In Vitro Mutagenesis For Selection Of Novel Germplasm In Brassica Napus

1. Ultraviolet (UV) irradiation, mutagenic agents ethylmethane sulphonate (EMS) and sodium azide (NaN₃) and herbicide pyribambenz-propyl (PP) were applied to cotyledons of two Brassica napus cultivars Zheshuang 758 and Zheshuang 6 in vitro. Cotyledons were exposed to the UV radiation for different time intervals (30, 60, 120, 180, 240 sec), and the results showed that UV had some promotive effect on cotyledon regenerations. The highest rates of callus induction and plant regeneration were observed from UV treatment of 120 and 60 sec in genotypes Zheshuang 758 and Zheshuang 6 respectively. There was obvious difference in the rates of callus induction and plant regeneration between the treatment of EMS concentration and the control. However, with the increase in concentrations and prolongation of treatment intervals, the rate of induced calli and rate of regenerated plants decreased progressively in both genotypes. After NaN₃ treatment, the rate of plant regeneration callus induction was not much affected or even higher than that of the control. As for the plant regeneration, there was obvious difference between the treatment of NaN3 concentration (100μM) and the higher treatment of NaN₃ concentration (500μM). There was no obvious difference between the treatment of shorter time (5h) and the longer time (24h) in both genotypes. Apart from control, all the cotyledon regeneration was the highest after PP treatment (10mg/L, 7h).2. Ultraviolet (UV) irradiation, mutagenic agents ethylmethane sulphonate (EMS) and sodium azide (NaN₃) were applied to isolated microspores and microspore-derived embryos of B. napus in vitro. In the UV light irradiation experiment, the isolated microspore-derived embryos were exposed for 10, 30,60 and 120 sec. Embryo germination showed a sharp decrease with increasing UV exposure and only 24.69% embryos were survived following an exposure of 120 s of genotype h23, which is lower than control (96.18%). Embryo germination was also decreased when isolated microspores and microspore-derived embryos were treated by UV irradiation. The mutagenic treatments, NaN₃ (10, 100, 500μM) and EMS (5, 20, 100 mg/L) were applied to the embryos at early cotyledonary stage for various time intervals (5, 12 and 24 h; 2, 6 and 12 h). Many fertile microspore-derived embryos died after the applying of the two chemical mutagens. Embryo germination was also decreased with the treatment of these two mutagens. Surprisingly, in this study the application of 10 and 100μM NaN₃ had a strong promotive effect on the rate of embryo germination and plant regeneration, with the significant increase over the control. When embryos developed they were transferred to solid 1/2MS medium supplemented with 2 mg/L 6-BA where regenerated plants took place. The regenerated plants from microspore-derived embryos were treated with colchicine (150 mg/L, 30h) to produce DH plants. These DH plants were gradually adaopted to the field condition, and their agronomic traits and yield components were recorded along with their conventional populations.3. Glucosinolate contents in different varieties and different organs of Brassica napus were determined by the palladium chloride method, meanwhile, the releationship of contents between seeds and other organs and their difference between leaves of doubled haploid (DH) populations and control. The results showed that glucosinolate content in 956 is the highest among seeds of all the varities, the same to pericarp in 202, root in 201 and stem in 956, and their contents were 81.45, 48.05, 54.33 and 37.94 μmol/g, respectively. Apart from roots'contents, all the glucosinolate contents were the lowest in variety 308. The correlations of all 4 agronomic traits were analyzed, and it was observed that there was a positive correlation between root and pericarp, with a correlation coefficient of 0.6579, while a negative correlation was observed between the seeds and pericarp. In addition, there was significant difference of glucosinolate contents between control and microspore-derived plants. The results showed that in vitro mutagenesis may affect glucosinolate content in Brassica napus, which became higher in three varieties (1142, 1170 and 1200) and could also become lower in 1167.