Tissue Culture Of Altemanthera Ficoidea Cv. "Ruliginosa" And Its Genetic Transformation By Agrobacterium Rhizogenes

Tissue culture and plantlet regeneration from leaf explants of A.Ficoidea cv."Ruliginosa" and its genetic transformation with Agrobacterium rhizogenes ATCC 15834 were investigated as well as changes in the contents of sucrose, nitrogen and phosphate in liquid medium during the hairy root cultures. The main results were as follows:Light green calli could be formed from all the leaf explants of A.Ficoidea cv."Ruliginosa" when cultured for 20 days on MS+6-BA1.0mg/L+4-PU 1.0mg/L+ NAA 0.lmg/L. Adventitious shoots could be developed from the calli at the frequency of 45.31% on MS medium supplemented with 1.0 mg/L 6-BA and 0.4 mg/L NAA. Adventitious shoots could be rooted and formed regenerated plants on 1/2 MS medium supplemented with NAA0.4mg/L.Hairy roots could be induced from the basal surface of the explants 8 days after inoculation of A.Ficoidea cv."Ruliginosa" leaf explants with A.rhizogenes ATCC 15834. The percentage of rooted leaf explants was 92.5% 21 days after infection, but the inducing frequency of hairy roots was 53.8%. Hairy roots were highly branched and had a plenty of white root hairs. All the hairy roots of A.Ficoidea cv."Ruliginosa" could have a rapid autonomous growth on phytohormone-free liquid or solid MS medium. The transformation of hairy roots was confirmed by PCR amplification of rolB and rolC genes of Ri plasmid from A.rhizogenes.The green callus was developed at the induction rate of 68.75% from hairy roots cultured onto MS medium supplemented with 6-BA 2.0mg/L,4-PU 2.0mg/L and NAA 0.5mg/L.Adventitious buds can be observed to form gradually from the calli on MS medium supplemented with 6-BA2.0mg/L and NAA0.8mg/L.When cultured in liquid medium, the growth period of A.Ficoidea cv."Ruliginosa" hairy roots could be divided into three phases: lag phase, rapid-growth phase and plateauphase. Changes in contents of sucrose, nitrogen and phosphate in liquid medium were determined during the whole culture of hairy roots. The results showed that the consumption rate of sucrose in the medium was positively related to the growth rate of hairy roots. Nicrate nitrogen and phosphate in liquid medium were quickly consumed at the lag phase of the growth, the consumption rate was 16.71 μ g/ml d and 1.67μ g/m) d, respectively; but during the rapid-growth phase, its consumption rate was decreased. The content of ammonium nitrogen in the medium reduced rapidly during the lag phase, but showed a slightly increase during the rapid-growth phase. The pH value in the medium was reduced gradually with time during the whole culture of hairy roots.