H2S and COS Gas Exchange of Transgenic Potato Lines with Modified Expression Levels of Enzymes Involved in Sulphur Metabolism

Different transgenic potato lines were generated for improving the nutritional value of tubers by an advanced perception of their sulphur metabolism. So far no data exist about possible implications for plant health and stress resistance. Metabolite analysis revealed that modifications of enzymes involved in sulphur metabolism were necessarily not reflected in distinctly altered thiol contents. The release of H₂S by plants is putatively involved in pathogen resistance, because of its fungi‐toxic mode of action. The emission of H₂S was determined in 16 potato lines with modified expression level in ATP sulphurylase (ATPS), serine acetyltransferase (SAT), O‐acetylserine(thiol)lyase (OASTL), homoserine kinase (HSK) and threonine synthase activities. The emission significantly increased by factor 7 in one of the ATPS antisense lines and by factor 8 in one of the OASTL antisense lines. A strong increase in H₂S emissions was observed in transgenic plants based on the potato cultivar White Lady, which expressed the Escherichia coli SAT. In addition, the exchange of COS was determined in relation to genetic modifications. Generally, plants act as a sink for COS, but all transgenic lines expressing the E. coli HSK and one of the ATPS antisense lines emitted COS indicating to strong changes in the metabolism of these plants. Such alterations in the gas exchange of transgenic potato plants will most likely also affect their resistance against biotic and abiotic stress.     

转录因子ABP9基因过表达对植物生长发育的影响分析

【研究目的】为分析玉米ABA/逆境响应转录因子ABP9基因过量表达对植物生长发育的影响以利用该基因开展抗逆分子育种。【方法】本研究构建了35S启动子驱动ABP9组成型表达的植物表达载体，获得了过表达ABP9基因的拟南芥转基因株系，并在正常生长条件下，比较了转基因植株和野生型在种子萌发、营养生长和生殖生长阶段的形态和生理变化，分析了可能的分子机制。【结果】结果表明，正常生长条件下，转基因植株与野生型相比表现出萌发，幼苗生长以及开花阶段的生长抑制；气孔开度减小，叶片内源ABA含量降低；ABA信号传导基因表达增强，而ABA合成基因表达降低；而且这些效应在ABP9基因表达相对较强的转基因株系中表现更明显。【结论】说明ABP9基因过量在正常生长条件下即诱导转基因植株产生耐逆反应，抑制植株的生长发育。因此，在利用ABP9基因进行转基因抗逆育种时须考虑控制ABP9基因适时适量表达。     

Enhancement of drought resistance and biomass by increasing the amount of glycine betaine in wheat seedlings

Drought is a major agricultural menace reducing crop productivity and limiting the successful realization of land potential throughout the world. Therefore, breeding common wheat with improved drought-tolerance via genetic manipulation is of great importance. We have introduced the betA gene encoding choline dehydrogenase from Escherichia coli into common wheat (Triticum aestivum L.) by Agrobacterium-mediated transformation. Various levels of expression of the betA gene were confirmed by RT-PCR among the transgenic lines and different levels of glycine betaine accumulation were detected in these lines. Several wheat transgenic lines with different betA expression levels in the T3 generation and wild-type (WT) were selected to test their performance under drought stress conditions. Water deficit in plants caused a reduction in photosynthesis and activity of the PSII complex and resulted in increased accumulation of osmolytes. Drought stress also led to lower membrane stability along with much higher activities of superoxide dismutase and peroxidase in all wheat lines. However, wheat lines that were transgenic for the betA gene were less injured and exhibited greater root length and growth compared with the WT. It was concluded that the amount of injury to the wheat plants was negatively correlated with the level of accumulation of glycine betaine, and the glycine betaine acted as an important osmoprotectant in transgenic plants to improve root growth, and enhance the resistance of transgenic plants to drought stress.     

The Experimental and Commercial Release of Transgenic Crop Plants

With advances in recombinant DNA methods and transformation procedures, it is possible to transfer genes into crop plants from unrelated plants, microbes and animals. Many of the modifications being carried out, or envisaged, are for disease and pest resistance, product quality and tolerance to environmental stress, but there are additional opportunities to modify crops to give specialized products for industrial or pharmaceutical use. Some of the characteristics of transgenic plants are considered, including: transgene copy number, position, expression, stability, pleiotropy, selectable marker genes and somaclonal variation. There have been several hundreds of field trials with transgenic plants, and the first transgenic varieties are likely to be approved for commercial production in 1993. Before releasing transgenic plants, it is necessary to carry out a risk assessment to determine whether the transgenic variety will behave differently from a conventionally bred variety. Assessment procedures are being harmonized internationally by various organizations. There is a growing commitment to apply these genetic modification methods to crops in developing countries, as genes relevant to their crops and environments become available.     

