In vitro seed germination and propagation of endemic scented orchid - Vanda hastifera
Vanda hastifera is listed under endangered orchid species of Appendix II in The Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) 2002. It is an endemic orchid to Borneo which has large and sweetly scented flowers. The objective of this study was to develop prot...
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| Format: | Thesis |
| Language: | English English |
| Published: |
2012
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| Subjects: | |
| Online Access: | https://eprints.ums.edu.my/id/eprint/41601/1/24%20PAGES.pdf https://eprints.ums.edu.my/id/eprint/41601/2/FULLTEXT.pdf |
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| Summary: | Vanda hastifera is listed under endangered orchid species of Appendix II in The Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) 2002. It is an endemic orchid to Borneo which has large and sweetly scented flowers. The objective of this study was to develop protocols for in vitro seed germination and micropropagation of V. hastifera . The techniques for seed germination, protocorm proliferation, protocorm development, and Protocorm-like body (PLB) induction were established by manipulating various factors such as basal media (Knudson (KC), Mitra, Murashige and Skoog (MS) and Vacin and Went (VW)), complex additives (coconut water, tomato juice, banana pulp, potato homogenate, peptone and yeast extract), plant growth regulators (PGRs) (6- benzylaminopurine (BAP), Kinetin, α-Naphtaleneacetic acid (NAA), Indole-3-butyric acid (IBA), and Indole-3-acetic acid (IAA)) and sugars (sucrose, glucose, fructose and galactose). Results showed that KC medium promoted seed germination up to 77.84% (obtained after 150 days of culture) followed by MS medium at 30.75%, and no germination was observed in VW medium. However, both basal media (KC and MS) delayed seed germination and also produced dead protocorms indicating the insufficiency of nutrient contents to support seed germination. Therefore, complex additives were added in the basal medium to increase the percentage of seed germination and shorten the time for seed germination. The fastest seed germination was observed in 10% (v/v) potato homogenate (19 days), followed by 0.2% (w/v) yeast extract and 10% (v/v) banana pulp (22 days), 10% (v/v) tomato juice (26 days), 10% (v/v) coconut water (28 days), and 0.2% (w/v) peptone (29 days). Observation after 150 days showed that seed germination increased up to 99.21% and 98.74% in medium supplemented with potato homogenate and tomato juice, respectively. Meanwhile, germinated protocorms in 0.2% (w/v) yeast extract medium failed to survive. The use of sucrose in the medium was found to promote higher seed germination as compared to glucose and fructose. The highest percentage of seed germination (86.13±1.90%, growth index value 281.75) was observed in 1% (w/v) sucrose. However, addition of galactose inhibited seed germination. In protocorm proliferation study, treatments with NAA and BAP combinations were more effective in promoting protocorm proliferation compared to single PGRs. Addition of 0.5 mg/l NAA+2.0 mg/l BAP had intensely induced protocorm proliferation up to 91.19±7.07% and induced 9.20±0.56 new protocorms after 150 days of culture. Meanwhile, single ctyokinins (BAP and kinetin) promoted protocorm proliferation while auxins (NAA, IAA, and IBA) promoted shoot and root rather than multiplication. The highest number of protocorm proliferation using single PGRs was obtained in 1.0 mg/l BAP (80.00% protocorms proliferated and produced 7.36±1.52 new protocorms). In the study of nutrients strength, KC medium at various nutrient strengths (¼X, ½X, 1X) was found to support protocorm proliferation better than Mitra (¼X, ½X, 1X) and MS (¼X, ½X, 1X, 2X) media. Of all of these, full strength KC medium was found to be the best medium strength (88.00% protocorm proliferated and produced 9.79±1.05 new protocorms), while, MS medium at all nutrient strengths was not suitable for the protocorm proliferation. Sucrose had been identified as the best sugar to support protocorm proliferation followed by glucose and fructose. The highest number of proliferated protocorm (79%) and new protocorm (10.50±1.86) was obtained on medium treated with 2% (w/v) sucrose. In protocorm development study, complex additives were able to shorten the time to induce protocorm development as compared to control. The fastest protocorm development was observed in media treated with peptone (70 days), followed by tomato juice, coconut water, potato homogenate, banana pulp and yeast extract. After 120 days, all the protocorms cultured in 0.2% (w/v) peptone developed into seedlings. This treatment induced the highest number of leaves (4.48±0.10) and roots (3.43±0.17). Treatments with banana pulp and yeast extracts were not suitable to support protocorm development of V. hastifera. Sucrose had been recognized as the best sugar to support protocorm development followed by glucose and fructose. Addition of 2% (w/v) sucrose enhanced the development of all the protocorms into seedlings after 70 days, and induced the highest number of leaves (4.56±0.44) and roots (3.24±0.34) formation. In PLBs induction study, PLBs were induced at the cut-end site of the leaf explants, they were not formed on the surface or the tip of the explants. Leaf base explant was found to produce more PLBs compared to leaf tip explants. Treatment with PGRs induced PLBs as early as 37 days after culture in medium containing 4.0 mg/l NAA+4.0 mg/l BAP (64.00% leaf base explants produced 10.45±2.47 PLBs) after 60 days of culture. Meanwhile, for single PGRs treatment, BAP had superior effect than kinetin, NAA, IAA and IBA. The highest number of PLB induction was observed in 2 mg/l of BAP (induced 32.00% leaf base explants, 3.15±2.98 PLBs) after 49 days of culture. The addition of IAA and IBA into the basal medium caused inhibited on PLBs induction. |
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