Transformation of potatoes with the potato leafroll virus coat protein gene.
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Date
1995
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Abstract
Potato leafroll virus (PLRV) is one of the most destructive potato viruses in South
Africa. In order to establish resistance against PLRV in commercial potato cultivars, the
coat protein (CP) gene of the virus was previously isolated, cloned and subcloned into
the plant expression vector pBI121 in both the sense and antisense orientations
(BURGER, unpublished results). The pBI121 constructs containing the PLRV-CP gene
were subsequently transferred to Agrobacterium tumefaciens LBA 4404 in a triparental
mating process with the helper plasmid pRK2013. Two A. tumefaciens- mediated
transformation methods for potatoes were investigated, viz. vacuum infiltration and leaf
disk transformation. In addition, optimal transformation and regeneration conditions
were identified for potato cultivars Late Harvest and BP[1] In total, 27 transgenic potato
lines containing the PLRV-CP, β-glucoronidase (GUS) and nptII (neomycin
phosphotransferase II) trans genes were generated under kanamycin selection. Transgenic
plants grown in the glasshouse appeared to be phenotypically normal, and no differences
in ploidy level in comparison to non-transformed plants could be established.
Stable transgene insertion into the genome of the transgenic plants was verified using
PCR and Southern blot analysis. Expression of the GUS transgene was investigated
using a fluorometric assay (JEFFERSON et al. 1987), and it was found that orientation
of the inserted PLRV-CP gene upstream from the GUS gene had a direct influence on
the levels of GUS expression. The expression of the PLRV-CP gene was analysed using
DAS-ELISA and immunoblot detection. Coat protein could not be detected in either
assay. RNA dot blots were used successfully to show PLRV-CP expression in
transgenic potato plants at the mRNA level.
Description
Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.
Keywords
Potatoes--Diseases and pests., Transgenic plants., Potatoes--Genetic engineering., Theses--Botany.