Genetic transformation studies and scale up of hairy root culture of Glycyrrhiza glabra in bioreactor
Arun Kumar Kukreja
Suman Preet Singh Khanuja
Bhartendu Nath Mishra
Financial support: fellowship by Council of Scientific and Industrial Research (CSIR), Government of India to the author Shakti Mehrotra.
Keywords: Agrobacterium, bioreactor, Glycyrrhiza, scale-up, transformed roots.
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).