Effects of antibiotics on the elimination of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> from loblolly pine (<Emphasis Type="Italic">Pinus taeda</Emphasis>) zygotic embryo explants and on transgenic plant regeneration

Three antibiotics were evaluated for their effects on the elimination of Agrobacterium tumefaciens during the genetic transformation of loblolly pine ( Pinus taeda L.) using mature zygotic embryos as targets. Agrobacterium tumefaciens strains, EHA105, GV3101, and LBA 4404, all harbouring the plasmid pCAMBIA1301, which carries the selectable marker gene, hygromycin phosphotransferase ( hpt) controlled by the cauliflower mosaic virus 35S promoter and terminator, and the uidA reporter gene (GUS) driven by the cauliflower mosaic virus 35S promoter and the terminator of nopaline synthase gene, were used in this study. Exposure to 350 mg l^-1 carbenicillin, claforan, and timentin respectively for up to 6 weeks did not eliminate the Agrobacterium, while antibiotics at 500 mg l^-1 eradicated them from the co-cultivated zygotic embryos. All three antibiotics increased callus growth and shoot regeneration at 350 and 500 mg l^-1 each, but reduced callus growth and shoot regeneration at 650 mg l^-1 when compared with controls. Putative transgenic calli were selected for continued proliferation and differentiation on 4.5 mg l^-1 hygromycin-containing medium. Transformed calli and transgenic plants produced on a selection medium containing 4.5 mg l^-1 hygromycin were confirmed by GUS histochemical assays, by polymerase chain reaction (PCR), and by Southern blot analysis. These results are useful for future studies on optimizing genetic transformation procedures in loblolly pine.     

植物激素和抗生素调节的转基因火炬松愈伤组织的生长和分化

包含质粒载体pBI121的农杆菌菌株LBA4404被用于转化火炬松的成熟合子胚。质粒载体pBI121含有胭脂碱合成酶基因的启动子驱动的新霉素磷酸转移酶基因（npt Ⅱ)和花椰菜花叶病毒的35S启动子驱动的GUS基因。器官发生的转基因愈伤组织和转基因的再生植株已经获得,并经GUS组织化学染色、聚合酶链式反应和Southern杂交分析证实。植物激素（BA/IBA）和抗生素对转基因愈伤组织的生长和分化的影响被研究。500mg/L羧苄青霉素和2mg/LBA、0.5mg/L IBA的组合导致转基因愈伤组织的生长增加54.2%,分化增加45.7%。500mg/L Claforan和2mg/L BA、0.5mg/L IBA的组合导致转基因愈伤组织的生长增加40.8%,分化增加38.7%。高浓度的植物激素和抗生素降低了转基因愈伤组织的生长和分化。实验结果有助于建立一个高效的农杆菌介导的火炬松遗传转化系统,也有助于未来针叶树的遗传转化研究。     

转苏云金杆菌毒蛋白基因玉米植株的获得及其抗虫性分析

玉米( Zea mays L.)转化成功与否与基因型密切相关.在转化过程中,除少数模式品种能够形成再生频率较高且易转化的Ⅱ型愈伤组织外,大多数栽培品种往往只能够形成再生频率较低且不易转化的Ⅰ型愈伤组织.因此探索Ⅰ型愈伤组织的诱导及其转化条件,提高转化效率,对直接改良玉米优良自交系具有重要意义.应用基因枪转化技术将苏云金杆菌( Bacillus thuringiensis ) cry1Ac3基因导入玉米优良自交系E28及340的Ⅰ型胚性愈伤组织中,经过膦丝菌素(PPT)或潮霉素(HygB)筛选,获得了再生植株.经PCR检测、Southern blot分析及Bt毒蛋白ELISA检测证实,外源基因已整合到玉米基因组中,并已获得表达.抗虫性分析结果表明,部分转基因玉米植株对玉米螟虫有较强的抗性.还比较了PPT和HygB两种筛选剂的筛选效果,表明PPT筛选的抗性愈伤组织的再生频率要高于HygB筛选的再生频率.     

Genetic transformation, recovery, and characterization of fertile soybean transgenic for a synthetic Bacillus thuringiensis cryIAc gene.

