大豆子叶节对潮霉素敏感性研究

标记基因的利用是筛选和鉴定转基因植株的有效方法,使用潮霉素能抑制植物的生长.选用3个基因型大豆为材料,以褐化率、分化率为指标,研究了不同质量浓度潮霉素对大豆子叶节分化和丛生芽生根的影响.结果表明不同基因型大豆对潮霉素的敏感性不同,潮霉素对大豆子叶节适宜筛选浓度黑农35为6 mg·L-1、合丰35和合丰25为4 mg·L-1;丛生芽生根筛选浓度黑农35为2 mg·L-1、合丰35和合丰25为1 mg·L-1.     

大豆子叶节遗传转化体系的优化研究

为了提高大豆转化效率,建立一个稳定高效的大豆遗传转化体系,选择了4个代表性基因型大豆子叶节为外植体,以子叶节和丛生芽GUS瞬时表达率为指标,优化了大豆遗传转化过程。结果表明:在侵染阶段分别进行超声波和抽真空辅助处理,GUS染色显示,4个基因型品种处理不同时间后遗传转化效率均有不同程度提高,超声波30 s或者抽真空2 min后农杆菌的侵染效率提高最为明显,转化效率分别提高8%~42%、16%~41%。在芽诱导前期进行PPT浓度筛选,4种基因型的最适PPT浓度均为0.2 mg·L~(-1),转化效率提高18%~33%。     

不同大豆基因型再生性及对农杆菌敏感性的研究

采用农杆菌介导的子叶节遗传转化方法对16个大豆基因型进行转化,以丛生芽分化率和GUS阳性率作为指标,比较不同大豆基因型再生性及对农杆菌敏感性的差异.同时对大豆遗传转化再生过程中的种子萌发所需6-BA浓度、草丁膦(PPT)筛选压力等因素进行研究.结果表明:萌发培养基中添加适宜浓度的6-BA能显著提高丛生芽的分化率；不同大...     

大豆子叶节再生影响因素的研究

为了建立一个大豆子叶节高效再生体系用于大豆的遗传转化，选用10个东北主栽大豆品种（Glycine max(L.)Merr.）的子叶节作为外植体，研究了基因型、植物激素的浓度、超声波辅助处理时间…等6种影响大豆子叶节再生的因素。结果表明合丰35、合丰25、黑农40再生效率较高，平均每个外植体分化出的丛生芽数分别为6.69、6.32和5.98；确定了丛生芽分化阶段所需的BA浓度，合丰35、合丰25为1.5mg/l，黑农40为1.2mg/l。适宜的外植体大小为保留全部子叶。作为遗传转化时的除菌剂，头孢唑啉钠（Cef）在500mg/l时对子叶节的再生无显著影响。当遗传转化时使用的超声波辅助处理时间小于12秒时，对子叶节再生无显著影响。     

影响农杆菌介导大豆子叶节遗传转化因素的研究

利用GUS基因的稳定性和特异性及在植物体中瞬时表达的特点,对农杆菌介导大豆子叶节遗传转化过程中的一些影响因素(激素浓度、筛选剂浓度、基因型和农杆菌菌株)进行研究.结果表明:芽诱导阶段6-BA浓度为1.6mg· L-1时诱导效率较高;适宜的草铵膦筛选浓度为3 mg· L-1;同时对24个大豆基因型的再生能力和对农杆菌菌株...     

大豆子叶节丛生芽的诱导研究

选用吉大豆1号、吉大豆2号、吉林47和东农42共4个基因型的大豆子叶节作为外植体进行离体再生,筛选出丛生芽诱导率较高的大豆基因型吉林47。在此基础上,以吉林47的大豆子叶节为外植体,研究了外植体消毒时间、发芽时间和条件对大豆子叶节再生的影响,并设计正交试验对芽诱导及伸长阶段的激素配比进行了研究。得出吉林47较适合氯气灭菌时间为6～10 h,最适萌发条件为16 h光+8 h暗光周期条件下培养5～7 d。最终确定芽诱导阶段添加6-BA浓度为2 mg.L-1、IBA浓度为0.1 mg.L-1;14 d后继代伸长阶段添加IAA浓度0.3 mg.L-1、GA浓度为0.5 mg.L-1。     

