High frequency production of microspore derived doubled haploids (DH) and its application for developing low glucosinolate lines in Indian Brassica juncea

摘要

The Indian varieties of Brassica juncea are high in erucic acid (EA) and glucosinolate (GSL) contents and are, therefore,recessive genes and hence, can be conveniently handled only through the production of doubled haploids. We developed an efficient protocol for the production of microspore derived doubled haploids in B. juncea, with reasonably high embryogenic and very high embryo conversion frequencies and applied it in a backcross breeding programme for the introgression of low glucosinolate trait from an un-ad apted canola quality B. juncea line Heera to a popular Indian variety Varuna. The parents differed significantly in their average embryogenic response and that of the F1DH was close to Varuna Microspores treated with colchicine at the initiation of culture for 24h showed 65-70% doubled haploid production. Maintenance of high humidity in the immediate environment of the cultured microspores was crucial for the induction of embryogenesis. Addition of activated charcoal after colchicine treatment improved the embryogenic frequency. Subjecting the fully differentiated embryos to ABA at low temperature (15°C) for physiological maturation eliminated embryo malformations such as secondary embryogenesis,The doubled haploids were transferred to the field without any prior hardening with a mortality rate of about 10%. Till date,following this protocol through five backcross generations coupled with stringent selections, we have developed several low GSL (≤10 μmol/g of seed) lines in B. juncea.