自地黃毛狀根再生其基因轉殖植株與其型態特徵比較之研究

Abstract

基因轉殖技術提供了經基因改良的高等植物許多的可能性，最近幾年，將建構的外來基因穩定地嵌入植物細胞中並表現的技術已獲得卓越的進展，尤其是以農桿菌(Agrobacterium)為媒介的基因傳遞系統已發展成熟並被廣為利用。到目前為止，利用農桿根群菌(Agrobacterium rhizogenes) Ri質體所誘導的毛狀根已被證明是重要藥用植物生產二次代謝產物之有效方法。 本篇論文中針對以農桿根群菌感染地黃(Rehmannia glutinosa)葉片誘導的毛狀根作再生基因轉殖植株條件之探討，並觀察其型態特徵、比較轉殖植株與野生植株之型態差異。地黃毛狀根誘導自以農桿根群菌感染地黃葉片，並培養於White’s培養基。在黑暗下培養於不含任何植物荷爾蒙之培養基中，3%的地黃毛狀根自然生成不定芽，自毛狀根發生的不定芽在16小時光照週期、培養於不含任何植物生長激素之1/2 MS培養基環境下，能促成根系快速生長及再生植株的形成。實驗中並利用聚合酶連鎖反應(PCR)針對再生植株中rol基因的嵌入作確認，亦發現此自毛狀根誘導之基因轉殖植株相較於野生植株而言有許多型態特徵發生改變，例如葉片皺縮、頂芽優勢減少、根系分支多、生物量大、多屬鬚根，不同於野生植株根系以主根為主，而在轉殖植株之葉片及莖上發現有不定根發生的現象亦顯示rolABC基因同時存在、表現並發生協同增效作用的可能性，種種型態特徵的改變亦反應rol基因的嵌入所導致之型態修飾。論文中亦針對梓醇成分的生產作探討，發現實驗使用之二個月生的再生植株中梓醇(catapol)含量甚少，但新合成的成分及可大量生產之化合物的確認 都值得進一步的探討。

Transgenic technology provides enormous possibilities for genetic improvement of higher plants. In the last few years, remarkable progress has been made in the technology for stable integration and expression of engineered foreign genetic information in plant cells. In particular, an Agrobacterium-mediated gene delivery system has been developed and widely used. So far, the hairy roots induced by Ri plasmid of Agrobacterium rhizogenes have been proved to be an efficient means of producing secondary metabolites in pharmaceutically important plants. Plant regeneration from hairy roots induced by infection with Agrobacterium rhizogenes in Rehmannia glutinosa (Chinese foxglove) and characterization of its morphology were studied in this report. Hairy roots were induced from leaf segments of Chinese foxglove when cultured on White’s medium after infection by A. rhizogenes. Hairy roots spontaneously gave rise to adventitious shoots at a frequency of 3% when cultured on growth regulator-free medium in the dark. Rooting of hairy root-derived adventitious shoots and therefore regeneration of transgenic plants occurred easily on growth regulator-free half-strength MS solid medium at a 16 h photoperiod. The stable introduction of rol genes into Rehmannia plants was confirmed by PCR. Transgenic plantlets showed considerable differences in their morphology when compared to the corresponding wild-type plants. Plants derived from hairy roots had wrinkled leaves, reduced apical dominance, and numerous fibrous roots with abundant lateral branches instead of the thickened taproots in non-transformed plants. Adventitious roots were induced in leaves and stems of transformed plants. It has been suggested that rolABC genes acted synergistically in promoting hairy roots from Chinese foxglove leaves and stems. The differences observed reflect the modification of morphological characters by introduction of rol genes. Hairy root culture and its transgenic plant-based catapol production was also qualitatively analyzed. Although these 2-months-old plantlets were found to be unfavorable for catapol production, novel compounds synthesized were valuable to be studied in the future.