两种不同的水稻标记基因删除技术的比较

Cre/loxP位点特异性重组系统是一种利用cre重组酶的瞬时表达来调控位于两个loxP位点之间DNA序列的删除系统。以反义LOX-3基因作为目的基因,以bar为选择标记基因,应用热激启动子驱动的Cre/loxP系统构建标记基因诱导删除型载体,得到转基因植株后,对比两种不同的热激方法中选择标记基因bar的删除效率,发现以生长到四叶期的转基因水稻植株为热激材料,标记基因删除效率较高,达到83.3％,因此,确定此方案为水稻标记基因删除的最佳方案。     

短光低温不育水稻宜D S成熟胚培养的研究

对短光低温不育水稻宜D S成熟胚在组织培养中的愈伤组织诱导和分化进行了研究,结果表明:愈伤组织诱导率与绿苗分化率之间无对应关系;诱导培养基的成分对转分化后的愈伤组织有后效作用,2,4-D与6-BA和NAA搭配使用比单独使用2,4-D诱导的愈伤组织更易于分化;同时还发现,不同诱导培养基诱导的愈伤组织在转分化后对分化培养基的要求不同.     

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。     

洁净DNA转化获得2mG2-epsps基因单拷贝整合的抗草甘膦水稻

洁净DNA转化是基因枪介导外源基因表达框导入植物的转化技术,能从根本上消除载体框架序列对转基因植株的不利影响。2mG2-epsps基因是具有重要育种价值的草甘膦除草剂抗性基因。以日本晴为材料,研究了草甘膦对水稻愈伤组织生长及分化的影响,采用洁净DNA转化技术将2mG2-epsps基因表达框导入水稻。结果表明:1)草甘膦对水稻愈伤组织的生长及分化有明显的抑制作用,当草甘膦浓度为2mmol/L时,愈伤组织绿苗分化率为18.97%,较对照71.67%显著降低。2)基因枪介导2mG2-epsps基因表达框转化水稻时,经草甘膦筛选获得抗性愈伤后,在植株再生培养基中去除筛选剂利于抗性愈伤的分化,转化率为17.20%。经Southern杂交分析,2mG2-epsps基因表达框均以单拷贝整合到受体基因组。52.17%(12/23)转基因株系可耐受12~50mmol/L的草甘膦。     

水稻成熟胚盾片诱导愈伤组织再生体系的建立

采用水稻成熟胚盾片诱导愈伤组织作为分化再生的外植体，通过优化激素组合和调节培养基渗透压，建立了适合水稻遗传转化的高效再生体系，将水稻成熟胚诱导愈伤组织分化再生的过程划分为3个不同时期，即成熟胚盾片愈伤组织诱导，胚性细胞诱导保持，胚性愈伤组织分化再生，以CultureI(LS 2,4-D2.0mg/L)诱导成熟胚盾片愈伤组织，愈伤组织剥离后接种于Culture II-3(LS 2,4-D 2.5mg/L 山梨醇3g/L)上诱导胚性愈伤组织，继代后接种于Culture Ⅲ-2(MS NAA 0.5mg/L 6-BA 1.0mg/L Kinetin 2.0mg/L 山梨醇8g／L）上分化成苗，培养结果表明，该再生体系所获得的胚性愈伤组织分化再生率均在60％－80％之间，利用本研究建立的高效再生体系建立水稻外源基因遗传转化系统，使转化过程中所得转化愈伤组织高效再生成苗，从而顺利获得转化植株，可以提高水稻外源基因转化的效率，为水稻品种的定向遗传改良提供优良的遗传转化技术体系。     

利用基因枪法将Bt基因导入玉米优良自交系

利用基因枪法将Bt基因导入玉米优良自交系501和C111.轰击后的幼胚转至附加Bialaphos浓度为10 mg/L的选择培养基上进行愈伤组织的诱导,继代筛选3轮,每轮3周.得到的抗性愈伤组织在不含Bialaphos的分化培养基上进行绿苗分化,501获得了20株转化再生植株,C111获得了10株转化再生植株,PCR鉴定结果表明Bt基因已整合到玉米自交系501和C111的基因组中。     

