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-1IAA 20 mg·L-1Hyg 300 mg·L-1Cb)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-1Hyg)上诱导生根.通过PCR和Southern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.     

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。     

口蹄疫抗原决定簇融合基因转化生菜的研究

植物疫苗具有生产简单、成本较低、使用安全方便、易贮存等优点,是目前植物基因工程的一个研究热点。本实验通过农杆菌介导的遗传转化,以O型和A型口蹄疫抗原决定簇融合基因O21-O14-A21-HBcAg转化生菜。通过筛选,获得潮霉素抗性愈伤组织,经胚状体再生得到56棵抗性苗。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。     

盾叶薯蓣花药培养及单倍体植株的获得

以处于单核期的盾叶薯蓣花药为材料,经愈伤组织途径成功获得单倍体盾叶薯蓣新材料。对盾叶薯蓣花药培养、植株再生过程的研究表明:来自不同居群的外植体对花药愈伤组织诱导有显著影响;W14是适合花药愈伤组织诱导的基本培养基;花药愈伤组织增殖和分化的适宜培养基为:MS基本培养基 BA2.0mg/L IAA0.2mg/L;生根培养基为添加了IBA2.0mg/L、NAA0.4mg/L和0.5g/L活性碳的MS培养基。以流式细胞仪和根尖染色体压片法检查花培植株的结果表明,有8%～12%的个体为单倍体。实验结果为进一步开展单倍体育种及相关理论研究提供了材料和技术基础。     

根癌农杆菌介导蓝猪耳转化的影响因素研究

研究了根癌农杆菌介导蓝猪耳转化的影响因子。结果表明,以蓝色花类型蓝猪耳5~6周的叶片为外植体,在OD₆₀₀为0.5~0.6的活化菌液中浸染5~10min,暗培养4d后,在愈伤组织诱导培养基(MS+BA 1.0mg/L+2,4-D 0.1mg/L)上生长14d,获得抗性愈伤组织;经芽诱导培养基(1/2MS+BA 1.0mg/L+NAA 0.1mg/L)培养28d得到抗性芽;生根培养基(1/2MS)上培养14d得到抗性植株。经PCR检测证实外源基因已整合到蓝猪耳基因组中,转化率达13%~14%。Cef和Hyg浓度对转化影响较大,转化的不同阶段其适宜浓度不同。     

狭叶薰衣草的离体培养及挥发性成分的分析

为建立新疆狭叶薰衣草(Lavandula angustifolia)的快速繁殖体系,以种子、茎、叶为外植体,对种子萌发、愈伤组织诱导、丛芽分化和生根的最适培养条件进行了研究;用水蒸气蒸馏法提取狭叶薰衣草挥发油,采用气相色谱-质谱法测定挥发油成分。结果表明,种子浸泡的适宜时间为6 h,切开种皮培养,出芽时间最少为6 d;诱导种子出芽的适宜培养基为MS+6-BA2 mg/L;以茎为外植体诱导愈伤组织效果较好,适宜培养基为MS+6-BA 2 mg/L+2,4-D 1 mg/L;诱导分化丛芽的适宜培养基为MS+6-BA 1 mg/L+NAA 0.5 mg/L;生根的适宜培养基为1/2MS+NAA 1 mg/L+6-BA 0.5 mg/L;盆栽薰衣草和无菌苗薰衣草的挥发油主要成分相差较大,离体培养的薰衣草的主要挥发性成分有叶绿醇、丁香油烃、氧化石竹烯等。     

蔓花生组织培养和植株再生的条件优化

应用MS为基本培养基,通过各种培养条件和不同激素配比,探讨蔓花生组织培养及其植株再生条件的优化。结果显示:幼叶为最佳的外植体,在幼叶愈伤诱导过程中,不超过12h光照,光照强度在21.6μmol.m-2.s-1均可;诱导愈伤组织的适宜培养基为MS+0.5mg/L6-BA+0.2mg/L2,4-D或MS+0.5mg/L6-BA+2mg/LNAA;最适的分化培养基为MS+1mg/LTDZ+2mg/L6-BA+0.5mg/LNAA;最适的生根培养基1/2MS+1mg/LNAA+1mg/LPP333。     

