The creation of sugar beet transgenic plants expressing bar gene

The parameters of transformation using Agrobacterium tumefaciens EHA 105 for 5 domestic sorts and lines of sugar beet (Beta vulgaris L. var. saccharifera (Alef) Krass) were optimized. The system of transgenic tissue selection based on resistance to phosphinothricin, allowing to avoid the appearing of chimeric shoots among initial transformants was developed. The transgenic plants of sugar beet sorts Ramonskaya single seed 47, L’govskaya single seed 52 and RMS 73, and LBO 17 and LBO 19 lines expressing the gene of phosphinothricin acetyl transferase bar have been obtained. The resistance of these sorts and lines to the effect of phosphinothricin in vitro has been shown.     

Transgenic lines of melon (Cucumis melo L. var. makuwa cv. ‘Silver Light’) expressing antifungal protein and chitinase genes exhibit enhanced resistance to fungal pathogens

The oriental melon (Cucumis melo L. var. makuwa cv. ‘Silver Light’) is an important fruit crop in the tropical and subtropical regions. However, oriental melon production is severely decreased by fungal diseases. In this study, antifungal protein (AFP) and chitinase (CHI) fusion genes were introduced into oriental melons to control fungal diseases caused by Rhizoctonia solani and Fusarium oxysporum. Transformation of oriental melon (Cucumis melo L. var. makuwa cv. ‘Silver Light’) with Agrobacterium tumefaciens strain LBA4404 containing antifungal protein (AFP) and chitinase (CHI) fusion genes under the control of the cauliflower mosaic virus (CaMV) 35S promoter and neomycin phosphotransferase (nptII) gene as a selectable marker was performed. Cotyledon explants of oriental melon were inoculated by Agrobacterium suspensions with pBI121–AFP–CHI and cultured in a regeneration medium. After regeneration, genomic DNA polymerase chain reaction (PCR) was conducted to confirm the presence of putative transgenic shoots. Southern blot analysis confirmed that the AFP–CHI fusion gene was incorporated into the genomic DNA of the PCR-positive lines. RT-PCR analysis showed that the AFP–CHI fusion gene was expressed in the individual transgenic lines. Western blot analysis revealed the accumulation of CHI protein in leaves. A segregation analysis of the T₁ generation confirmed the inheritance of the transgene. Our results demonstrated that the AFP–CHI fusion gene was effective in protecting the transgenic melon plants against fungal disease caused by Rhizoctonia solani and Fusarium oxysporum.     

Expression of baculovirus anti-apoptotic genes p35 and op-iap in cotton (Gossypium hirsutum L.) enhances tolerance to verticillium wilt

Background

Programmed cell death plays an important role in mediating plant adaptive responses to the environment such as the invasion of pathogens. Verticillium wilt, caused by the necrotrophic pathogen Verticillium dahliae, is a serious vascular disease responsible for great economic losses to cotton, but the molecular mechanisms of verticillium disease and effective, safe methods of resistance to verticillium wilt remain unexplored.

Methodology/Principal Findings

In this study, we introduced baculovirus apoptosis inhibitor genes p35 and op-iap into the genome of cotton via Agrobacterium-mediated transformation and analyzed the response of transgenic plants to verticillium wilt. Results showed that p35 and op-iap constructs were stably integrated into the cotton genome, expressed in the transgenic lines, and inherited through the T₃ generation. The transgenic lines had significantly increased tolerance to verticillium wilt throughout the developmental stages. The disease index of T₁–T₃ generation was lower than 19, significantly (P<0.05) better than the negative control line z99668. After treatment with 250 mg/L VD-toxins for 36 hours, DNA from negative control leaves was fragmented, whereas fragmentation in the transgenic leaf DNA did not occur. The percentage of cell death in transgenic lines increased by 7.11% after 60 mg/L VD-toxin treatment, which was less than that of the negative control lines''s 21.27%. This indicates that p35 and op-iap gene expression partially protects cells from VD-toxin induced programmed cell death (PCD).

