A grobacterium-mediated genetic transformation in hot chilli (Capsicum annuum L. var. Pusa jwala)
Introduction
Recent advances in genetic engineering of plants have evoked great interest in developing modern technology for crop improvement. In many countries efforts have been made at cloning genes for use potential in agriculture and introducing them gradually into plants of important crop species. Consequently, many transgenic plants have been successfully produced which showed remarkable results such as resistance to chemicals, pests and disease [1].
Chilli is a spice cum vegetable of commercial importance. In India, the production of chilli has declined from 779 thousand tonnes during 1992–93 to 730 thousand tonnes during 1993–94, the overall percentage in decrease is −6.25. Consequently, the export of chilli fell during the 1994–95 season [2]. There are many factors which contributed to this decline. The most important among them are the diseases caused by viruses, bacteria, fungi and insects. Spraying of fungicides and pesticides can control the diseases to some extent, however, effective resistance against several destructive pathogens is still not possible. Efforts are being made to produce disease resistant plants through genetic engineering.
Genetic transformation through Agrobacterium tumefaciens is now a routine procedure for introducing foreign genes into many plant species including several vegetable crop plants such as tomato, brinjal, Brassica etc. 3, 4, 5. The two most important pre-requisites for the success of the method are the availability of a plant regeneration system from the explants and suitable method for transformation. Although chilli belongs to the Solanaceae family, whose members are easily amenable to tissue culture and transformation practices, it is highly recalcitrant. Several reports of plant regeneration either through organogenesis 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16or embryogenesis 17, 18are evident. However, these reports are genotype-specific and consequently, the regeneration protocol as well as viable transformation has to be established for each commercial cultivar for exploiting the potential of genetic engineering. Recently transformation has been reported 19, 20in sweet pepper with viral coat protein gene and herbicide resistant gene.
In this study, we show the successful establishment of plant regeneration and genetic transformation with marker genes (GUS and NPT II) from the cotyledonary leaves of chilli var. Pusa jwala.
Section snippets
Establishment of aseptic plants
Seeds of Capsicum annuum L. cv. Pusa jwala were surface disinfected with the fungicide, Bavistin, for 1 h and rinsed repeatedly with running tap water. The seeds were sterilized for 2 min in 0.1% HgCl2 and then rinsed in several changes of sterile distilled water. The seeds were germinated in a magenta box containing solidified MS basal medium [21].
Culture condition and media
The cotyledonary leaves from 3 week old seedlings were used as explants and inoculated on the MS medium with 2% sucrose and supplemented with
Regeneration
The composition of the media used in the regeneration of shoot buds and their frequency of induction from the cotyledonary explants are shown in Table 1. On the media containing BAP and IAA, the maximum regeneration frequency of 72.9% was obtained with 10 mg/l BAP and 1 mg/l IAA. Our results are consistent with those in previous reports in which BAP and IAA were used successfully to regenerate shoot buds 6, 7, 8, 9, 10, 11, 12, 13, 14, 16. However, the highest frequency of 90.9% of shoot buds
Acknowledgements
The corresponding author is grateful to Council of Scientific and Industrial Research (CSIR) for sanctioning the project entitled ‘In vitro development of transgenic plants in tomato and Capsicum’.
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