Callus Culture of Eucalyptus grandis x urophylla and Preliminary Studies on Organogenesis and Agrobacterium-mediated Transformation

雜交桉之癒合組織培養及其器官形成與基因轉殖之初步研究

陳振榮(Zenn-Zong Chen) ； 蔡錦瑩(Jeen-Yin Tsay) ； 鍾振德(Jeng-Der Chung)

《台灣林業科學》 11卷1期 (1996/03) Pp. 43-52

https://doi.org/10.7075/TJFS.199603.0043

摘要

利用試管內經多芽體誘導所生產之雜交桉(Eucalyptus grandis x urophylla)植株，取其葉片、節及側芽，用以建立癒合組織培養。雖然最佳之癒合組織形成係得自葉片之培養，同樣也可以得自節與芽之培養。癒合組織增殖之最佳培養基為MS添加1mg/L kinetin及10mg/L IBA。當癒合組織培養在含有IBA或NAA的培養基時，根的再生很容易發生，但是其發生頻率會隨著BA濃度的增加而下降。芽體的形成僅僅偶爾發生自癒合組織培養在木本植物培養基添加12mg/L BA或MS培養基添加14mg/L BA。此一組織培養方法被進一步利用於農桿菌中介基因轉殖技術之發展。基因轉殖是取雜交桉之葉片做為培植體，分別與兩種雙質體的農桿菌共同培養而完成。兩種菌株皆含有編碼製造neomycin phosphotransferase (NPT Ⅱ)與β-glucuronidase (GUS)的轉移核酸序列。培植體與農桿菌之CIB542菌株共同培養後，並初步利用40mg/L kanamycin sulfate篩選轉殖的癒合組織，而獲得31.3%的轉殖率。切割自擬轉殖癒合組織團的細胞群，經初步利用組織化學染色法檢定GUS基因的表現情行。對於組織化學染色檢定顯示正確反應之癒合組織系，則進一步萃取其細胞之總核酸，並經複製而且檢視都具有NPT Ⅱ基因的790bp核酸片段，以及GUS基因的800bp核酸片段。此外，從組織化學染色法檢定呈現正確反應之癒合組織系所再生之轉殖根系，經置於染液中也呈現同樣的藍色反應。這些結果顯示已經將外來GUS基因引進雜交桉，而且可能穩定的嵌入雜交桉之染色體基因組。

關鍵字

組織培養 ； 器官形成 ； 基因轉殖 ； 農桿菌 ； 報告基因

並列摘要

Leaf pieces, nodes and axillary buds of in vitro plantlets micropropagated via multiple shoot induction were used to establish callus cultures of Eucalyptus grandis x urophylla. Callus formation, although best on culture of leaf pieces, could be obtained with nodes and buds as well. Callus proliferation was optimal on MS medium containing 1 mg/L kinetin and 10 mg/L indolebutyric acid (IBA). Root regeneration from calli was commonly observed in the presence of IBA or naphthaleneacetic acid (NAA), but declined in frequency with an increased benzylaminopurine (BA) concentration. Shoot formation only occasionally occurred on a woody plant medium (WPM) containing 12 mg/L BA or MS medium containing 14 mg/L BA. This tissue culture method was further used to develop Agrobacterium-mediated transformation which was accomplished by co-cultivation of leaf pieces with 2 disarmed, binary strains of Agrobacterium tumefaciens. Both strains harbored T-DNA sequences encoding neomycin phosphotransferase (NPT II) and β-glucuronidase (GUS). A good level of transformation (31.3%) was attained by inoculating explants with the Agrobacterium strain CIB542 when kanamycin sulfate at 40 mg/L was primarily used to select and maintain transformed callus lines - Transformation was primarily verified by histochemical staining of GUS expression in cell aggregates dissected from putative transformed callus clumps. Moreover, a 790bp fragment of the NPT II gene and an 800bp fragment of the GUS gene were amplified from total cellular DNA isolated from callus lines that stained positive in histochemical assay. Roots regenerated from the positive-staining callus lines appeared bluish in the staining solution. These above findings indicate that it is possible to introduce and stably integrate the foreign GUS gene into the genome of E. grandis x urophylla.

並列關鍵字

tissue culture ； organogenesis ； gene transfer ； Agrobacterium tumefaciens ； β-glucuronidase