Initiation and maintenance of suspension cultures

Friable calli derived from seedling roots of three genetic stocks, A188 Pr and r-r, were transferred into liquid MS basal media containing 0.5-4 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D), 2,4,5-trichlorophenoxy acetic acid (2,45-T) and 2,4,5-trichloropropionic acid (2,4,5-P) to establish fine cell suspensions for studying growth rate and effect of hormones and genotypes. After a week, the suspension mostly consisted of cell aggregates (2mm) and few single cells (5-10%). Of the four concentrations of 2,4-D tested, 2 mg/l exhibited good separation of the callus mass into fine suspensions of small aggregates and single cells (20-40%); whereas the degree of separation was less in other concentrations as seen by larger aggregates (2mm) and frequency of single cells was low (4-5%). The two analogues of 2,4-D, i.e. 2,4,5-T and 2,4,5-P, were not effective in inducing a fine suspension compared to 2,4-D as evidenced by fresh weights (128, 10.2 and 20.1 mg) and dry weights (15.5, 2.3 and 4.31 mg). Differences were observed, however, in the size of cell aggregates. 2,4,5-P was found to be superior over 2,4,5-T in having small cell aggregates (2-4%) whereas in the former, only aggregates were observed. Larger aggregates had revealed loosely connected smaller cells. The cells were of mostly parenchymatous type and revealed cytoplasmic strands. As subculture proceeded, the rounded cells predominated and formed into aggregates, whereas the elongated cells remained undivided, suggesting association of cell types in differentiation. A188 exhibited good cell suspension compared to other genotypes.

P Suprasanna, K.V. Rao and G.M. Reddy

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