Vanilla is an emerging cash crop in Taiwan, and its domestic cultivation area reached 20 hectares in 2020. Vanilla comes from the cured vanilla beans, which are the pods of the orchid plant Vanilla planifolia Andrews. In response to domestic requirements of vanilla production, related research on physiology, cultivation, genetics, and breeding is still awaiting exploration. This study investigated the pod/seed development, pollen tube growth, the optimal protocols for asymbiotic germination, the induction of lateral shoots and polyploidy, and the vanilla curing process. Results are summarized as below.
1. The pod and seed development study showed: (1) After successful hand-pollination, ovaries began to enlarge and elongate rapidly. The pod length increased steadily and reached the maximum size at 35 DAP (days after pollination), while the diameter of the pod reached the maximum size at 49 DAP. (2) The 45-DAP immature seeds have developed globular embryos and the thickened non-lignified cell wall at the outermost layer of the outer seed coat. After 60 DAP, the cell wall of the outermost layer of the outer seed coat became lignified and finally compressed into a thick envelope. These features match the significant decreases of immature seed germination percentage after 60 DAP.
2. The pollen germination and pollen tube development study indicated: (1) BK medium without sucrose is the best for in vitro pollen germination, and 25℃- 30℃ is the optimum temperature. (2) During vanilla flowers opened, the pollen activity was highest at midnight and had no significant reduction before 9 am, but decreased significantly after 12 pm. (3) High temperature significantly affects pollen activity: treatment at 35°C for 1 hour would have harmful effects, including abnormal pollen tubes and stunt growth of pollen tube, but the growth of pollen tube can be restored at 25°C. The longer the high-temperature treatment, the worse the harmful effects; treatment at 40°C for 1 hour causes severe damage to the pollen tube and unable to recover. (4) For the in vivo germination trials, the pollen tubes germinated one day after pollination, grows to the base of styles at 5 to 7 DAP, entered the ovary at 14 DAP, arrived in the middle of the ovary at 21 DAP, and finally reached the base of the ovary at 28 DAP. Pollen tubes enter ovules to fertilize at approximately 21 DAP. The development of fertilized ovules into seeds in an ovary is progressive: seeds near the joint between the style and ovary developed earlier than seeds near the distal end of the ovary. This study describes the flowering habit of vanilla and the observation of pollen tube growth after pollination, which can be used as a reference for field management and likely helps improve the success rate of pod setting.
3. The micropropagation study revealed showed: (1) mature seeds soaking in 4 % sodium hypochlorite solution for 75 - 90 min would increase germination significantly. (2) immature seeds at 45 days after pollination (DAP) had the highest germination percentage. (3) Plantlets in the medium containing 1 mg mL-1 BA for 45 d had the best effect to induce lateral shoots. Each node could produce 5.2 buds. These buds can produce roots in the basal medium without growth regulators and move out after two months subculture. (4) Treating liquid medium with 0.625 mM - 1.2 mM colchicine for three days can successfully induce polyploid. Two independent tetraploid plants were obtained.
4. The study of vanilla curing illustrated that: (1) the major component β‐glucovanillin started accumulating at 24 WAP. The cumulative efficiency of glucovanillin slowed down after 34 DAP and reached the highest cumulative content at 42 DAP. The cured beans which harvested at 38 DAP had the highest vanillin content. (2) The ideal killing condition was at 65℃ for 3 min while reducing the temperature or killing duration in order to prevent over shaded beans from heat damage. (3) Vanilla beans can keep vanillin content when sweated and dried at 40℃. (4)Vanillin content advanced quickly during the first 5 days after killing and slowed down from the 5th to 7th day. Vanillin content reached the highest level from the 8th to 10th day during the curing process and could not increase any more at the same condition. Overall, the condition established in this study can help promote curing process and produce beans which had similar vanillin content to abroad.