绿色荧光蛋白基因mgfp4在水稻愈伤组织中的瞬时表达

采用基因枪方法将适用于高等植物的绿色荧光蛋白基因ｍｇｆｐ４转化到水稻愈伤组织之中,获得高效瞬时表达,在荧光显微镜和激光共聚焦显微镜下观察到强烈绿色荧光。绿色荧光蛋白ｍＧＦＰ４生色团形成迅速,转化４ｈ后就能观察到绿色荧光,并且持续时间较长,２ｄ后才基本消退。     

水稻悬浮系细胞的建立及植株再生

水稻（辽盐9）种子在LS＋2,4－D（2,4-二氯苯氧乙酸）2．0～3．0mg/L的培养基上进行诱导培养,经3～4次继代后,即可得到淡黄色颗粒性愈伤组织;将该种愈伤组织在LS＋2,4－D2．0～3．0mg／L＋LH（水解乳蛋白）300mg/L的液体培养基上进行振荡培养,经4～5次继代后,即可建立起良好的细胞悬浮系;培养基中附加2．0～2．5mg／LKT,使愈伤组织和悬浮细胞均可再生,其再生率分别为15．9％～17．9％和18．1％～20．8％。     

农杆菌介导转化水稻成熟胚影响因素研究

实验对蜀恢881、蜀恢527、中花9号等7个水稻品种的成熟胚为受体材料，研究了影响农杆菌转化水稻频率的几个重要因素，实验结果表明：CC培养基是籼稻成熟胚愈伤组织的最适诱导培养基，而NB培养基是粳稻品种成熟胚的最适诱导培养基；诱导培养基中添加ABA并不能显著提高籼稻和粳稻成熟胚愈伤组织的诱导率，但在ABA为3mg／LN可以明显改善愈伤质量；适当的低温预处理可以不同程度提高诱导率；农杆菌菌株EHA105对水稻愈伤组织的转化能力优于LBA4404和AGL1；在分化时，采用适宜的激素配比有利于提高分化率。     

抗除草剂旱稻转基因植株的获得

以旱稻丹粳旱５－５５、６－２４、６－３７的悬浮细胞及未成熟胚为受体材料，采用基因枪法将含ＢＡＲ基因的ｐＤＭ３０２质粒ＤＮＡ导入旱稻细胞中，经ＰＰＴ筛选获得抗性愈伤组织，筛选出的愈伤组织在分化培养基上再生出完整的旱稻转基因植株。Ｓｏｕｔｈｅｒｎ分子杂交分析表明，外源ＢＡＲ基因已整合到水稻基因组内，抗除草剂试验结果表明，转化植株对０．０１％Ｂａｓｔａ（有效成分为ＰＰＴ）有一定程度的抗性，说明外源ＢＡＲ     

Introduction of rice chitinase gene into wheat via low energy Ar+ beam implantation

A chitinase gene (RCH8) in plasmid vector pCAMBIA1308 was delivered into 3 wheat cultivars (Yangmai 158, Wan 9210, Wanmai 32) by low energy Ar⁺ beam-mediated method. Preliminary calli from treated mature embryos were first selected on hygromycin (Hm, 20 or 30 mg/L) containing medium. After the resistant calli formed, they were transferred to the regeneration medium with 10 or 20 mg/L Hm. All the three wheat varieties obtained transgenic plants. PCR and PCR-Southern assays showed that most plants regenerated from the resistant calli were positive transgenic plants. Southern blot of the positive green plants confirmed stable integration of alien DNA into wheat genome. The plant transformation frequencies varied with the variety and ion dose implanted. Wanmai 32 possessed the highest transformation frequency, reaching 3.8% at a suitable implantation dose. The transformation frequency of Yangmai 158 and Wan 9210 varied from 0.5% to 2.5% and from 0.5% to 1.4%, respectively. Progeny test for resistance to wheat scab showed that the leaf extract of R₁ generation inhibited the growth of wheat scab strain R₀ and F₁₅.     

