Effects of Heat Shock and 2, 4-D Treatment on Morphological and Physiological Characteristics of Microspores and Microspore-Derived Doubled Haploid Plants in Brassica napus L.

Stresses such as heat shock, starvation, or osmotic is essential to lead isolated microspores towards embryogenesis. Despite the effectiveness of stresses in embryogenesis, they exert adverse effects on metabolism and growth of the regenerated plants. The effects of heat shock and 2,4-D treatment on total protein content of treated microspores, morphological and physiological characteristics of the doubled haploid (DH) plants were assessed. Buds containing mid- to late- uninucleate microspores were used for microspore culture. Microspores were isolated and cultured in NLN-13 medium and incubated at 30ºC for 14 days or treated with 2,4-D (35 mg.L-1) for 30 min to induce embryogenesis. Microspore-derived embryos were transferred onto B5 medium for plantlet regeneration. Ploidy level of the regenerated plantlets was determined using Partec flow cytometry. Spectrophotometric readings were carried out at 490, 663 and 645 nm to determine Chl a-b and carotenoids contents. TRIzol and cetyl-three-ethyl-ammonium bromide (CTAB) were used for protein extraction from microspores and leaves. Length and width of stomata and pollen grains were also photographed using light microscope (Olympus). Applied stressors significantly reduced total protein content of treated microspores however, protein content and concentration of chlorophyll a and b of the DH plants were only increased by heat shock treatment when compared with the donor plant ‘Hyola 420’. In contrast, carotenoids were not affected by applied stressors. Longer and wider stomata were observed by 2,4-D treatment but, the length of pollen grains was significantly decreased following heat shock and 2,4-D treatment. Total protein content of cultured microspores, concentration of chlorophyll a and b, length and width of stomata of microspore-derived doubled haploid plants were significantly affected by the type of inductive stresses. However, carotenoids were more stable and not affected by applied stressors.