Summary of SKM's MSc Thesis submitted to the Indian Agricultural Research Institute, New Delhi

Tobacco (Nicotiana tabacum) and soybean (Glycine max) cells were cultured in vitro and attempts were made to synchronize cell divisions in these systems.

Callus cultures of both these species were grown on solid MS medium and a variation of this containing basal MS+2.0mg NAA and 0.2mg kinetin per litre.Batch propagated suspension cultures of tobacco cells were grown in liquid medium containing basal MS +2mg NAA and 0.2mg kinetin per litre and of soybean cells, in a similar medium containing 0.1mg instead of 0.2mg kinetin per litre.The proliferating cells in the suspension cultures were in clusters of varying sizes. Synchronization was tried by different kinds of starvation-replenishment treatments.Advanced stationary phase cells transferredinto fresh medium at low inoculum density yielded partial synchrony: a peak mitotic index of 6.8 per cent, after a lag of 41 hours, was obtained in the case of tobacco suspension culture cells and in the case of soybean suspension culture cells, the first peak mitotic index of 6.8 per cent was obtained after a lag of 31 hours, followed by another small peak of 3.5 per cent mitotic index after another 40 hours.

Tobacco callus cells deprived of kinetin by incubating them in kinetin-free medium and subsequently provided with kinetin, showed remarkable synchrony of the first two sets of divisions that took place in rapid succession, within a period of 11 hours.The second set of divisions was less synchronous than the first. The kinetin starved cells had mostly taken up, elongated tubular shape and when induced to divide,gave rise to uniseriate filaments of cells, which later formed 3-dimensional colonies.Deprivationof kinetin and its subsquent replenishment in suspension cultures of both tobacco and soybean gave good synchrony of the first wave of divisions, with the mitotic indices reaching as high as 19.6 per cent and 18.4 per cent in the case of tobacco and soybean cell suspensions, respectively.In one trial with tobacco suspension culture cells,a second, though diminished peak of mitotic activity was obtained by the same technique.

Combining stationary phase starvation with kinetin deprivation followed by transfer into fresh complete medium yielded a peak mitotic index of 15.4 per cent after a lag of 33 hours in the case of soybean suspension culture cells,but did not result in any synchrony in the case of tobacco suspension culture cells, when very few divisions were obtained up to 60 hours after their transfer into complete medium.

Sucrose starvation in soybean suspension culture cells and its subsquent replenishment resulted in two groups of cells dividing synchronously, one nine hours after the other, indicating that sucrose starvation had caused arrest at two different points. This treatment in the case of tobacco suspension culture cells did not reveal any synchrony.

Cytological studies revealed a sequential pattern of chromosomal condensation during the preparatory and early stages of mitosis, that was very helpful in recognising cells at the G2-Mitosis boundary, when chromosomal threads begin, to appear,which proved to be much more prolonged than the other mitotic phases.A transient chromosomal condensation (typical of late G2) phase which did not lend to normal mitotic condensation,was noticed in the case of stationary phase cells transferred to kinetin-less medium,indicating that kinetin is required for the transition from G2 to the mitotic phase.

Synchrony at the level of the two nuclei of a binucleate cell was excellent, in that they both divided simultaneously.Also when small groups of cells were taken into consideration, remarkable synchrony of divisions were observed;but the overall synchrony at the population level was never as high as in isolated groups, due to the heterogeneous composition of the suspension cultures, which included proliferating as well as non-dividing cells in aggregates of varying sizes.Chromosome number counts revealed ploidy changes in the cultured cells of both tobacco and soybean.