小麦幼胚再生培养体系优化及优良转化受体基因型的筛选

为建立适于优良小麦品种遗传转化的高效组织培养体系,以20个优良小麦推广品种为材料,对以幼胚为外植体的愈伤诱导、分化和生根壮苗培养基进行筛选.结果表明,添加500 mg/L水解酪蛋白、100 mg/L脯氨酸、100 mg/L谷氨酰胺,并以40 g/L麦芽糖为碳源的MXD2培养基的出愈率最高,愈伤质量最好.以大量元素减半的1/2 MX为基本分化培养基,添加1 mg/L ZT和0.5 mg/L IAA对愈伤组织诱导分化的效果最好,分化率最高达到83.0%.以1/2 MX培养基中添加0.5 mg/L IAA和0.5 mg/L MET的生根效果最好,且移栽成活率高.不同基因型的组织培养特性差异显著,筛选到扬麦158、南农9918、济麦20、宁麦9号等8个基因型可作为优良的转基因受体材料,并初步建立起适合于它们的植株再生体系.     

大麦幼胚离体培养条件的建立

为了建立一个适于优良大麦品种遗传转化的高效组织培养体系,对两个大麦地方品种晋引6号和来色衣的未成熟胚离体培养条件进行了研究。结果表明,在M S基本培养基上添加ABA 0.5 m g/L、CH 300m g/L能够很好地抑制幼胚过早发芽,并促进植株再生;添加2,4-D 2 m g/L也能够很好地促进幼胚愈伤组织的诱导和分化;添加ZT 1.0 m g/L和IAA 0.1 m g/L,对愈伤组织诱导分化的效果比较理想,分化率平均达到86.6%。     

茶树叶片组织培养的初步研究

用MS培养基附加以不同激素的种类及浓度的组合配比,探讨茶树叶片组织培养中对愈伤组织、胚状体、芽和小植株分化的能力。结果表明,连续用7、8号两组培养基作继代培养,新形成的愈伤组织都具有分化芽的能力。当这种愈伤组织转移到4号培养基上培养,可获得胚状体或胚性细胞团的分化,进而形成正常芽和苗。若将上述新形成的愈伤组织转移到9号培养基上培养,可使芽体有根的分化,从而得到健壮苗株。说明茶树叶片的组织培养中诱导愈伤组织,分化胚状体和小植株的形成所需要的外源激素的种类组合及浓度配比上存在明显差异。     

珂字棉312组织培养体系的建立

以珂字棉312为试验材料,以无菌苗的下胚轴为外植体诱导愈伤,并将愈伤组织置于不同培养基下诱导分化,初步建立棉花组织培养体系。结果表明:一是对珂字棉312组织培养体系建立较好的各阶段培养基分别是愈伤组织诱导培养基为MS+0.1 mg/L 2,4-D+0.5 mg/L KT+30g/L葡萄糖+8 g/L琼脂,胚性愈伤组织的诱导培养基为MS+30 g/L葡萄糖+2.5 g/L phytagell,分化培养基为MS+0.1 mg/L IAA+30 g/L葡萄糖+2.5 g/L phytagell,生根培养基为MSB5(双倍KNO_3,无NH_4NO_3)+0.1 mg/L GA3+30 g/L葡萄糖+2.5 g/L phytagell;二是各培养基调节pH值为5.8,培养温度为28℃时能够获得植株再生体系。     

