大岩桐SsCYC在圓葉菸草的表現及性狀觀察與其組織培養再生系統之優化

Abstract

大岩桐(Sinningia speciosa)是著名的園藝植物，野生大岩桐具兩側對稱的花型；然而園藝栽培種中，輻射對稱花型的品種因大而美麗的花型而被保留並且大量地栽培。在模式植物金魚草中，CYCLOIDEA 是調控花朵兩側對稱發育的關鍵基因。在實驗室先前的研究中，發現 CYCLOIDEA 的同源基因(SsCYC)在輻射對稱花型品系中有一個小片段的核酸缺失(ΔSsCYC)，很可能是造成兩側對稱花型發育模組喪失而轉變成為輻射對稱花型的原因。本研究將兩側對稱花型大岩桐的SsCYC基因轉殖進入菸草進行過量表現，和已知CYC基因能影響細胞面積、數目、調控器官大小的功能類似：菸草的花朵長度縮短、開口直徑下降、增加側芽、葉片蜷曲和植株的矮化。而過量表現有輻射對稱花的 ΔSsCYC，沒有性狀改變，代表核酸缺失使基因功能喪失。本研究也探討了大岩桐的組織誘導再生技術和農桿菌的感染條件，以期建立一個高效且穩定的基因轉殖系統。實驗結果顯示，當 MS 培養 基 中 擁 有 0.1 ppm naphthalene-acetic acid (NAA) 和 1 ppm 的6-benzylaminopurine (BA)時，會有最佳的再生率：以 6 mm 直徑葉切片為材料時可以獲得 86%的再生率，以 5 mm 長度的葉柄切塊為材料時可以獲得 56%的再生效率。更進一步地，本研究發現葉柄切塊在培養基上的擺放方向對於再生率有很大的影響，只有倒立或是水平放置的葉柄切塊可以成功地再生，推測可能和原本葉柄內部生長素向基性的分布模式有關。另外，本研究也發現再生的芽起源於葉片深處單一的維管束薄壁細胞，這和其他苦苣苔物種的起源於表皮細胞或是球型毛絨基座細胞不同。為了要找到最佳的農桿菌感染條件，本研究利用 GUS 報導基因的表現作為成功感染的標記，結果發現年輕的幼苗相較於成熟的葉切片或是葉柄切塊有較佳的感染效果，尤其是其子葉和第一對初生葉片，顯示大岩桐的幼苗相當有潛力發展成為良好的農桿菌基因轉殖材料。進一步透過石蠟切片發現感染的位置為表皮、葉肉和球型絨毛的頭狀細胞，然而這些組織是否可以誘導成再生苗仍有待進一步的實驗。另外，大岩桐的癒傷組織可能也是良好的基因轉殖材料。本研究為大岩桐 SsCYC 基因如何影響花部對稱的功能做了初步的探討。

The native varieties of Sinningia speciosa (Gesneriaceae) bear zygomorphic flowers, but in horticultural varieties, large size showy actinomorphic flowers are selected due to human’s preference. CYCLOIDEA has been demonstrated to have a major genetic control in zygomorphy in Antirrhinum. In actinomorphic varieties, we found a small fragment deletion in its CYCLOIDEA homologue (∆SsCYC), which might indicate that the reversal to actinomorphy is a SsCYC loss of function mutant. I introduced CYC homologues from both zygomorphic cultivar (SsCYC) and actinomorphic cultivar (ΔSsCYC) into Nicotiana benthamiana, a closely related species to Sinningia speciosa, to verify whether ΔSsCYC has any effect on floral phenotype. I found that ectopic expression of SsCYC causes shorter longitudinal length of flowers, smaller floral opening diameters, induction of axillary shoots, curled leaves and dwarfism, agreed with CYC’s putative effects on cell proliferation or expansion. However, no visible phenotypic change could be observed in ΔSsCYC overexpression lines. I also optimized the genetic transformation system in Sinningia speciosa, focusing on tissue regeneration and Agrobacterium infection conditions. The MS medium supplied with 0.1 ppm naphthalene-acetic acid (NAA) and 1 ppm 6-benzylaminopurine (BA) was the best for shoot regeneration in both leaf and petiole explants. Eighty six percent and 56% regeneration rates were obtained from 6 mm diameter leaf explants and 5 mm petiole explants, respectively. Moreover, the orientation of petiole explants must be up-side down or horizontal to induce the shoot regeneration, which might relate to the endogenous basipetal distribution of auxin inside the petiole vascular tissue. It was found that the regenerative shoots of explants initiated from a single vascular parenchyma cell deep inside the regenerated tissue. This is different from other reported cases in Gesneriaceae species, in which their regenerative shoots usually originated from an epidermis cell or a glandular trichome basal cell. To explore whether Agrobacterium infection can enter regenerative tissue, transient transformation using GUS reporters was applied. In contrast to mature leaf or petiole explants, I found that young seedlings, especially those in cotyledonary stage or with the first pair of primary leaves have much higher success of transformation. This opens an opportunity that young seedlings are potential material for transformation. By paraffin sections, the positive signals of transformation were seen in epidermis, mesophyll and glandular trichome head cells but not inside the regenerative shoots. Alternatively, the induced callus tissue might be a better transformation material. This study provides a preliminary study on the functions of SsCYC genes and guidelines for further optimization of transformation system of Sinningia speciosa.