可可癒傷組織培養系統之建立與應用

Abstract

可可屬於熱帶多年生樹種，是許多國家重要的經濟作物，其種子可作為製作巧克力、藥品及化妝品的材料。可可的雜交育種需要十數年，因此，利用組織培養以縮短育種時間與基因轉殖漸被採用。然而可可組織培養大多是利用花瓣、雄蕊或珠心做為培植體，再進一步誘導成為植株，但可可從種子栽培到成樹開花約需二到三年的時間，無助於縮短育種時間。因此，若能以葉做為培植體則可以減少獲得新植株所需的時間，目前甚少實驗利用可可葉進行可可組織培養。且大多試驗僅探討胚成為植株之階段，癒傷組織生成之階段研究甚少。此外，癒傷組織常作為利用農桿菌轉殖基因的材料。故本研究的目的，期望能提高可可葉之癒傷組織誘導率，並利用癒傷組織為材料，探討進行農桿菌轉殖基因及細胞懸浮培養的可能，最後更希望能以本系統誘導出體胚進一步成為植株。本論文以可可成樹或可可幼苗之未成熟葉與成熟葉為試驗材料，並依葉片與葉柄分別進行癒傷組織之誘導。獲得癒傷組織後，利用含pCAMBIA1301載體之農桿菌EHA105感染癒傷組織進行基因轉殖。另外將癒傷組織進行懸浮培養，最後則嘗試將癒傷組織誘導成為植株。結果顯示，以MS培養基誘導可可葉產生癒傷組織，其植物生長調節劑濃度為7.2 μM 2,4-D + 2 μM kinetin時約有76%之成功率。葉柄培植體長度以每段0.5至1.0公分為佳，葉片與培養基接觸面正反皆可。可可幼苗之葉柄誘導率較佳，但仍較可可花器組織之癒傷組織誘導率 (達90%以上) 為低。成熟葉柄之癒傷組織可利用農桿菌成功轉殖報導基因GUS。葉柄癒傷組織懸浮培養兩個月可建立均質的懸浮細胞系。可可葉柄癒傷組織誘導植株再生，多停留在胚的初始階段甚或產生白絲狀不成熟胚。

Cacao (Theobroma cacao L.) is a tropical perennial species and an important economic crop in many developing countries. The cacao seeds can be used as the materials for making chocolate, drugs and cosmetics. It needs more than decades to achieve crossing-breeding, thus using tissue culture to shorten the breeding time is getting attention. Moreover, tissue culture can be used to obtain transgenic plant. Generally, most of studies use petal, staminode and nucellus as explants for inducing plantlet. However, cacao cultivation from seed into flowering stage takes about two to three years, which cannot help to shorten the breeding time. Therefore, if the leaf can be the explants for callus induction, the time requiring for new plantlet can be reduced. There were little reports using leaf as explants. And most of the studies focus on the embryo stage, not much on the callus formation stage. Besides, callus is an excellent material for Agrobacterium-mediated transformation. The aim of this study is to improve the callus induction rate on cacao leaf, and use it for Agrobacterim-mediated transformation and cell suspension. And hope it can become a plantlet from somatic embryo. In the thesis, cacao tree or cacao seedlings’ immature leaf and mature leaf were used as the material to induce callus. After that, the Agrobacterium tumefaciens EHA105 containing pCAMBIA1301 vector infection was used of gene transformation and explores the feasibility of the callus into suspension culture. Finally, the possibility for plantlet regeneration was test. The results showed that MS medium can be used to induce callus, and using 7.2 μM 2,4-D and 2 μM kinetin as plant growth regulators had 76% success rate. Petiole length use 0.5 to 1 cm much better. Leaf contact surface with medium can be front or back side. The callus induction rate of cacao seedling’ petiole is better, but still lower than cacao flower (more than 90%). Agrobacterium-mediated transformation of the reporter gene GUS was successful on mature petiole callus. Petiole callus suspension culture can create a homogeneous suspension cell lines. The plant regeneration stays in the initial stage of the embryo or even white trichomes.