Non-ionic surfactants improved microspore embryogenesis and plant regeneration of recalcitrant purple flowering stalk (Brassica campestris ssp. chinensis var. purpurea Bailey)

As Brassicaceae species are mostly cross-pollinated, breeding homozygous parental lines by traditional approaches is time-consuming and costly. Alternatively, microspore culture has been widely applied to produce double haploid lines in a short time. This study aimed to establish a highly efficient microspore culture protocol for purple flowering stalk. Among the five genotypes studied, the highest and lowest embryo induction rates were observed in J18 and J17 (13.5 and 7.67 embryos per bud, respectively). Microspores of genotypes J17 and J18 were successfully induced to produce embryos in NLN-13 medium, but the frequency of microspore embryogenesis was low. Three non-ionic surfactants (Pluronic F-68, Triton X-100, Tween-20) were evaluated independently for their effect on microspore embryogenesis of purple flowering stalk. Microspores of the two genotypes were cultivated in NLN-13 medium supplemented with different concentrations (0.0001%, 0.001%, 0.01%, 0.1%, 0.5%, and 1% (w/v)) of the three non-ionic surfactants to enhance microspore embryogenesis and plant regeneration. In both genotypes, supplementation with any of the three non-ionic surfactants at 0.0001% significantly increased the frequency of microspore embryogenesis; furthermore, at that concentration, Tween-20 significantly increased the number of plants regenerated from induced embryoids by 29.9% and 30% in J17 and J18, respectively. Moreover, the rate of double haploid formation among regenerated plants of the five genotypes was above 60%, which allowed the creation of 93 double haploid lines.