麻竹組織培養培植體消毒及癒傷組織誘導之探討

Abstract

麻竹（Dendrocalamus latiflorus）為臺灣分布最廣泛且適應力良好之竹種，也是臺灣重要的竹類經濟作物。但麻竹遭受竹嵌紋病毒（Bamboo mosaic virus, BaMV）危害嚴重，阻礙了臺灣的竹產業發展。再者，現今常用的育苗法具有諸多限制，使麻竹產業發展面臨瓶頸。 至今已有諸多學者成功建立麻竹組織培養系統。其中因組織培養需於無菌環境中進行，良好的消毒條件成為建立系統的首要環節。但因麻竹培植體消毒困難，以常規的消毒藥劑難以將附於培植體表面之微生物及內生菌完全去除，因故多數學者使用消毒能力佳的HgCl2作為消毒藥劑。然而HgCl2易造成環境汙染，因此本研究選用對環境傷害較小的消毒藥劑，重新尋找麻竹組織最佳的消毒方式。並將培植體用於誘導癒傷組織，尋得癒傷組織誘導與增生之最適條件。 本試驗以麻竹含芽枝條進行試驗，結果顯示使用高濃度藥劑搭配短時間消毒，並且培養於含抗生素培養基，能有效地降低感染率至20%以下，同時不影響芽生長。進一步以麻竹筍作為試材，誘導麻竹癒傷組織。結果顯示，單獨使用1% NaOCl消毒45 min後以無菌水潤洗，培養在1/2 MSp680 + 2.41 mg/L Picloram培養基避光培養可得麻竹癒傷組織。同時也發現添加100 μg/ml Cefotaxime + 100 μg/ml Timetin於培養基可降低感染率。另以麻竹已抽芽枝條經2% NaOCl消毒30 min，輔以超音波震盪消毒15 min後，經3次無菌水潤洗後，置於添加50 μg/ml Cefotaxime及50 μg/ml Timetin培養基中，可得良好的消毒效果。培植體在不照光條件下，培養於1/2 MSp680 + 2.21 mg/L 2,4-D培養基結果最佳，誘導率達82.8%。然而癒傷組織生長仍需依附於培植體，單獨培養皆未能持續增生。 本結果可作為未來進一步研究麻竹癒傷組織形成及再生之基礎，並於可見之未來應用於深入探討細胞生長變化及其機制，提供未來竹類生長相關研究及應用之基石。

Ma bamboo (Dendrocalamus latiflorus) is an important commercial crop of Taiwan and it is the most distributed and well adapted bamboo specie. However, it is commonly infected by the Bamboo mosaic virus (BaMV) which has severely hindered the development of bamboo production. Furthermore, common breeding methods of bamboo have many limitations which pose challenges for the industry of ma bamboo. Many successful ma bamboo tissue culture systems have been established. In tissue culture system, explants should be cultured in aseptic condition. Therefore, having a good sterilization method is the very first and an important step of system establishment. However, complete sterilization of microorganisms and its endophytes remain inefficient using common disinfectants. Research shows that HgCl2 is the most widely used disinfectant, yet it causes heavy environmental pollution. This study aims to find the optimal sterilization method that use disinfectants with less environmental impact and seek the best condition for ma bamboo callus induction. Using the nodal branch of ma bamboo as material, results show that sterilization at high concentrations of disinfectant for a short time combined with antibiotic-supplemented mediums can lower contamination rates by more than 80% without affecting nodal growth. Ma bamboo shoots were also used to induce callus. Results show that there was successful callus induction when ma bamboo shoots were treated with 1% NaOCl for 45 min, washed thoroughly with sterilized distilled water, and cultured in 1/2 MSp680 medium that was supplemented with 2.41 mg/L Picloram in the absence of light. It was also found that adding 100μg/ml Cefotaxime, and 100μg/ml Timetin to the medium reduced contamination rates. The lowest contamination rate was observed when the ma bamboo budding branch was treated with 2% NaOCl for 30 min, sonication for 15 min, three thorough washes in sterilized distilled water, and cultured in a medium containing 50 μg/ml Cefotaxime and 50 μg/ml Timetin. The best callus induction rate (82.8%) occurred when the budding branch was cultured on 1/2 MSp680 medium supplemented with 2.21 mg/L 2,4-D in the absence of light. Despite these observations, the growth of callus still depended on explants and could not survive alone. These results can be used as basis for further study of ma bamboo regeneration system, callus induction and reproduction. And be applied to understand the changes and mechanisms of cell growth, providing the cornerstone of future research and application of bamboo growth in the foreseeable future.