Rice Blast Resistance of Transgenic Rice Plants with Pi-d2 Gene

Resistance to rice blast of transgenic rice lines harboring rice blast resistance gene Pi-d2 transformed from three different expression vectors of pCB6.3kb, pCB5.3kb and pZH01-2.72kb were analyzed. Nine advanced-generation transgenic rice lines with Pi-d2 gene displayed various resistance to 39 rice blast strains, and the highest disease-resistant frequency reached 91.7%. Four early-generation homozygous transgenic lines with Pi-d2 gene exhibited resistance to more than 81.5% of 58 rice blast strains, showing the characteristic of wide-spectrum resistance. The transgenic embryonic calli selected by the crude toxin of rice blast fungus showed that the callus induction rate of immature embryo from transgenic rice plants decreased as the concentration of crude toxin in the culture medium increased. When the concentration of crude toxin reached 40%, the callus induction rate of immature embryo from transgenic lines was 49.3%, and that of the receptor control was 5%. The disease incidence of neck blast of the transgenic rice lines in fields under induction was 0% to 50%, indicating that the rice blast resistance of transgenic rice lines is much higher than that of the receptor control.     

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.     

Infection of Early and Young Callus Tissues of Indica Rice BPT 5204 Enhances Regeneration and Transformation Efficiency

A rapid and reproducible method to develop transgenic plants with enhanced transformation efficiency using Agrobacterium has been developed for the elite indica rice variety BPT 5204. Different rice calli aged from 3 to 30 d were co-cultivated with pre-incubated Agrobacterium suspension culture （LBA4404： pSB1, pCAMBIA1301） and incubated in dark for 3 d. Based on the transient GUS gene expression analysis, 6-day-old young calli showed high transformation frequency followed by 21-day-old ones. Thus, both 6-and 21-day-old calli were used for assessing the stable transformation efficiency. It was observed that the 6-day-old young transformed calli showed about 2-fold higher regeneration frequency when compared with 21-day-old calli. The transformation efficiency was enhanced for young calli to 5.9% compared with 0.8% of the 21-day-old calli. Molecular and genetic analysis of transgenic plants （To） revealed the presence of 1-2 copies of T-DNA integration in transformants and it follows Mendalian ratio in T1 transgenic plants. From the present study, it was concluded that the development of transgenic rice plants in less duration with high regeneration and transformation efficiency was achieved in BPT 5204 by using 6-day-old young calli as explants.     

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.     

基因枪法转化籼稻有关因素的评价

 利用基因枪轰击水稻未成熟胚及未成熟胚诱导来源的愈伤组织,对国内外16个籼稻品种进行了转化。设置不同的基因枪转化参数,并在培养基中添加各种植物激素、甘露醇以及2-N-吗啉-乙基磺酸(简称MES)来改善继代过程中籼稻愈伤组织的生长状态。基因枪转化的操作参数（包括金粉浓度和DNA含量、基因枪氦气压力、轰击受体的不同状态）对转化频率有重要影响;在继代培养基中添加MES（500 mg/L）作为缓冲剂,可以减少籼稻愈伤组织褐化现象的发生;在诱导和继代培养基中补加NAA(0.5 mg/L）,KT(0.2 mg/L)可改进愈伤组织质量,进而提高再生频率;在继代培养基中补加低浓度的 ABA(1 mg/L)和甘露醇（20 g/L）,能普遍改善愈伤组织生长状况,减少褐化现象的产生,得到较多结构致密的颗粒状抗性愈伤。获得了再生的转化植株,经检测表现出对Basta的抗性。     

农杆菌介导法将抗草甘膦基因转入玉米自交系

利用农杆菌介导法将抗草甘膦基因(ssu-G₂-aroA)转入优良玉米自交系R18-599和齐319,侵染后的愈伤组织转至潮霉素浓度为10 mg/L的筛选培养基上,继代筛选3轮,每轮3周;得到的抗性愈伤组织在含有潮霉素浓度为3 mg/L的分化培养基上进行分化,R18-599获得30株再生植株,齐319获得18株再生植株。经PCR鉴定后共得到7株转化抗性植株(4株R18-599、3株齐319)。经PCR-Southern及RT-PCR检测结果表明,ssu-G₂-aroA基因已稳定整合到了玉米基因组中,并在RNA水平上得到表达。