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Cell biological manipulations for transfer of the Mi root-knot nematode resistance gene from tomato to potato

Improvement of potato (Solanum tuberosum), an important crop for food consumption and biomass has until recently been confined to conventional breeding approaches. Root-knot nematodes (Meloidogyne spp) are serious pests of crop and horticultural plants, including potato, in tropical and sub-tropical regions of the world. The Mi resistance gene derived from Lycopersicon peruvianum has been introduced into cultivated tomato. This gene confers resistance to three major species of root-knot nematodes. Tomato lines containing the Mi gene have been obtained and were tested for functional resistance to root-knot nematodes by infection with nematode juveniles in glasshouse tests. The presence of the Mi gene in resistant tomato lines was also confirmed using two biochemical methods: (1) by staining of specific bands on polyacrylamide gels (PAGE) for an acid phosphatase-I I enzyme linked to the Mi gene, and (2) the presence of an amplified DNA fragment of 570 hp using specific PCR primers (REX-Fl and REX-F2) which amplify a DNA sequence closely linked to the Mi gene. For tissue culture experiments, potato and tomato shoot cultures were established, and larger potato leaves were induced by treatment with silver thiosulfate (STS) to inhibit ethylene production. Potato and tomato protoplasts were isolated from these shoots and a protoplast-to-plant regeneration system for potato cv. Delaware was established. Conditions required to induce electrofusion between potato and tomato protoplasts were optimised. However, the Mi resistant tomato genotypes were not responsive in culture, and hybrid plants were not generated. As a result, procedures for direct gene transfer of the Mi gene were studied. Optimum conditions for direct gene transfer ( electroporation) of potato cv. Delaware were established and calli were obtained from Delaware protoplasts after electroporation with tomato DNA (line BTN815). These were analysed by PCR. To improve the frequency of transformants that might contain Mi DNA, DNA enriched with low copy repetitive sequences was obtained by passage through a hydroxyapatite column. This work forms the basis for further experiments to transfer DNA fragments, which include the Mi gene, into potato by direct gene transfer. In further cell biological studies, transgenic plants containing a Parasponia promoter-glucuronidase insert were analysed for expression of the reporter gene when infected with root-knot nematodes. The reporter gene was down-regulated in early stages of giant cell development, but strongly up-regulated in mature giant cells. 
 
The results of the research were discussed in relation to various options available to introduce resistance to root-knot nematodes into potato, using cell biological transfer or genetic engineering of natural or synthetic resistance genes.