基因鎗轉殖台農67號水稻影響因子研究

Optimization Parameters of Particle Bombardment for Tainung 67 Rice (Oryza sativa L.)

夏奇鈮(Chi-Ni Hsia) ； 顏信沐(Hsin-Muh Yen) ； 曾東海(Tung-Hai Tseng) ； 卓緯玄(Woei-Shyuan Jwo) ； 王強生(Chang-Sheng Wang)

《中華農業研究》 50卷3期 (2001/09) Pp. 1-11

https://doi.org/10.29951/JARC.200109.0001

摘要

本研究以本省最普遍栽培之水稻品種一台農67號之成熟種子為材料，利用基因鎗(Biolistic PDS1000/He device, Bio-Rad)進行基因轉殖。將成熟種子去殼後培養於添加2, 4-D之培養基中誘導胚性癒合組織增生，並以此材料測試影響基因鎗轉殖效率之各項因子，轉殖效率評估以GUS基因之暫時表現率為依據。評估之因子包括：培植體預培養天數、微載體種類及用量、高滲透壓處理及轉殖質體對轉殖之影響；基因鎗測試之條件有：微載體至射擊目標之飛行距離(6、9 cm)，大載片至擋片之距離（4、8及12 mm）。初步結果顯示，以預培養2~7日之胚性癒合組織逐日測試轉殖率，以預培養7日之培植體表現最佳；培植體於轉殖前4小時至轉殖後24小時以高滲透壓培養基處理，有助於轉殖效率之提高；比較金、鎢粒子對轉殖率在轉殖後48h並無不同，但轉殖後120 h取樣，則金粒子組有較高之藍點數；鎢粒子用量可減少至1/3標準用量對轉殖率並無影響；利用基因鎗在使用1, 100 psi裂碟片、大載片至擋片之距離為4 mm、微載髁至射擊目標之飛行距離6或9 cm之設定，GUS基因表現之平均藍點數均可達15~20個／培植體。

關鍵字

水稻 ； 基因鎗 ； 轉殖

並列摘要

The objective of this study was to establish a genetic transformation system using biolistic bombardment (Biolistic PDS 1000/He device, Bio-Rad) for the most popular rice cultivar 'Tainung 67' in Taiwan. Embryogenic calli were induced from mature seeds on the medium containing 2, 4-D and they were used as material for bombardment. Transformation efficiency was evaluated by the transient expression of /β-glucuronidase gene. The tested parameters included: the effect of preculture duration for explants, the type of microcarriers, density of microcarrier particles used for DNA preparation, the effects of osmotic treatments, and plasmids with various constructions. Parameters of biolistic device including the flight distance of microcarriers (6 and 9 cm) and macrocarriers (4, 8, and 12 mm) were investigated. The results showed that 7-day-precultured embryogenic calli had the best responses for bombardment among 2-7 day' preculture duration. Enhanced transformation efficiency was found in the explants treated with high osmotic medium 4h prior to till 24 h after bombardment. Particles of gold and tungsten were both found suitable for rice transformation and there was no adverse effect when tungsten particles were reduced to 1/3 standard amount for DNA precipitation on transient GUS gene expression. However, it was found that gold particle had higher GUS expression at 120 h post-bombardment. A preliminary microprojectile transformation system with an average of 15-20 blue spots/explant GUS expression was routinely obtained for 'Tainung 67' by using the particle gun with 1,100 psi rupture disk, 6 or 9 cm microcarrier flight distance and 4 mm of macrocarrier flight distance setting.

並列關鍵字

Rice Oryza sativa L. ； Particle gun ； Transformation