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

The present study was undertaken with two main objectives: (1) to standardise procedures for efficient culture of protoplasts from four plant species, viz. Nicotiana glauca, N.glutinosa, Brassica oleracea var. capitata and Vigna radiata and (2) to find out a procedure for efficient fusion of protoplasts from diverse sources, in order to achieve a high frequency of heterokaryon formation.

Protoplasts isolated from 3-day precultured leaves of N. glauca divided regularly in KM8p medium of Kao and Michayluk (1975) and MSM medium which is a modification of the Murashige and Skoog (1962) formulation. A minimum plating density of 10 (to the power 4) protoplasts/ml was requried in either of these media. The response in ON medium of Ohyama and Nitsch (1972) was poor. Plants could be regenarated from protoplast derived calli which were initiated in MSM medium but not from those grown in KM8p medium.

Nicotiana glutinosa callus protoplasts were fractionated into two populations based on whether or not they floated in the MSM medium. The heavier population showed better plating efficiency than the lighter one, when protoplasts were isolated from young or old calli. For any given fraction, the protoplasts from younger calli wer more responsive than those from older ones,although the yield of protoplasts from the latter was more.

A plating density of 5x10 (to the power 4) was found suitable in the case of the three media tried, viz., KM8p medium, MSM medium and MSG medium which is an adaptation of the MSM medium with glucose in the place of mannitol. There was no significant difference among the response of N. glutinosa protoplasrs in these media. The protoplast derived calli failed to regenerate plants, just as the source callus in this system.

Brassica oleracea var. capitata precultered leaves yielded more protoplasts per gramme tissue, than leaves which were not precultured. The protoplast yield increased with the duration of preculture upto 5 days of the latter. There was a similar effect of the duration of preculture on the plating efficiency of the protoplasts. The lighter and heavier fraction protoplasts, that were separated in the same fashion as mentioned above in the case of N.glutinosa, revealed distinct morphological differences. The proportion of lighter fraction protoplasts increased with the duration of preculture. There was no large differences in the plating efficiencies of the two fractions.

A density of plating around 10 (to the power 5) protoplasts/ml was critical. The presence of glutamine in the medium enhanced the plating efficiency significantly. KM8p medium gave inferior results when compared to the 1/2MSM (gln) medium or its derivatives. Callus recovery was better on agar solidified medium than in liquid medium. Rooting of protoplast derived calli was very frequent and was initiated at an early stage. Shoot regeneration was comparatively rare and delayed.Shoots could be obtained only on the shoot culture medium of Binding (1974) supplemented with activated charcoal.All the other shoot differentiation media, including the above without activated charcoal, failed to support shoot regeneration.

Protoplasts from Vigna radiata hypocotyls could also be separated on the basis of their bouyant densities.The heavier ones were smaller in size and possessed more starch grains compared to the lighter ones.The plating efficiencies of these two protoplast types were by and large similar;but the same was found to decrease with an increase in the age of the hypocotyl.The response in KM8p was most superior, followed by that in 1/2MSM (gln+ malate) medium.Macroscopic colonies could be recovered on agar solidified medium but not in liquid medium.No shoot regeneration was obtained though root formation was common in the protoplast derived calli.

Anucleate protoplasts were formed in substantial numbers in Vigna radiata hypocotyls during isolation.It was experimentally shown that subprotoplast formation as a result of plasmolysis was dependent on the osmolarity of the medium as well as on the degree of elongation of the cells.

In protoplast fusion experiments the ‘twin-drop’ method of Hein et al.(1983) was found to be most suitable followed by the ‘rolling-tube’ method of Harms and Potrykus (1978b), whereas the procedural steps as recommended by Kao (1978) or Menczel et al. (1981) gave unsatisfactory results. The frequency of bicell aggregates and of heterokaryons (wherever identifiable) were higher in combinations of closely related fusion partners than in those of distantly related partners. The mixing of parental cytoplasms in identifiable heterokaryons was evident within 12 hours in culture. Sustained divisions, beyond the first few, were not obtained in any of the combination of fusion partners.

The heterokaryon frequency could be slightly increased and unfused protoplasts of one of the parents could be eliminated by using parental protoplasts having distinctly different bouyant densities and centrifuging them in a discontinuous density gradient after the fusogenic treatment.Physical elimination of one of the parental set of protoplasts from a mixture of ‘hybrid’ and parental protoplasts would facilitate the selection of the hybrid fusion products, especially if the other parent is inactivated by some means like anti-metabolite treatment or irradiation.