氯化锂作为转基因大豆筛选剂的研究

以东农50大豆子叶节为材料,以大豆子叶节丛生芽黄化率和丛生芽高度为考察指标,确定氯化锂作为转基因大豆筛选剂的最佳浓度和筛选临界观察时间。在此基础上,对转基因大豆丛生芽进行GUS染色,并对转化植株进行PCR检测。通过对受体植株进行氯化锂叶片涂抹,确定适宜的表型筛选浓度。结果表明,延迟7 d进行氯化锂筛选,筛选浓度为60 mmol.L-1为宜,最佳筛选天数为10 d。抗氯化锂丛生芽的GUS染色阳性率为80%,确定叶片涂抹法的适宜筛选浓度为700 mmol.L-1,并且经氯化锂筛选得到的转基因植株后代可以正常生长发育。     

大豆科丰14子叶节遗传转化体系的优化

科丰14是一个适应性较广的大粒型大豆品种,以农杆菌介导的子叶节法为转化途径,研究农杆菌侵染时间、6-BA浓度及培养基中添加硫代硫酸钠对转化率的影响,以建立适宜科丰14的子叶节遗传转化体系。结果表明:当农杆菌侵染外植体时间为15 min、丛生芽培养基中6-BA的浓度为1.50 mg·L-1时产生的丛生芽率最高,可达72.92%;在共培养基中加入1 mmol·L-1的硫代硫酸钠能有效防止外植体褐化,提高丛生芽率;利用GUS基因染色技术检测转化效果,表明在大豆常规转化体系的基础上,上述措施可提高科丰14的转化效率。     

农杆菌介导的大豆子叶节遗传转化技术流程及操作要点

大豆遗传转化效率低,是大豆转基因育种亟待解决的重要问题。以大豆子叶节为外植体采用农杆菌介导法,系统地研究了大豆子叶节遗传转化各技术环节的操作要点,包括工程菌液制备、无菌苗获得及外植体制备、农杆菌的侵染及共培养、不定芽诱导、不定芽伸长、根诱导、驯化与移栽、抗性植株的获得及转基因植株的检测等,建立了一套较稳定、高效的大豆子叶节遗传转化体系。并利用该体系,将野生大豆耐盐碱相关基因A1对大豆品种绥农28、合丰50、合丰55、东农50进行遗传转化,系统地探讨了大豆基因型、影响T-DNA的转移效率的各环节、筛选剂浓度及不同基因型转化效率等几个重要因素。     

"小粒黄"大豆子叶节丛生芽的诱导

为建立大豆子叶节高频再生体系,以"小粒黄"大豆子叶节为外植体,研究了萌发天数、6-BA及TDZ浓度对大豆子叶节丛生芽诱导的影响.结果表明: 随着萌发天数的增加丛生芽诱导率下降,萌发7 d的子叶节外植体对丛生芽诱导率最高,达到82.6%.附加不同浓度6-BA 和 TDZ的1/2MSB5的基本培养基上均能诱导出丛生芽,但丛生芽诱导率也不同,以1.5 mg·L-16-BA 和 0.2 mg·L-1 TDZ配比时,丛生芽诱导率最高,达到85.7%.     

根癌农杆菌介导的大麦茎尖转化研究

为了建立农杆菌介导的大麦茎尖遗传转化技术,以我国大麦主推品种鄂大麦9号、鄂大麦32122为材料,以农杆菌介导法转化大麦茎尖,经PPT筛选获得了转抗除草剂基因的大麦株系。PCR鉴定表明,以鄂大麦9号茎尖为受体,筛选获得4个转基因植株,阳性率为0.59%;以鄂大麦32122茎尖为受体,筛选获得10个阳性植株,阳性率为1.96%。转基因T1代Southern杂交分析证实,外源基因确已整合到大麦基因组中,已鉴定的转基因株系均含单个转基因拷贝,不同转基因株系整合的位点不同。因此大麦茎尖可作为农杆菌转化的受体。本研究结果为拓宽大麦遗传转化受体提供了技术和方法。     

小麦苗期抗旱相关形态指标的灰色关联度分析

为了系统研究小麦苗期形态指标与抗旱性的关系并筛选有效的抗旱性指标,使用PEG-6000模拟干旱胁迫,采用灰色关联度分析法分析了28份小麦品种苗期干旱胁迫下的苗高、最长根长、根数、地上部鲜重、地上部干重、根鲜重、根干重、叶片数、根冠比等9个形态指标与抗旱系数的关联程度,并以所得加权抗旱系数对各品种进行聚类分析.结果显示,各形态指标与抗旱系数的关联度依次为:叶片数(0.817)>苗高(0.766)>地上部干重(0.746)>地上部鲜重(0.729)>根数(0.699)>根干重(0.688)>根鲜重(0.681)>根冠比(0.645)>最长根长(0.399).研究还表明,9个形态指标可分为叶片相关因子、地上部生物量因子和根部生物量因子三大类,地上部生物量因子的关联度大于根部生物量因子;不能只通过差异显著度检验来判断某一指标是否可以用作抗旱性鉴定;聚类结果能较好地反映各小麦品种的地域分布.因此,进行小麦品种苗期抗旱性鉴定时,应加强对地上部形态指标的选择和鉴定,以提高选择效率.     

