Comparison of anther and microspore culture in androgenic embryogenesis and regeneration of broccoli (Brassica oleracea L. var. italica P.)

Abstract

The aim of this study was to compare the efficiency of broccoli anther and microspore culture methods for doubled haploid (DH) lines production. We evaluated the main influencing factors and optimized the culture methods to improve embryo induction and plant regeneration for efficient doubled haploid production in broccoli breeding. Six broccoli hybrids were used in this study. Our results show that generally, the efficiency of androgenic embryogenesis and regeneration in microspore culture is higher than that in the anther culture. Moreover, the microspore culture eliminated the possibility of plantlets coming from diploid tissue. In this study, the four-day cold pre-treatment yielded the highest number of embryos in both anther and microspore culture methods; the embryo yield at 32.5°C for 24 h was the highest in anther and microspore culture. Optimal plating densities were 30 anthers per dish in anther culture and 4×10⁵ microspores per ml in microspore culture. In androgenic embryo production, the PG-96 medium proved to be more effective than NLN medium. Sucrose concentration at 10% for anther culture and 13% (w/v) for microspore culture was recommended. A total of 70 regenerants were obtained from three genotypes including doubled haploids, haploids and aneuploids.

Keywords: Anther, broccoli, doubled haploid, microspore, plant regeneration