Various aspects of the micropropagation of strawberry (Fragaria ananassa Duch.) from shoot tips and from single mesophyll cells were investigated. 1. The conditions affecting shoot proliferation were studied in two types of tip culture experiments : one without subculturing for 60 days, the other with subculturing at 10 day intervals. The basal medium used was that of Murashige and Skoog (1962). IBA (0.5 muM) was always present in the medium. No callus formation occurred during these experiments. 2. The effect of BA (0-40 muM) was found to be different in the two types of experiments. In the experiments involving continuous subculture, the shoot proliferation rate increased with BA concentration up to 5 muM BA both at low (2 watts/m2) and at high (13.5 watts/m2) light intensities. 3. Shoot tip proliferation was preceded by a lag phase of up to 30 days when the shoot tips were obtained from plants grown on BA free medium. The length of this lag phase was dependent on the length of time that the donor plants spent in BA free medium. 4. The presence of myoinositol stimulated shoot tip proliferation while glycine was less effective although it did not suppress proliferation. Interestingly, nicotinic acid, pyridoxine HC1., and thiamine HCl., when present together reduced the shoot tip proliferation rate. Therefore, these three vitamins are not required in the medium when micropropagation of strawberry is the objective. 5. Sucrose was found essential for proliferation and the optimum concentration was in the range of 3 - 6%. The response to sucrose varied with the light intensity. At 2 watts/m2 light intensity, 0.75% sucrose and at 13.5 watts/m2 light intensity, 3% sucrose resulted in maximum proliferation in long term cultures. In the experiments with continuous subculture, 6% sucrose was found best. 6. For shoot tip proliferation light was required. No multiplication occurred when the shoot tips were cultured in the dark. Shoot tip proliferation increased with light intensity; 13.5 watts/m2 was the highest light intensity used; this supported the maximum rate of shoot proliferation. 7. Rooting of in vitro produced shoot tips occurred readily in simple media. Maximum rooting occurred on a medium containing MS inorganic salts. The extent of rooting was directly related to light intensity. No root formation occurred in the dark, or at low light intensity, or in sucrose free medium. 8. Roots were formed directly when shoots without roots were transferred to the soil, when the shoots were taken from MS inorganic salts with 2% sucrose. In the greenhouse, mist was found necessary for the first 7 days for the survival of plants. 9. Callus was established from aseptically produced roots and 2,4-D was most effective while NAA and IAA was moderately and least effective respectively. Kinetin was little better than BA in stimulating callus initiation. Organogenesis did not occur in callus produced on roots. 10. Mesophyll cells of strawberry were isolated mechanically. Cells were isolated from aseptically produced plants maintained either on MS inorganic salts (Appendix A, A to C inclusive) with 2% sucrose or on whole modified MS medium (Appendix A, A to N inclusive containing 1 muM BA and 3% sucrose). 11. Optimum plating efficiences of the order of 60 - 70% were obtained in cultures supplemented with a range of organic additives, including kinetin and 2,4-D, grown on filter sterilized media at low light intensities and at low sucrose concentration; at 25° or 30°C. Autoclave sterilization, high osmotic pressure, additional sucrose, and high light intensities separately or together have all reduced the plating efficiencies. 12. In some of the treatments, cell division led to the formation of cell clusters without any change of media. Groups of these cell clusters have been visible to the naked eye within 60 days. These groups of cell clusters did not develop further on changing the medium. 13. The viability of mesophyll cells from strawberry, Asparagus sprengeri and from Macleaya cordata was assessed by observing the exclusion of various dyes from the cells. The penetration of some commonly used dyes, such as Evan's blue into the cells was found to be dye concentration dependent. (Abstract shortened by UMI.).
|Date of Award||1981|