Effects of genotype and culture conditions on microspore embryogenesis in radish (Raphanus sativus L.)

Radish (Raphanus sativus L.), an important annual or biennial root vegetable crop, is widely cultivated in the world for its high nutritive value. Isolated microspore culture (IMC) is one of the most effective methods for rapid development of homozygous lines. Due to imperfection of the IMC technology system, it is particularly important to establish an efficient IMC system in radish. In this study, the effects of different factors on radish microspore embryogenesis were investigated with 23 genotypes. Buds with the largest population of late-uninucleate-stage microspores were most suitable for embryogenesis, with a ratio of petal length to anther length (P/A) in buds of about 3/4 ~ 1. Cold pretreatment was found to be genotype specific, and the highest microspore-derived embryoid (MDE) yield occurred for treatment of the heat shock of 48 h. In addition, the supplement of 0.75 g/L activated charcoal (AC) could increase the yield of embryoids. It was found that genotypes, bud size, as well as temperature treatments had significant effects on microspore embryogenesis. Furthermore, somatic embryogenesis–related kinase (SERK) genes were profiled by reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis, which indicated that they are involved in the process of MDE formation and plantlet regeneration. The ploidy of microspore-derived plants was identified by chromosome counting and flow cytometry, and the microspore-derived plants were further proved as homozygous plants through expressed sequence tags-simple sequence repeats (EST-SSR) and genetic-SSR markers. The results would facilitate generating the large-scale double haploid (DH) from various genotypes, and promoting further highly efficient genetic improvement in radish.