水稻懸浮細胞培養之研發與利用白葉枯病菌篩選抗白葉枯病品系之新策略

Developments of Rice Cell Suspension Culture and a Novel Strategy for Screening New Resistant Lines to Rice Blight Disease Caused by Xanthomonas oryzae pv. oryzae

古書瑪(RESTU RIZKYTA KUSUMA)

指導教授 ： 鄭秋雄 博士 ； 林宜賢 博士 ； Liliek Sulistyowati, Ph.D

國立屏東科技大學/農學院/植物醫學系所/碩士(2013年)

https://doi.org/10.6346/NPUST.2013.00127

摘要

稻米為全球半數以上人口之主食，在亞洲地區水稻白葉枯病為水稻栽培過程中重要的限制因子，而組織培養之發展有助於解決這些問題。在細胞懸浮培養技術中可能發生體細胞無性系變異而產生新的具有高潛力之改良作物。本研究中，開發包含初代培養細胞懸浮系統之台梗九號稻，並建立一新的篩選平台選出具有抗水稻白葉枯病之品系。首先將培養於含3%蔗糖及2 mg/L 2,4-D之CS-1培養基4週之癒傷組織行懸浮細胞建立培養2週之培養細胞做為材料，以CS-1培養基為基礎培養基，探討碳源、植物生長調節劑、氮素、維生素及調整培養基（新舊培養液之比例）等對細胞增殖之影響。結果發現，調整培養基組成中之碳源、植物生長調節劑、氮素、維生素及調整培養基等有助於水稻初代細胞培養。CS-1調整培養基（新鮮培養基與舊培養基之比為1：1）內含3%蔗糖、0.5%葡萄糖、0.05%果糖及2 mg/L 2,4-D行初代培養3週，細胞增殖約1倍。隨後利用CS-1此培養基透過水稻白葉枯病菌之培養過濾液篩選新的抗水稻白葉病之抗性細胞系，會產生大量活性氧化物(reactive oxygen species,ROS)後，新的抗性細胞系在細胞識別水稻白葉枯病病原菌相關分子(pathogen-associated molecular pattern, PAMPs)。篩選初期利用1 %的水稻白葉枯病培養過濾液添加於水稻細胞懸浮培養液未增加篩選環境壓力。進一步將存活之細胞分離至24孔微孔盤中，經CS-1培養基培養一週後將部份細胞取出添加10 %水稻白葉枯病培養過濾液培養1小時並評估激活化氧(ROS)累積之情形，可將細胞ROS產生的情形分成三個等級，結果顯示在三次篩選後有強烈的ROS產生細胞株的比例由33 %增加到67 %同時，此些細胞株的細胞可強烈產生ROS的比例最高可達75 %。此結果說明篩選過的細胞品系在三輪篩選中可更為穩定產生高濃度的ROS，將這些產高ROS之細胞品系，行體胚分化誘導為植株，在水稻白葉枯病的感染過程中，細胞辨識後若能產生高量的ROS，並造成過敏性反應。這些新篩選出來的水稻細胞品系可能具有抗水稻白葉枯病之抗性潛力。研發水稻細胞初代懸浮培養系統，進一步建立新的篩選策略，將來將有機會獲得對水稻白葉枯病有抗性之新的水稻細胞品系。

關鍵字

水稻 ； 細胞懸浮培養 ； 水稻白葉枯病 ； 活性氧化物 ； 篩選 ； 抗性細胞

並列摘要

Rice is the staple food for more than half of the world population. Bacterial blight (BB) disease is one of the serious problems in rice production, especially in Asia. Development of cell culture system will help to solve some problems. As an alternative of cell suspension technologies, cell suspension culture has produced somaclonal variants with higher potential to screen novel lines for crop improvement. In this study, we developed a rice cell suspension system in primary culture and further screen resistant cell lines of Oryza sativa var. japonica cv. Taiken 9 to bacterial blight disease caused by Xanthomonas oryza pv.oryzae (Xoo). The culture cells obtained from the callus, cultured on CS-1 medium containing 3 % sucrose and 2 mg/L 2,4-D for 4 weeks, were offered as the materials and CS-1 medium as the basal medium for establishment culture of cell suspension for 2 weeks, and the effects of carbohydrate source, plant growth regulator, nitrogen, vitamin and conditioned medium on cell proliferation were evaluated. The results indicated that rice cell suspension in primary culture can be improved by modifying the composition of medium, such as carbohydrate source, plant growth regulator, nitrogen, vitamin and conditioned medium. The proliferation cell was signifcantly increased 1-fold in 3 weeks of primary culture in CS-1 conditioned medium (fresh/spent medium ratio 1:1) containing 3 % sucrose, 0.5 % glucose, 0.05 % fructose and 2 mg/l 2,4-D. This medium was further used to screen the bacterial leaf blight-resistant cell lines through applying culture filtrate of Xoo. In this strategy, we attend to screen a novel line which could produce high amounts of reactive oxygen species (ROS) while the variant could recognize pathogen-associated molecular patterns (PAMPs) from Xoo. In the begining, 1 % of Xoo filtrates were applied as environmental pressure in cell suspension culture. Then, the survival cells were further separated into 24-wells microplate in modified CS-1 medium for 1 week. After 10 % of Xoo filtrate was applied in cell suspension for 1 hour, the ROS production in cells was evaluated. Results showed 33 % cell lines were strong ROS-producing, two cell lines were selected and cultured for second round screening. The ratio of strong ROS-producing cell lines was increased up to 67 % in the third round screening. We observed that most the cells in selected cell lines were high to medium ROS-producing. The strong ROS-producing cell lines in third round screening can be further cultured for plant regeneration. The rice cell lines with high ROS production may have potential of resistant cell lines against Xoo. In this study, we developed a rice cell suspension system in primary culture and further establish a new screening strategy to obtain novel disease resistant cell lines against bacterial blight pathogen.

並列關鍵字

rice ； suspension cell culture ； Xanthomonas oryzae pv. oryzae ； reactive oxygen species ； screening ； resistant cell lines

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