Genetic engineering of potato cultivars for potato tuber moth resistance

Potato tuber moth Phthorimaea operculella (Zeller) is a major insect pest of potato (Solanum tuberosum L) throughout the world, including New Zealand. This thesis investigates two genetic engineering strategies to develop potato cultivars resistant to this pest using cry genes and genes encoding biotin-binding proteins. The crystal proteins of Bacillus thuringiensis are specifically toxic to larvae of certain Lepidoptera, Coleoptera and Diptera. Two cry genes (cry1Ac9 and cry9Aa2), with a series of modifications to their codon use, have been transferred to a range of potato cultivars by an Agrobacterium-mediated transformation system. The transformation efficiency of recovered putative transgenic lines determined that cultivars Iwa and Red Rascal are both highly amenable for transformation and regeneration response. Molecular analysis and larvae feeding trials on potato lines transgenic for either the modified cry1Ac9 or cry9Aa2 genes identified 38 and 31 transgenic lines respectively with normal phenotypic appearance in a containment greenhouse and significantly less leaf damage compared with the non-transgenic controls. To investigate an alternative approach for PTM resistance a chimeric avidin gene, with the expressed protein targeted to the vacuoles of plant cells using a signal peptide from the PPI-I gene from potato, was transformed using three explant sources from cultivars Iwa and Red Rascal. This identified leaf explants as the most convenient and productive explants source for potato transformation relative to stems and roots. Complete larvae mortality was observed in 65% from potato lines transgenic for the avidin gene. A great effect on the phenotypic appearance of the transgenic lines expressing the avidin gene was evident. Avidin concentrations >0.8 nmole/gm of fresh weight, as determined by ELISA, either markedly inhibited larval growth rates or induced complete larval mortality. The field evaluation of 84 potato lines transgenic for either the modified cry1Ac9 and cry9Aa2 genes identified 15 phenotypically normal potato lines transgenic for the cry1Ac9 and 21 for cry9Aa2 genes with significantly reduced larval growth and similar tuber yield to nontransgenic controls