19 research outputs found
Genetic transformation studies and scale up of hairy root culture of Glycyrrhiza glabra in bioreactor
The study was undertaken to induce hairy roots in Glycyrrhiza glabra
in leaf explants and to optimize the nutritional requirement for its
growth kinetics at shake flask and bioreactor level. Pathogenecity of
Agrobacterium depends upon transformation ability of strain and age,
type, and physiological state of explants. Agrobacterium rhizogenes
strain K599 was used to infect leaf explants of G. glabra. Explants of
different age groups were obtained from 2 to 5 weeks old in vitro grown
cultures. Bacterial strain K599 could induce hairy roots in 3 and 4
weeks old leaf explants cultured on B5, MS, NB and WP basal semi-solid
medium. Leaf explants of 2 and 5 weeks old culture were not responsive
to bacterial infection in terms of hairy root induction. Maximum
transformation frequency (TF) of tested bacterial strain was 47%
obtained in 3 weeks old explants after 25 days of incubation on MS
basal semi solid medium. NB and B5 both media composition showed 20% of
transformation frequency after 28 and 38 days respectively. WP medium
did not support induction of roots in cultured leaf explants infected
with A. rhizogenes strain K599even after 50 days of incubation.
Further, when all the four media combinations were tested for root
growth it was found that though WP was not responsive for hairy root
induction, yet all four basal media supported hairy root growth and a
gradual increase in fresh weight biomass was observed with an increase
in culture duration. However amongst all, the NB medium composition
supported best growth of hairy roots followed by MS, B5 and WP media.
About 20 times increase in root biomass on fresh weight basis was
recorded after 45days of culture in NB medium. Initial inoculum of
roots (0.18 g. F.wt./ flask) containing 50 ml of liquid culture medium
produced 3.59 g (F. wt.) biomass. A fast growing hairy root clone G6
was grown in a 5 l capacity mechanically agitated bioreactor provided
with a nylon mesh septum. After 30 days of sterile run, 310 g of root
biomass was harvested from the bioreactor culture vessel, recording
about 20 times increase over initial inoculum (16.0 g)
Not Available
Not AvailableThe molecular basis of antifungal toxin production by a fluorescent Pseudomonas strain EM 85 was studied. The bacterial strain showed in vitro inhibition of growth of Rhizoctonia solani. NTG mutagenesis of the wild type strain helped in the isolation of a antifungal-toxin-defective mutant AN 21. A genomic library of the wild type strain was constructed in the cosmid vector pLAFR 1 and maintained in an E. coli background. Complementation analysis with cosmid library resulted in the isolation of a cosmid clone which complemented the defective character in the mutant AN 21. The size of the complementing DNA fragment was found to be 23.5kb.Not Availabl