Effects of brassinolide on microspore embryogenesis and plantlet regeneration in pakchoi (Brassica rapa var. multiceps)

Hybrids are often superior to conventional varieties because of their high yield and strong stress resistance. The production of homozygous inbred lines is an important prerequisite for hybrid seed production; however, they may require more than six years by traditional breeding methods. Microspore culture is an attractive approach to obtain homozygous doubled haploids (DHs) in a short period. However, low embryogenesis frequency and high rates of embryo callus or mortality remain the main problems of this method in pakchoi. In this study, four different concentrations (0.01, 0.02, 0.04, 0.08 mg L-1) of brassinolide (BR) were added into NLN liquid medium and used to examine the effect of microspore embryogenesis frequency and plant regeneration rate in three genotypes of pakchoi. The results showed that 0.01 mg L-1 BR was the optimum concentration for genotype 'XM2', significantly increasing the frequency of embryogenesis induction and plant regeneration by 2.05-fold and 1.25-fold, respectively, compared with those in the control treatment; there was also an observed 0.56-fold decrease in the rate of callus formation. For both 'XM1' and 'XM3', 0.02 mg L-1 BR was the most optimum concentration, resulting in a 3.41-fold and 6.39-fold increase in embryogenesis frequency compared with that in the control treatment; the frequency of plant regeneration was more than 74%. Meanwhile the rate of callus formation decreased 0.28-fold and 0.59-fold, respectively. The average spontaneous doubling rate of the three genotypes was 68.50%, whereas the lowest spontaneous doubling rate of the genotype 'XM2' was 65.8%. For 'XM1', 'XM2' and 'XM3', we obtained 202, 144 and 85 DHs, respectively. Excellent horticultural traits were identified in DH lines from XM2, including increased leaf number and bright leaf color. All DH lines derived in this study were self-incompatible lines and showed high stability and consistency, which will effectively accelerate the application of microspore technology in hybrid breeding.