大岩桐(Sinningia speciosa)農桿菌轉殖系統之建立

Abstract

大岩桐(Sinningia speciosa)是今日廣為栽培的觀賞植物。其栽培歷史悠久，故品種眾多，各品種之間也不難找到一些變異，花形的變異更是明顯，例如花市常見的輻射對稱品系便是現在最普遍的。因此大岩桐可以作為我們探究控制花部對稱性候選基因的材料。一旦建立了有效的農桿菌轉殖系統，這些基因的功能則會漸漸浮出水面。雖然先前已經有學者針對大岩桐的轉殖發表過文章，但是該文獻著重的是基因功能分析，因此有需要進一步針對大岩桐轉殖的部分作探討。我們先將無菌組培的大岩桐葉片切成0.5平方公分大小的葉片，在使用農桿菌菌株LB4404進行感染。感染之後以潮黴素(Hygromycin) 5 mg/L進行篩選。使用的載體是pCAMBIA1301，其中的beta-glucuronidase(GUS)帶有內含子。除此之外，我們又發現，在含MS、2 ppm BA和0.2 ppm NAA的培養基上，大岩桐的葉子切片能透過這樣的組培方式在兩個星期後分化出小型的愈傷組織，並且在一個禮拜後分化成不定芽，並長根繼續生長，再將這些不定芽移植至於菌環境以外的培養土上，仍能繼續生長。運用同一個轉殖系統轉殖後，在120片外體植片中，分化出14棵不定芽，經gDNA PCR檢測GUS基因，證實其中五株確定為轉殖株。另外RT-PCR結果又顯示在這五株轉植株中，全都表現GUS基因，轉殖成功率為4.16%。這是繼貓臉苦苣苔(Kohleria sp.)後，第二種苦苣苔科植物的轉殖系統，可作為往後研究計畫的基礎技術，進而得以在演化發育的研究領域中進行花部控制、發育或是逆境反應基因的功能性研究。

Sinningia speciosa is an ornamental plant species that is widely cultivated. Many cultivars, however, generated a range of floral mutations such as Peloria (actinomorphy). It thus allows us to examine candidate genes that control floral symmetry. These functions should be able to be clarified if a suitable transformation system is established. Although there was a transformation protocol published on S. speciosa, yet it mainly focused on genetic analysis. Due to this reason, a more detailed transformation should be clarified. We therefore chose Agrobacterium, strand LBA4404, with medium Hygromycin as a selective agent, to infect leaf explants after they were sectioned into the size of 0.5 cm2. We also found out that S. speciosa has the capability to regenerate vigorously through vegetative cuttings after being placed on MS medium containing 2 ppm of BA and 0.2 ppm of NAA. So the major optimizing factor is on Agrobacterium infections. New plantlets induced from shoots were beta-glucuronidase(GUS)-stained and confirmed with gDNA PCR and RT-PCR in order to predict the rate of success. Out of all 120 explants, 14 regenerants were collected and five of which was successfully transformed as confirmed by PCR using GUS primers, while all of which showed GUS expression as confirmed by RT-PCR, resulting in a transformation rate of 4.16%. This project shall serve as an important mile stone in building a reliable transformation protocol in Gesneriaceae, allowing functional validations of several candidate floral developmental genes in evo-devo, thus assists any possible future project which could be brought out by our laboratory.