| Summary: | 碩士 === 國立中興大學 === 生命科學系所 === 98 === This study is on cryopreservation of micropropagation of Blumea balsamifera (L.) DC., a Taiwan native medicinal plant, by vitrification the shoot tips. This thesis investigates the treatment of different precultures on sucrose medium and the injury of explants by plant vitrification solution. The goal of this thesis is to achieve germplasm conservation and sustainable use.
B. balsamifera (L.) DC. was cultured on the proliferation medium for 30 to 40 days. Excised 1-2 mm buds were placed on different treatments of PVS2, LS solution, and preculture. The established protocol was that buds were treated with 0.5 M sucrose medium for 3 days, loaded to LS 60 min at 25℃, and dehydrated to PVS2 90 min at 0℃ for long-term storage. After one week, buds were rewarmed rapidly, and then transferred to petri dishes containing 1/2 MS medium, supplemented with 0.2 mg L-1 BA, 0.04 mg L-1 IBA, and activated carbon 3g L-1, as a recovery medium for 1 month. The protocol provides the survival rate of 50%. The preculture treatment decreased the relative water content, water potential, and osmotic potential and increased soluble sugar and soluble protein content of the buds. The result indicated that plantlets of B .balsamifera (L.) DC. underwent osmotic adjustment during preculture. Regression analysis of the relative water content, osmotic potential, soluble protein, and sucrose content of buds to the survival rate after cryopreservation showed that the suitable sucrose content improved survival rate of B.balsamifera (L.) DC. shoot tips.
The result of ion leakage assay of buds for cryopreservation process showed the rate achieved 95% and the regeneration rate was lower than 20% after plant vitrification solution. The treatment of PVS2 caused low survival rate of B. balsamifera (L.) DC. buds for cryopreservation. The survival rate of buds by PVS3, as a plant vitrification solution, were lower than by PVS2. The survival rate of buds were 0% by the treatment of air drying for cryopreservation. Thus cryopreservation of B.balsamifera (L.) DC. of shoot tips was not suitable by PVS3 or dessciation treatments. On recovery treatment of buds, the result showed 60 % recovery rate of buds grown in the recovery medium containing 1/2 MS medium, supplemented with 0.2 mg L-1 BA and 0.04 mg L-1 IBA. The composition of recovery medium is another key factor of survival after cryopreservation.
In conclusion, the optimal protocol for cryopreservation of B. balsamifera (L.) DC. in this study was precultured on 0.5 M sucrose medium for 3 days, treated with LS for 60 min and PVS2 for 90 min, and recovered in 1/2 MS medium containing 0.2 mg L-1 BA and 0.04 mg L-1 IBA. This protocol could achieve 60% survival rate.
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