Genetic engineering for pest resistance in potato (Solanum tuberosum L.)

Abstract

Insect pests of potato cause significant losses of yields under field and storage conditions. Conventional breeding techniques for targeting genes of interest is usually laborious and time-consuming. An alternative way for targeting genes of interest into the plant genome consists of Agrobacterium-mediated gene transfer. This thesis studied Agrobacterium-mediated transformation to transform two potato cultivars with an avidin gene anticipated to confer insecticidal activity against certain lepidopteran insects.

A successful gene transfer into plants requires a high regeneration response of transformed cells of the target explant used for plant transformation. Twenty four potato cultivars, each represented by three explant sources (leaves, stems, and roots), were screened for their regeneration response on a standard regeneration medium. From this study 'Red Rascal' was found to have an outstanding regeneration response.

A maximum of 50 shoots regenerated from leaves, stems, and roots of this cultivar were recovered. A field trial was established with these plants to study a possible effect of the explant source used for plant regeneration on the frequency of somaclonal variation. A similar frequency of somaclonal variation was observed in plants regenerated from all three explant sources considered. This indicates that somaclonal variation is independent from the explant source used for plant regeneration.

After determining the outstanding regeneration response of cv. 'Red Rascal', this cultivar together with a standard cultivar ('Iwa') were transformed using an Agrobacterium binary vector bearing the gene encoding for avidin protein. The avidin protein is known for its insect antimetabolite properties. Both cultivars were successfully transformed with the avidin gene. Surprisingly, 'Red Rascal' showed the lowest overall transformation efficiency (0.12 transformants/explant). In contrast, transformation efficiency of 'Iwa' reached 0.81 transforrnants/explant. All transformed plants rooted on kanamycin-containing medium. Six transgenic lines were challenged with neonate larvae of the potato tuber moth (Phthorimaea operculella). These were three lines from cultivar 'Iwa' and three lines from cultivar 'Red Rascal'.

The transgenic status of putative transgenic lines was successfully determined by means of the polymerase chain reaction. All true transgenic lines (those that rooted on kanamycin-containing medium and showed the band corresponding to the avidin gene after PCR) successfully controlled growth of potato tuber moth neonate larvae. The growth index of larvae fed on transgenic lines was significantly inhibited by the presence of the avidin gene compared to the growth index of larvae fed on leaves of non-transgenic plants.