Study of different methods and factors affecting rapeseed microspore culture technique as well as responsiveness of commercial rapeseed cultivars to this technique.

Isolated microspore culture method and regeneration of microsporederived embryos is one of the new breeding methods in rapeseed. In this research project, different factors affecting microspore embryogenesis and doubled haploids production in various rapeseed genotypes (including spring and winter genotypes) were studied. In the first experiment, the aim was to optimize microspore embryogenesis and regeneration in order to introduce an applied protocol. In genotypes Hyola 420 and Hyola 401, the frequency of microspores-derived embryos was the highest when microspores subjected to heat shock (30oC) for 14 and 10 days respectively. In Topas, the highest embryogenesis frequency was obtained when heat shock applied to microspores at 32,5 oC for 1 day. In Hyola 308, microspore embryogenesis could not be achieved under heat shock. For regeneration optimization, embryos in cotyledonary stage were transferred to B5 medium supplemented by GA3(0,01 mg/l) in dark condition for 10 days at 4 oC and then transferred to B5 medium without GA3 under the light condition at 25 oC. For doubling, plantlets were treated with colchicine at 3,4gr/l for 1/5 hours. In other experiments, the effect of new chemical stresses on microspore embryogenesis induction was studied that their results are given separately. In the second experiment, microspores of Brassica napus L. cvs. Topas and Hyola 420 were subjected to 2¡4-D at 4 concentrations 15, 25, 35 and 45 mg/l for 15-45 minutes. . The results showed that embryogenesis and regeneration of microspores stressed with 2¡4-D were comparable to heat shock. The highest yield of torpedo stage embryos was achieved at 15 mg/l 2¡4-D for 30 minutes among 2¡4-D treatments and the best frequency of normal plantlets was produced when 2¡4-D (25 mg/l for 30 minutes) applied to the microspores. In the last experiment, three chemical stresses i.e. trifluralin, oryzalin and pronamid were applied to isolated microspores in hybrid Hyola 401. The results showed that the new chemical stresses can induce microspore embryogenesis. Although in model plant rapeseed their efficiency is lower compared to the heat shock, however it is proposed that in species whose microspores are sensitive to classical stresses, the new chemical stresses could be effective for embryogenesis induction. According to the results obtained, in addition to introduction an applied protocol for doubled haploid production in rapeseed, the new stresses (2¡4-D, trifluralin, oryzalin and pronamid ) are introduced for induction of embryogenesis in microspore as the first report.