Genetic evaluation of Agrobacterium rhizogenes mediated hairy root induction on Stevia rebaudiana L. Bertoni
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Date
2011
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Birsa Agricultural University, Kanke, Ranchi, Jharkhand
Abstract
Hairy root cultures produced by Agrobacterium rhizogenes Conn mediated transformation,
have been widely accepted as an important experimental system for plant physiological and
molecular research. It has also been found promising for producing useful secondary plant
metabolites due to its many well known advantages as fast growth rate, high genetic stability,
and regeneration ability. Stevia rebaudiana L. Bertoni is a herbaceous perennial plant of the
Asteraceae family. It is a natural sweetener plant known as “Sweet Weed”, “Sweet Leaf”,
“Sweet Herbs” and “Honey Leaf”, which is estimated to be 300 times sweeter than cane sugar.
The leaf extract of this plant has been used traditionally in the treatment of diabetes.
This work compiled the in vitro hairy root induction on node and shoot tips of
S.rebaudiana and their molecular characterisation through PCR using gene specific primers.
To get aseptic explants micropropogation was carried out. Contamination free culture
was established by treating shoot tip explants with 0.1% HgCl2 for 5 minutes followed by their
inoculation in MS medium supplemented with BAP 1.0 mg/l, AdSO4 50 mg/l and citric acid 1.0
mg/l.
After 48 hour of incubation of Agrobacterium rhizogenes at 28ºC in YEB broth (OD 0.6)
supplemented with 50.0 mg/l rifampicin, bacterial pellets were obtained and resuspended in MS
broth that further used for co-cultivation. For inducing hairy root, 48 hours co-culture was found
to be best (Shweta Kumari, 2010) and inoculated into the MS medium devoid of any
phytohormone.
Molecular characterisation of hairy roots was done through PCR using rolC and aux1
gene specific primer. For this, genomic DNA from hairy root of shoot tips and node were
isolated using standard CTAB method. On PCR, amplification of the specific gene was noted in
the transformant at 487 bp for rolC and 815 bp for aux1 respectively. On the other hand, non
transformant did not show any amplification corresponding to the gene specific primers.
Description
Genetic evaluation of Agrobacterium rhizogenes mediated hairy root induction on Stevia rebaudiana L. Bertoni
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