumpkin (Cucurbita pepo L.) seedlings were inoculated with Agrobacterium rhizogenes and cultured on hormone-free Murashige and Skoog (MS) medium. At the site of inoculation, transformed hairy roots were successfully induced by using wild strains 8196 (mannopine-type) and 15834 (agropine-type). After a subsequent transfer on a solid MS medium without hormones, roots obtained by transformation with strain 15834 failed to form stable hairy root cultures, while several hairy root lines were established with strain 8196. Three hairy-root lines, Cp1, Cp2 and Cp31 have spontaneously generated callus with embryo-like structures after more than three years of growth on the solid medium. The callus proliferation was more frequent when the autoclaving of nutrient medium, pH 5.7, was prolonged to 30 min. Separated calli continued to proliferate and generated embryos with abnormal morphology. The combination of indole-3-acetic acid and benzylaminopurine had a favourable influence on embryogenesis and organogenesis in Cp31 callus line. The Southern analysis of Cp31 roots and embryos DNA confirmed the presence of T-DNA of Agrobacterium rhizogenes.

Agrobacterium rhizogenes; callus proliferation; embryogenesis; plant growth regulators; transformation

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