Conclusion/Significance

Verticillium dahliae can trigger plant cells to die through induction of a PCD mechanism involved in pathogenesis. This paper provides a potential strategy for engineering broad-spectrum necrotrophic disease resistance in plants.     

Increased resistance to fungal wilts in transgenic eggplant expressing alfalfa glucanase gene

The wilt diseases caused by Verticillium dahliae and Fusarium oxysporum are the major diseases of eggplant (Solanum melongena L.). In order to generate transgenic resistance against the wilt diseases, Agrobacterium-mediated gene transfer was performed to introduce alfalfa glucanase gene encoding an acidic glucanase into eggplant using neomycin phosphotransferase (npt-II) gene as a plant selection marker. The transgene integration into eggplant genome was confirmed by Polymerase chain reaction (PCR) and Southern blot analysis and transgene expression by the glucanase activity and western blot analysis. The selected transgenic lines were challenged with V. dahliae and F. oxysporum under in vitro and in vivo growth conditions, and transgenic lines showed enhanced resistance against the wilt-causing fungi with a delay of 5–7 days in the disease development as compared to wild-type plants.     

High-Level Expression of Sporamin in Transgenic Chinese Cabbage Enhances Resistance Against Diamondback Moth

This study introduced the protease inhibitor-encoding gene sporamin from sweet potato together with the wound-responsive promoter pMSPOA into the Chinese cabbage (Brassica campestris ssp. chinensis var. parachinensis) through Agrobacterium tumefaciens-mediated transformation. Transgenic lines were confirmed via PCR detection of both the sporamin and hygromycin gene fragments in the cloning vector. The expression level of sporamin in the different lines was determined using RT-PCR analysis. Leaves from four individual transgenic lines including T0-2, T0-3, T0-4 and T0-5, possessing higher levels of sporamin than control plants, were then used to conduct insect feeding studies. Second instar larvae of diamondback moth (Plutella xylostella L.) were used in feeding assays. All four transgenic lines demonstrated high levels of resistance against diamondback based on average body weight of total incubated larvae following feeding assays and amount of leaf damage.     

Functional role of rice germin-like protein1 in regulation of plant height and disease resistance

The functional role of rice (Oryza sativa) germin-like protein1 (OsGLP1) was elucidated through development of transgenic plants involving endogenous gene silencing in rice and heterologous gene expression in tobacco. Usually, the single copy OsGLP1 gene in rice plant was found to be expressed predominantly in green vegetative tissues. The transgenic rice lines showed significant reduction in endogenous OsGLP1 expression due to 26 nt siRNA-mediated gene silencing, displayed semi-dwarfism and were affected seriously by fungal diseases, compared to the untransformed plant. Structural homology modeling predicted a superoxide dismutase (SOD) domain in OsGLP1 protein which upon over-expression in transgenic tobacco plant clearly documented SOD activity. Our observations on the maintenance of cell dimension, cell wall-associated localization particularly in the sub-epidermal tissues and the SOD activity of OsGLP1 could explain its functional role in regulation of plant height and disease resistance in rice plant.     

StP5CS基因转化结球甘蓝的研究

为研究StP5CS基因在结球甘蓝中的耐盐作用,以结球甘蓝下胚轴为外植体,采用农杆菌介导法将耐盐基因StP5CS和抗除草剂Bar基因导入结球甘蓝基因组中,在双丙氨膦的筛选下扩繁、生根,共获得了36株抗性植株。PCR扩增和Southern印迹杂交检测表明:目的基因StP5CS和Bar基因已经成功导入结球甘蓝基因组中。RT-PCR检测表明:StP5CS基因在转录水平也有表达。转基因植株耐盐试验结果显示:高浓度盐处理(400mmol/L NaCl)下,对照植株整株枯死,而转基因植株仍能正常生长;转基因植株的SOD活性、脯氨酸含量和相对膜透性均随盐浓度的升高呈上升趋势,均在400mmol/L NaCl处理下达到最大。结果表明转基因植株对高盐环境有一定的耐受性。     

