Sugarcane has become an increasingly important crop in recent years, with South Africa featuring as one of the prominent producers. This has led to a significant growth in the South African sugarcane industry, translating into an increased demand for planting material. Although this demand is now satisfied by recent biotechnological advancements such as protocols for somatic embryogenesis to increase the production of planting material, such techniques are limited as a result of the progressive loss of the embryogenic potential of calli over time. In order to facilitate management of this material, it is desirable to develop a protocol for the long-term storage of the germplasm. This study reports on investigations of the different parameters that influenced the cryoprocess in attempts to develop a protocol for the successful cryopreservation of sugarcane somatic embryos of the 88H0019 variety. Experiments were carried out to determine in vitro culture conditions for successful induction of somatic embryos via both the direct and indirect routes of micropropagation. A suitable regeneration medium for plantlet establishment pre- and post-cooling was established (Chapter 2). Investigations were also carried out to ascertain the responses of somatic embryos to both rapid and slow dehydration techniques (Chapter 3). Finally, several cooling techniques (both slow and rapid), were applied, on partially dehydrated somatic embryos, either without, or after cryoprotection, in an attempt to achieve survival after cryopreservation of the somatic embryos (Chapter 4). Both directly- and indirectly-derived somatic embryos were converted, most successfully, on full strength Murashige and Skoog medium without addition of plant growth regulators. The initial mean water contents of directly- and indirectly-derived somatic embryos were not significantly different from each other (8.38±0.19 g g-1 and 8.45±0.33 g g-1 [dry mass basis], respectively). The percentage conversion at these water contents was also not significantly different; 97% for directly- and 98% for indirectlyinduced embryos. Slow dehydration by culture on a series of media with increasing concentrations of sucrose (from 0.2 M to 1.2 M) for a period of 48 h each was the most effective technique, with water content being reduced to 0.94±0.03 g g-1 and 0.95±0.02 g g-1 after dehydration on media containing 1.0 M sucrose, while maintaining between 98% and 100% conversion, respectively. Of the various cryoprotectants tested, proline and casamino acid had the least adverse effects on the somatic embryos. The encapsulation-vitrification cooling technique was the most efficient of all techniques employed. The best conditions involved encapsulation of embryo clumps in a solution of MS medium with 3% (w/v) Na-alginate and loading solution containing 2 M glycerol plus 0.4 M sucrose, followed by infiltration and dehydration at 0°C for various time intervals (0, 5, 10, 15, 20, 25, 30 min) with 1 ml PVS2 solution and thereafter, rapid immersion in liquid nitrogen. Under such conditions, 30% of the cryopreserved somatic embryos retained viability, going on to form callus from which shoots and roots were produced. Although somatic embryos of sugarcane of the local variety 88H0019 have proved to be recalcitrant to cryopreservation, the results obtained with explants that had been processed by encapsulation-vitrification suggest that this approach may be worth pursuing and refining. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2009.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/1045 |
| Date | January 2009 |
| Creators | Jaimangal, Ashika. |
| Contributors | Pammenter, Norman W., Berjak, Patricia. |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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