大岩桐基因轉殖系統之優化

Abstract

大岩桐是十分著名的園藝植物，因其花色及花型上的多變而受到大眾喜愛。大岩桐基因體小(300 Mbp)、短的生活史、自交親和性高及組織培養上容易再生等優點，皆支持其發展成研究花部發育機制的模式物種，為此有必要以基因轉殖之技術，輔佐了解相關調控花發育基因之功能性，但目前仍沒有明確的轉殖流程可遵循，因此本研究之目的為優化農桿菌及基因槍的轉殖條件。農桿菌轉殖中 GUS 訊號顯示以 1 ppm BA 前處理大岩桐三週大幼苗三天並與農桿菌菌株 EHA 105 共配養五天的處理方式下可得到暫時性的轉殖率 78.3 %，同樣條件下可獲得 17.2 %的再生率及 2.1 %的轉殖率。GUS 報導基因顯示大岩桐四週大幼苗在基因槍氦氣壓力 900 psi 下，6 公分及 9 公分的距離可分別獲得 58.1 % 與 21.6 % 的暫時性轉殖率。兩者轉殖的效率顯示農桿菌轉殖策略優於基因槍。另一方面，癒傷組織因生長快速容易再生適合做為轉殖的材料，本研究也成功從大岩桐葉片中以0.1 ppm 2,4-D 和 1 或 2 ppm BA 搭配 25 mM 或 50 mM 的山梨糖醇誘導出胚性癒傷組織(embryogenic callus)。本研究優化農桿菌介導的轉殖條件將有助於大岩桐調控花部發育基因功能性分析研究。

Sinningia speciosa is a popular houseplant because of its big flower with a remarkable diversity in colors, patterns and shapes. S. speciosa has a small genome size (300 Mb), short life cycle, self-compatible, easily propagated in tissue culture therefore is emerging as a model plant for flower development studies. However, a reliable genetic transformation system is not available in S. speciosa. To this end, the Agrobacterium mediated transformation and particle bombardment transformation were tested in this study. Transient GUS expression assay showed that 3 days pre-culture of three weeks old seedlings on medium supplied with 1 ppm BA and co-culture for 5 days with Agrobacterium strain EHA105 achieved an overall transient transformation rate of 78.3%. Under these optimized conditions, the regeneration rate is 17.2 % and the transformation rate is up to 2.1 %. Another approach is particle bombardment transformation for optimizing genetic transformation system. In GUS transient assay, it was found that under helium pressure 900 psi, at distance 6 and 9 cm displayed the transient transformation rate of 58.1 % and 21.6 % respectively. The transformation efficiency of two approaches demonstrated that Agrobacterium-mediated transformation is better than particle bombardment transformation. Because callus grows rapidly and regenerate easily, it serves as a good material for transformation. I also successfully induced embryogenic callus with 0.1 ppm 2, 4-D and 2 ppm BA plus 25 or 50 mM sorbitol in the medium. Callus transformation rate will be tested further. This study optimized the transformation protocol for studying gene regulation and gene function in S. speciosa.