Effects of genotype and sodium p-nitrophenolate on microspore embryogenesis and plant regeneration in broccoli (Brassica oleracea L. var. italica)

Abstract

Isolated microspore culture has been applied in breeding to obtain doubled haploids (DH) and therefore shorten the time of breeding process; however, the production of pure broccoli lines using DH produced in isolated microspore culture has not been sufficiently developed. This study aimed to establish an effective microspore culture protocol for broccoli for hybrid breeding purposes. Microspores of thirteen broccoli genotypes were successfully induced to form embryos in NLN-13 liquid medium. Three genotypes, BB1, BB4, and BB6, were cultured in NLN-13 medium containing sodium p-nitrophenolate (SPN) ranging from 0 to 0. 2 mg•L-1, to improve microspore embryogenesis and plantlet regeneration without callus development. The addition of 0.01–0.2 mg•L-1 SPN enhanced the frequency of microspore embryogenesis. Adding 0.1 mg•L-1 SPN yielded the maximum number of embryos per bud in BB1, BB4, and BB6, which were 1.92-, 2-, and 2.1-folds higher than the control, respectively. The suitable concentration (0.1 mg•L-1) of SPN application could enhance the frequency of direct plantlet regeneration for broccoli. Further, average spontaneous diploidization rate for BB1, BB4, and BB6 was 51.28%. This is the first study to utilize SPN in improving the frequency of microspore embryogenesis and plantlet regeneration, and offer an efficient method for the large production of DH lines that needed in hybrid seeds and genetic study in broccoli.