提高农杆菌转化水稻频率几个因素的研究

以6个重要的籼稻和粳稻品种为材料,研究了影响农杆菌转化水稻频率的几个重要因素。结果表明,对于籼稻品种而言,CC培养基是愈伤组织的最适诱导培养基;添加1.0~2.0 mg/L ABA能有效地改善愈伤组织的质量;在继代培养基中添加山梨醇能改善愈伤组织的生长状态,降低愈伤组织的褐化。而对于粳稻品种来说,适宜的诱导培养基是NB培养基,添加ABA对愈伤组织的改善效果不明显,对于多次继代的愈伤可以适当增加蔗糖浓度以提高抗性愈伤率。此外,在筛选时期适当加大筛选压力,分化时去掉筛选压力,壮苗时加适当的选择压力,可提高转化率。在分化时,采用适宜的激素配比有利于提高分化率。应用所优化的农杆菌转化系统,获得了一些有价值的转化植株。     

Factors Affecting Agrobacterium-Mediated Transformation Efficiency in Rice

Several important factors affecting the efficiency of Agrobacterium-mediated rice transformation were studied with several predominant commercial indica and japonica rice cultivars. As far as indica rice callus was concerned, CC medium was the best and the quality of callus was improved with the addition of 1.0 to 2.0 mg/L ABA. It decreased the percentage of browning calli and improved the callus growing state by addition of a certain amount of sorbitol to the subculture medium. NB medium was the best for callus initiation of japonica rice, but the improvement in the quality of callus of japonica subspecies was not obvious by adding ABA. During the period of subculture, to a certain degree, increasing the sucrose concentration could improve the proportion of hygromycin resistant calli. Furthermore, the transformation efficiency would be higher by applying selection pressure in the selection stage, removing selection pressure during the plantlet differentiation period and applying selection pressure again during seedling hardening period. Besides, suitable combination of plant hormones was beneficial for callus differentiation. An efficient /Agrobacterium-mediated rice transformation system had been established for several rice cultivars and a lot of transgenic rice plants had been obtained.     

Factors Affecting Agrobacterium-Mediated Transformation Efficiency in Rice

Several important factors affecting the efficiency of Agrobacterium-mediated rice transformation were studied with several predominant commercial indica and japonica rice cultivars. As far as indica rice callus was concerned, CC medium was the best and the quality of callus was improved with the addition of 1.0 to 2.0 mg/L ABA. It decreased the percentage of browning calli and improved the callus growing state by addition of a certain amount of sorbitol to the subculture medium. NB medium was the best for callus initiation of japonica rice, but the improvement in the quality of callus of japonica subspecies was not obvious by adding ABA. During the period of subculture, to a certain degree, increasing the sucrose concentration could improve the proportion of hygromycin resistant calli. Furthermore, the transformation efficiency would be higher by applying selection pressure in the selection stage, removing selection pressure during the plantlet differentiation period and applying selection pressure again during seedling hardening period. Besides, suitable combination of plant hormones was beneficial for callus differentiation. An efficient Agrobacterium-mediated rice transformation system had been established for several rice cultivars and a lot of transgenic rice plants had been obtained.     

茶树快速育苗方法研究

针对茶树无性系育苗周期长以致不适应茶产业快速发展需求的问题,本研究开发了一种基质育苗技术,将育苗周期由传统的14-18个月缩短到2个月,加速茶树育种进程.同时,对40个不同茶树品种的扦插发根特性进行了比较研究,证明'浙农121'发根最快,而'竹枝春'发根最困难,为品种推广提供有用资料.     

