未經冷馴化之草莓莖頂超低溫冷凍保存研究

Study on the Cryopreservation of Non-cold Hardening Fragaria × ananassa Duch. Shoot Tips

蕭翌柱(Yih-Juh Shiau) ； 陳冠文(Guan-Wen Chen)

《台灣農業研究》 63卷1期 (2014/03) Pp. 57-67

https://doi.org/10.6156/JTAR/2014.06301.05

摘要

草莓（Fragaria × ananassa Duch.）屬於薔薇科（Rosaceae）多年生高價值經濟作物，並且有超過70個以上的國家進行栽培與量產，因此進行種原的長期保存益顯重要。本試驗的主要目的，在於建立未經冷馴化草莓（「桃園1號」）最適宜的超低溫冷凍保存處理方法。培植體在尚未浸入液態氮前的預處理結果如下：（1）莖頂培植體（1－2 mm^3）預培養於添加0.3 M蔗糖之MS基本鹽類培養基14 d後的平均存活率為100%；（2）將填充溶液（LS）中的蔗糖濃度自0.6 M提升至1.6 M並且滲透壓保護90 min，莖頂的平均存活率仍可達到100%；以及（3）使用植物玻璃質化溶液2（PVS2）脫水60 min後之莖頂平均存活率達到96.7% ± 4.7%，此百分比顯著高於PVS2脫水180 min（60.0% ± 14.1%）和240 min（46.7% ± 17.0%）的處理組。經PVS2脫水後，每10顆含莖頂的藻膠球及0.3 mL新鮮的PVS2被置入1個2 mL容量的抗凍管中，然後直接投進-196℃的液態氮至少保存1 h，經液態氮凍存後的抗凍管，在38℃恆溫水槽中迅速回溫60 s，此種含有莖頂的藻膠球繼代培養14 d後的再生率可達到80%。本研究各項試驗結果，將有助於建立及簡化草莓超低溫冷凍保存的程序。

關鍵字

草莓 ； 超低溫冷凍保存 ； 填充溶液 ； 滲透壓保護 ； 植物玻璃質化溶液2

並列摘要

Strawberry (Fragaria × ananassa Duch.) is a high economic value perennial crop. It belongs to the Rosaceae family and is distributed in more than 70 countries. Therefore, the research on long-term storage of germplasm is important. The major objective of this study was to establish an optimum cryopreservation method of non-cold hardening Fragaria × ananassa Duch. cv. 'Taoyuan No.1'. The results obtained on pretreatment of explants prior to plunge into liquid nitrogen (LN) were as follows: (1) The survival rate of shoot tips (1-2 mm^3) was 100% when explants precultured on MS basal medium supplemented with 0.3 M sucrose for 14 d. (2) To increase sucrose levels from 0.6 M to 1.6 M in loading solution (LS) and then osmoprotected for 90 min, the survival rate of shoot tips remained 100%. (3) Survival rate of shoot tips achieved 96.7% ± 4.7% when treated with Plant Vitrification Solution 2 (PVS2) dehydration for 60 min. This percentage was significantly higher than treatments of PVS2 dehydration for 180 min (60.0% ± 14.1%) and 240 min (46.7% ± 17.0%). After PVS2 dehydration treatment, 10 shoot tip beads with 0.3 mL fresh PVS2 were transferred to a 2 mL cryotube and then plunged directly into LN for at least 1 h. After storage in LN, cryotubes were rapidly rewarmed in a water bath at 38℃ for 30 s. The final average regeneration rate of shoot tip beads (80%) was achieved after subcultured for 14 d. These research results appear promising for establishing and simplifying cryopreservation procedure of strawberry.

並列關鍵字

Fragaria × ananassa Duch. ； Cryopreservation ； Loading solution ； Osmoprotection ； Plant Vitrification Solution 2