籼稻胚性愈伤组织继代培养基体系优化及愈伤组织褐化原因剖析  

Optimization of Cultivation Conditions for Calli Subcultures of Indica Rice(Oryza sativa ssp. indica) and Study on the Mechanism of Calli Browning

作　　者：毛小辉[1,2] 肖开转[2,3] 王进兰 郑燕梅[1,2] 蔡秋华[2] 张建福[1,2,3] 谢华安[1,2,3] 

机构地区：[1]福建农林大学生命科学学院,福州350002 [2]福建省农业科学院水稻研究所/农业部华南杂交水稻种质创新与分子育种重点实验室/福州(国家)水稻改良分中心/福建省作物分子育种工程实验室/福建省水稻分子育种重点实验室/福建省物种质创新与分子育种省部共建国家重点实验室培育基地/杂交水稻国家重点实验室华南研究基地/水稻国家工程实验室,福州350003 [3]福建师范大学生命科学学院,福州350108

出　　处：《农业生物技术学报》2016年第9期1319-1327,共9页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展规划(973)项目(No.2013CBA01405-7);国家高技术研究发展计划(863)项目(No.2014AA10A603和No.2014AA10A604);国家转基因农作物新品种培育重大专项(No.2015ZX08001-002和No.2015ZX08001-006);福建省属公益类研究所专项(No.2014R1021-15和No.2015R1021-7);福建省财政专项-福建省农业科学院创新团队项目(No.CXTD-1-1301)

摘　　要：农杆菌(Agrobactrium tumefaciens)介导的转化技术已广泛应用于粳稻品种(Oryza sativa ssp.japonica)中,但迄今未能找到适合于籼稻品种(O.sativa ssp.indica)高效遗传转化的农杆菌介导转化体系,籼稻品种的愈伤组织在继代过程中的严重褐化是最主要的制约因素之一。本研究以籼稻品种93-11为材料,通过响应面设计分析方法,进行二次多项式回归拟合,预测数学模型,研究2,4-二氯苯氧基乙酸(2,4-dichlorophenoxyacetic acid,2,4-D)浓度、N6-呋喃甲基腺嘌呤(kinetin,KT)浓度和碳源中蔗糖和麦芽糖的比例对籼稻品种愈伤组织诱导的影响,确定了最适宜93-11成熟胚诱导愈伤组织的培养基成分:以MS为基本培养基,添加2.92 mg/L 2,4-D、0.59 mg/L KT、16.37 g/L蔗糖和13.63 g/L麦芽糖;并以此优化培养基继代籼稻愈伤组织。为了研究93-11愈伤褐化的原因,采用组织学与解剖学方法,观察93-11正常愈伤组织和褐化愈伤组织。结果表明,两者的表观结构存在较大差异,正常愈伤组织表面粗糙,凹凸不平;褐化愈伤组织表面结构看似光滑,但是放大后发现这种平滑结构是由于所有细胞失去规则圆形变为无活性扁平状,且细胞不易成团,细胞之间空隙小而少形成的。用q RT-PCR技术分析了褐化相关基因在褐化愈伤组织中的表达情况,发现切伤后与褐化相关的基因生长素输入载体1基因(auxin1,AUX1)和蔗糖非依赖1蛋白激酶2基因(sucrose non-fermenting 1-related nrotein kinase 2,Sn RK2)的表达量虽然都比较低,但是在93-11成熟胚诱导的正常愈伤组织和褐化愈伤组织中存在明显差异,而酚类物质氧化途径中与酚类物质合成的基因苯丙氨酸解氨酶基因(phenylalanine ammonia-lyase,PAL)和多酚氧化酶基因(polyphenol oxidase,PPO)在正常愈伤组织还是褐化愈伤组织中都没有检测到表达量。发现组织培养切伤是愈伤组织褐化的主要原因,为进一步阐明愈伤组织的褐化机理提供参Agrobacterium-mediated transformation technology has been widely used in japonica rice （Oryza sativa ssp. japonica） varieties, but so far inaccessible to indica rice（O, sativa ssp. indica） varieties, mainly attributed to calli browning in following regeneration, which has severely hindered the application of transformation in indica rice. In this study, cv. 93-11, a typical indica variety, was used and the mathematical model was established by central composite design combined with response surface methodology and the media component for callus subculture were optimized. Results showed that MS media was more suitable for primary callus induction than N6, B5, N6B and NMB, which callus browning was significant inhibited through observation and comparison. Based on MS media, the effects of single factors such as hormone, carbon source were analyzed through single factor experiment. It was found that the best 2,4-dichlorophenoxyacetic acid （2,4-D） was 3 mg/L, kinetin （KT） was 0.5 rag/L, sucrose was 15 g/L and maltose was 15 g/L. And then Box-Behnken design was used to obtain the best value. The determination coefficient R2 of the model was 0.9 476 and the optimal combination of media component for inducing yellow, compact and organized calli was 2,4-D 2.92 rag/L, KT 0.59 rag/L, sucrose 16.37 g/ L and maltose 13.63 g/L. Histological and anatomical analysis revealed the remarkable morphological differences between normal calli and brown calli： The surface of normal calli was quite irregular and was full with distinctive globular nodules and the globular nodules were full with tightly packed cells. The somatic embryogenesis consisted of white, sheet- shaped non- embryogenesis structures could be descried from the yellowish opaque sectors of a calli. In contrast, the smooth surface of brown calli was due to the fact that nearly all cells distorted from round shape to flat shape and the inactivation cells were unlikely to occur globular nodules, qRT-PCR revealed that the transcript levels of artificial w

关 键 词：籼稻 93-11 组织培养 褐化 原因分析 

分 类 号：Q944.6[生物学—植物学]