l-Ascorbic acid sodium salt promotes microspore embryogenesis and chromosome doubling by colchicine in ornamental kale (Brassica oleracea var. acephala)

Isolated microspore culture (IMC) represents a potential alternative technique in the plant breeding process, as it allows the effective production of doubled haploid (DH) homozygous lines. However, the implementation of this technique is limited by a low rate of embryogenesis, high level of embryo death, and low frequency of chromosome doubling. Thus, we investigated the effects of using different concentrations of L-ascorbic acid sodium salt (VcNa), which has never been applied for kale, to enhance the embryogenesis and regeneration by IMC. Specifically, 1 to 5 mu M VcNa was added to the NLN-13 medium of four kale genotypes, while control was grown on VcNa-free medium. Overall, 1-4 mu M VcNa at pH 5.84 increased embryogenesis, with 4 mu M VcNa being the optimum concentration (12.92-fold increase). The proportion of embryo deaths declined when using appropriate VcNa concentrations. To increase the frequency of chromosome doubling, an artificial chromosome doubling protocol was developed for kale microspore-derived haploids. This protocol involved dipping roots of haploid plantlets in colchicine solution and adding colchicine treatment to solid Murashige and Skoog (MS) medium. Optimum chromosome doubling of haploids was achieved by dipping their roots in 750 mg/L colchicine solution for 4-6 h and 1000 mg/L colchicine solution for 2 h (doubling for nearly 50% of haploids). In conclusion, this study delineated an effective tissue culture process in promoting chromosomal ploidy of microspore-derived regenerated plants, allowing more microspores to be maintained that have excellent ornamental characteristics through crossbreeding. Key message Doubled haploids were produced by microspore culture and optimum L-ascorbic acid sodium salt concentrations increased embryogenesis. Artificial chromosome doubling by colchicine solution improved the doubling rate by nearly 50%.

Automated summary

Isolated microspore culture (IMC) is a potential alternative technique in plant breeding for producing homozygous lines. However, low embryogenesis rate, high embryo death rate, and low chromosome doubling frequency limit its implementation. This study investigated the effects of L-ascorbic acid sodium salt (VcNa) concentrations on embryogenesis and regeneration in kale using IMC, and developed an artificial chromosome doubling protocol for kale microspore-derived haploids. The results showed that 4 μM VcNa at pH 5.84 was the optimum concentration for embryogenesis, and dipping haploid plantlet roots in colchicine solution at specific concentrations and durations achieved nearly 50% chromosome doubling rate. This study provides an effective tissue culture process for promoting chromosomal ploidy of microspore-derived regenerated plants.