Somatic embryos of jack, a Glycine max (L.) Merrill cultivar, were transformed using microprojectile bombardment with a synthetic Bacillus thuringiensis insecticidal crystal protein gene (Bt cryIAc) driven by the 35S promoter and linked to the HPH gene. Approximately 10 g of tissue was bombarded, and three transgenic lines were selected on hygromycin-containing media and converted into plants. The recovered lines contained the HPH gene, but the Bt gene was lost in one line. The plasmid was rearranged in the second line, and the third line had two copies, one of which was rear-ranged. The CryIAc protein accumulated up to 46 ng mg-1 extractable protein. In detached-leaf bioassays, plants with an intact copy of the Bt gene, and to a lesser extent those with the rearranged copy, were protected from damage from corn earworm (Helicoverpa zea), soybean looper (Pseudoplusia includens), tobacco budworm (Heliothis virescens), and velvetbean caterpillar (Anticarsia gemmatalis). Corn earworm produced less than 3% defoliation on transgenic plants, compared with 20% on the lepidopteran-resistant breeding line GatIR81-296, and more than 40% on susceptible cultivars. Unlike previous reports of soybean transformation using this technique, all plants were fertile. To our knowledge, this is the first report of a soybean transgenic for a highly expressed insecticidal gene.     

植物抗毒素转化水稻和转基因植株的生物鉴定

用基因枪法转化了水稻（ＯｒｙｚａｓａｔｉｖａＬ．）６个材料的未成熟胚、成熟胚及胚性愈伤组织。质粒ｐＳＳＶｓｔ１和ｐＶＥ５＋是由葡萄中分离出的编码芪类合成酶的植物抗毒素基因与３５Ｓ或它自己的启动子组成。Ｇ４１８（１００～１５０ｍｇ／Ｌ）或潮霉素（５０ｍｇ／Ｌ）筛选后,经ＰＣＲ、Ｓｏｕｔｈｅｒｎｂｌｏｔ或Ｄｏｔｂｌｏｔ分析证明的转基因植株共５４株。对转基因植株及其后代进行了稻瘟病和白叶枯病的抗性鉴定。初步结果表明,芪类合成酶基因可以提高转基因植株及后代的抗性。     

Biolistic transformation of highly regenerative sugar beet (Beta vulgaris L.) leaves

Leaves of greenhouse-grown sugar beet (Beta vulgaris L.) plants that were first screened for high regeneration potential were transformed via particle bombardment with the uidA gene fused to the osmotin or proteinase inhibitor II gene promoter. Stably transformed calli were recovered as early as 7 weeks after bombardment and GUS-positive shoots regenerated 3 months after bombardment. The efficiency of transformation ranged from 0.9% to 3.7%, and stable integration of the uidA gene into the genome was confirmed by Southern blot analysis. The main advantages of direct bombardment of leaves to regenerate transformed sugar beet include (1) a readily available source of highly regenerative target tissue, (2) minimal tissue culture manipulation before and after bombardment, and (3) the overall rapid regeneration of transgenic shoots.     

Transgenic Tobacco Plants with a Fully Synthesized GFM CryIA Gene Provide Effective Tobacco Bollworm (Heliothis armigera) Control

GFM CrylA gene is a fully modified synthetic gene derived from insecticidal crystal prorein gene of Bacillus thuringiensis Berliner (Bt). It was synthesized based on the codon usage of plant genes instead of changing the primary sequences of amino acids of insecticidal crystal protein (ICP) gene of Bacillus thuringiensis Htibner. To test the function of the synthetic GFM CrylA gene, we introduced the GFM CrylA gene into tobacco plant cells via an Agrobacterium tumefacieus (Smith et Townsedn) Conn binary vector system. As expected, the GFM CrylA gene is expressed under control of the cauliflower mosaic virus (CaMV) 35S promoter and allows efficient production of lepidopteran insectspecific toxic proteins in the transformed tobacco plants. Bioassays using transgenic tobacco plants with tobacco bollworm showed that the transgenic tobacco plants expressing proteins of GFM CrylA gene had effective control to tobacco bollworm. In this paper the authors firstly report the complete synthesis of GFM CryIA gene and the construction of plant expression vector pGBI4AB. The authors performed introduction of the synthetic GFM CrylA gene into the tobacco plants, and the integration of GFM CrylA gene into tobacco genome was confirmed by Southern blot analysis of the tobacco genomic DNA. The gene was efficiently expressed in the transgenic tobacco plants and effective tobacco bollworm control was verified by the insect-bioassays.     