欧李“大磨盘”丛生芽的诱导及生根技术

以欧李"大磨盘"的茎尖为外植体,1/2MS和MS为基本培养基,研究了不同生长激素对启动培养的影响,不同培养基对丛生芽增殖的影响,初步建立了欧李"大磨盘"丛生芽的诱导及生根技术。适宜"大磨盘"的启动培养的生长素为IBA,其最佳丛生芽的培养基为MS+6-BA1.0mg/L+NAA0.01mg/L+IBA0.2mg/L,最佳分化培养基是MS+6-BA0.5mg/L+NAA0.01mg/L+IBA0.2mg/L,最佳生根培养基是1/2MS+IBA0.5mg/L+NAA0.1mg/L。     

意大利生菜转化体系建立及EV71抗原基因表达初探

以意大利生菜(Lactuca sativa L.var.ramosa Hort.)为试材,农杆菌LBA4404叶盘法转化子叶,通过植物组织培养技术,筛选获得再生体系最佳选择压为Kan 50 mg/L,最佳抑菌剂为Carb 400 mg/L,生菜子叶抗性愈伤组织诱导的最佳植物生长调节剂条件为6-BA 0.4 mg/L+NAA 0.15 mg/L或6-BA 0.3 mg/L+NAA 0.2 mg/L,其中在6-BA 0.3 mg/L+NAA 0.2 mg/L的培养基上诱生的抗性愈伤组织后续生芽效果较好。根据GenBank序列,优化设计合成适合植物表达的肠道病毒71型(Human enterovirus 71,EV71)C4亚型P1和3CD基因,运用基因重组技术,同时克隆入植物表达载体pCAMBIA2301,构建共表达植物表达载体pCAMBIA2301-P1-3CD。以农杆菌介导的叶盘法转化意大利生菜子叶,对叶片PCR阳性的抗性株进行蛋白质表达检测,Western-blot实验表明,两株抗性株叶片中表达出具有生物学活性的EV71抗原蛋白质,证明了利用生菜表达EV71抗原的可行性,为利用植物生物反应器生产抗EV71口服疫苗提供了理论和实验依据。     

三倍体毛白杨组织脱分化培养与植株体再生

采用毛白杨三倍体幼叶作为外植体进行组织培养,以MS为基本培养基获得了再生植株。从NAA和IAA与6-BA间进行的18个正交试验中,选择出适宜脱分化培养基MS+6-BA 0.5mg/L+NAA 0.1mg/L,对三倍体毛白杨愈伤组织诱导率为87.6%。5种生长素与6-BA配比的再分化培养基中,12MS+IAA 0.1mg/L+6-BA 0.1mg/L对不定芽的分化诱导率可达到68.5%;MS+6-BA 0.5mg/L+NAA 0.01mg/L的培养基可使单芽直接增殖出7.4个芽。在MS+IBA 1.0mg/L的生根培养基上,试管苗的生根率可达85.7%。     

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 .     

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.     

Development of anAgrobacterium-mediated transformation system for regenerating garland chrysanthemum (Chrysanthemum coronarium L.)

Although efficient shoot regeneration and selection are essential for genetic transformation mediated byAgrobacterium, success has been limited with the garland chrysanthemum (Chrysanthemum coronarium L.). In this study, we developed a useful protocol for shoot regeneration with leaf disk explants. The optimal concentrations of NAA and BA were 0.2 mg L⁻¹ and 0.5 mg L⁻¹, respectively. To optimize the selection system for regenerating plants from genetically transformed tissues, we tested the effects of four antibiotics (kanamycin, hygromycin, carbenicillin, and cefotaxime). Among them, 5 mg L^-1 hygromycin proved adequate as a selectable marker, whereas 500 mg L^-1 carbenicillin was effective in eliminating excessiveAgrobacterium after co-cultivation. Transgenic plants were obtained by first co-culturing garland chrysanthemum leaf disks withA. tumefaciens strain EHA105, which harbors plasmid pRCVII containing the hygromycin resistance (hpt) and β-glucuronidase (GUS) genes. After the transgenic plants were confirmed via Southern analysis, they were rooted in soil and appeared phenotypically normal. Our report is the first to describe the optimum conditions for producing transgenic plants of this species.     

