| Pear is one of the most important temperate fruit crops. It belongs to the Rosaceae family, and is widely grown in temperate and sub-tropical regions of the world. There are many problems in the traditional cross-breeding of pear tree, including a long juvenile period, high levels of genetic heterozygosity, and the need for a large area to screen ideal cultivars. These seriously hamper the process of pear breeding. With the development of plant genetic engineering, Agrobacterium-mediated genetic transformation method was widely used in crop breeding. It can make some economic value exogenous gene(s) integrate into the crop, to improve desirable traits for target plants. An efficient regeneration system is thought to be crucial to the success of plant genetic engineering, since the efficiency of Agrobacterium-mediated transformation is considered to be dependent on two primary factors, one being the regeneration ability of the infected tissue, and the other, the infection efficiency of Agrobacterium.In this research, we have studied the effects of a series of factors (basic media, plant growth regulators, explants inoculation methods, leaf ages dark period and AgNO3) in the inducing adventitious shoot of Dangshansu pear. Then on the basic of optional regeneration system obtained above, we established an efficient and quick Agrobacterium-mediated transgenic system of Dangshansu pear, with which the VpSTS and VpPR10 gene from Chinese wild Vitis pseudoreticulata accession Baihe-35-1 were respectively transformed into the Dangshansu pear genome. The details were as follows:1. Establishment of the regeneration system from leaves of Dangshansu pear in vitro Compared with MS, 1/2MS, QL, NN69, WPM and B5 medium, NN69 medium was more suitable for Dangshansu pear leaf regeneration.The combination of BA 3.0 mg/L and IBA 0.2 mg/L has a good role in promoting shoot regeneration.A certain period of dark culture can also promote the regeneration of Dangshansu pear adventitious buds, among which 3-week dark culture treatment was the best. There was a significant difference in the leaf shoot regeneration rates with various leaf ages. Among them, the leaves with 25-d ages displayed the highest regeneration rate. It was also found that leaf abaxial face down in contact with the medium and regeneration medium supplemented with 0.5 mg/L of AgNO3 have significant impact on shoot regeneration Dangshansu pear. 2. Construction of the plant expression vector pWR-II-VpPR10A nucleotide sequence carrying promotor ED35s, Omega TMV translation enhancer and nopaline synthase terminator was cut from plasmid pWR306 and specifically cloned intoHindⅢ/EcoRⅠdigested plasmid pCAMBIA1300 to construct intermediate vector pWR-II. The segment of VpPR10 gene from the recombinant plasmid pGEM-T-VpPR10 was specifically cloned into BamHI/ SalI digested pWR-II to construct the plant expression vector pWR-II-VpPR10. The recombinant plant expression vector were introduced into Agrobacterium strain EHA105 using the improved freezing-thawing method.3. Establishment of an efficient Dangshansu pear transformation systemIn the transformation of Dangshansu pear, the critical concentration of hygromycin selection was 5 0 mg/L and the inhibitory concentration of cefotaxime was 300 mg/L. Some factors that affect the transformation of Dangshansu pear were studied, including bacterium concentration, infecting time, co-culture time and AS concentration. An optimal transformation protocol was obtained and described as followings: OD600=0.5, 5 min for infection, 48 h for co-culture, 100μM AS in co-culture medium, and 5-d delayed selection. The best induction root was achieved by two-step rooting method. First, the plantlets in vitro were incubated on ASH medium supplemented with 80 mg/L PG and 20 mg/L IBA and cultured in the dark for 7 days. Second, the shoots were transferred to the same medium with 80 mg/L PG in the normal light conditions. As a result, 91.7% of the rooting rate was obtained under above conditions.4. Molecular detection of transgenic plantsHygromycin-resistant transgenic pear plants were validated using polymerase chain reaction (PCR), RT-PCR and Southern blot analysis. The result indicated that the VpSTS and VpPR10 gene have been integrated into the Dangshansu pear genome. The positive plants transformed with VpSTS gene were further analyzed using high performance liquid chromatography (HPLC) method. The result displayed that the resveratrol contents of 3 tested transgenic pear plants were 1.50μg/g, 2.137μg/g, 1.931μg/g (fresh weight), whereas non-transgenic plants were not detected in resveratrol. |
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