提高苹果基因转化效率的研究

采用根癌农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ）强株系ＥＨＡ１０５（ｐ３５ｓＧＵＳ－ｉｎｔｒｏｎ）研究了影响‘皇家嘎拉’苹果（Ｍａｌｕｓ ｄｏｍｅｓｔｉｃａ Ｂｏｒｋｈ．）外植体的β－葡萄糖醛酸酶（ＧＵＳ）基因的瞬时、稳定表达水科和转基因植株的再生。证明在培养基生长素（ＩＢＡ、ＮＡＡ）存在的条件下，外植体的ＧＵＳ基因的瞬时表达水平提高了３～４倍，而共培养两周后稳定表达水平提高２     

培育苹果转基因植株的研究

采用“皇家嘎拉”苹果白化面外植体,通过根癌农杆菌ＥＨＡ１０５介导,证明了培养基中存在生长素可促进基因转化,转化效率比对照提高２倍以上。将外植体下ＥＨＡ１０１株系在含有生长素的２基中共培养,获得了转基因植株。采用ＰＣＲ分析和ＳｏｕｔｈｅｒｎＢｌｏｔ核酸杂交,确定ＧＵＳ基因已整合到苹果植株的染色体上。组织化学染色确定了ＧＵＳ基因在杆株体内的表达。     

含ipt基因生长素调控基因根癌农杆菌转化牵牛萌动种胚同工酶分析

以牵牛萌动种胚为受体,用含有３５Ｓ启动子－Ｇｕｓ基因－ｉｐｔ基因－Ｎｏｓ基因的根癌农杆菌ＬＢＡ４４０４３及含有３５Ｓ启动子－Ｇｕｓ基因－生长素调控基因－Ｎｏｓ基因的根癌农杆菌ＬＢＡ４４０４进行转化。通过对转化及对照植株苗期叶的同工酶分析,发现转化植株叶的过氧化物酶同工酶及酯酶同工酶谱带来对照植株有明显差异。转化频率较高。间接验证了外源ＤＮＡ的导入,说明同工酶分析方法可做为转基因植株早期筛选的方法之     

赤霉素和细胞分裂素调控苹果果实早期生长发育机理的研究

采用流式细胞术（ＦＣＭ）及酶联免疫法（ＥＬＩＳＡ）从细胞学和激素生理学的角度研究了ＧＡ４／７、ＢＡ及ＣＰＰＵ调节苹果果实早期发育的可能机理。ＧＡ４／７（５０ｍｇ／Ｌ）抑制了苹果果肉细胞分裂,未提高果实内源细胞分裂素（ＺＲｓ,ｉＰＡ）含量推测它主要通过促进工而促进果实生长,ＢＡ（５０ｍｇ／Ｌ）和ＣＰＰＵ（１２．５ｍｇ／Ｌ）可以显著促进苹果幼果的细胞分裂,ＣＰＰＵ的细胞分裂素活性较ＢＡ高,ＢＡ和ＣＰＰ     

Ri质粒介导TMV和CMV外壳蛋白基因转化甜椒研究

利用发根农杆菌（Ａｇｏｂａｃｔｅｒｉｕｍ ｒｈｉｚｏｇｅｎｅｓ）的Ｒｉ质粒介导的二元载体,将ＰＢＴＣ－８质粒上的ＴＭＶ－ｃｐ和ＣＭＶ－ｃｐ基因导入甜椒中,用共培养的方法,诱导愈伤组织,经卡那霉素的抗性筛选,进一步的培养获得再生植株,经ＰＣＲ和ＥＬＩＳＡ的检测呈阳性反应,转化植株田间人工接种为抗性,证明ＴＭＶ－ｃｐ和ＣＭＶ－ｃｐ基因导入甜椒植株并已表达。     

沙田柚遗传转化的探讨

采用含ｎｏｓ基因和人工合成柞蚕抗菌肽Ｄ基因ＡＰ－Ｄ基因的根癌农杆菌对沙田柚外植体进行转基因研究,与根癌农杆菌共培养产生的芽,苗经Ｋｍ敏感性筛选,获得了若干转基因植株,并对这些植株进行了报告基因ｎｏｓ表达的电泳检测,同位素标记、ＡＰ－Ｄ基因为探针的点印迹杂交,外源ＡＰ－Ｄ基因的ＰＣＲ扩增和Ｓｏｕｔｈｅｎ杂交。结果显示,报告基因ｎｏｓ、抗菌肽Ｄ基因已转入沙田柚植株细胞中。     

