Study of Protocol on the Cryopreservation of Taiwan Native Medicinal Herbs Mosla scabra (Thunb.) Wu & Li and Laggera alata (D. Don) Sch. Bip. ex Oliver by Vitrification.
| Autor: | Jing-Kai Lin, 林經剴 |
|---|---|
| Rok vydání: | 2009 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 97 This study examined the pretreatment protocols for cryopreservation of in-vitro grown apical buds of two Taiwan native medicinal herbs Mosla scabra (Thunb.) Wu & Li and Laggera alata (D. Don) Sch. Bip. ex Oliver. In the meanwhile, the apical buds were used to examine the changes in water status and ultrastructure in order to correlate with the effects of cryopreservation procedure. The plantets of M. scabra were precultured for 7 days on hormone-free solidified Muashige and Skoog medium (MS medium) supplemented with 0.3 M sucrose. The excised shoot tips were treated with loading solution (LS) for 60 min and dehydrated with a highly concentrated plant vitrification solution 2 (PVS2) for 120 min. Following this protocol, we achieved the best survival rate of 80.45 ± 6.75 %. The plantets of L. alata were precultured for 21 days on MS medium supplemented with 0.4 M sucrose. The excised shoot tips were treated with LS for 120 min and PVS2 for 180 min. Following this protocol, we achieved the best survival rate of 77.71 ± 4.03 %. The physiological analyses were performed using shoot tips of M. scabra which have been precultured on hormone-free MS medium and supplemented with 0.3 M sucrose for 7 days. The relative water content reduced from 88.02 % to 79.38 %, and the amounts of soluble sugar and soluble protein increased from 13.82 mg g-1 to 35.86 mg g-1, and 4.08 mg g-1 to 5.73 mg g-1, respectively. The osmotic potential and water potential reduced from -0.81 to -1.79 MPa, and -0.92 to -1.77 MPa, respectively. This results showed the preculture procedures greatly enhanced the freezing or dehydration tolerance and improved the survival rate of cryopreservation shoot tips. The apical meristem of M. scabra shoot tip during preculture showed dense cytoplasm and slight plasmolysis. TEM ultrasctuctural examination of shoot tips after cryopreservation revealed the cells had high nucleo-cytoplasm ratio and slight plasmolyzed at first few layers of cells in apical meristem. The estimated interval between plasma membrane and cell wall was 168.42 nm. The 4 to 5 layers of cells below apical meristem had high degree of plasmolysis and the interval between plasma membrane and cell wall was 795.83 nm. This result showed the survived cells after cryopreservation were mainly localized in apical meristem or leaf primordia and subsequently regenerated into plantlets. This study provided an optimal pretreatment protocol for cryopreservation and the results showed greatly improved water relations and ultrastructural changes in of M. scabra and L. alata. This study was one step forward to develop long-term cryopreservation technology. |
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