Year: 2015 | Month: June | Volume 8 | Issue 2
An Efficient Regeneration and Genetic Transformation Protocol of Coleus forskohlii using Biolistic Gun
DOI:10.5958/2230-732X.2015.00029.7
Abstract:
An efficient selection and plant regeneration protocol for biolistic gun transformation using leaf derived callus of Coleus forskohlii has been developed. Highest regeneration frequency 90% with 50 shoots per callus clump was obtained on Murashige and Skoog (MS) media supplemented with benzylaminopurine (BAP) 2.0 mg L-1 + naphthalene acetic acid (NAA) 0.5 mg L-1 The rate of shoot multiplication was increased with each subculture. Rhizogenesis was obtained on the same media composition. The in vitro raised plants were established successfully in sand and cocopeat (1:1). Callus of C. forskohlii was bombarded using biolistic gun with pABC plasmid DNA which contains β-glucuronidase (GUS) reporter gene and Arabidopsis thaliana white brown complex homologs (AtWBC19) as selectable marker gene. Kanamycin in the shoot induction medium was compared qualitatively and quantitatively for its efficiency as a selection agent for the selection and regeneration of transgenic plants after biolistic gun transformation. Kanamycin levels at or above 50mg L-1 completely inhibited growth of untransformed shoots. The integration of selectable marker gene GUS and AtWBC 19 into the genome of transgenic plants was confirmed using isoenzymatic GUS assay and polymerase chain reaction (PCR) respectively. These results pave the way for the transformation of Coleus forskohlii with desirable genes.
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