New insights into glucosinolate-myrosinase system through production and characterization of homozygous lines of transgenic MINELESS plants

Transgenic MINELESS Brassica napus plants have been produced where selectively myrosin cell idioblasts were induced to undergo controlled cell death (ablation) leading to production of seeds with a severe reduction of myrosinase. Although myrosinase activity in MINELESS was very low as compared to Westar, a high degree of variation was observed. In order to overcome this variability, microspore culture was performed which resulted in production of haploid MINELESS plants. Sterile haploids from these were treated with colchicine, and transgenic double haploids with full fertility were produced, hence resulting in good seed setting. Specific myrosinase activity was very low and considerably constant in MINELESS homozygous seeds. Immunoblot analysis further revealed absence of 70 and 75kDa isoforms of myrosinase. Targeted myrosin cells stained with toluidine blue and labelled with fluorescein-isothiocyanate appeared degraded/empty when examined by light and confocal microscopes. No significant difference in total glucosinolate was observed between Westar and MINELESS. Headspace Solid-Phase Micro-extraction analysis showed reduced levels of glucosinolate degradation products in MINELESS seeds for most of the compounds. Westar and MINELESS also exhibited significant variations for various growth parameters, like plant height, leaf traits and biomass accumulation. In addition, various yield parameters also reflected alterations for MINELESS plants. Significant reductions were noticed in seed yield and harvest index of MINELESS plants, accounted by decreased number of seeds per silique. Contrarily, 1000-seed weight of MINELESS plants showed increase over Westar. Pure seed material from these homozygous lines will be of immense importance for future functional genomics and plant defence studies.