Optimizing Tissue Culture Techniques for Virus Elimination in Grapevines
Microshoot tip tissue culture is the method of choice to eliminate virus infection from valuable grapevine clones. However, survival rates are often low or highly variable. The objectives of this research were 1) to optimize tissue culture protocols to increase shoot tip survival and growth rate, increasing efficiency of tissue culture treatment; and 2) to investigate the use of small single node cuttings for virus elimination. A new protocol was found which was a clear improvement over those used in the past, greatly increasing survival and efficiency of tissue culture treatment for virus elimination in grapevines. Four media were used in various sequence for a total of five different protocols. The effect of virus status on survival was studied utilizing the new protocol. We found no significant difference between healthy or virus-infected source materials in percent survival or percent root development in culture, although incidence of disease symptoms was significantly higher in plants cut from virus-infected source plants. The virus elimination rate in this study was 97%. For objective #2, we have found a significant percentage of “escapes” among relatively large explants cultured from virus infected plants. This suggests that as virus detection techniques become more sensitive it may be possible to regenerate clean stock far more efficiently using relatively large explants. We have found that plants continue to test negative for viruses by ELISA. This is further indication that the virus has been successfully eliminated. A woody index test is in progress to confirm this.