Genetic Transformation With Resistant To Disease And Insect And Their Resistant Evaluation In Brassica Napus L.

Oilseed rape (Brassica napus L.) is one of the most important oil crops throughout the world. Diseases and insects have been a major threat to the yield of rapeseed. Hence, the resistance against fungal diseases and insects in oilseed rape has always been the breeding goals of researchers. Plant genetic engineering techniques, resistant germplasm resources, genetic transformation through foreign resistant genes into cultivars and directional selection of agronomic quality characters in serial propagation have produced high quality transgenic materials, as well as enriching the germplasm resources of resistant rapeseed. Transformation through Agrobacterium-mediation has been widely adopted in transgenic plant studies. Transformation efficiency in rapeseed is affected by many factors. Therefore it is very important to establish a high efficient genetic transformation system for rapeseed. In the experiment, the influencing factors of transformation system of leaf petioles were studied, and also we have analyzed the foreign genes expression by keeping in mind the laws of genetic segregation through molecular techniques. The main results are listed as follows:1. Leaf-petiole regeneration system of oilseed rape (B. napus L., cv. Zheshuang 758) was established to study the effect of concentrations and combinations of exogenous hormone, AgNO3 and sucrose on leaf-petiole explants regeneration efficiency. The results showed that the quality of calli was the best when 2.0 mg/L 2,4-D, with its induction frequency being 89.6%. Excessive 2,4-D resulted in calli malformation and affected the differentiation ability of the regenerated shoot. In calli differentiation stage, shoot regeneration frequency of calli reached its peak at 53.4% in the differentiation MS medium containing 3.0 mg/L 6-BA+0.1 mg/L NAA, superior to other combinations. In this experiment, browning frequency of leaf-petiole was decreased significantly by the addition of 2.5 mg/L AgNO3 to the medium. The frequency of shoot regeneration reached its peak (86.7%) when the medium was supplied with 3.0 mg/L 6-BA+0.1 mg/L NAA+2.5 mg/L AgNO3. A certain degree of vitrification of regenerated plants occurred in the experiment, while sucrose concentration of 10 mg/L could maintain their growth, even depress vitrification of rape plants. Hormones showed a significant positive effect on root induction. The frequency of root induction was 91.1% on the root-inducing medium supplemented with 4.0 mg/L IAA.2. Effect of Agrobacterium infection, acetosyringone, antibiotic and its screening concentrations on Agrobacterium-mediated transformation efficiency and shoot regeneration were studied by establishment of transformation systems using leaf-petiole explants of Brassica napus L. cv. Zheshuang 758. The result showed that acetosyringone (AS) had no effect on leaf-petiole expansion frequency of explants, and rot of explants occured easily on highly concentrations of Agrobacterium tumefaciens infection. Analysis of variance indicated that both of them had no interactive effect on expansion of leaf-petiole explants. The optimum concentration of carbenicillin for A. tumefaciens was 500 mg/L, which had little effect on callus induction and differentiation of leaf-petiole explants, with the ratio being 75.2% and 65.1%, respectively. In root-inducing stage, adding cephamycin at 100 mg/L to the medium could decrease the effect of A. tumefaciens on plants regeneration, but not for rooting. Hygromycin (5.0 mg/L) inhibited the growth of non-transformed regeneration plants completely, even caused browning to death, whereas 32 regenerated plants were obtained on such lethal concentration. The frequency of shoot regeneration on co-cultivation medium containing 100μmol/L AS (3.9%) was significantly higher than those without AS (2.0%), which showed that addition of phenolic compounds such as acetosyringone to the medium were beneficial to the transformation of T-DNA in co-cultivation.3. Detection and identification of the effect of resistance to fungus and insect for transformants. Twenty-seven of 32 regenerated plants which came from 600 leaf-petioles were detected by polymerase chain reaction, indicated that the efficiency of transformation was 4.5%. Eight plants were selected randomly for Southern and Northern hybridizations, four plants (T0-11, To-14, T0-16 and T0-26) having single copy, others being multi-copies. A stable and high expression level of sporamin gene was confirmed using Northern hybridization technique in transformants by wounding initiation. These eight To plants were selected to carry out the in vitro inoculation bioassays with Lepidoptera larvae (Plutella xylostella) and fungus (Sclerotinia sclerotiorum). The results revealed that the average body weight of P. xylostella was from 11.1 mg to 19.2 mg after 4 days inoculation, with survival ratio ranged from 5% to 25%, which was significantly lower than that of control (40.3 mg and 100%, respectively). The growth of fungal could be inhibited greatly by transformants after 2 days inoculation, with spot diameter ranged from 0.73 cm to 2.17 cm, which was significantly shorter than that of control (2.87 cm). These results indicated that sporamin and chitinase PjChi-1 showed a consistent resistance to fungus and insect in transformants.4. Analyses of inheritance patterns and identification of resistance against fungus and insect were carried out in T1 progeny. Two genes (sporamin and chitinase PjChi-1) in T1 progeny of four single-copy To transformants(To-11, To-14, To-16 and To-26) were characterized. PCR detection revealed that the segregation ratio of positive and negative plants was 3:1, which agreed with the Mendelian rules throughχ2 test. Meanwhile, the results also confirmed that the integration pattern of exogenes of four To transformants was single copy. A stable and high expression of foreign genes on wounding initiation in T1 progeny was proved by RT-PCR and Northern hybridization test, and there was no significant difference among progeny. Artificial inoculation of 10 positive plants (T1-11) with Lepidoptera larvae (P. xylostella) and fungus (S. sclerotiorum) in in vitro bioassays, showed that the resistance was close to that of parental plants. Their average body weight was from 19.6 mg to 25.3 mg after inoculated 4 days, with survival ratio ranged from 20% to 40%, which was significantly lower than that of control (47.3 mg and 100%, respectively). The spot diameter of progeny leaves from ten positive plants was from 1.03 cm to 1.93 cm, smaller than that of control (3.17 cm). In addition, the average spot diameter was 1.57 cm, whereas the resistance was equal to that of parental plants (1.17 cm). These results demonstrated that foreign target genes had a stable inheritance and high inducted expression in progeny without significant variances, which kept a strong resistance against fungus and insect of B. napus.In summary, it is suggested that the establishment of Agrobacterium-mediated efficient genetic transformation regeneration system has some significant value regarding disease and insect resistance for transgenic B. napus. The transformants could provide a new resistant germplasm resource and theoretical foundation for B. napus breeding program and practice applications.