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VOL. 6, ISSUE 6 (2021)
A simple and efficient Agrobacterium-mediated gene transformation in leaf explant of Coleus forskohlii (Briq)-GUS as a reporter gene
Authors
P Senthil Kumar, A Lakshmi Prabha
Abstract
An efficient selection and plant rejuvenation protocol for Agrobacterium mediated gene transformation using leaf explant of Coleus forskohlii has been developed. Leaf explants from 30 days old in vitro plant were inoculated with EHA105 bacterial strain (carrying NPT II and GUS gene).Among the tested factor T-DAN transfer of plants, the EHA105 strain and addition of the Aceto-syringone to the co-cultivation medium increased the transformation efficiency. The factors affecting the frequency of transient gene expression were optimized, including leaf age, Agrobacterium concentration, infection time, Kanamycin concentration and co-cultivation period. The highest efficiency (32%) was obtained in 4 day pre-culture, 1.0 OD bacterial suspension culture, were sub-cultured on selection medium (MS medium with KIN 1mg/l + NAA 0.1mg/l +Kanamycin 50mg/l) after 3-day co-culture with Agrobacterium. Kanamycin levels above 50mg/l completely inhibited growth of untransformed region of the plants. The integration of selectable marker gene (NPTII) and reporter gene (GUS gene) into the genome of transgenic plants was confirmed using histoenzymatic GUS assay and polymerase chain reaction (PCR) respectively. These results pave the way for the transformation of Coleus forskohlii with desirable genes and should provide novel opportunity for the genetic improvement of more production of secondary metabolite in medicinally important of C.forskohlii.
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Pages:1196-1201
How to cite this article:
P Senthil Kumar, A Lakshmi Prabha "A simple and efficient <em>Agrobacterium</em>-mediated gene transformation in leaf explant of <em>Coleus forskohlii (Briq)</em>-GUS as a reporter gene ". International Journal of Botany Studies, Vol 6, Issue 6, 2021, Pages 1196-1201
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