Research on the key factors influencing isolated microspore culture in broccoli×cabbage hybrid

YANG Yanyan; ZHANG Yanfeng; HU Tixu

doi:10.16861/j.cnki.zggc.2025.0138

Abstract：The broccoli × cabbage hybrid materials can produce various gamete types during meiosis,generating new resources with significant variation.This contributes to optimizing the isolated microspore culture system of broccoli × cabbage hybrids,obtaining high-quality broccoli double haploid(DH)plants,and enriching germplasm resources.Isolated microspore culture was applied to F1hybrids derived from five broccoli and one cabbage line to investigate the morphologi-cal characteristics of flower buds at different developmental stages and analyzed the effects of key culture factors-including genotype,sucrose concentration,cold pretreatment duration of buds at 4℃,and heat shock duration of microspores at 32 ℃on the embryogenesis rate.The results showed that for all five F1 materials,when the P/A(petal length/anther length)ratio of the flower buds were between 0.8 and 0.9,the proportion of microspores at the uninucleate late stage reached 60％-70％,indi-cating the optimal sampling period.All five materials produced embryoids,with embryo induction rates ranging from 0.52 to 16.79 embryoids per bud.Material H1(23ZXQ1 ×LC8)exhibited the highest embryo induction rate at 16.79 embryoids per bud and was subsequently used for further pretreatment experiments.Pretreating microspores in N17 medium(NLN liquid medium containing 17％sucrose)resulted in the highest embryo induction rate of 8.93 embryoids per bud;Pretreating flower buds at 4℃ for 3 days yielded the highest embryo induction rate of 10.01 embryoids per bud.Treating microspores at 32 ℃for 3 days achieved the highest embryo induction rate of 17.96 embryoids per bud.In conclusion,P/A ratio between 0.8 and 0.9 corresponds to the highest proportion of microspores at the uninucleate late stage in broccoli × cabbage flower buds and can serve as the criterion for bud sampling.To significantly enhance embryo induction efficiency,flower buds should be pre-treated at 4 ℃ for 3 days,followed by pretreatment of the isolated microspores in N17 medium at 32 ℃ for 3 days.