GENETIC TRANSFORMATION STUDIES IN BELL PEPPER (Capsicum annuum L.)
Loading...
Date
2009
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
ABSTRACT
The research work was conducted to standardize a protocol for genetic transformation in bell
pepper. Plant regeneration studies were carried out using two types of explants viz hypocotyl and cotyledon.
The cotyledon explants showed high frequency of shoot regeneration (80.95%) on MS medium
supplemented with 6.0 mg/l BA and 0.3 mg/l IAA as compared to hypocotyl (only 8%) on MS medium
supplemented with 4.5 mg/l BA and 0.1 mg/l IAA. The shoots regenerated after 25-30 days. The
regenerated shoots were in the form of profuse rosette, which elongated to about 2-2.5 cm on MS medium
supplemented with 2.25 mg/l BA and 2 mg/l GA3. MS medium containing 0.25 mg/l IAA was found to be best
for root regeneration (50%). Regenerated plantlets were acclimatized successfully. During kanamycin
sensitivity test, kanamycin concentration as low as 20 mg/l was found to be inhibitory to the growth of bell
pepper cells. For genetic transformation, disarmed Agrobacterium tumefaciens LBA 4404 strain containing a
reporter -glucuronidase (gus) gene in binary vector (pBI 121) system along with kanamycin resistance
gene (npt-II) for selection in both bacteria and plant was used for co-cultivation experiment to transfer gus
and npt-II genes in bell pepper cells (cotyledon and hypocotyl explants). After co-cultivation only the
transformed cells were able to grow on selective shoot regeneration medium which formed callus and
regenerated shoots, whereas non transformed cells/explants died on the selective medium. The elongation
of the putative transgenic shoots was achieved on selective elongation medium. Root regeneration in the
putative elongated transgenic shoots was obtained on selective root regeneration medium. Out of the total
15 putative transgenic shoots developed, 5 were randomly selected for PCR analysis using designed
primers for npt-II and gus genes. Only two shoots/plantlets showed amplified gene separately indicating the
presence of npt-II and gus gene(s) in the transgenic shoots/plantlets. Expression of the gus gene was
confirmed by GUS assay. The PCR positive shoots/plantlets have shown the expression of gus gene as
well. A protocol for genetic transformation in bell pepper has been standardized.