Development of adjustable male sterile plant in broccoli by antisense DAD1 fragment transformation

Abstract

Male sterile line is very important in hybrid seed production in broccoli (Brassicaoleracea L. var. italica). A fragment of BoiDAD1F was amplified from this specie using primers designed against the conserved DAD1F fragment of DAD1 genes inArabidopsis thaliana and Brassica rapa. The BoiDAD1F sequence was 88 and 99% similar to the homologous genes in Arabidopsis and B. rapa, respectively. Theantisense BoiDAD1F was then cloned onto pBI 121 vector to make the transformation construct, pBI-antiBoiDAD1F. Cotyledons with petiole were inoculated with Agrobacterium tumefaciens harboring the pBI-antiBoiDAD1F plasmid. Positive transformation was confirmed using polymerase chain reaction (PCR) and Southern blots. Twenty individual transformants were obtained, of which 15 showed suppressed DAD1 mRNA level. Pollens produced by the male sterile transgenic plants germinated poorly (≤ 10%). No seeds were produced on the flowering transgenic plants, and the siliquae were empty, or the few shrinked seeds, which failed to germinate. When pollinated with wild type plant pollen, the siliquas of the transgenic plants enlarged to produce normal seeds. No morphological differences were observed between the transgenic and wild type plants, except a few abnormal flowers on transgenic ones. To restore the fertility, exogenous JA were applied by dipping the floral buds into an aqueous solution containing 500 μM MeJA and 0.05% Tween 20 after removal of the opened flowers on the inflorescence. The treated flower produced normal pollens which germinated on the medium, and were fertile. T1 population consisted of fertile and sterile plants in the ratio of 3:1. The T2 population comprised of both complete fertile and sterile lines. These results confirmed the Mendel model of separation of the antisense RNA insert in the transgenic plants. Key words: Brassica oleracea L. var. italica, DAD1, adjustable male sterility, jasmonic acid and antisense RNA.