保加利亚尖椒再生及遗传转化条件的优化

为了提高辣椒子叶不定芽的伸长率和遗传转化效率,本研究以保加利亚尖椒子叶为外植体,通过正交试验分别对影响保加利亚尖椒子叶不定芽伸长的激素组合以及遗传转化参数进行了优化。结果表明:诱导不定芽伸长的最佳激素组合为0.2mg/LIAA+1.0mg/LGA3+0.1mg/LPBU,不定芽伸长率最高为60%;以5mg/L潮霉素为选择压,预培养时间为4d、共培养时间为2d、侵染时间为20min时,诱导的抗性不定芽比率最高。本研究建立的辣椒再生及遗传转化体系为辣椒转基因研究奠定一定的基础。     

影响花椰菜农杆菌介导转化因素的研究

以花椰菜赛雪的带柄子叶为外植体,以MS为基本培养基,GUS基因为报告基因,分析了遗传转化过程中的影响因子,如预培养时间、农杆菌菌液浓度、侵染时间、共培养时间、乙酰丁香酮浓度、延迟筛选时间等对外植体瞬间表达和稳定表达的影响。结果显示,以花椰菜的带柄子叶为外植体,预培养2d,农杆菌菌液为OD6000.3～0.4,侵染8min,共培养2d,乙酰丁香酮浓度为100μmol/L,延迟筛选7d,卡那霉素筛选压为5mg/L为最优的遗传转化方案,转化率最高可达35.7%。另外,GUS瞬间表达率和转化率并不存在绝对的相关性,但瞬间表达分析仍然可以作为外源基因进入受体细胞的指示。花椰菜农杆菌介导转化方案的优化研究为芸薹属蔬菜高效遗传转化提供了技术保障,有利于芸薹属蔬菜遗传育种与种质创新研究。     

农杆菌介导的大豆高频遗传转化

大豆(Glycine max(L.)Merrill)遗传转化目前常用的两种方法为农杆菌介导的子叶节转化系统和基因枪介导的体细胞胚转化,但这两种转化系统都存在转化频率低、难于重复及依赖于特定的基因型等问题.为了提高农杆菌介导的大豆子叶节的转化频率,采用了一种基于bar基因作为筛选标记基因的固体-液体筛选系统,与农杆菌共培养3d的大豆子叶节在MS添加2 mg/L 6-BA和5 mg/L的glufosinate的筛选培养基培养2周后,再转到含有0.01 mg/L TDZ和2mg/L glufosinate的液体培养基中筛选,并每周更换一次培养液.得到的再生芽首先经GUS分析为阳性后再转入生根培养基得到完整转化植株,然后通过Southern杂交分析证实外源基因整合到大豆基因组,转化植物含有1～2个基因拷贝数.该转化系统具有转化频率高、转化周期短以及不依赖于大豆基因型等优点,对影响该转化系统的一些因子进行了讨论.     

农杆菌介导的大豆高频遗传转化

大豆(Glycinemax(L.)Merrill)遗传转化目前常用的两种方法为农杆菌介导的子叶节转化系统和基因枪介导的体细胞胚转化,但这两种转化系统都存在转化频率低、难于重复及依赖于特定的基因型等问题。为了提高农杆菌介导的大豆子叶节的转化频率,采用了一种基于bar基因作为筛选标记基因的固体-液体筛选系统,与农杆菌共培养3d的大豆子叶节在MS添加2mg/L6-BA和5mg/L的glufosinate的筛选培养基培养2周后,再转到含有0.01mg/LTDZ和2mg/Lglufosinate的液体培养基中筛选,并每周更换一次培养液。得到的再生芽首先经GUS分析为阳性后再转入生根培养基得到完整转化植株,然后通过Southern杂交分析证实外源基因整合到大豆基因组,转化植物含有1~2个基因拷贝数。该转化系统具有转化频率高、转化周期短以及不依赖于大豆基因型等优点,对影响该转化系统的一些因子进行了讨论。     

液泡转化酶反义基因对番茄的遗传转化

以'中蔬4号'番茄的子叶为试材,通过农杆菌介导法遗传转化,将液泡转化酶反义基因导入再生植株,经PCR和Southern斑点杂交检测证明,5株转化植株基因组中整合有目的基因.遗传转化最佳条件为:在附加1.5 mg/L 6-BA和0.1 mg/L IAA的MS培养基上再生培养,外植体预培养2 d,菌液浓度OD600=0.5,侵染时间5 min,共培养2 d.遗传转化后,对整合有目的基因的再生番茄叶片液泡转化酶活性测定,表明液泡转化酶活性明显受到抑制.获得的转基因植株为进一步研究液泡转化酶基因的功能奠定了基础.     

