ISHS Acta Horticulturae 528: VII International Symposium on Grapevine Genetics and Breeding
DEVELOPMENT OF TRANSGENIC GRAPEVINE ROOTSTOCKS WITH GENES FROM GRAPEVINE FANLEAF VIRUS AND GRAPEVINE LEAFROLL ASSOCIATED CLOSTEROVIRUSES 2 AND 3 |
| Authors: | S. Krastanova, K.S. Ling, H.Y. Zhu, B. Xue, T.J. Burr, D. Gonsalves |
| Keywords: | Embryo differentiation, regeneration, callus, heterografting, micrografting |
| Abstract:
Grapes are the most widely grown fruit crop in the world with grapevine fanleaf virus (GFLV) and grapevine leafroll viruses (GLRV), being the two most damaging and widespread viruses that affect them. Grapevine fanleaf virus is transmitted by a soilborne nematode and is a nepovirus of great economic importance. At present, effective insecticides and nematicides are either not available because they cause environmental pollution. Grapevine leafroll associated closterovirus 3 (GLRaV-3) is transmitted by mealybugs, however, there is no evidence of vector transmission of GLRaV-2. Development of rootstocks resistant to GFLV and GLRV should provide an alternative solution to control these important virus diseases. The goal of this work is to introduce resistance genes from nepoviruses and closteroviruses into grapevine rootstocks species using the pathogen-derived resistance approach. Immature flower buds were harvested in May-June from grapevines in the field and in March from woody canes that had been forced in greenhouse. Anthers at different stages were isolated and plated on MS medium containing 2,4-D and BA. Callus initiation and proliferation was performed for 1–3 months from 3309 C, Riparia Gloire, MGT 101-14, 110 Richter, Rupestris St. George and 5C Teleki. Clusters were kept in the dark at 28°C. Depending on the genotype, embryogenic callus culture were induced in 12–47% of the isolated anthers. Embryogenic calli were transferred onto different media (with or without hormones) in order to induce embryo differentiation. Early stage embryogenic calli were cocultivated with Agrobacterium tumefaciens strain C58Z707 or LBA4404 containing different constructs of virus genes. The gene constructs include translatable, antisense or nontranslatable coat protein genes from GFLV, GLRaV-3 and GLRaV-2. A gene sequence in an open reading frame of GLRV-3 was also used. Over 24 independent transformations were performed. After 2–3 months under selection on kanamycin, secondary embryogenic calli were recovered and were regenerated in MS basic medium without hormones. The plantlets were transferred to rooting medium. Numerous putative transgenic lines have been developed. Moreover, grapevine plants regenerated through somatic embryogenesis appeared morphologically normal, showing no somaclonal variation. Characterization of transgenic plants are being carried out using different techniques, including NPTII ELISA, DAS-ELISA for coat protein expression, GUS assay and PCR analysis and Southern blot. Analysis of plants expressing the translatable coat protein gene of GFLV shows various levels of expression. Resistance to GFLV is being evaluated by different inoculation techniques including micrografting, heterografting with GFLV infected Chenopodium quinoa, in vitro green grafting and woody grafting. | |
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