Regeneration of transgenic loblolly pine (Pinus taeda L.) from zygotic embryos transformed with Agrobacterium tumefaciens

Embryos of 24 open-pollinated families of loblolly pine (Pinus teade L.) were used as explants to conduct in vitro regeneration. Then, Agrobacterium tumefaciens strain GV3101 harboring the plasmid pPCV6NFHygGUSINT was used to transform mature zygotic embryos of seven families of loblolly pine. The frequency of transformation varied among families infected with A. tumefaciens. The highest frequency (100%) of transient beta-glucuronidase (GUS)-expressing embryos was obtained from family 11-1029 with over 300 blue spots per embryo. Expression of the GUS reporter gene was observed in cotyledons, hypocotyls, and radicles of co-cultivated mature zygotic embryos, as well as in callus and shoots derived from co-cultivated mature zygotic embryos. Ninety transgenic plants were regenerated from hygromycin-resistant callus derived from families W03. 8-1082 and 11-1029. and 19 transgenic plantlets were established in soil. The presence of the GUS gene in the plant genome was confirmed by polymerase chain reaction. Southern blot, and plant DNA/T-DNA junction analysis. These results suggest that an efficient A. tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for future studies on transferring economically important genes to loblolly pine.     

Agrobacterium tumefaciens-mediated transformation of cauliflower,Brassica oleracea var. botrytis

We have developed an efficient and simpler method for genetic transformation and regeneration of cauliflower, Brassica oleracea var. botrytis plants. Explants from 4-day old seedlings were inoculated and cocultivated with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector with the neomycin phosphotransferase-II gene under the regulatory control of nopaline synthase promoter and terminator sequences, permitting transformed shoots to be selected on kanamycin containing medium. After three months rooted transformed plantlets were successfully transferred and grown under glasshouse conditions. Higher numbers of transformed plants were obtained from cotyledon than hypocotyl explants, presumably indicating cotyledons of cauliflower are more amenable to genetic transformation. Integration and expression of the introduced transgene were analysed by DNA gel blot and PCR analysis and NPT-II expression assay. Factors influencing transformation efficiency include explant age, concentration of bacterium used for infection, duration of infection and cocultivation with Agrobacterium. Transgenic plants of three commercial genotypes of cauliflower were produced using this method. We also show that introduction of antisense Bcp1 (pollen-specific gene) linked to a pollen-specific promoter (Lat52) resulted in the expected sterility of 50% pollen carrying this transgenic construct.     

转双抗虫基因烟草的研究

用改造的雪花莲凝集素基因GNAmm与合成的苏云金芽孢杆菌(Bt)毒蛋白cry1Ac基因构建了带有双价基因的植物表达载体,在该表达载体中这两个基因的转录分别受笋瓜PP2启动子(SPP2P)和CaMV 35S启动子的调控。通过根癌土壤杆菌介导转化法,获得了一批抗卡那霉素的转化再生烟草植株。PCR检测及基因组DNA Southern blot\,Slot blot杂交分析的结果表明Gna基因和Bt基因已整合到烟草总DNA中。用Bt毒蛋白抗血清进行Western blot分析,转基因植株均有Bt杀虫蛋白的不同程度的表达。对转化再生烟草的虫试结果表明,在所受试的19株烟草中60%的植株上的棉铃虫在5天内死亡率达到100%,而且存活幼虫的生长发育受到明显抑制;蚜虫抑制生长试验表明,多数转化再生植株具有较强的抗蚜活性,平均能够抑制桃蚜50%～60%的蚜口密度,有的高达80%以上。以上结果表明利用这两个改造过的抗虫基因可以获得既抗虫又耐蚜的转双抗虫转基因植物。     

Rice Transformation with a Phytoalexin Gene and Bioassay of the Transgenic Plants

Immature embryos, mature embryos and embryogenie ealli of 6 rice ( Oryza sativa L. ) materials were transformed with particle bombardment. The plasmids pSSVsfl and pVE5 + were used, both containing the phytoalexin gene from grapevine coding for stilbene synthase, but driven by 35S and its own promoter respectively. Through resistance selection for G418 ( 100 to 150 mg/L) or hygromycin (50 mg/L), 54 independent transgenic plants were isolated and further assessed by PCR, Southern blot and Dot blot analyses. The transgenic plants and their progenies were tested for resistance to blast ( Pyricularia oryzae) and bacterial blight of rice ( Xanthomonas oryzae). Preliminary results indicated that the stilbene synthase gene could enhance the resistance of transgenic plants and their progenies to both pathogens.     

