IN VITRO REGENERATION AND TRANSFORMATION OF MUSKMELON (Cucumis melo L.) WITH ALTERNATIVE MARKER GENE
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Date
2009-07-22
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University of Agricultural Sciences GKVK, Bangalore
Abstract
Selection of transgenic plants after transformation is a very important task.
Selectable Marker Genes (SMGs) presently in use consist of mostly ‘bacterial originated’
antibiotic resistant marker genes like nptII or herbicide resistant bar gene. Use of these
marker gene consist of potential risk of horizontal transfer of these genes from transgenic
plants to non- harmful bacteria/plants leading to the development of antibiotic resistant
strains or development of superweeds. In this regard efforts have been made to use ‘plant
originated’ AtWBC19 gene as selectable marker gene.
Standard tissue culture technique is an essential part of successful transformation.
Considering good tissue culture response, Muskmelon has been used for transformation.
Standardization of growth regulators for direct regeneration (DR) from zygotic embryos
and somatic embryogenesis (SE) from cotyledonary explants were carried out. Muskmelon
varieties used in this study were Hara Madhu, Punjab Sunheri and Eden Gem. Hormonal
concentration of 1 mgL-1 BAP for direct regeneration and 4.0 mgL-1 of 2,4-D with 0.2
mgL-1 of thidiazuron for somatic embryogenesis was standardized. Among genotypes Hara
Madhu and Eden Gem were considered good for tissue culture. Calli obtained from SE and
zygotic embryos were used as explants for Agrobacterium and Biolistic transformation.
After transformation explants were regenerated on medium containing standardized
hormonal concentration. 100 mgL-1 of Kanamycin was found to be optimum for selection
of transformed explants. Biolistic transformation showed high transformation efficiency
(91% GUS positive explants) over Agrobacterium mediated transformation (20%).
Transformation of muskmelon varieties with AtWBC19 gene was carried out for
testing kanamycin resistance given by targeted gene. The presence of transgene (2.18 kb)
was confirmed through PCR using gene specific primers.
Thus, the use of plant originated selectable marker genes in genetic engineering
may provide a practical alternative to current bacterial marker genes, in terms of risk of
horizontal gene transfer of resistance genes.
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