Occasional loss of expression of phosphinothricin tolerance in sexual offspring of transgenic oilseed rape (Brassica napus L.)

The commercial and economic value of genetically modified crops is determined by a predictable, consistent and stable transmission and expression of the transgenes in successive generations. No gene inactivation is expected after selfings or crosses with non-transformed plants of homozygous transgenic oilseed rape plants if the expression of the transgene in homozygous or hemizygous nature in such plants is stable. The segregation ratios of phosphinothricin (PPT) tolerance in successive generations of selfings and mutual crosses of a few independent transgenic PPT-tolerant oilseed rape plants indicated a dominant, monogenic inheritance. In within-variety and between-variety crosses no transgene inactivation was observed. However, after selfings and backcrosses with non-transgenic oilseed rape infrequent loss of the expression of the PPT tolerance transgene was observed independent from its homozygous or hemizygous nature. Molecular analysis of PPT-susceptible plants showed that the loss of expression was due to gene inactivation and not to the absence of the transgene. Methylation and co-suppression are mechanisms that might cause reduced or even loss of expression of the transgene in later generations. The implications of this observation for seed multiplication of varieties and breeding activities with transgenic oilseed rape are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transmission of a Bacillus thuringiensis cry3Aa transgene from diploid to tetraploid potato using 4x-2x hybridization: effect of ploidy increase on transgene expression and implications for TPS hybrid production

The study examined the effect of ploidy elevation through unreduced gametes on transgene expression in potato. Tetraploid transgenic progenies were obtained from one tetraploid potato cultivar crossed with 2n pollen producing diploid clones harbouring an exogenous transgene (cry3Aa). Both single‐ and multiple‐insert diploid transgenic lines that were regenerated by Agrobacterium tumefaciens leaf disc inoculation were used in crosses. A DAS‐ELISA system and no‐choice feeding bioassay enabled characterization of the parental lines as either ‘high’ or low’ expressers of the Cry3Aa protein. High Cry3Aa expression was observed for both single‐insert transgenic diploids and their 4x‐2x progeny. On the contrary, 68% of 4x‐2x progeny derived from a multiple‐insert, diploid transgenic had significantly reduced Cry3Aa expression compared with the parent, with 32% demonstrating nearly complete silencing of the transgene. Multiple copies of a transgene, like homologous native genes, may be susceptible to transgene silencing following polyploidization. Therefore, incorporation of exogenous transgenes into a true potato seed (TPS) production system is feasible if a single‐insert diploid parent is used. Gene‐centromere mapping of the cry3Aa transgene demonstrated that a non‐transgenic refuge might be naturally created in a TPS hybrid system through genetic recombination.     

Field evaluation of transgenic Brassica napus lines carrying a seed-specific chimeric 2S albumin gene

The agronomic performance of transgenic Brassica napus lines carrying chimeric 2S albumin seed protein genes was studied under field conditions. A total of 37 transgenic lines were compared to non-transformed lines. Plant development, morphological characteristics, yield parameters and seed composition were considered. Only a few transgenic lines differed from the control, and then only for particular parameters: four lines had altered leaf morphology, two differed in germination efficiency, one differed in yield, and three differed in some parameters of seed composition. These differences were not correlated with the presence or expression of the transgene and, thus, were more probably linked to cell- or tissue-culture effects, or to residual variation present in cv. ‘Drakkar’. A statistical interaction between variations in a limited number of seed composition parameters and cell haploid culture was observed. While the expression of the chimeric 2S albumin gene was not as high as hoped, this study demonstrates that transgenic lines can be isolated in which the intrinsic morphological and agronomic properties of the original lines are maintained.     

脱水素基因转化的矮牵牛对干旱胁迫的反应

将厚叶旋蒴苣苔脱水素(Dehydrin)基因BDN1置于CaMV35S启动子的调控之下,并插入于表达载体pCAMBIA3300中,通过根癌土壤杆菌(Agrobacterium tumefaciens)介导导入矮牵牛(Vetunia Hybrida),经PCR及Southem杂交表明BDN1已转入矮牵牛基因组中,Northem实验证明,BDN1基因在矮牵牛中在RNA水平有较强的表达。通过对转BDN1基因矮牵牛的保水力和低温离子渗漏的测定,初步验证转基因矮牵牛对水分胁迫的能力在一定程度上有所增加。     

Transgenic cotton, expressing Amaranthus caudatus agglutinin, confers enhanced resistance to aphids

To evaluate the possible antiaphid function of Amaranthus caudatus agglutinin (ACA) in allogenetic plants, transgenic cotton plants expressing the ACA gene under the control of a phloem‐specific promoter were generated via Agrobacterium‐mediated gene transformation. Based on the results of Southern blot analyses, six plants with single or lower copy transgene and favourable agronomic traits were selected for further studies. ACA expression levels ranged from 0.02% to 0.45% of total soluble protein as determined by Western blot analysis in the six selected transgenic plants. Insect bioassays using nymphs of cotton aphid (Aphis gossypii Glover) showed that five of the six transgenic plants significantly inhibited the population growth of cotton aphid, with the highest inhibition rate of 64.5%. These results shed some new light on the antiaphid function of the ACA gene as well as the promising application of the gene for obtaining aphid‐resistant transgenic cotton plants to reduce the yield loss and honeydew contamination of fibre by aphids.     