番茄抗青枯病品系(种)在恶劣条件下的抗性反应

对番茄青枯病菌（Ｐｓｅｕｄｏｍｏｎａｓ ｓｏｌａｎａｃｅａｒｕｍ）具有抗性的１６个番茄品系（种）在温室条件下，分别测定第３和第７星期两个苗龄的植株对青枯病菌的抗性。结果表明：苗龄３星期与苗龄７星期植株的抗性无显著相关性；参试的大部分抗病品系在恶劣条件下（高温、高湿、高病菌接种体）抗性会丧失；提出ＧＡ１５６５－２－４ＢＷＴ和７５Ｗ－ＮＲＳ－１是两个高抗的抗病品系；可作为高温、潮湿地区抗病育种较为理想的抗源。     

西藏的腹菌纲真菌资源

西藏的腹菌纲真菌资源ＴＨＥＲＥＳＯＵＲＣＥＳＯＦＦＵＮＧＩＯＦＧＡＳＴＥＲＯＭＹＣＥＴＥＳＩＮＸＩＺＡＮＧ徐阿生（西藏高原生态研究所八一８６００００）ＸｕＡｓｈｅｎｇ（ＩｎｓｔｉｔｕｔｅｏｆＰｌａｔｅａｕＥｃｏｌｏｇｙ，ｔｉｂｅｔ，Ｂａｙｉ８６０００...     

2,4—D对苹果新根发生及其超微结构的影响

２，４—Ｄ对苹果新根发生及其超微结构的影响杨洪强，黄天栋，束怀瑞（山东农业大学园艺系，泰安２７１０１８）关键词苹果；２，４—Ｄ；根；超微结构Ｅｆｆｅｃｔｓｏｆ２，４－ＤｏｎＲｏｏｔＦｏｒｍａｔｉｏｎａｎｄＳｕｂｍｉｃｒｏｓｔｒｕｃｔｕｒｅｏｆＮｅｗＲ...     

花叶万年青的组织培养

以“夏雪”品种为试材,研究花叶万年青的组培快繁,结果表明,无菌材料的获得以ＭＳ＋ＢＡ５＋ＩＢＡ０．２为最佳。快速繁殖阶段以ＭＳ＋ＢＡ５＋ＡＤ（１－１０）＋ＩＢＡ（０．１－０．２）＋３０（Ｇ／Ｌ）糖为最佳,６－ＢＡ与ＩＢＡ的比值在１０～５０倍为宜。生根前一代用ＭＳ＋ＢＡ（１－５）＋ＩＢＡ（０．１－０．２）＋３０（Ｇ／Ｌ）糖组合有利于芽苗长高,生根培养用１／２ＭＳ＋ＩＢＡ１＋６－ＢＡ０．１,在快繁阶段     

草莓主栽品种再生和转化的研究

 建立草莓主栽品种高效、稳定的离体再生体系和遗传转化体系, 获得了转基因植株。‘弗吉尼亚’和‘森嘎拉’的叶片离体再生芽频率达到100 %。试管苗叶片与农杆菌菌株EHA105 共培养3 d。共培养后的叶片在附加卡那霉素40 mg/L 的再生培养基上选择培养4周后, 外植体再生出转化芽, 弗吉尼亚的转化芽再生频率可达6. 8 %。采用组织化学染色法对随机选取的10 个GUS 基因转化植株进行基因表达测定, 结果5 个植株强烈表达GUS 活性。转bar 基因植株在附加除草剂草丁膦10 mg/L 的培养基上能够正常分化, 在田间对草丁膦表现出强烈抗性。转基因植株开花、结果正常。     

