Exploring new transgenes for pyramiding resistance to potato tuber moth in potato

Abstract

Potato tuber moth, Phthorimaea operculella (Zeller) (PTM), is a major insect pest of potato crops. The larvae mine into foliage and exposed tubers, making them difficult to control with insecticide applications or cultural methods. This thesis describes strategies to genetically engineer potato plants resistant to PTM. It includes the construction of chimeric cry genes, the insertion of these genes into standard binary vectors, and the subsequent transformation of potato using Agrobacterium-mediated gene transfer. Multiplex-PCR was performed to confirm the presence of the selectable marker gene and specific cry gene(s) in all regenerated lines. Multiplex RT-PCR was performed to confirm the transgene expression in transgenic lines. Southern blot and ELISA analyses were performed for transgenic lines to reveal the gene copy number and Cry protein level respectively.

In transgenic potato it is often desirable to couple high-level expression in foliage with no expression in the edible tubers, especially for resistance to pests that primarily infest foliage. To accomplish this, a light inducible Lhca3 promoter for transcriptional control of cry1Ac9 and cry9Aa2 genes for resistance to PTM was successfully used. Significantly inhibited larval growth of PTM on excised greenhouse-grown leaves was observed in 51 % of the cry1Ac9-trangenic lines and 84% of the cry9Aa2-transgenic lines. RT-PCR analysis identified several transgenic lines with high levels of cry gene mRNA in leaves and no to low levels in tubers.

This thesis has also demonstrated the effectiveness of two further cry genes, cry1Ba1 and cry1Ca5, for PTM resistance in transgenic potato. This is important to assemble a suite of genes capable of effecting PTM control to allow future evaluation of different approaches toward deployment of PTM resistance management strategies. Over 90% of the cry1Ca5-transgenic lines gave 100 % larval mortality of PTM on excised greenhouse-grown leaves and tubers bioassays. 40-50% of the cry1Ba1-transgenic lines gave 50 to 100% of larval mortality of PTM on excised greenhouse-grown leaves and tubers bioassays.

An experimental approach to simulate cry gene pyramiding in potatoes was designed and used to assess the interaction of three different cry genes, cry1Ac9, cry9Aa2 and cry1Ba1 genes, in pair wise combinations. It is important to investigate how the expression of two dissimilar Bt toxins interact to confer insect resistance in transgenic plants before developing pyramided plants. The results showed that all combinations of the three cry genes were largely consistent with additive impacts on PTM larvae, although results from the combination of the cry1Ac9 and cry9Aa2 genes were suggestive of slight synergistic effects. Potato lines transgenic for both cry1Ac9 gene and cry9Aa2 gene were developed to investigate the effects of gene pyramiding. The feasibility of two strategies for Agrobacterium-mediated trans gene pyramiding in potato were also evaluated.

The results of this thesis demonstrated that potatoes transgenic for cry genes offer a valuable approach to develop elite plant material for potential use as an additional component in integrated pest management of PTM.