含编码类铁氧还双亲性蛋白ap1基因的转基因水稻植株的获得

利用基因枪法将含有潮霉素抗性基因（hpt),gusA报告基因和ap1基因的2个质粒（pJIMB15和pBiSAP1)共同转化同转化由粳稻品种鄂宜105号种 子在胚诱导的愈伤组织（2－3周龄）。ap1基因编码一种双亲性的蛋白。该蛋白能延缓因假单孢菌感染所引起的非寄主植物中的过敏反应。经过2轮潮霉素（30mg/L)筛选,抗性愈伤组织被转入含30mg/L潮霉素的再生培养基中再生植株。从轰击的186块愈伤组织中共再生出32株独立的转基因水稻植株（转化率为17．2％）,PCR／Southern blot分析显示84％的转基因植株含有所有3个基因。     

籼稻“成恢448”组培体系的建立

对籼稻"成恢448"愈伤组织的诱导和分化过程进行了优化条件的探索.通过正交实验设计了不同的激素配比,结果显示:最佳的愈伤组织诱导条件为MS培养基加入1.0 mg/L 2,4-二氯苯氧乙酸(2,4-D),4.0 mg/L萘乙酸(NAA)和2.0 mg/L激动素(KT);最佳的分化条件为MS培养基加入1.0 mg/L NAA,2.0 mg/L 6-苄氨基腺嘌呤(6-BA)和0.5 mg/L KT.     

三叶半夏细胞悬浮培养和植株再生的研究

三叶半夏幼嫩叶片在MS+2,4-D2.0mg/L+BA0.5mg/L培养基上一个月内可以诱导出愈伤组织.取颜色鲜黄的愈伤组织进行悬浮振荡培养,经3～4次继代培养得到分散性好的细胞悬浮系.悬浮培养过程中细胞生长曲线呈S型,培养12～16d达对数生长期,细胞鲜重在第18d达到最大值.悬浮液pH值的变化在对数生长期同生长曲线一致.将细胞悬浮液过滤得到的小细胞团接种于MS+NAA0.2mg/L+BA1.0mg/L分化培养基上,40d分化出小植株.     

萱草(Hemerocallis hybrida)再生植株过程中根的诱导

利用1／2MS培养基作为基本培养基，附加不同浓度的NAA作为外源激素，分别对萱草愈伤组织块和无根再生进行根的发生诱导试验，结果：愈伤组织上均不易生根，而无根再生苗根的诱导效果较好，特别是在含有0．075mg/L NAA的培养基上，生根数量和生根率均最佳，为工厂化生产提供了相关的技术资料。     

Establishment of a genetic transformation system for maize inbred P9-10

It was found that the supplement of 10 -4 mol/L AgNO3 in N6 medium enhanced the induction of type Ⅰ embryogenic calli from immature embryos of maize inbred P9-10. After high-osmotic treatment, the induced calli was taken as transformation recipient to be bombarded with plasmid pMG6 carrying synthetic Bt gene by PDS-1000/He genegun. A total of 14 resistant calli were obtained after screening subsequently on the mediums with gradually increasing selective pressure. 10 plants were regenerated from the resistant calli on 3 different induction media, and 8 out of the 10 regenerated plants were confirmed to be integrated with Bt gene by PCR and Southern blotting analysis. Results of ELISA showed that the Bt-protein content in the leaves of the transgenic plants varied between 20-200 ng/g fresh weight.     

大豆胚芽尖再生体系的建立

以'西豆3号'等3个大豆品种为材料,以胚芽尖为外植体,研究种子萌发时间、植物生长调节剂对不定芽分化的影响以及生长调节剂对不定芽生根的影响,建立高效再生体系.结果表明种子在1/2MS培养基上萌发24 h后,将胚芽尖接种在BM+5 mg/L BA培养基上培养24 h,然后转入BM+0.4 mg/L BA+0.4 mg/L IBA培养基诱导不定芽,出芽率达64.91%～68.89%;将长3～5 cm的不定芽在BM+0.5 mg/L BA+1.5 mg/L IBA培养基中培养7 d后,转入BM基本培养基中生根,生根率达22.22%～26.32%.     