Proteomic analysis of stress-related proteins in transgenic broccoli harboring a gene for cytokinin production during postharvest senescence

Our previous study revealed a cytokinin-related retardation of post-harvest floret yellowing in transgenic broccoli (Brassica oleracea var. italica) that harbored the bacterial isopentenyltransferase (ipt) gene. We aimed to investigate the underlining mechanism of this delayed post-harvest senescence. We used 2D electrophoresis and liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry for a proteomics analysis of heads of ipt-transgenic and non-transgenic inbred lines of broccoli at harvest and after four days post-harvest storage. At harvest, we found an accumulation of stress-responsive proteins involved in maintenance of protein folding (putative protein disulfide isomerase, peptidyl-prolyl cis-trans isomerase and chaperonins), scavenging of reactive oxygen species (Mn superoxide dismutase), and stress protection [myrosinase-binding protein, jasmonate inducible protein, dynamin-like protein, NADH dehydrogenase (ubiquinone) Fe-S protein 1 and stress-inducible tetratricopeptide repeat-containing protein]. After four days’ post-harvest storage of non-transgenic broccoli florets, the levels of proteins involved in protein folding and carbon fixation were decreased, which indicates cellular degradation and a change in metabolism toward senescence. In addition, staining for antioxidant enzyme activity of non-transgenic plants after post-harvest storage revealed a marked decrease in activity of Fe-superoxide dismutase and ascorbate peroxidase. Thus, the accumulation of stress-responsive proteins and antioxidant enzyme activity in ipt-transgenic broccoli are most likely associated with retardation of post-harvest senescence.     

Agrobacterium-mediated co-transformation of rice using two selectable marker genes derived from rice genome components

A method for Agrobacterium-mediated co-transformation of rice (Oryza sativa L.) was developed using rice-derived selection markers. Two T-DNAs were efficiently introduced into separate loci using selectable marker gene cassettes consisting of the mutated acetolactate synthase gene (mALS) under the control of the callus-specific promoter (CSP) (CSP:mALS) and the ferredoxin nitrite reductase gene (NiR) under the control of its own promoter (NiR P:NiR). The CSP:mALS gene cassette confers sulfonylurea herbicide resistance to transgenic rice callus. The NiR P:NiR construct complements NiR-deficient mutant cultivars such as ‘Koshihikari’, which are defective in the regulation of nitrogen metabolism. In the present study, the CaMV35S:GUS and CaMV35S:GFP gene cassettes were co-introduced into the ‘Koshihikari’ genome using our system. Approximately 5–10 independent transgenic lines expressing both the GUS and GFP reporters were obtained from 100 Agrobacterium co-inoculated calli. Furthermore, transgenic ‘Koshihikari’ rice lines with reduced content of two major seed allergen proteins, the 33 and 14–16?kDa allergens, were generated by this co-transformation system. The present results indicate that the generation of selectable antibiotic resistance marker gene-free transgenic rice is possible using our rice-derived selection marker co-transformation system. Key message An improved rice transformation method was developed based on Agrobacterium-mediated co-transformation using two rice genome-derived selectable marker gene cassettes.     

Constitutive expression of a fusion gene comprising Trigonella foenum-graecum defensin (Tfgd2) and Raphanus sativus antifungal protein (RsAFP2) confers enhanced disease and insect resistance in transgenic tobacco