甜蛋白基因MBLII 对莴苣的遗传转化

 以4 日龄莴苣(Lactua sativa L.) 无菌苗子叶为外植体, 通过根癌农杆菌介导,成功地进行了马槟榔甜蛋白基因MBLII 对莴苣的遗传转化。抗生素浓度和子叶外植体与农杆菌的共培养时间是影响转化的重要因素。附加卡那霉素( Kan) 50 mg/ L 的诱芽培养基MS I(MS+ NAA 0. 1 mg/ L+ 6􀀁BA 0. 1 mg/ L+ 羧卞青霉素500 mg/ L) 最适于侵染后子叶外植体的诱芽培养。在外植体与农杆菌共培养0~ 7 d 的范围内, 以共培养3 d 最佳( 生芽率58. 3%, 白化率29%) 。1~ 2 cm 再生芽移入诱根培养基MS II (MS+ NAA 0. 05 mg/ L+ Kan 50 mg/ L+ 羧苄青霉素300 mg/ L) 中, 诱根率可达100%。获得的抗性植株经组织化学及PCR 特异扩增鉴定和统计, 7. 6%阳性。Southern blot 结果证明MBL II 基因已整合到莴苣基因组中。     

新红星苹果离体高效再生体系的建立

为了建立新红星苹果离体高效再生体系,以苹果品种新红星为试材,研究了不同基本培养基、激素质量浓度、外植体类型以及暗处理时间对其再生频率的影响。结果表明3种外植体的最适基本培养基均为MS培养基;叶片的再生效果最好,其次为黄化茎段,茎段最差,最高再生频率分别为74.1%,51.9%,42.6%;叶片和茎段的最佳暗处理时间为3周,黄化茎段的最佳暗处理时间为2周。新红星苹果再生的最佳外植体为叶片,最佳培养基为MS+TDZ1.0mg/L+NAA0.3mg/L+蔗糖30g/L+琼脂6.0g/L,最佳暗处理时间为3周。     

抗菌肽MB39基因导入‘皇家嘎啦’苹果及其四倍体植株的培育

 采用农杆菌介导法, 将抗菌肽MB₃₉基因转入‘皇家嘎啦’苹果, 获得了7 个转基因株系。通过秋水仙素诱变染色体组工程育种技术, 由转化体叶片获得了20 个四倍体植株。经PCR 检测和Southern Blotting 杂交证明, 抗菌肽MB₃₉基因已经整合到皇家嘎啦苹果的染色体组中。转基因株系TR21、TR23 提高了对火疫病的抗性。采用流式细胞技术( Flow Cytometry) 确定植株为四倍体。     

Shoot regeneration and genetic transformation by Agrobacterium tumefaciens of Hydrangea macrophylla Ser. leaf discs

A reproducible procedure was developed for genetic transformation of Hydrangea macrophylla Ser. cv. Blaumeise by Agrobacterium tumefaciens following the development of an efficient regeneration system using leaf discs excised from 12 to 15 weeks old meristem-derived vitroplants. Explants were cultivated on solid B5 medium complemented with maltose 110 mM, BAP 10 μM and NAA 0.5 μM. A low light regime of 17 μmol m⁻² s⁻¹ improved regeneration frequency up to 86%. For transformation, leaf discs were inoculated and co-cultivated with two disarmed A. tumefaciens strains, EHA 101 and LBA 4404, both carrying the binary vector pFAJ3000 which contained the nptII selectable gene and the GUS reporter gene. A pre-culture period of 3 days and a short co-cultivation duration (1 day) improved the efficiency of transformation. Inoculation of only 10 min with agitation including (or not) vacuum infiltration was sufficient. If selection on kanamycin containing medium was applied after a 2 weeks culture period on shoot regeneration medium, the percentage of explants forming kanamycin-resistant shoots increased from 3.3 to 13.3%. Integration and expression of the introduced transgene were confirmed by histochemical GUS assay, PCR and Southern blot analysis. Flowering of transgenic plants in glasshouse occurred 10 months after acclimatization.     

