Abstract: 【Objective】To efficaciously use Ogura cytoplasmic male sterility (CMS) hybrids in breeding and provide resources for genetic improvement of germplasm, broccoli restorers were developed by transforming the matching fertility restorer gene of Rfo from radish into the leading cultivar Ogura CMS line of ‘Nai Han You Xiu’ (named as SFB45).【Method】The sequence of CDS with its preceding promoter of Rfo were synthesized and the pRfo::Rfo plant expression vector was constructed to infect the cotyledon with petiole and hypocotyl explants of the SFB45 through Agrobacterium-mediated transformation method. After regeneration and screening of herbicide resistant seedlings, transgenic plants were identified by detection of the Bar resistant marker. The fertility of transgenic plants were observed at flowering stage. The pollen viability of SFB45 and the transgenic plants were analyzed using the alexander stain. Total RNA of <3 mm and >3 mm flower buds from SFB45 and the transgenic plants were isolated and transcribed into cDNA. The Rfo specific primers were designed and RT-PCR was performed to analyze the expression levels of Rfo and also the genes related to tapetum and pollen wall development in flower buds of SFB45 and its restorer lines.【Results】A total of 10 transgenic plants were obtained by genetic transformation. Among which, the fertilities of 8 were restored to varying degrees with the average pollen viability ranging from 84.2% to 90.4%. RT-PCR analysis showed that Rfo was expressed in flower buds of fertility restored plants. Key regulators of tapetum development (DYT1 and TDF1) and the essential gene (ACOS5) for the synthesis of sporopollenin, a major component of the pollen wall were significantly up-regulated in <3 mm flower buds of Ogura CMS restore lines. Tapetum degradation related gene AMS and tetrad callose wall and pollen outer wall development related genes CalS5 and CYP703 were significantly up-regulated in >3 mm flower buds of the restore lines. Analysis of the self- and hybrid-crossing progenies of the positive transgenic lines R-1, R-3 and R-6 showed that the introduced Rfo could be stably inherited, which was consistent with the Mendelian inheritance.【Conclusion】The fertility restorer lines of Ogura CMS SFB45 were obtained by genetic transformation of Rfo and the integration of Rfo into the genome of SFB45 recovered the expression of genes associated with tapetum and pollen wall development.

Key words: Broccoli, genetic transformation, Ogura CMS, Rfo, tapetum and pollen wall development-related genes