雄性二倍体毛白杨再生体系的构建和遗传转化的研究

【目的】建立雄性二倍体毛白杨再生体系,构建稳定的遗传转化方法,为进一步研究毛白杨基因功能提供试验平台。【方法】以雄性二倍体毛白杨的幼嫩茎段和叶片为外植体,1/2 MS为基本培养基,通过调整6-BA、IAA和TDZ激素浓度进行再生体系筛选;采用农杆菌EHA105介导叶盘法,控制预培养时间、菌液浓度、侵染时间、共培养时间和卡那霉素筛选浓度,进行遗传转化;以幼嫩叶片原生质体为受体细胞,PEG介导转化荧光标记基因EGFP,进行瞬时表达。【结果】雄性二倍体毛白杨再生过程包括继代、芽伸长和生根3个阶段,其培养基组分分别为1/2 MS+0.5 mg/L 6-BA+0.5 mg/L NAA+0.005 mg/L TDZ、1/2 MS+0.5 mg/L 6-BA+0.3 mg/L NAA和1/2 MS+0.3 mg/L NAA+0.5 mg/L IBA,该条件下生根率为96.7%,增殖系数为4.47。遗传转化过程包括预培养、农杆菌侵染、共培养、抗性筛选和生根5个阶段,其中预培养为12 h、农杆菌浓度OD₆₀₀为0.4、侵染时间为20 min、共培养时间为24 h、卡那霉素(30 mg/L)筛选45 d和抗性苗生根20 d。试验共获得86株抗性植株,其中14株分子鉴定结果为阳性。在40% PEG4000的介导下,EGFP基因瞬间转化效率为50%。【结论】雄性二倍体毛白杨再生周期短、遗传转化稳定,是杨树基础研究的理想材料。本研究拓宽了杨树遗传转化体系,为杨树分子辅助育种提供了新途径。     

Transgenic rice homozygous lines expressing GNA showed enhanced resistance to rice brown planthopper

Mature seed-derived calli from two elite Chinese japonica rice (Oryza sativa L.) cultivars Eyi 105 and Ewan 5 were co-transformed with two plasmids, pWRG1515 and pRSSGNA1, containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β-glucuronidase gene (gusA) and the snowdrop (Galanthus nivalis) lectin gene (gna) via particle bombardment. 61 independent transgenic rice plants were regenerated from 329 bombarded calli. 79% transgenic plants contained all the three genes, revealed by PCR/Southern blot analysis. Western blot analysis revealed that 36 out of 48 gna-containing transgenic plants expressed GNA (75%) at various levels with the highest expression being approximately 0.5% of total soluble protein. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From the R2 generations whose R1 parent plants showing 3:1 Mendelian segregation patterns, we identified five independent homozygous lines containing and expressing all the three transgenes. Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to rice brown planthopper (Nilaparvata lugens, BPH) by decreasing BPH survival and overall fecundity, retarding BPH development and declining BPH feeding. These BPH-resistant lines have been incorporated into rice insect resistance breeding program. This is the first report that homozygous transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis-based selection, conferred enhanced resistance to BPH, one of the most damaging insect pests in rice.     

Agrobacterium-mediated transformation of herbicide resistance in creeping bentgrass and colonial bentgrass

Embryogenic calli were induced from the seeds of creeping bentgrass ( Agrostis palustris Huds. ) cv. Regent and colonial bentgrass ( Agrostis Tenuis Sibth. F1. Oxen. ) cv. Tiger. The embryogenic calli were precultured on fresh medium for 4-7 days and then co-cultivated with Agrobacterium tumefaciens, LBA4404,which contains plasmid vector-pSBGM harboring bar coding region, synthetic green fluorescent protein (sGFP) coding region and matrix attachment region (MAR) . After 3 days of co-cultivation, the calli were washed thoroughly and transferred to MS medium containing 2 mg/L of 2, 4-D, 12-15 mg/L phosphinothricin (PPT) and 250 mg/L of cefotaxime. After 2-3 months of selection, the actively growing calli of ‘Regent‘ and ‘Ti-ger‘ were transferred to MS medium with 12-15 mg/L PPT and 250 mg/L cefotaxime for regeneration. The putative transformants were maintained on MS medium with 3 mg/L PPT for long period but control died within 1 month. After establishing in greenhouse, the transformants also showed strong resistance to 0.4 % of herbi-cide Basta but control plants died within 2 weeks. Under confocal microscope, both young leaves and roots showed significant GFP expression. PCR analysis revealed the presence of a DNA fragment of GFP gene at the expected size (380 bp) in the transformants and its absence in a randomly selected control plant.     

