农杆菌介导的柳枝稷遗传转化体系的优化

doi:10.3864/j.issn.0578-1752.2016.01.007

摘要/Abstract

摘要： 【目的】为建立农杆菌介导的柳枝稷高效再生及遗传转化体系。【方法】试验以柳枝稷品种Alamo、Performer及Blackwell的成熟种子为外植体诱导愈伤组织。愈伤诱导6周后，借助解剖镜观察愈伤形态，去除愈伤组织外层含水量大、海绵状愈伤组织，挑选位于愈伤组织中心位置的核状、紧实型愈伤置于继代培养基培养。暗培养3周后，在解剖镜下挑选结构松脆、生长旺盛的愈伤组织按细胞系继代增殖培养。该类愈伤组织易于分化，属单子叶植物愈伤组织分类中的第Ⅱ型。经两次继代增殖培养后，可以获得足够的愈伤组织进行再生和遗传转化研究。为优化柳枝稷Ⅱ型愈伤组织的农杆菌侵染流程，试验比较了3种农杆菌侵染流程下的转基因效率。真空和干燥处理（VD）：将装有愈伤组织及菌液的50 mL离心管置于真空泵中抽真空10 min（压力-0.8MPa），28℃, 80 r/min慢摇20 min后弃菌液，将愈伤组织堆放于3层滤纸上干燥处理2 h，随后将愈伤组织转入含有500 μL无菌水的2层滤纸上进行共培养；冷处理加真空和干燥处理（CVD）：侵染前将愈伤组织置于3%麦芽糖、300 μmol·L^-1谷氨酰胺（Gln）溶液中冰浴20 min，然后依VD流程侵染，共培养阶段用MP培养基代替无菌水；渗透冷处理加真空和干燥处理（PVD）：用6%的麦芽糖溶液替代CVD中3%麦芽糖溶液。通过比较Gus染色及PCR阳性率来评价三种方法的转化效率。【结果】愈伤挑选方法不仅从柳枝稷低地型品种中挑选得到再生率达95%以上的Ⅱ型愈伤细胞系，也首次获得高地型品种Blackwell的Ⅱ型愈伤组织，分化率达50%。不同转化方法评价过程中发现，低地型品种AlamoⅡ型愈伤组织采用CVD农杆菌侵染流程转化效率最高，达72%，显著高于侵染流程VD（53%）和PVD（44%）。应用CVD转化法，Performer转化率达96%；首次成功进行了高地型品种Blackwell的遗传转化，转化效率为5.6%。【结论】本研究建立了借助解剖镜挑选柳枝稷易于再生的Ⅱ型愈伤组织的方法，该方法适用于柳枝稷不同生态型品种；农杆菌转化过程中采用CVD侵染流程可显著提高转基因效率。本研究首次报道了柳枝稷Ⅱ型愈伤组织的挑选流程，建立了适合不同生态型柳枝稷的高效再生及遗传转化体系，首次获得高地型品种Blackwell的转基因植株，为柳枝稷遗传改良及其功能基因研究奠定基础。该方法也适用与其他难于再生的单子叶植物再生及遗传转化体系的建立。

关键词: 柳枝稷, 高地型品种, Ⅱ型愈伤组织, 遗传转化, 农业杆菌介导

Abstract: 【Objective】 To establish a high-efficiency plant regeneration and transformation procedure of switchgrass.【Method】mature seeds of three different switchgrass (Panicumvirgatum L.) varieties were used as explants for callus induction. After six weeks of callus induction, the phenotype of the callus was observed under a dissecting microscope, we discarded the out-layer sponge-like, watery callus and selected the core-like, highly compact callus that lies in the middle of callus clump for further subculture, after the sub-culture was in the dark for three weeks, calluses grew fast, compact and grainy like were selected for further culture, calluses generated from one seed as a cell line and were kept together. This type of callus was highly regenerable, and was characterized as Type Ⅱ callus of monocot plant. After two rounds of selection, enough type Ⅱ callus could be obtained for plant regeneration and transformation. To optimize the procedure of Agrobacterium-mediated transformation, transformation efficiencies under three different Agrobacterium-infection procedures were evaluated. They are vacuum-drying treatment (VD): That was in a 50 mL centrifuge tube, the calluses that merged in Agrobacterium suspension were given a vacuum treatment (-0.8 Mpa) for 10 min, then slight agitated in a incubator-shaker at 80 r/mim, 28℃ for 20 min. Then drainage the bacteria and dry on a sterilized filter paper tower, the calluses were transfer onto two layers of filter paper soaked with 500 mL sterile water in a petri dish for co-cultivation. Cold combined with vacuum-drying treatment (CVD): calluses were given a cold treatment on ice in a solution of 3% maltose and 300 μmol·L^-1 of glutamine (Gln) for 20 min before conducted as VD process. A slightly different procedure was used instead of sterile water MP liquid medium was used for cocultication. Osmotic treatment combined with the cold and vacuum-drying treatment (PVD): 6% maltose was used in CVD process. The transgenic transformation efficiencies were evaluated by GUS staining assay and PCR tests.【Result】A regeneration efficiency of over 95% was reached for the type Ⅱ callus selected from the lowland switchgrass cultivar “Alamo” and “Performer”; 50% was reached for upland cultivar “Blackwell”. The transformation rate of switchgrass cultivar “Alamo” reached to 72% under CVD procedure, significantly higher than that was obtained under the procedure VD (53%) and PVD (44%). Under CVD procedure, transformation efficiency of lowland cultivars “Performer” reached to 96.7%; for the upland cultivar “Blackwell”, the transformation efficiency reached to 5.6%. This was the first time of obtaining the transgenic plant of the upland cultivar “Blackwell”.【Conclusion】Highly regenerative type Ⅱ calluses can be obtained from different ecotypes of switchgrass with a stereo-microscope. Agrobacterium- mediated transformation efficiency could be significantly improved under a CVD procedure for infection. This was the first report on how to select the type Ⅱ callus derived from mature seeds of switchgrass. A high-efficiency plant regeneration and transformation procedure that was suitable to lowland and upland switchgrass cultivars was developed, which made a foundation for switchgrass genetic improvement and functional gene research. The method presented here is also helpful in establishing a plant regeneration and transformation system for other recalcitrant monocot.

Key words: switchgrass, upland cultivars, typeⅡ callus, Agrobacterium-mediated transformation, Agrobacterium-mediated

刘燕蓉，岑慧芳，严建萍，张万军. 农杆菌介导的柳枝稷遗传转化体系的优化[J]. 中国农业科学, 2016, 49(1): 80-89.

LIU Yan-rong, CEN Hui-fang, YAN Jian-ping, ZHANG Wan-jun. Optimizing of Agrobacterium-Mediated Transformation of Switchgrass Cultivars[J]. Scientia Agricultura Sinica, 2016, 49(1): 80-89.

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