影响农杆菌介导的大豆子叶节遗传转化的因素

利用携带pCAMBIA1301质粒（含hpt和gus基因）的超毒根癌农杆菌菌株EHA105对大豆子叶节外植体进行遗传转化,研究了影响农杆菌介导的大豆子叶节遗传转化的因素。研究结果表明．农杆菌侵染液和共培养培养基中添加200μmok/L乙酰丁香酮和50mg／L抗坏血酸可以有效促进农杆菌对大豆子叶节的转化。农杆菌与子叶节共培养后羧苄青霉素（250mr／L）和头孢霉素（100mg／L）结合使用能有效抑制农杆菌过度繁殖并提高转化芽诱导频率;在转化细胞的分化和转化芽伸长过程中,改进的筛选策略可以明显改善对转化芽的筛选效果,从而提高转化频率。应用优化后的转化体系．获得了3个国内大豆主栽品种的转基因植株,PCR阳性植株频率为3．8％～7．6％。转化植株叶片总DNA的PCR和Southern blot实验表明,T-DNA上的外源基因已经整合到大豆基因组中。     

改善培养条件以利于番茄的遗传转化

通过改善培养条件可以促进番茄的遗传转化。本实验从六个番茄杂交组合亲本中筛选出两个分化频率较高的品种,费洛雷德与402;它们子叶比下胚轴分化频率高些,共培养后子叶也比下胚轴易于培养与分化,因此宜选用子叶作为外植体;激素宜用 NAA 与 BA,且较合适的浓度分别为0.2mg/L,2mg/L;最好是从子叶基部处切下子叶,染菌时也只把基部的     

双价抗病基因导入籽瓜的遗传转化研究

以籽瓜主栽品种新籽瓜2号、五原籽瓜等为研究材料,用苗龄1-3d的子叶切块作外植体,不定芽诱导培养基为MS 6-BA2mg/L IBA 0.1mg/L,芽伸长培养基为MS KT0．2mg／L,诱导生根的培养基是MS IAA0．5mg／L。籽瓜遗传转化研究结果表明农杆菌感染时间以10min,共培养时间以2d为合适。共获得抗卡那霉素100mg／L的试管苗41个,PCR检测呈阳性的有15株,转化率为34．9％。该研究初步建立了籽瓜的遗传转化体系和转化体的检测体系。     

农杆菌介导的紫茎泽兰遗传转化研究

以紫茎泽兰为受体材料,GUS基因为报告基因,对农杆菌介导的遗传转化条件及影响因素进行研究,建立了农杆菌介导的紫茎泽兰遗传转化体系。结果表明,将未经过预培养的幼叶外植体在OD600为0.4的稀释菌液中浸泡12min,共培养3d后,转移到加有卡那霉素50mg/L(筛选压)和羧苄青霉素250mg/L的分化培养基上,经过20d外植体直接分化出不定芽,诱导生根成苗。经PCR和组织化学染色鉴定,可稳定获得较高的阳性转化率。     

中国传统菊花品种‘小林静’再生及转化体系的建立

以中国传统菊花品种‘小林静’叶片为外植体,建立了‘小林静’较好的再生体系及遗传转化体系.结果表明,‘小林静’叶盘最适不定芽分化培养基为MS+2.0 mg/L 6-BA+1.5 mg/L NAA,不定芽分化率为92.76％,平均再生不定芽数为2.3767个;试管苗最佳生根培养基为1/2MS,生根率达100％.移栽采用灭菌的蛭石,再生苗移栽成活率达90％以上.采用根癌农杆菌C58C1介导的叶盘转化法进行‘小林静’的遗传转化试验,农杆菌OD600=0.5-0.6,侵染10 min后,将叶片外植体接种到MS+2.0 mg/L 6-BA+1.5mg/L NAA的培养基中黑暗共培养2d,之后转接到附加10 mg/L硫酸卡那霉素和400 mg/L羧苄青霉素的分化筛选培养基中进行转化细胞的筛选,待长出抗性芽后转接至生根培养基中进行培养,最终建立了菊花品种‘小林静’的遗传转化体系.     

不同抗生素对雪莲愈伤组织生长的影响

以雪莲叶片为外植体,探讨了卡那霉素(kanamycin,Kan)、潮霉素(hygromycin B,Hyg)、羧苄青霉素(car-benidillin,Car)3种抗生素对雪莲愈伤组织诱导、生长及分化的影响,以确定农杆菌介导的遗传转化研究中筛选剂和抑菌剂的最适浓度。结果表明:40mg/L的卡那霉素已抑制雪莲愈伤组织生长,当卡那霉素为50mg/L时,愈伤组织的生长基本停止;8.0mg/L潮霉素能够有效抑制雪莲愈伤组织的生长,当潮霉素为20mg/L时则生长的愈伤组织块较小、褐化、甚至死亡。同时,低浓度(0.5～2.0mg/L)的潮霉素可以提高雪莲愈伤组织的分化率;作为农杆菌抑菌剂,不同浓度羧苄青霉素对雪莲愈伤组织生长的影响差异极显著,当羧苄青霉素的浓度超过400mg/L时对雪莲愈伤组织的出愈及生长均有明显的抑制作用。     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of niger [<Emphasis Type="Italic">Guizotia abyssinica</Emphasis> (L. f.) Cass.] using seedling explants