GUS和HPT基因的基因枪法转化水稻

以水稻（丹粳－５号）愈伤组织为受体材料，采用基因枪法将含有ＧＵＳ和ＨＰＴ基因的ＣＭ２质粒导入水稻细胞．经过５０ｍｇ／Ｌ的潮霉素筛选，获得抗性愈伤组织，并在同样筛选压力下诱导分化成苗，获得抗性苗．共获得１７个抗性克隆．ＧＵＳ组织化学检测表明，有１２个克隆为ＧＵＳ阳性，ＧＵＳ和ＨＰＴ基因共表达率为７０％左右．Ｓｏｕｔｈｅｒｎ分子检测证明ＨＰＴ基因已整合到水稻基因组中．     

驱蚊香草愈伤组织再生植株的诱导与培养

驱蚊香草(Pelargonium citrosumVanleenii)具有较强的净化空气作用,因其所分泌的挥发性成分香茅醇、香茅醛具有驱蚊功效而广泛栽培.利用愈伤组织再生技术对其增殖进行研究.叶柄愈伤组织诱导芽分化最适培养基为MS BA 1.0 mg/L NAA 0.1 mg/L 2,4-D 0.05 mg/L,可使诱导分化率达87%;增殖继代最适培养基为MS BA 1.0 mg/L NAA 0.1 mg/L,增殖系数达9.79;生根最适培养基为1/2 MS NAA 0.1 mg/L,生根率达100%.     

桂竹香花托诱导再生植株的研究

用桂竹香带子房花托,在附加6-BA2.00～3.00mg/L+NAA0.1～0.5mg/L的MS培养基上诱导出愈伤组织后,转接到附加6-BA0.2mg/L+NAA0.01mg/L的MS培养基上,分化出的丛生芽状态最佳,丛生芽在附加NAA0.5mg/L的1/2MS培养基中可诱导出多而粗壮的根.     

农杆菌介导的DREB1A基因转化多花黑麦草及其转化体系的优化

采用携带卡那霉素抗性基因nptII和GUS基因的ubiquitin启动子驱动的表达载体pBI121／DREB1A的根癌农杆菌AGL1, 对多花黑麦草幼胚来源的胚性愈伤组织进行了遗传转化,并优化了各种影响因素。胚性愈伤组织经根癌农杆菌感染和共培养后,用50mg／L巴龙霉素筛选抗性愈伤组织,待抗性愈伤组织在IB分化培养基上分化成苗后用25mg／L卡那霉素进一步筛选再生植株, 获得了部分抗性植株。抗性植株的总DNA用DREB1A基因的特异引物进行PCR检测,转化频率为2.14％,PCR-Southern blot进一步验证了转化植株基因组中含有该外源基因。各种影响转化效率因素的优化实验表明,当转化时菌液浓度的OD600为2.0、侵染时间为1h、共培养时间为2d、共培养温度为21℃及在共培养期间使用乙酰丁香酮等,均可明显提高转化频率。     

濒危药材红芽大戟组培快繁影响因素研究

【目的】研究濒危药材红芽大戟(Knoxia corym bosa Willd)最适宜的组培快繁方法,为规模化生产红芽大戟提供依据。【方法】选取红芽大戟不同组织器官为外植体,采用不同配方的培养基培养,研究影响外植体诱导分化、增殖和生根的因素,筛选获得最佳培养基。【结果】外植体采用茎尖优于叶轴、叶片,茎尖接种于MS+1.5mg/L 6-BA+0.15mg/L NAA,萌芽率为100%,MS+3.0mg/L 6-BA+0.30mg/L NAA最适用于继代增殖培养,1/2MS+2.0mg/L IBA+0.2mg/L NAA最合适生根培养。【结论】选取合适的外植体,采用适宜的培养条件和培养基,可以有效地进行红芽大戟的组培快繁。     

水稻基因整合平台系统供体质粒pURKMT的构建

提高水稻产量,改良稻米品质是育种学家广泛研究的课题．随着现代生物技术的发展,水稻已成为植物基因工程的重要研究对象．许多实验室已成功地建立了一系列供外源基因转化水稻的系统．但是这些转化系统主要应用Ｔｉ质粒衍生的载体,通过Ｔ－ＤＮＡ左右两端的序列将目的基...