<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.     

Green fluorescent protein as a visual selection marker for coffee transformation

The green fluorescent protein (GFP) was used as a visual selectable marker to produce transgenic coffee (Coffea canephora) plants following Agrobacterium-mediated transformation. The binary vector pBECKS 2000.7 containing synthetic gene for GFP (sgfp) S65T and the hygromycin phosphotransferase gene hph both controlled by 35S cauliflower mosaic virus CaMV35S promoters was used for transformation. Embryogenic cultures were initiated from hypocotyls and cotyledon leaves of in vitro grown seedlings and used as target material. Selection of transformed tissue was carried out using GFP visual selection as the sole screen or in combination with a low level of antibiotics (hygromycin 10 mg/L), and the efficiency was compared with antibiotics selection alone (hygromycin 30 mg/L). GFP selection reduced the time for transformed somatic embryos formation from 18 weeks on a hygromycin (30 mg/L) antibiotics containing medium to 8 weeks. Moreover, visual selection of GFP combined with low level of antibiotics selection improved the transformation efficiency and increased the number of transformed coffee plants compared to selection in the presence of antibiotics. Molecular analysis confirmed the presence of the sgfp-S65T coding region in the regenerated plants. Visual screening of transformed cells using GFP by Agrobacterium-mediated transformation techniques was found to be efficient and therefore has the potential for development of selectable marker-free transgenic coffee plants.     

Regeneration of transgenic plants from two indica rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) cultivars using shoot apex explants

We have established a reproducible procedure for transformation of shoot apices and regeneration of transgenic plants for two indica rice cultivars, white ponni (WP) and Pusa Basmathi 1 (PB 1). Four-day-old shoot apex explants were transformed by cocultivation with Agrobacterium tumefaciens strain EHA 101 harbouring a binary plasmid pRIT1. The vector contained an improved hygromycin phosphotransferase (hpt) gene for hygromycin resistance driven by actin 1 promoter and the reporter gene beta-glucuronidase intron (INT-GUS) controlled by CaMV 35S promoter. Rice shoots were induced on media containing 0.1 mg/l napthalene acetic acid (NAA), 1.0 mg/l kinetin (kn), 1.0 mg/l N(6)-benzyleaminopurin (BAP), 300 mg/l casaminoacid, 500 mg/l proline, 50 mg/l hygromycin and 500 mg/l cefotaxime. Transgenic plants were raised in pots and seeds were collected. Histochemical and polymerase chain reaction (PCR) analyses of field established transgenic rice plants and their offsprings confirmed the presence of GUS gene. Integration of T-DNA into the genome of putative transgenics was further confirmed by southern analysis. The transformation efficiency of WP was found to be ranging from 5.6 to 6.2% whereas in the case of PB1, it was from 7 to 8%. Progeny analysis of these plants showed a pattern of classical Mendelian inheritance for both hpt and GUS gene.     

Thansgenic peanut plants obtained by particle bombardment via somatic embryogenesis regeneration system

INTRODUCTIONArachiS hypogaea L., Peanut or groundnut, isan importal commercial crop worldwide. It provides an excellellt source of protein and other nutrients. Its production and quality can be severelyimpacted under stressful growing conditions such ascdriate factors, pests and diseajses. Genetic engineering provides a prospective way to reduce certainproblems by transferring individual genes for pestresistance or other traits into elite germplasm of acultiVated species. Thansgenic pea…