A protocol was developed for Agrobacterium-mediated genetic transformation of niger [ Guizotia abyssinica (L.f.) Cass.] using hypocotyl and cotyledon explants. Hypocotyls and cotyledons obtained from 7-day-old seedlings were co-cultivated with Agrobacterium tumefaciens strain EHA101/pIG121Hm that harbored genes for beta-glucuronidase (GUS), kanamycin, and hygromycin resistance. Following co-cultivation, the hypocotyl and cotyledon explants were cultivated on MS medium containing 1 mg/l 6-benzylaminopurine (BA) for 3 days in darkness. Subsequently, hypocotyl and cotyledon explants were transferred to selective MS medium containing 1 mg/l BA, 10 mg/l hygromycin, 10 mg/l kanamycin, and 500 mg/l cefotaxime. After 6 weeks, hypocotyls and cotyledons produced multiple adventitious shoot buds, and these explants were subcultured to MS medium containing 1 mg/l BA, 30 mg/l hygromycin, and 30 mg/l kanamycin. After a further 3 weeks, the explants (along with developing shoot buds) were subcultured to MS medium containing 1 mg/l BA, 50 mg/l kanamycin, and 50 mg/l hygromycin for further selection. Transgenic plants were obtained after rooting on half-strength MS medium supplemented with 0.1 mg/l alpha-naphthaleneacetic acid, 50 mg/l kanamycin, and 50 mg/l hygromycin and were confirmed by GUS histochemical assay and polymerase chain reaction analysis. Genomic Southern blot hybridization confirmed the incorporation of the neomycin phosphotransferase II gene into the host genome.     

Effects of hygromycin on cotton cultures and its application in <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated cotton transformation

We have evaluated the effects of the antibiotic hygromycin B on cotton (Gossypium hirsutum L.) callus induction, callus proliferation, and seed germination. Nontransgenic cotyledon and hypocotyl showed obvious variance in tolerance to hygromycin. Cotyledons were more sensitive to hygromycin than hypocotyls. Hygromycin at 7.5 and 20 mg l⁻¹ completely inhibited callus initiation from cotyledon and hypocotyl explants, respectively. Nontransformed calli did not grow on media supplemented with 10 mg l⁻¹ hygromycin and were killed at 15 mg l⁻¹. In seed germination assay, the presence of 20 mg l⁻¹ hygromycin significantly suppressed shoot and root elongation of seedlings. This hygromycin concentration was applied to select regenerated transgenic plantlets and their progenies. Based on these results, we developed an efficient hygromycin selection protocol for Agrobacterium-mediated cotton transformation and regeneration.     

生菜遗传转化体系的建立及转基因研究

以散叶生菜大速生(Lactuca sativavar.capatataL.)为试材,以MS为基本培养基,采用不同激素配比,确定生菜高效诱芽培养基为MS 1.5mg/L6-BA 0.2mg/LIAA;抗生素敏感性试验表明,筛选培养基中适宜的潮霉素选择压为20mg/L,抑菌剂羧苄青霉素的适宜质量浓度为300mg/L;通过根癌农杆菌介导的叶盘法将携带O型和A型口蹄疫抗原决定簇融合基因O21-O14-A21-HBcAg转入大速生散叶生菜,对部分抗性植株进行PCR和PCR-Southern杂交检测,证实目的基因已经成功整合到生菜基因组中。RT-PCR检测初步表明,O21-O14-A21-HBcAg基因可以在生菜中正常转录表达。     

Enhancing the frequency of somatic embryogenesis following <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of immature cotyledons of soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merrill.]

Summary A characteristic phenotype of highly embryogenic explants along with the location of embryogenesis- and transformation-competent cells/tissues on immature cotyledons of soybean [Glycine max (L.) Merrill.] under hygromycin selection was identified. This highly embryogenic immature cotyledon was characterized with emergence of somatic embryos and incidence of browning/necrotic tissues along the margins and collapsed tissues in the mid-region of an explant incubated upwards on the selection medium. The influences of various parameters on induction of somatic embryogenesis on immature cotyledons following Agrobacterium tumefaciens-mediated transformation and selection were investigated. Using cotyledon explants derived from immature embryos of 5–8 mm in length, a 1∶1 (v/v; bacterial cells to liquid D40 medium) concentration of bacterial suspension and 4-wk cocultivation period significantly increased the frequency of transgenic somatic embryos. Whereas, increasing the infection period of explants or subjecting explants to either wounding or acetosyringone treatments did not increase the frequency of transformation. An optimal selection regime was identified when inoculated immature cotyledons were incubated on either 10 or 25 mgl⁻¹ hygromycin for a 2-wk period, and then maintained on selection media containing 25 mgl⁻¹ hygromycin in subsequent selection periods. However, somatic embryogenesis was completely inhibited when inoculated immature cotyledons were incubated on a kanamycin selection medium. These findings clearly demonstrated that the tissue culture protocols for transformation of soybean should be established under both Agrobacterium and selection conditions.     