Plant defensins are small, basic cysteine-rich peptides that can inhibit the growth of a broad range of fungi or bacteria at micro-molar concentrations. They have been introduced as transgenes into different species to enhance host resistance to pathogens. In this study, a fusion gene of two defensins, Trigonella foenum-graecum defensin 2 (Tfgd2) and Raphanus sativus antifungal protein 2 (RsAFP2) fused by a linker peptide of a polyprotein precursor from Impatiens balsamina was introduced into tobacco (Nicotiana tabacum var. Xanthi) via Agrobacterium-mediated leaf section transformation. Putative transgenic plants were confirmed by PCR analysis and integration of the fusion gene was confirmed by Southern blotting. RT-PCR analysis showed that the fusion gene was expressed in several confirmed transgenic plants. Western blotting analysis of crude protein extracts from leaves of the transgenic plants with anti-Tfgd2 and anti-RsAFP2 antibodies exhibited an 8 and 9 kDa bands corresponding to size of the fusion gene and confirmed the expression of fusion protein. When the leaves of transgenic plants were challenged with Rhizoctonia solani and Phytophthora parasitica var. nicotianae pathogens, they showed enhanced levels of disease resistance along with resistance to the generalist herbivore, Spodoptera litura larvae compared to control. Our results demonstrate that Tfgd2–RsAFP2 fusion protein is effective in protecting the transgenic plants against fungal and insect pathogens.     

Stable expression of exogenous imported <Emphasis Type="Italic">sporamin</Emphasis> in transgenic Chinese cabbage enhances resistance against insects

Cultivating insect pest-resistant varieties is one of the most effective ways to prevent or mitigate pest infestation in Chinese cabbage (Brassica campestris ssp. chinensis). Via the agrobacterium tumefaciens-mediated transformation method, this study introduced the protease inhibitor encoding gene sporamin into two widely cultured cultivars ‘Youdonger’ and ‘Shanghaiqing’, of the common variety of Chinese cabbages (B. campestriss ssp. chinensis var. communis), getting transgenic plants with high sporamin expression. In vitro insect bioassays indicated that, compared with the wild type plants, the transgenic plants exhibited improved resistance to diamondback moth (Plutella xylostella L.) The analysis of inheritance pattern of exogenous sporamin in the progenies of single copy insertion transgenic lines demonstrated that sporamin could be inherited and expressed stably in transgenic progenies. Field survey of the insect resistance under the normal culture condition confirmed the enhanced resistance in transgenic progenies to diamondback moth. Our results strongly suggest that sporamin is an efficient candidate gene for insect-resistant genetic engineering in Chinese cabbage.     

Overexpression of rice thaumatin-like protein (Ostlp) gene in transgenic cassava results in enhanced tolerance to Colletotrichum gloeosporioides f. sp. manihotis

Cassava (Manihot esculenta Crantz) is the most important staple food for more than 300?million people in Africa, and anthracnose disease caused by Colletotrichum gloeosporioides f. sp. manihotis is the most destructive fungal disease affecting cassava production in sub-Saharan Africa. The main objective of this study was to improve anthracnose resistance in cassava through genetic engineering. Transgenic cassava plants harbouring rice thaumatin-like protein (Ostlp) gene, driven by the constitutive CaMV35S promoter, were generated using Agrobacterium-mediated transformation of friable embryogenic calli (FEC) of cultivar TMS 60444. Molecular analysis confirmed the presence, integration, copy number of the transgene all the independent transgenic events. Semi-quantitative RT-PCR confirmed high expression levels of Ostlp in six transgenic lines tested. The antifungal activity of the transgene against Colletotrichum gloeosporioides pathogen was evaluated using the leaves and stem cuttings bioassay. The results demonstrated significantly delayed disease development and reduced size of necrotic lesions in leaves and stem cuttings of all transgenic lines compared to the leaves and stem cuttingss of non-transgenic control plants. Therefore, constitutive overexpression of rice thaumatin-like protein in transgenic cassava confers enhanced tolerance to the fungal pathogen C. gloeosporioides f. sp. manihotis. These results can therefore serve as an initial step towards genetic engineering of farmer-preffered cassava cultivars for resistance to anthracnose disease.     

