Keshavachandran, RKarmarkar, Shirish HariKAU2020-01-132020-01-132001http://krishikosh.egranth.ac.in/handle/1/5810140291PGThe present study was undertaken at the Department of Plantation Crops and Spices, College of Horticulture, Kerala Agriculture University, Vellanikkara during May 1999 to October 2000 The study was undertaken to standardize the procedure to genetically transform Holostemma ada-kodien and induce hairy roots it was also envisaged to standardise the biochemical techniques for the estimation of secondary metabolites in the roots of Holostemma. Hairy roots were induced by infection of Holostemma explants with a gram negative soil bacterium, Agrobacterium rhizogenes. Leaf segments, shoot buds, intermodal segments, seedling hypocotyls and callus were used as explants for hairy root induction. Among them, the seedling hypocotyls showed highest potential for hairy root induction followed by shoot buds. Leaf segments, intermodal segments and callus did not induce hairy roots. Different strains of Agrobacterium rhizogenes viz PcA4, 15834, A4, 8196 and 2659 were evaluated for their ability to induce hairy roots in Holostemma explants. The strain PcA4 showed the highest potential for hairy root induction, followed by strains 15834 and A4. The strains 8196 and 2659 did not induce hairy roots. Direct inoculation of bacteria on wounds induced hairy roots on seedling hypocotyls only. Co-culture of wounded explants with bacteria induced hairy roots on seedling hypocotyls and shoot buds. In the direct inoculation method, the nature of bacterial inoculum and the intensity of bacterial inoculum applied on wounds influenced the transformation. Bacterial cell suspensions when applied on wounds induced transformation. Less intensity of bacterial inoculum when applied on wounds gave greater transformation frequencies and vice versa. In the co-culture method, the intensity of bacteria present during co-culture, the co-culture time and the shaker speed influenced the transformation. Comparative evaluation of varying intensity of bacterial population during co-culture and the co-culture time showed that, both less intensity of bacterial inoculum with more co-culture time and vice versa showed almost similar transformation frequencies (14.40 % and 14.51 % respectively). The shaker speed of 100 rpm gave the highest transformation percentage than 50 rpm speed. Shaker speed of 150 rpm did not induce any transformation. Application of different concentration of NAA prior to direct inoculation of bacteria on wounded explants did not aid in transformation. Addition of 2 mg 1-1 NAA in the co-culture medium, however, increased the transformation frequency from 25 per cent to 75 per cent. Photoperiod influenced the transformation frequencies. A photoperiod of 16 h light was found to be the best for hairy root induction in Holostemma. Media influenced the hairy root induction. Full strength MS medium favoured hairy root induction while ½ strength MS medium did not favour hairy root induction. Hairy roots were induced in a period of one to four weeks in all the treatments. The induced hairy roots showed altered phenotypes. The hairy root obtained directly from explants without NAA treatment were whitish, hairy and showed negative geotropism. The hairy roots obtained after NAA treatment were brownish yellow and were induced from calli formed on wounds after infection. Hairy roots obtained on infection with strains PcA4, 15834 and A4 showed the presence of agropine confirming their transformed nature. Normal roots did not show presence of opine (s). Hairy roots showed greater sensitivity to lower concentrations of NAA than the normal roots. At 10-9 M concentration of NAA the hairy roots showed lateral branch formation. The tubers and in vitro induced callus of Holostemma were tested for the presence of amino acids, essential oils, triterpenoids and sterols. Six amino acids were found in the root tubers while an additional amino acid (Rf 0.48) that may be L Proline or Cysteine MHC was found in the callus. Root tubers and callus showed the presence of essential oils, triterpenoids and sterols. Root tubers showed the presence of more number of essential oils and triterpenoids than the callus. The callus showed the presence of new terpene compounds. The two sterols present in root tubers and calli were identified to be α –amyrin and β –amyrinennullThesis