| Vitis pseudoreticulata glyoxal oxidase (VpGLOX) was previously isolated from the Chinese wild vine Vitis pseudoreticulata accession'Baihe-35-1'during a screen for genes that are upregulated in response to infection with grapevine powdery mildew(Uncinula necator, PM). In the present study, a possible function of VpGLOX for defence against PM was investigated using Agrobacterium-mediated transient expression. After optimizing agro-infiltration, VpGLOX was transiently overexpressed in leaves of either PM-susceptible (accession'6-12-2') or PM-resistant (accession'6-12-6') plants;'6-12-2','6-12-4','6-12-6', which belongs to segregating populations derived from a Vitis pseudoreticulata'Baihe-35-1'×Vitis vinifera cv.'Carignane'cross, Vitis pseudoreticulata W.T.'Shangnan-2', Vitis pseudoreticulata W.T.'Hunan-1','Carignane', and'Baihe-35-1'were used for Agrobacterium-mediated genetic transformation by a variety of genetic transformation methods, and then the putative transformed grape lines were confirmed. The maily results were as follows:1. The efficiency of transfection was verified using aβ-glucuronidase (GUS) reporter and was found to comprise most leaf areas regardless of the initial leaf position on leaves of the third to the sixth leaves beneath the apex from 6-week old in vitro grown plants. The third and fourth leaves beneath the apex were most fit for infiltrated since they were fully expanded and vigorous. Upon infection with U. necator, clear differences were observed with respect to hyphal development between agro-infiltrated leaves and control groups of both, the susceptible and the resistant, genotypes. At 12 dpi, the overexpressed'6-12-2'and'6-12-6'presented sparse hyphae compared to the profuse fungal growth in control'6-12-2'and'6-12-6'. The expression of VpGLOX was followed by real-time PCR in both genotypes. Whereas'6-12-2'expression was found to increase only in transfected leaves and remained transient,'6-12-6'appeared a second peak later in transfected leaves.2. Regeneration systerm from leaf and petiole of'6-12-2'and'6-12-6'using TDZ and NAA was studied. The results were: adventitious bud from leaves of'6-12-2'could regenerate on 1/2MS medium with 1.0 mg·L-1 TDZ and 0.15 mg·L-1 NAA, but little regenerating rate 1.11%;adventitious bud from petioles of'6-12-6'could regenerate on 1/2MS medium with 2.0 mg·L-1 TDZ and 0.15 mg·L-1 NAA, regenerating rate of 1.67%. The medium fitted for roots inducing was 1/2MS + 0.15 mg·L-1 IBA + 0.02 mg·L-1 NAA.3. Somatic embryogenesis and plant regeneration from flowers of'Hunan-1'and'Shangnan-2'were studied.'Shangnan-2'cultured on medium NN69 + 1.0 mg·L-1 2,4-D + 2.0 mg·L-1 6-BA were best for callus induction, with the induction rate of 8.06%. Then they were transferred to embryo proliferation medium supplementing combination of hormones, 6-BA,NOA and IAA, which turned out to be 0% formation of the somatic embryos. Transient-expression of green fluorescent protein (GFP) gene in transgenic flowers at different developmental stages showed that floweral dip at anthers'uninucleate pollen stage, or infection after 1 week on somatic embryos formation and proliferation, the trangenic rate could be over 93.33%.4. The study on in-planta Agrobacterium-mediated transgenic efficiency on the flowers and fruits of'6-12-2'and'6-12-6'were performed. The DNA from seeds, peels and seedling leaves were extracted. But none of them could be detected the putative VpGLOX by PCR analysis.5. In this study, shoot apical meristem explants of'6-12-2','6-12-4','6-12-6','Shangnan-2'and'Baihe-35-1'were used for Agrobacterium-mediated genetic transformation. For the micropropagation shoots,which was determined should be in liquid C2D4B medium at 80 rpm constant orbital shaking. Then cultures were placed on fresh solidified C2D4B medium with 0.5 mg·L-1 GA. Besides, it did not need to be subjected to a dark growth phase before transformation. For the in vitro internode explants, which could grow well on 1/2MS medium with 2.0 mg·L-1 TDZ and 0.5 mg·L-1 NAA.6. The highest transformation frequency was obtained when explants were infected for 10 min with the concentration of Agrobacterium tunefaciens OD (600 nm) reached a value of 0.4, and then co-cultivated for 3 d.7. Polymerase chain reaction (PCR) and PCR-Southern blot analyses were used to confirm putative transformed grape lines. Up to forty-five micropropagation shoots of'6-12-2', seventeen micropropagation shoots of'6-12-4', ten micropropagation shoots of'6-12-6', three micropropagation shoots of'Baihe-35-1', four micropropagation shoots of'Carignane', produced stable transgenic plants.8. One of'6-12-2'and one of'Baihe-35-1'have been domesticated and transplanted. |
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