Agrobacterium tumefaciens-mediated transformation to alter ethylene and cytokinin biosynthesis in broccoli

Broccoli (Brassica oleracea var. italica) deteriorates rapidly following harvest. Postharvest treatment of broccoli with 6-benzylaminopurine delays senescence, whilst exogenous ethylene has been shown to accelerate this process following harvest. To alter ethylene biosynthesis, broccoli was transformed, using Agrobacterium tumefaciens-mediated transformation, with an antisense ACC oxidase gene from broccoli driven by the asparagine synthetase promoter from asparagus. In addition, broccoli was transformed with the chimeric gene construct SAG12-IPT to alter cytokinin biosynthesis during harvest-induced senescence. Transformation was achieved using both hypocotyl and cotyledonary petiole explants. The presence of an antisense ACC oxidase gene enhanced transformation efficiency, but Ag⁺ incorporated into the medium did not. The transgenic nature of these plants was confirmed by PCR and Southern analyses.     

The rice <Emphasis Type="Italic">OsLOL2</Emphasis> gene encodes a zinc finger protein involved in rice growth and disease resistance

Arabidopsis LSD1-related proteins that contain LSD1-like zinc finger domains have been identified to be involved in disease resistance and programmed cell death. To investigate the potential role of LSD1-related gene in rice (Oryza sativa L.), we cloned an LSD1 ortholog, OsLOL2, from the rice cDNA plasmid library. The OsLOL2 gene is predicted to encode a polypeptide of 163 amino acids with two LSD1-like zinc finger domains with 74.5% identity to those of LSD1. Southern blot analysis indicated that OsLOL2 was a single-copy gene in the rice genome. Transgenic rice lines carrying the antisense strand of OsLOL2 with decreased expression of OsLOL2 had dwarf phenotypes, and the dwarfism could be restored by exogenous GA₃ treatment, suggesting that the dwarfism was the result of a deficiency in bioactive gibberellin (GA). In agreement with this possibility, the content of endogenous bioactive GA₁ decreased in the antisense transgenic lines. Expression of OsKS1, one of the genes encoding for GA biosynthetic enzymes, was suppressed in the antisense transgenic lines. Sense transgenic lines with increased expression of OsLOL2 were more resistant to rice bacterial blight, while antisense transgenic lines were less resistant to rice bacterial blight. The OsLOL2-GFP (green fluorescence protein) fusion protein was localized in the nucleus of cells of transgenic BY2 tobacco (Nicotiana tabacum L.). These data suggest that OsLOL2 is involved in rice growth and disease resistance.     

Transformation of LTP gene into Brassica napus to enhance its resistance to Sclerotinia sclerotiorum

Rapeseed (Brassica napus L.) is one of the most important economic crops worldwide, and Sclerotinia sclerotiorum is the most dangerous disease that affects its yield greatly. Lipid transfer protein (LTP) has broad-spectrum anti-bacterial and fungal activities. In this study, B. napus was transformed using Agrobacterium tumefaciens harboring the plasmid-containing LTP gene to study its possible capability of increasing plant’s resistance. First, we optimized the petiole genetic transformation system by adjusting the days of explants, bacterial concentrations, ratio of hormones, and cultivating condition. Second, we obtained 8 positive plants by PGR analysis of T0 generation. The PGR results of T₁ generation were positive, indicating that the LTP gene had been integrated into B. napus. Third, T₁ transgenic plants inoculated by detached leaves with mycelia of S. sclerotiorum showed better disease resistance than non-transformants. Oxalic acid belongs to secondary metabolites of S. sclerotiorum, and several studies have demonstrated that the resistance of rapeseed to oxalic acid is significantly consistent with its resistance to S. sclerotiorum. The result from the seed germination assay showed that when T₁ seeds were exposed to oxalic acid stress, their germination rate was evidently higher than that of non-transformant seeds. In addition, we measured some physiological changes in T₁ plants and control plants under oxalic acid stress. The results showed that T₁ transgenic plants had lower malondialdehyde (MDA) content, higher super oxide dismutase (SOD), and peroxidase (POD) activities than non-transformants, whereas disease resistance was related to low MDA content and high SOD and POD activities.