Insect Control and Dosage Effects in Transgenic Canola Containing a Synthetic Bacillus thuringiensis cryIAc Gene

Zygotic hypocotyls of canola (Brassica napus L.) cv Oscar, cv Westar, and the breeding line UGA188-20B were transformed with a truncated synthetic Bacillus thuringiensis insecticidal crystal protein gene (Bt cryIAc) under the control of the cauliflower mosaic virus 35S promoter using Agrobacterium tumefaciens-mediated transformation. Fifty-seven independently transformed lines were produced, containing 1 to 12 copies of the transgenes. A range of cry expressors was produced from 0 to 0.4% Cry as a percentage of total extractable protein. The Brassica specialists, the diamondback month (Plutella xylostella L.) and the cabbage looper (Trichoplusia ni Hubner), were completely controlled by low-, medium-, and high-expressing lines. Whereas control of the generalist lepidopteran, the corn earworm (Helicoverpa zea Boddie), was nearly complete, the other generalist caterpillar tested, the beet armyworm (Spodoptera exigua Hubner), showed a dose response that had a negative association between defoliation and cry expression. These plants were produced as models for an ecological research assessment of the risk involved in the field release of naturalized transgenic plants harboring a gene (Bt) that confers higher relative fitness under herbivore-feeding pressure.     

Expression of a Bacillus thuringiensis cryIA(c) gene in transgenic peanut plants and its efficacy against lesser cornstalk borer

The invasion of peanut (Arachis hypogaea L.) pods and seeds by aflatoxin-forming species of Aspergillus is linked to injury by the lesser cornstalk borer and frequently causes a severe reduction in crop quality. The lesser cornstalk borer is susceptible to the lepidopteran-active Bacillus thuringiensis insecticidal crystal protein. We have introduced a codon-modified Bacillus thuringiensis cryIA(c) gene into peanut using microprojectile bombardment. The toxin-coding region of a Bt cryIA(c) gene was reconstructed for expression in plants and the resulting 3.4 kb gene cassette (promoter: 1.8 kb coding: 3) was directly cloned into the BglII site of plant transformation vectors. The vectors contained the hph gene, conferring resistance to the antibiotic hygromycin. Somatic embryos initiated from immature peanut cotyledons of two cultivars were used as the target for bombardment. DNA from hygromycin-resistant embryogenic cell lines, regenerated plants, and a progeny plant showed the presence and integration of hph and Bt genes by PCR and/or Southern blot analyses. ELISA immunoassay of the CryIA(c) protein from the hygromycin-selected plants showed the expression of CryIA(c) protein up to 0.18% of total soluble protein. Insect feeding bioassay of transformed plants indicated various levels of resistance to the lesser cornstalk borer, from complete larval mortality to a 66% reduction in larval weight. A negative correlation between percent survival or larval weight and the amount of Bt CryIA(c) protein was recorded indicating in general that the higher the protein level the lower the survival or larval weight of the insect. Based on leaf bioassay, transformation of peanut with vectors containing the Bt cryIA(c) gene may be effective in protecting the peanut plants from damage by lepidopteran insect larvae of lesser cornstalk borer     

Development of Cotton leaf curl virus resistant transgenic cotton using antisense ßC1 gene

Cotton leaf curl virus (CLCuV) is a serious pathogen causing leaf curl disease and affecting the cotton production in major growing areas. The transgenic cotton (Gossypium hirsutum cv. Coker 310) plants were developed by using βC1 gene in antisense orientation gene driven by Cauliflower mosaic virus-35S promoter and nos (nopaline synthase) terminator and mediated by Agrobacterium tumefaciens transformation and somatic embryogenesis system. Molecular confirmation of the transformants was carried out by polymerase chain reaction (PCR) and Southern blot hybridization. The developed transgenic and inoculated plants remained symptomless till their growth period. In conclusion, the plants were observed as resistant to CLCuV.     

Transformation of cucumber tissues by microprojectile bombardment: identification of plants containing functional and non-functional transferred genes.

The microprojectile bombardment method was used to transfer DNA into embryogenic callus of cucumber (Cucumis sativus), and stably transformed cucumber plant lines were obtained. A total of 107 independently regenerated cucumber plants were assayed for the presence and expression of the transferred Nos-NPTII gene (encoding nopaline synthase-neomycin phosphotransferase II). Genomic blot hybridization analyses showed that a high percentage (16%) of the cucumber plants were transformed with Nos-NPTII; however, only about 25% of these transgenic plants expressed Nos-NPTII. Inactivity of Nos-NPTII in many of the transformed cucumber plants may be associated with the transfer of multiple copies of Nos-NPTII. PCR and genomic blot hybridization analyses were used to show that the transferred gene was inherited in the subsequent plant generation.     

