Construction of an antisense CYP86MF gene plasmid vector and production of a male-sterile Chinese cabbage transformant by the pollen-tube method

Abstract

SummaryA plant expression vector pBIA9-AMF containing an antisense fragment of the CYP86MF gene and the tapetum-specific A9 promoter was constructed. Plasmid vectors were introduced by floral-dipping and pollen-tube transformation methods to Chinese cabbage pak-choi (Brassica campestris ssp. chinensis (L.) Makino var. communis Tsen et Lee, syn. B. rapa ssp. chinensis (L.) Makino var. communis Tsen et Lee) and flowering Chinese cabbage (B. campestris ssp. chinensis (L.) Makino var. parachinensis (Bailey) Tsen et Lee). Results showed that KanR seedlings could be obtained by the pollen-tube method through germination tests of T1 progeny seeds, but not by the floral-dipping method. One of the two KanR seedlings proved that the antisense fragment of the CYP86MF gene was integrated into the Chinese cabbage genome by PCR amplification and Southern blotting. Northern hybridization indicated that the CYP86MF gene, under the A9 promoter, was inhibited in the transformant, and self-infertility was found in the transgenic plantlets after self-pollination. Comparison of floral organs and the morphology of microspores by scanning electron microscopy of transformant and wild-type plantlets, showed no significant differences except that microspores from the transgenic plant were covered in a layer of an unknown substance. We suggest this layer might be the cause of the male-sterile phenotype induced by the antisense fragment of the CYP86MF gene.