异源表达棉花GhPRP5基因增强了拟南芥对盐和ABA的敏感性

富含脯氨酸的蛋白(proline-rich proteins, PRPs)代表一类富含脯氨酸和羟脯氨酸的细胞壁结构蛋白质,最先在伤害诱导的胡萝卜贮藏根中被发现。越来越多的证据显示这类蛋白在应答多种生物和非生物胁迫中起作用。我们之前从棉花cDNA文库中分离了一个命名为GhPRP5的编码富含脯氨酸蛋白的基因,为研究其功能,构建了GhPRP5的过量表达载体,转化拟南芥,获得GhPRP5高表达的8个株系的纯合体。在正常培养条件下,转基因株系和野生型种子的萌发率一致,但盐胁迫和ABA处理显著抑制了转基因拟南芥株系种子的萌发。盐胁迫条件下野生型的绿苗率明显高于转基因拟南芥株系,与正常生长条件相比,ABA处理抑制转基因拟南芥主根伸长的程度更大。利用Quantitative RT-PCR技术分析几个胁迫相关标记基因的表达情况表明,盐和ABA诱导了RD29A、RD29B和KIN1的表达,但诱导水平在转基因株系和野生型中不一样,说明GhPRP5参与调控拟南芥胁迫相关标记基因的表达,但具体参与的胁迫应答信号传导途径仍需进一步研究。     

Transgenic soybean with low phytate content constructed by <Emphasis Type="Italic">Agrobacterium</Emphasis> transformation and pollen-tube pathway

The expression of a microbial phytase in transgenic plants may create a new biochemical pathway that mobilizes its endogenous phytate and release inorganic phosphate from it, so that more phosphorus is available for plant growth. In this study, transgenic soybean plants were generated via both Agrobacterium transformation and pollen tube pathway with the PhyA gene of Aspergillus ficuum. The optimal concentrations of plant hormones including N₆-benzylaminopurine (BAP), gibberellin (GA₃) and indole-3-butyric acid (IBA) were tested based on their effectiveness on promoting the growth of transgenic explants. Genomic PCR results and Southern blot hybridization analysis showed that transgenic soybean plants selected for resistance to kanamycin contained the phyA transgene. The transgenic soybean plants with phyA gene integrated in their genome exhibited lower amount of phytate in different soybean tissues including leaf, stem and root, which indicated that engineering crop plants with a higher expression level of heterologous phytase could improve the degradation of phytate and potentially in turn mobilize more inorganic phosphate from phytate and thus reduce phosphate load on agricultural ecosystems.     

Improved salt tolerance and seed cotton yield in cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) by transformation with <Emphasis Type="Italic">betA</Emphasis> gene for glycinebetaine synthesis

Homozygous transgenic cotton (Gossypium hirsutum L.) plants that accumulated glycinebetaine (GB) in larger quantities were more tolerant to salt than wild-type (WT) plants. Four transgenic lines, namely 1, 3, 4, and 5, accumulated significantly higher levels of GB than WT plants did both before and after salt stress. At 175 and 275 mM NaCl, seeds of all the transgenic lines germinated earlier and recorded a higher final germination percentage, and the seedlings grew better, than those of the WT. Under salt stress, all the lines showed some characteristic features of salt tolerance, such as higher leaf relative water content (RWC), higher photosynthesis, better osmotic adjustment (OA), lower percentage of ion leakage, and lower peroxidation of the lipid membrane. Levels of endogenous GB in the transgenic plants were positively correlated with RWC and OA. The results indicate that GB in transgenic cotton plants not only maintains the integrity of cell membranes but also alleviates osmotic stress caused by high salinity. Lastly, the seed cotton yield of transgenic lines 4 and 5 was significantly higher than that of WT plants in saline soil. This research indicates that betA gene has the potential to improve crop’s salt tolerance in areas where salinity is limiting factors for agricultural productivity.     