Development of transgenic sorghum for insect resistance against the spotted stem borer (Chilo partellus)

Transgenic sorghum plants expressing a synthetic cry1Ac gene from Bacillus thuringiensis (Bt) under the control of a wound-inducible promoter from the maize protease inhibitor gene (mpiC1) were produced via particle bombardment of shoot apices. Plants were regenerated from the transformed shoot apices via direct somatic embryogenesis with an intermittent three-step selection strategy using the herbicide Basta. Molecular characterisation based on polymerase chain reaction and Southern blot analysis revealed multiple insertions of the cry1Ac gene in five plants from three independent transformation events. Inheritance and expression of the Bt gene was confirmed in T₁ plants. Enzyme-linked immunosorbant assay indicated that Cry1Ac protein accumulated at levels of 1–8 ng per gram of fresh tissue in leaves that were mechanically wounded. Transgenic sorghum plants were evaluated for resistance against the spotted stem borer (Chilo partellus Swinhoe) in insect bioassays, which indicated partial resistance to damage by the neonate larvae of the spotted stem borer. Reduction in leaf damage 5 days after infestation was up to 60%; larval mortality was 40%, with the surviving larvae showing a 36% reduction in weight over those fed on control plants. Despite the low levels of expression of Bt -endotoxin under the control of the wound-inducible promoter, the transgenic plants showed partial tolerance against first instar larvae of the spotted stem borer.     

Transient expression from microprojectile-mediated DNA transfer in pinus taeda

Summary Transfer of plasmid DNA to Pinus taeda L. (loblolly pine) cotyledon cells by microprojectile bombardment has been demonstrated using beta-glucuronidase (GUS). GUS histochemical staining indicated active enzyme in localized centers (blue spots) 24 hours after bombardment. GUS expression declined during subsequent culture, but remained detectable in meristematic tissue 62 days post-bombardment, however, transgenic shoots were not recovered. Localized GUS expression events resulted predominantly from single-cell events containing one microprojectile. The staining pattern was complex, with indigo found both in the central target cell and in adjacent cells. Cellular damage sustained by GUS-positive cells ranged from undetectable to sufficiently extensive to cause cell death. Microprojectile bombardment provides a useful method to assay transient gene expression in loblolly pine and has potential for the production of transgenic plants in pine.     

饲料原料中转基因成分的PCR检测

采用PCR检测方法从饲料的主要原料豆粕、玉米蛋白粉中成功地检出启动子35S (35S-promoter,originated from cauliflower mosaic virus)、终止子NOS (nopaline synthase-terminator,derived from Agrobacterium tumefaciens)、耐除草剂基因EPSPS(5-enolpyruvylshikimate-3-phosphate synthase)和抗虫基因CryIA（b）(delta-endotoxin,evolved from Bacillus thuringiensis subsp.kurstaki)等转基因成分,并通过扩增玉米自身蛋白基因Zein( a protein extracted from corn gluten)及大豆自身基因Lectin(chitin-binding protein)的引物和阴阳性对照、阴阳性质控,避免假阳性、假阴性结果。该方法已在口岸进口饲料原料转基因检测中得到初步应用。 Abstract:Based on the heterogenous genes usually used in transgenic crops,the PCR technique was performed with primers derived from CaMV 35S promoter(35S-promoter,originated from cauliflower mosaic virus),NOS terminator(nopaline synthase-terminator,derived from Agrobacterium tumefaciens),EPSPS(5-enolpyruvylshikimate-3-phosphate synthase) gene,and CryIA(b)(delta-endotoxin,evolved from Bacillus thuringiensis subsp.kurstaki)gene to detect transgenic agents from feed raw materials of soybean dregs and corn gluten meal,respectively.Endogenous corn Zein(a protein extracted from corn gluten) gene,soybean Lectin(chitin-binding protein) gene and negative,positive control were applied for avoiding false results.The method established here has been succeessfully applied in detecting transgenic elements in imported feed raw material.     

The Production of Transgenic Scots Pine (Pinus Sylvestris L.) via the Application of Transformed Pollen in Controlled Crossings

The study demonstrates the production of a transgenic Scots pine (Pinus sylvestris L.) seedling through the application of transformed pollen in controlled crossings. The pollen lots were transformed by particle bombardment, resulting in transient transformation frequencies varying from 15 to 49% of the germinated pollen grains, and bombarded pollen was used to pollinate megasporangiate strobili. Progeny was screened by histochemical, GUS assays, and selected seedlings were further analysed by PCR. PCR amplification revealed the presence of both the nptII and gusA genes in one seedling (23/237). Results were confirmed by Southern blot analysis. The morphology and growth of this transgenic seedling was normal. Although the transformation frequency of recovered plants was very low (1/14999), the present protocol suggests that production of transgenic Scots pine is possible without the use of any tissue culture methods or the involvement of marker genes, for selection of transformants.