# Induction and Regeneration of Microspore-Derived Embryos for Doubled Haploid Production in Cabbage (Brassica oleracea var. capitata)

## TL;DR

This study optimizes methods to produce doubled haploid cabbage plants using microspore culture, which can speed up breeding.

## Contribution

The study establishes an efficient system for microspore-derived embryo regeneration and doubled haploid production in cabbage.

## Key findings

- Optimal flower bud size (4.0 ± 0.5 mm) maximized microspore yield and embryo formation.
- Heat shock treatment at 32.5 °C for 24–48 h was crucial for embryogenesis.
- 50% of regenerated plants were spontaneous doubled haploids with no genetic polymorphism.

## Abstract

Cabbage (Brassica oleracea L. var. capitata) is an important leafy vegetable crop, and the development of homozygous parental lines is essential for F1 hybrid breeding. Isolated microspore culture (IMC) provides a rapid approach for producing haploid and doubled haploid (DH) lines. However, its efficiency in cabbage remains highly dependent on genotype, donor plant growth conditions, and culture conditions. This study aimed to optimize key factors affecting microspore embryogenesis and plant regeneration in a Korean green cabbage (‘SJ-Ca 13’) and to evaluate the ploidy and genetic characteristics of regenerated plants. Microspore yield and embryogenesis were strongly influenced by flower bud size. Bud size of 4.0 ± 0.5 mm yielded the highest number of microspores (4.17 × 104 per bud) and exclusively produced microspore-derived embryos (2.33 embryos per Petri dish), whereas smaller or larger buds failed to induce embryogenesis. Heat shock treatment at 32.5 °C was essential for embryogenesis, with 24 or 48 h of treatment inducing embryo formation, while prolonged exposure (72 h) completely inhibited embryogenesis. Efficient shoot regeneration was achieved when microspore-derived embryos were cultured on semi-solid MS medium with reduced salt strength (1/3×) and higher agar concentration (1.0%), resulting in the highest shoot regeneration rate. Ploidy test revealed that 50% of regenerated plants were spontaneous doubled haploids. SSR analysis using 26 markers detected no genetic polymorphism among regenerated plants. Overall, this study establishes an efficient IMC and regeneration system for cabbage and demonstrates its potential for rapid DH line production to support cabbage breeding programs.