Establishment of Genetic Transformation System and Transgenic Studies in Lettuce ( Lactuca sativa var. capatata)

In this experiment , the effects of different hormone concentration on shoot regeneration from cotyledon explants are investigated . The results show that MS medium supplied with 1.5 mg/L 6-BA and 0.2 mg/L IAA is the suitable shoot regeneration. The experiment on sensitivity of cotyledon to hygromycin B and carbenicillin shows that the suitable hygromycin concentration for selecting transgenic tissue is 20 mg/L and the carbenicillin concentration is 300 mg/L. Fused gene O₂₁ - O₁₄ - A₂₁ containing both type O and A FMDV is transferred into lettuce mediated by Ti plasmid of Agrobacterium tumafaciens. The results of PCR and PCR-Southern blotting indicated that the O₂₁ - O₁₄ - A₂₁ gene were successfully integrated into the genomes of some transformed lettuce plants. Expression of O₂₁ - O₁₄ - A₂₁gene in vtransformed lettuce plants was proved by RT-PCR analyses .     

影响农杆菌介导的麻疯树基因转化因素的研究(简报)

麻疯树(Jatropha curcas)为大戟科麻疯树属植物,是一种多年生木本油料植物。原产美洲,广泛分布于热带亚热带地区。因其种子含油量高达 60％,可作生物燃料之用,麻疯树是目前正在被开发利用的重要能源植物之一。除此之外,麻疯树的种子含有多种活性成分,有着重要的农药和医药价值;其生长能耐干旱贫瘠,可用于荒山造林。因此,麻疯树是一种具有多种用途、重要经济价值和学术研究价值的植物。目前对其研究多停留在植物组织培养、植物化学、毒理学、种植业方面等。     

Optimization of factors for efficient recovery of transgenic peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

De-embryonated cotyledon explants of peanut were co-cultivated under different conditions with Agrobacterium tumefaciens harbouring pIG121hm plasmid carrying intron-containing β-glucuronidase as a reporter while hygromycin phosphotransferase and neomycin phosphotransferase as selectable marker genes. Co-cultivation duration and temperature, various antioxidants and their concentrations, bacterial strains and explant characteristics (incised and non-incised) were examined either alone or in combinations for optimization of transient expression of the reporter gene. Up to 81% transformation was recorded when non-incised explants were co-cultivated with strain EHA101 for 5 days at 21°C on shoot induction medium containing 100 mg/L l-cysteine. Addition of the optimized concentration of augmentin (200 mg/L) along with cefotaxime (200 mg/L) to the shoot induction medium not only effectively eliminated bacterial growth, but also facilitated high frequency of shoot induction. The 40 mg/L hygromycin concentration prevented complete shoot regeneration of non-transgenic explants thus considered for the regeneration of transgenics. Resistant shoots were successfully transferred to soil either by grafting or in vitro rooting. Survival rate of the grafted shoots was nearly 100% in glass-house conditions. The optimized protocol took around 3 months to generate healthy plants. Polymerase chain reaction, Southern blot hybridization, histochemical tests, segregation and hygromycin-leaf assays of selected transgenic plants showed integration of the transgene into peanut genome. No chimeras were noticed during the study.     

口蹄疫抗原决定簇融合基因转化生菜及表达

以3-5天苗龄的散叶大速生生菜(Lactuca sativa var. capatata)无菌苗子叶为外植体, 通过根癌农杆菌介导, 将携带O型和A型口蹄疫抗原决定簇融合基因O21- O14 -A21-HBcAg导入生菜。研究结果表明, 含有20 mg.L-1潮霉素(Hyg)的S2 培养基(MS+1.5 mg.L-1 6-BA+0.2 mg.L-1 IAA+20 mg.L-1 Hyg +300 mg.L-1 Cb)为子叶外植体转化后诱导愈伤和芽再生的最适培养基, 经抗性筛选, 将抗性芽切下于S3 培养基(1/2MS+20 mg.L-1 Hyg)上诱导生根。通过PCR 和outhern杂交分析证明, 基因已经整合到生菜基因组中。RT-PCR检测初步表明, O21-O14 -A21-HBcAg基因可以在生菜中正常转录。