Koo, Geck Chin (2008) Development of cryopreservation protocols for paphiopedilum rothschildianum and phalaenopsis gigantea for long-term germplasm conservation program. Masters thesis, Universiti Malaysia Sabah.
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Abstract
Paphiopedilum rothschildianum and Phalaenopsis gigantea are two endangered orchids of Sabah. Both are currently protected and listed under the Convention in Trade on Endangered Species of Flora and Fauna (CITTES). Cryopreservation is an effective method for long term storage of plant materials. Hence, development of cryopreservation protocols for these two species will facilitate the long term conservation programmes of these orchids. In this study, protocorms and seeds were used as the explants. Preculture conditions, preculture durations and combinations of loading solution were evaluated for cryopreservation of protocorms of both species. The effect of PVS2 incubation period (0-90 min) on the viability of protocorms was evaluated. Protocorms were precultured in liquid medium supplemented with different concentrations of sucrose (0-0.SM) and precultured at different period (0-7 days). Different combinations of glycerol (0-2.SM) and sucrose (0-0.SM) were evaluated for loading solution. Protocorms of P. rothschildianum were used in developing encapsulation dehydration and encapsulation vitrification method. The viability of protocorms was determined using 2,3,5-triphenyl tetrazolium chloride (TTC) test. For the development of cryopreservation protocol of P. rothschildianum seeds, incubation time (0-5 hours) in PVS2 solution at 0°C, preculture conditions (0-0.SM sucrose), preculture durations (1-7 days) and storage durations in liquid nitrogen (2-24 weeks) were evaluated. The viability of the seeds was determined using TTC test and the germination ability was recorded. Protocorms of P. rothschildianum precultured in MS medium supplemented with 0.25M sucrose for 3 days gave the highest viability value while protocorms of P. gigantea in XER medium supplemented with 0.25M sucrose for 5 days showed the highest viability value prior to liquid nitrogen storage. Loading solution comprised of O.SM glycerol and 0.4M sucrose showed the highest viability value for protocorms of P. rothschildianum while the combination of O.SM glycerol and 0.2M sucrose resulted in the highest viability value for P. gigantea. Protocorms showed low viability in all incubation periods. Protocorms failed to grow even after two months on recovery media after cryopreservation. No regrowth of protocorms were observed after cryopreservation by both encapsulation-dehydration and encapsulation-vitrification methods. Seeds that were dehydrated with PVS2 solution for 5 hours gave the highest seed germination percentage (30%). Seeds precultured on medium supplemented with 0.4M sucrose and precultured for 7 days resulted in the highest seed germination percentages, which was 30% and 63%, respectively. No significant difference in seed germination was observed when seeds were stored in liquid nitrogen for 2 to 24 weeks.
| Item Type: | Thesis (Masters) |
|---|---|
| Keyword: | Paphiopedilum rothschildianum, Phalaenopsis gigantea, vitrification, Encapsulation dehydration, Encapsulation vitrification |
| Subjects: | Q Science > QK Botany > QK1-989 Botany > QK474.8-495 Spermatophyta. Phanerogams |
| Department: | INSTITUTE > Biotechnology Research Institute (BRI) |
| Depositing User: | DG MASNIAH AHMAD - |
| Date Deposited: | 19 Jun 2025 15:39 |
| Last Modified: | 19 Jun 2025 15:39 |
| URI: | https://eprints.ums.edu.my/id/eprint/44175 |
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