Optimizing protocol for efficient microspore embryogenesis and doubled haploid development in different maturity groups of cauliflower (B. oleracea var. botrytis L.) in India

Abstract

Microspore culture technique is widely used in Brassicas for development of haploids and doubled haploids (DH) in breeding programmes and genetic studies. Indian cauliflower, a group of vegetable Brassica; with tolerance to higher temperature and moisture, has been selected in India for last 200 years. In India, cauliflowers are grown throughout the year in different climatic zones based on their temperature adaptation. For the first time, we report microspore embryogenesis in different maturity groups of Indian cauliflowers. The main objective of our study was to optimize protocol for efficient microspore embryogenesis in different types of cauliflower cultivated in India. We studied different factors like bud size, developmental stage of the microspores, culture density and heat shock treatments for efficient microspore embryogenesis in four different genotypes representing four different maturity groups. Among different factors, the bud size and microspore developmental stage was found to be more critical for successful microspore embryogenesis. The optimum bud size for high frequency microspore embryogenesis for the two early maturity groups (September–October maturity) was 4.0–4.5 mm, whereas, it was 4.5–5.0 mm for the cultivars grown for November onwards harvesting. These optimized bud sizes contained the highest percent of viable microspores and maximum number of microspores at late uninucleate to early binucleate stage. The microspore density of 6–8 × 104 cells per mL was found most suitable for efficient microspore embryogenesis. A post-culture incubation of microspores at 30.0 °C/32.5 °C for 24 h followed by maintenance at 25 °C was found optimum for induction of microspore embryos in different maturity group of Indian cauliflower. The flow cytometry analysis of 646 microspore derived plants revealed that more than 60 % plants were spontaneous diploids and less than 15 % were haploids. However, the genotypes from different groups showed varied response in different culture density and temperature treatments. Thus, it is necessary to optimize genotype specific protocol for efficient microspore embryogenesis before its routine application. The protocol developed in this study will facilitate doubled haploids based breeding and genetic studies of all types of cauliflower grown in India.