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Potato transformation using Agrobacterium tumefaciens to study root-knot nematode (Meloidogyne spp.) resistance

Purwati, Rully Dyah (1996) Potato transformation using Agrobacterium tumefaciens to study root-knot nematode (Meloidogyne spp.) resistance. Masters by Research thesis, Murdoch University.

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Potato (Solanum tuberosum, L) is an important crop for food consumption and biomass which is susceptible to several pests and diseases, including root-knot nematodes {Meloidogyne spp.) Root knot nematodes are serious pests of food crop and horticultural plants in tropical and sub-tropical regions of the world. In order to apply molecular approaches to confer resistance to root-knot nematodes to new potato varieties, it is necessary to have optimum conditions for tissue culture and transformation. A major aim of the work reputed was to establish such conditions for potato varieties grown in Western Australia.

In this work, a shoot regeneration system for potato cvs. Delaware, Desiree, Kennebec, and Russet Burbank has been studied using tissue culture in vitro. Different explants (stem segments and leaf pieces) were cultured on two different callus induction and shoot regeneration media of different hormone composition. Results from this work showed that medium M1 (callus induction medium: MS medium, NAA (0.186 mg/l), BAP (2.25 mg/l) followed by shoot regeneration medium: MS medium, BAP (2.25 mg/l), and GA3 (5 mg/l} was the best overall medium composition for callus production and shoot regeneration. This system has been applied to generate transgenic plants. The two plasmids which were used in transformation studies with cvs. Delaware and Desiree, pAF1 and pAF2, contained the ∆0.3 and ∆0.6 sequences of the TobRB7 promoter linked to the gus reporter gene, and also contained a sequence for constitutive expression of the npt II gene which conferred resistance to kanamycin. Transformation using Agrobacterium tumefaciens and regeneration of transformed shoots of potato has resulted in regeneration of a 76 putative transgenic plants of Delaware and 72 transgenic plants of Desiree (see Appendix 1 and 2). The putative transgenic lines generated were analysed by PCR and Southern hybridisation to confirm their transgenic nature. Based on PCR analysis, more that 70% of the lines tested has the gus gene and all plants tested except 3 of cv. Desiree with pAF1 contained the kanamycin resistance gene. This is the first report of the production of transgenic plants of potato cv. Delaware.

Potato plants transformed with the pAF1 construct, containing ∆0.6TobRB7 promoter (pAF2) which has been reported to drive gus expression only in nematodeinduced giant cells, were infected with juveniles of the root-knot nematode Meloidogyne javanica. However, no gus expression was found in the transformed plants tested in this work. A number of factors can influence detection of the gus expression, for example, the size of cells and their metabolic activity, position and copy number of T-DNA insertions. Transgenic potato plants transformed with the ∆0.6TobRB7 promoter (pAF2) which is reported to drive gene expression in roots and in giant cells were evaluated by nematode infection. Most of the lines tested showed gus expression in the root tissues and giant cells of infected plants, and in root tissues of uninfected plants.

It is concluded that in future work transgenic plants resistant to root-knot nematodes (or other nematodes) may be generated by fusing the A0.6TobRB7 promoter to a gene which can disturb nematode migration of body structure or giant cells, without an effect on plant cells.

Item Type: Thesis (Masters by Research)
Murdoch Affiliation(s): School of Biological and Environmental Sciences
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: Thank you.
Supervisor(s): Jones, Michael and Potter, Robert
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