Regeneration and biotransformation of some members of the Cucurbitaceae.
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Date
1998
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Abstract
Five cultivars, all belonging to the family Cucurbitaceae, have been tested for the
ability to regenerate shoots or somatic embryos from cotyledonary explants. The
influence of several combinations of growth regulators on regeneration from
cotyledonary and other explants was tested.
No regeneration was obtained from the two cultivars Cucurbita maxima Duch. cv
A-Line and Cucurbitapepo L. cv Rolet. Somatic embryos developed on Cucurbita
maxima Duch. cv Chicago Waited, a Hubbard squash. A shoot regeneration
response was observed for the cultivar Cucumis sativus L. cv Ashley, but the
frequency was low and results could not be repeated in subsequent experiments. A
reliable shoot regeneration protocol was developed for Cucumis melo L. cv Hales
Best 36.
The influence of the antibiotics kanamycin sulphate and cefotaxime on shoot
regeneration from cotyledonary explants of Cucumis melo L. cv Hales Best 36 was
tested. The plasmid pBI121 was transferred from Escherichia coli strain HB101 into
Agrobacterium tumefaciens strain LBA4404 via a triparental mating. The plasmid
pBI121, contains the screenable marker gene β-glucuronidase (GUS) and the
selectable neomycin phosphotransferase-II gene (NPT-II) that confers kanamycin
resistance. Cotyledonary tissue was transformed using this Agrobacterium
tumefaciens transformation system. The influence of co-cultivation time, inoculation
time and the wound factor acetosyringone on transformation was established. Rooted
plantlets were regenerated from transformed cotyledonary tissue placed on
kanamycin supplemented regeneration media. Plantlets tested positive for the
presence of the GUS gene, using fluorometric and histochemical assays.
The developed protocol was used to transform Cucumis melo cv Hales best 36 with
the pat gene that provides resistance to the herbicide Ignite®. A selection medium
was developed containing phosphinothricin, the active ingredient of the herbicide Transformants were selected on this medium and five lines were recovered. These
plants were acclimatized and the herbicide resistance was confirmed in greenhouse
spray tests. The ploidy level of these plants was deduced from indirect evidence of
micro- and macroscopic characteristics that have been shown to have a correlation
with the chromosome number of melon plants.
The five lines were subjected to molecular analysis. The polymerase chain reaction
was used to give an indication of the transformed nature of the selected plants.
Agarose gel electrophoresis confirmed that the correct size band could be obtained
from the putative transformants and the presence of pat in the product was verified
using a non-radioactive system for nucleic acid analysis. Stable gene insertion into
the genome of the plant was verified with a Southern blot of the total genomic DNA.
This was achieved by hybridising a radioactively labelled ³²P probe specific for the
pat gene to a blot of restriction digested plant DNA.
Description
Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1998.
Keywords
Cucurbitaceae--Genetic engineering., Transgenic plants., Cucurbitaceae--Diseases and pests--Control., Theses--Botany.