Effect of two different strains of Agrobacterium rhizogenes on hairy root induction in Stevia rebaudiana L.Bertoni
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Date
2012
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Birsa Agricultural University, Kanke, Ranchi, Jharkhand
Abstract
The present work has been conceived to establish the protocol for Agrobacterium rhizogenes
Conn mediated hairy root induction on Stevia rebaudiana L.Bertoni, a valuable medicinal plant
as well as calorie free natural sweetener. The work focused on the induction of hairy roots using
two different strains of A. rhizogenes ATCC 15834 and MTCC 532. The transformed roots were
highly differentiated.
To obtain aseptic explants for transformation, micropropagation was carried out by inoculating
shoot tip of S. rebaudiana on MS media supplemented with 2mg/l BAP, 50 mg/l AdSO4 and
1mg/l citric acid.
Two different Agrobacterium rhizogenes strains (ATCC 15834 and MTCC 532) were tested to
investigate the ability for transformation and production of secondary metabolites. Both bacterial
strains ATCC 15834 and MTCC 532 were maintained at 28°C & 25°C respectively for 48h on
solid medium. The optimum growth of A. rhizogenes (OD 0.6) was observed after 48h of
incubation in rotary shaker in dark. In case of ATCC 15834, the media was supplemented with
50mg/l rifampicin. Bacterial suspension was prepared by inoculating single colony in liquid
medium followed by incubation for 48 h (ATCC 15834) and 24 h (MTCC 532). The selected
explants were wounded and dipped in the thick bacterial suspension. Co-cultivation was done for
different time periods. In case of ATCC 15834 the highest transformation frequency was found
to be 91.60% at 48 h co-culture after 24 days with the highest number of hairy roots (20.66) per
explants. While MTCC 532 showed the highest infection frequency (94.33%) along with the
highest number of hairy roots (24.33) per explants for 3 h co-culture after 24 days.
A. rhizogenes strain MTCC 532 was found to be better than ATCC 15834 both for
transformation frequency as well as number of hairy roots/ explants.
A comparative account of HPLC study using C18 separating column with isocratic mobile phase
revealed presence of active component stevioside at 210nm in in vitro roots of both strains as
well as in vivo roots of Stevia rebaudiana. The identification of stevioside in sample was done by
comparing the retention time.
Description
Effect of two different strains of Agrobacterium rhizogenes on hairy root induction in Stevia rebaudiana L.Bertoni
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