Application of tissue culture and molecular techniques in disease resistance breeding of grapevine

Abstract

Grapevines (Vitis vinifera Linnaeus.) are susceptible to a number of diseases which lead to reduced yields and shortened lifespans of vines. This study concerned two important diseases of grapevine, namely bacterial blight caused by Xylophilus ampelinus and powdery mildew caused by Erysiphe necator. The study had two aims, firstly it aimed to develop an in vitro phenotypic screening method for resistance to bacterial blight of grapevine using three varieties, namely Dauphine, Redglobe and C-3229, which is a cross between G4-682 (a breeding line) and Regal Seedless, to reduce the time required for disease resistance screening. Secondly, the study aimed at selecting individuals raised from self-pollinated Kishmish Vatkana (KV) which are homozygous for Ren1. Ren1 is a single, dominant gene conferring resistance to powdery mildew (PM) and is heterozygous in KV. These selected homozygous donors of Ren1 will ensure the transfer of resistance to powdery mildew to all progeny derived from these lines. Grape plants of two cultivars, Dauphine and Redglobe known to be tolerant and susceptible respectively, to bacterial blight were inoculated with VS20 isolate of X. ampelinus (1 x 108 CFU/ml) using scalpel and needle pricking methods for screening. The results revealed that both inoculation methods were successful in discerning resistance to grapevine bacterial blight. Both methods caused plants to develop necrosis at the point of inoculation (IP). However, the needle pricking method showed disease progression over time became worse as necrosis progressed to other parts of the plant and eventually resulted in wilting of plants. On the basis of these results, the needle pricking method was selected to assess C3229, a selection whose resistance reaction was unknown at the start of the study. C3229, like Redglobe, showed susceptibility to bacterial blight. Efficacy of the inoculation was verified with nested PCR analysis. Analysis of macerated samples inoculated with the bacteria confirmed the presence of X. ampelinus in the inoculation point and in areas away from the IP for all three varieties. This indicates some useful traits in Dauphine that have been linked with tolerance to bacterial blight and can be considered as basis for breeding tolerant varieties. The second part of the study involved developing homozygous Ren1 donors for breeding through self-pollinating KV. The population comprised 36 offspring. Genomic DNA from the leaves of the offspring were screened using SSR markers (UDV020, UDV124, VMC9h4.2, VMCNg4e10.1 and VVIP10) to identify offspring homozygous for the Ren1 gene. Among the 36 offspring, four were found to be off types, in other words, they were not from the self-pollinated KV, and rather KV crossed with another cultivar. Some individuals, 18 in total, were similar to the parent since they were heterozygous for Ren1, while five were found to be homozygous, for the recessive alleles. The latter individuals, although homozygous are excluded from the breeding programme as they will be susceptible to powdery mildew. Ten offspring were found to be homozygous for the Ren1 gene. These individuals were identified as suitable parents for breeding. These individuals will be planted out, thereafter, the plants showing the best fruits for breeding will be selected.