不同品种茶树生长对氮素浓度的响应差异

利用盆栽沙培法,研究了铁观音、福鼎大白茶、黄旦等6个基因型茶树对氮素浓度的响应差异。试验表明：（1）在低氮胁迫下茶树株高、根干重、地上部重、叶片SPAD值等显著降低;茶树根重、根冠比与茶树总生物量之间呈显著正相关（P<0.05）。（2）不同基因型茶树对氮素吸收利用能力差异较大,其中福鼎大白茶、春闺属低氮高效基因型。（3）低氮胁迫下,茶树根冠比显著增加,铁观音根冠比的变异幅度最大,为20.97%;基因型对茶树根冠比具有决定作用,不同基因型茶树根冠比的差异很大,如铁观音仅为0.71,春闺为1.81。（4）在低氮条件下,根冠比与叶片SPAD值可作为筛选耐低氮茶树品种的参考指标。     

根癌农杆菌介导的水稻纹枯病菌转化系统的建立

为建立水稻纹枯病菌的T-DNA插入诱变转化系统,以水稻纹枯病菌(Rhizoctonia solani AG-1ⅠA)强致病力菌株GD118为转化的初始菌株,从预诱导时间、共培养时间、共培养时乙酰丁香酮的浓度、共培养温度和共培养时固体诱导培养基(SIM)的pH值等5个方面对转化条件进行了优化,成功地建立了适合于水稻纹枯病菌根癌农杆菌介导转化(ATMT)的优化系统。这个优化系统的转化条件如下:以30μg/mL的潮霉素B作为转化子的筛选浓度,预诱导8h,共培养20h,共培养时固体诱导培养基上的乙酰丁香酮浓度为200μmol/L,共培养温度25℃,共培养时固体诱导培养基pH5.6～5.8。采用这个系统筛选到的转化子继代培养5代后,在含30μg/mL潮霉素B的PDA平板上仍表现明显的抗性。从得到的转化子中随机抽取10个,利用根据抗潮霉素hph基因设计的特异性引物进行PCR扩增,转化子均能扩增出500bp左右的预期条带;与此同时,以4个根癌农杆菌作为阳性对照,用根癌农杆菌Vir基因特异引物对转化子进行PCR扩增,以排除转化子受农杆菌污染所致的假阳性,结果表明:4个根癌农杆菌均能扩增出Vir基因条带(730bp),而10个转化子均未能扩增出相应条带。以上两个PCR扩增的实验结果清楚地表明,T-DNA已经插入到目标菌株GD118中。     

Divergent selection at ultra-low plant density for seed protein and oil content within soybean cultivars

Soybean [Glycine max (L.) Merr.] seed is a major source of protein for animal feed and oil for human consumption. Selection within elite soybean cultivars for the improvement of agronomic and seed traits is assumed to be ineffective due to the belief that cultivars are highly homogeneous. Previously reported data suggest that latent variation among the single plant selections within a cultivar exists and that mechanisms that generate de novo variation may also be present. The main objective of this study was to perform divergent single-plant selection at ultra-low plant density and investigate the presence of genetic variation for seed protein and oil within three elite soybean cultivars. A secondary objective was to investigate the variation for fatty acid composition. In 1995, single plants from the three cultivars were grown in a honeycomb design using a plant-to-plant spacing of 0.9 m. A total of 333 plants from ‘Benning’, 392 plants from ‘Haskell’, and 371 plants from ‘Cook’ were evaluated. Divergent single-plant selection for protein and oil content was performed to select a total of 20 plants for high or low protein and 20 plants for high or low oil from each cultivar. The selected plants were further evaluated in replicated row-plot experiments for 3 years. Our results indicate that single-plant selection at low plant density was successful in discovering significant variation for seed protein and oil within each of the three soybean cultivars. For protein content, the magnitude of intra-cultivar variation between the highest- and lowest-protein lines averaged 19 g kg⁻¹ across the three cultivars and ranged from 13 to 24 g kg⁻¹. For oil composition, the magnitude of variation between the most divergent lines averaged 12 g kg⁻¹ across the three cultivars and ranged from 9 to 14 g kg⁻¹. Significant variation among the selected progeny lines was also discovered for specific fatty acid composition. The magnitude of intra-cultivar variation averaged from 6 to 29 g kg⁻¹ across the five fatty acids of soybean. The genetic variation discovered within the soybean cultivars is most likely due to latent variation and/or newly created variation. Our data provide evidence that single-plant selection at ultra-low plant density within elite cultivars can be effective in improving the seed composition of a soybean cultivar.     

茶树新品种春雨1号、春雨2号区域适应性研究

在湖北武汉、四川成都、福建福安布点区试,以福鼎大白茶为对照,对春雨1号、春雨2号2个新品种进行了区域适应性试验.结果表明,春雨1号在物候期早于对照2 - 14天、发芽密度、产量均高于对照,制绿茶品质与对照持平,适于该三省及条件相似区域推广；春雨2号物候期总体迟于对照,武汉、成都发芽密度、产量高于对照,制绿茶花香浓郁,品...