Genetically manipulated pineapple: transgene stability, gene expression and herbicide tolerance under field conditions

Pineapple plants transformed with the bar gene for bialaphos resistance were evaluated for transgene stability, gene expression and tolerance to glufosinate ammonium, the active ingredient of the herbicide Basta^® X, under field conditions. Genetically modified plants of the cv. Phuket were micropropagated, rooted and established in a shade house before transfer to an experimental plot. Seven months after transfer to the field, plants were tolerant to 1600 ml/rai of the herbicide Basta^® X (stock concentration 15% w/v glufosinate ammonium), this being twice the dose recommended for field application of the herbicide. Genetically modified plants remained green and healthy following spraying with the herbicide. In contrast, non‐transformed pineapple plants of the same cv. became necrotic and died within 21 days of spraying with the herbicide at a reduced concentration of 800 ml/rai. Bar gene stability and expression in clonally‐derived plants were assessed by PCR, RT‐PCR and Southern analyses at 120, 210, 240, 270 and 380 days following transfer of the plants to the field. The bar gene was stable and expressed in transgenic plants throughout the duration of the trial. Fruit characteristics and yield were not affected by transgene introduction and expression. Transgenic plants tolerant to glufosinate ammonium should facilitate more effective weed control in pineapple plantations without damage to the crop.     

Amenability of castor to an <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated <Emphasis Type="Italic">in planta</Emphasis> transformation strategy using a <Emphasis Type="Italic">cry1AcF</Emphasis> gene for insect tolerance

Agrobacterium tumefaciens mediated in planta transformation protocol was developed for castor, Ricinus communis. Two-day-old seedlings were infected with Agrobacterium strain EHA105/pBinBt8 harboring cry1AcF and established in the greenhouse. Screening the T₁ generation seedlings on 300 mg L⁻¹ kanamycin identified the putative transformants. Molecular and expression analysis confirmed the transgenic nature and identified high-expressing plants. Western blot analysis confirmed the co-integration of the nptII gene in the selected transgenic plants. Bioassay against Spodoptera litura corroborated with high expression and identified five promising effective lines. Analysis of the T₂ generation plants proved the stability of the transgene indicating the feasibility of the method.     

Effect of genetic background on the target fatty acids of acyl-ACP thioesterase transgenes in Brassica napus

Acyl‐acyl carrier protein (ACP) thioesterase (TE) is involved in the biosynthetic fatty acid pathway of plants. Conventional canola lines transformed individually with the bay‐TE (Uc FatB1), elm‐TE (Ua FatB1), nutmeg‐TE (Mf FatB1) or Cuphea‐TE transgene (Ch FatB1), produce seed oil with modified fatty acid compositions. This study assessed the effects of genetic background, cytoplasm, maternal parent, and interaction of different TE transgenes, on the target fatty acids using F1 seeds and double haploid (DH) lines. The F₁ seeds were produced by crossing four TE transgenic parental lines and three non‐transgenic cultivars with distinct fatty acid compositions. The DH lines were developed from microspores of F₁ plants. DH lines from different crosses showed that genetic background does not have an effect on the relative levels of the target fatty acids of the same TE, indicating the stability of the substrate specificity of the TE within canola. However, significant effects of genetic background on the content of the major target fatty acids, lauric acid (C12:0) or palmitic acid (C16:0) depending on the TE, were observed. Expression of the TE in low erucic acid (C22:1) genotypes resulted in higher target fatty acid levels than expression in high C22:1 genotypes. Reciprocal crosses showed maternal effects, but not cytoplasmic effects. In addition, co‐expression of two different TE transgenes in the same seeds was observed. These results indicate the importance of selection for appropriate genetic backgrounds in order to maximize the expression of the target fatty acids of TE transgenes, and also indicate potential uses of TE co‐expression in modifying canola seed oil.     

Enhanced resistance to a lepidopteran pest in transgenic sugar beet plants expressing synthetic <Emphasis Type="Italic">cry1Ab</Emphasis> gene

Two diploid sugar beet genotypes of agronomical importance were transformed using Agrobactrium tumefaciens harboring pBI35Scry containing a synthetic cry1Ab gene. Leaf blade with attached shoot bases, a highly regenerative tissue, were used as explant substratum for transformation. PCR screening with cry1Ab-specific primers showed the presence of transgene in more than 50% of the regenerated kanamycin-resistant plants after treatment with the antibiotic. A transformation rate of 8.8–12.2% (depending on genotype) was achieved as revealed by genomic DNA dot blotting. The intact integration of transgene cassette into the genome was furthermore confirmed by Southern blot analysis. The expression of the cry1Ab gene encoding a truncated endotoxin (67 kDa) at about 0.1% of total soluble protein was achieved in the leaves of transgenic plants as shown by Western blot analysis. Bioassays under in vitro conditions with Spodoptera littoralis, one of the most important pests in sugar beet fields, demonstrated enhanced resistance against this pest. The inheritance of the inserted transgene was confirmed in F₁ plants obtained through crossing of T₀ plants with a cytoplasmic male sterile line. Transgenic plants are currently grown in a greenhouse and will be subjected to further bioassay analyses against other lepidopteran pests of sugar beet.