Somatic embryogenesis of Limonium sinense: a study on the regeneration efficacy via direct and indirect approach followed by Agrobacterium-mediated genetic transformation

An efficient indirect somatic embryogenesis and Agrobacterium-mediated transformation protocol for Limonium sinense has been established, wherein neomycin phosphotransferase II (npt II) and β-glucuronidase (GUS) genes were used as selectable and screenable markers, respectively. The efficiency of plantlet regeneration from transformed tissue was compared between direct embryogenesis from leaf and indirect embryogenesis from callus. Embryogenic callus (EC) was initiated from leaf explants on MS medium supplemented with 6.7 μM 2,4-D and 2.22 μM BA. The somatic embryos were induced, matured, and germinated when ECs were transferred onto MS medium supplemented with 4.44 μM BA and 1.07 μM NAA. Agrobacterium tumefaciens strain LBA 4404 containing the vector pBI121 was used for the transformation. Transient GUS expression frequency was evaluated and putative transgenic plants were successfully grown on culture medium in presence of kanamycin (80–100 mg L^?1). PCR analysis of putative transgenic plants confirmed the presence of GUS and nptII genes. The transformation efficiency obtained through indirect embryogenesis from calluses (4%) was much higher than through direct embryogenesis from leaf explants (0.9%).     

Adventitious shoot regeneration and Agrobacterium tumefaciens-mediated transformation of leaf explants of sweet cherry (Prunus avium L.)

Sweet cherry (Prunus avium L.) remains recalcitrant for genetic transformation due to the lack of efficient plant regeneration systems via organogenesis or somatic embryogenesis. In this study, in vitro shoot cultures were derived from a single mature embryo (open pollinated) of ‘Selah’ sweet cherry. Leaf explants were cultured on Woody Plant Medium supplemented with different plant growth regulators to induce shoot regeneration. The optimal regeneration at a frequency of 32.5% and an average of 1.1 shoots per explant occurred on the medium containing 4.54 µM thidiazuron (TDZ) and 2.95 µM indole-3-butyric acid (IBA). Transient transformation showed an efficient delivery of the β-glucuronidase (GUS) reporter gene (gusA) using Agrobacterium tumefaciens strain EHA105. Under the optimal gene delivery conditions, stable transformations were conducted using pGA643 and pBI-VcFT containing a blueberry FLOWERING LOCUS T (VcFT). A total of 500 leaf explants, 250 for each construct, were used for transformation. After 10-week selection, three leaf explants transformed with the pGA643 produced four kanamycin-resistant shoots, in which stable integration and expression of the nptII were confirmed by Southern blot and RT-PCR analysis, respectively. This study demonstrated that it was possible to produce stable transgenic sweet cherry using Agrobacterium tumefaciens-mediated transformation of leaf explants.     

萝卜遗传转化体系的建立

系统研究了影响根癌农杆菌介导的萝卜(Raphanus sativus L.)遗传转化的若干因素,建立了萝卜遗传转化体系:即切取萝卜带柄子叶外植体,先进行2 d预培养,用OD600为0.3~0.5的EHA105农杆菌菌液侵染5~7 min后,再进行5 d共培养,然后将外植体转接到MS+6-BA 6 mg.L-1+NAA 0.05 mg.L-1+Cef 500 mg.L-1的培养基上进行7 d延迟筛选,再将外植体转接到MS+6-BA 6 mg.L-1+NAA 0.05 mg.L-1+Cef 500 mg.L-1+Hyg 5 mg.L-1的培养基上进行10 d的抗性筛选。结果共获得126个抗性芽,其中8个抗性芽经PCR检测扩增出目的基因的启动子BcA9,约850bp,阳性率为6.3%,初步验证目的基因已经转入萝卜再生芽中。     

MAR调控的胰岛素样生长因子-Ⅰ转化甘蓝的研究

 采用根癌农杆菌介导法, 将表达框两翼构建了核基质结合区(matrix attachment region, MAR)的胰岛素样生长因子-Ⅰ ( Insulin-like Growth Factor-Ⅰ, IGF-Ⅰ) 基因导入甘蓝中, 在含卡那霉素的培养基上连续4次筛选, 通过10个批次的转化试验, 获得25株抗性植株, PCR检测均呈阳性, 随机选取5株进行基因组Southern杂交, 结果有3株出现了杂交带, 表明IGF-Ⅰ基因已成功整合到甘蓝基因组中。