Tissue culture specificity of the tobacco ASA2 promoter driving hpt as a selectable marker for soybean transformation selection

This study was carried out to determine if the tobacco anthranilate synthase ASA2 2.3 kb promoter drives tissue culture specific expression and if it is strong enough to drive hpt (hygromycin phosphotransferase) gene expression at a level sufficient to allow selection of transformed soybean embryogenic culture lines. A number of transformed cell lines were selected showing that the promoter was strong enough. Northern blot analysis of plant tissues did not detect hpt mRNA in the untransformed control or in the ASA2-hpt plants except in developing seeds while hpt mRNA was detected in all tissues of the CaMV35S-hpt positive control line plants. However, when the more sensitive RT-PCR assay was used all tissues of the ASA2-hpt plants except roots and mature seeds were found to contain detectable hpt mRNA. Embryogenic tissue cultures initiated from the ASA2-hpt plants contained hpt mRNA detectable by both northern and RT-PCR analysis and the cultures were hygromycin resistant. Friable callus initiated from leaves of ASA2-hpt plants did in some cases contain hpt mRNA that was only barely detectable by northern hybridization even though the callus was very hygromycin resistant. Thus the ASA2 promoter is strong enough to drive sufficient hpt expression in soybean embryogenic cultures for hygromycin selection and only very low levels of expression were found in most plant tissues with none in mature seeds.     

Expression of a feedback insensitive anthranilate synthase gene from tobacco increases free tryptophan in soybean plants

Soybean [Glycine max (L.) Merr.] embryogenic cultures were transformed by particle bombardment with the feedback-insensitive tobacco anthranilate synthase (AS) gene ASA2 driven by the CaMV 35S promoter and selected using hph as the selectable marker gene. Only one of eight regenerated lines that set seed and contained ASA2 expressed the gene highly and contained increased free tryptophan (Trp) levels in leaves, seeds and embryogenic cultures. Leaf extracts of the ASA2 expressing line contained about twice as much AS enzyme activity as the untransformed control and this activity was only slightly more feedback-insensitive. Amino acid analysis showed that both leaves and embryogenic tissue cultures of the ASA2 expressing line had four to five-times the normal levels of free Trp and slightly higher free tyrosine and phenylalanine. The seed total Trp content was only slightly increased. Metabolic profiling-analysis by GC-MS detected no other consistent differences. These studies show that the ASA2 gene can be expressed in soybean and that modest changes in Trp synthesis occurs.     

Expression of a fungal cyanamide hydratase in transgenic soybean detoxifies cyanamide in tissue culture and in planta to provide cyanamide resistance

Embryogenic tissue cultures of soybean were transformed by particle bombardment with a vector pCHZ-II that carries the coding sequence for cyanamide hydratase (Cah), an enzyme that converts toxic cyanamide to urea, from the soil fungus Myrothecium verrucaria. The Cah gene was driven by the constitutive Arabidopsis thaliana actin-2 promoter and terminated with its cognate terminator. This vector also carries the hygromycin phosphotransferase gene (hpt) driven by the potato (Solanum tuberosum) ubiquitin-3 promoter. Twelve individual lines of transgenic plants that were obtained under hygromycin selection expressed Cah mRNA and exhibited resistance to hygromycin in leaf tissue culture, while the untransformed tissues were sensitive. Cah enzyme activity was present in extracts of transformed leaves and embryogenic tissue cultures when measured by a colorimetric assay and the presence of the Cah protein was confirmed by enzyme-linked immunosorbent assay (ELISA). Cah expression detoxified cyanamide in leaf callus and embryogenic cultures as well as in whole plants as shown by cyanamide resistance. The Cah-expressing plants grew and set seeds normally indicating that the Cah enzyme activity did not affect soybean plant metabolism. We also describe a test whereby callus was formed on cultured leaf tissue in the presence of hygromycin or cyanamide only if the hpt or Cah gene was expressed, respectively. This test is a convenient and cost-effective way to follow the marker gene in the primary regenerated plants and subsequent generations, which is particularly reliable for the hpt gene expression using hygromycin.     

Regeneration of fertile transgenic indica (group 1) rice plants following microprojectile transformation of embryogenic suspension culture cells

Regenerable embryogenic suspensions of elite Indica (group 1) rice varieties IR24, IR64, IR72 and an advanced Indica rice breeding line IR57311-95-2-3 were established within 6–8 weeks from 3–4 week old calli derived from mature seeds. Transgenic rice plants were obtained by introducing a plasmid carrying genes encoding hygromycin phosphotransferase (hph, conferring resistance to hygromycin B) and ß-glucuronidase (uidA), both driven by the CaMV 35S promoter, via particle bombardment of embryogenic suspensions. The effect of osmotic conditioning on transformation was evaluated. Regenerated plants were resistant to hygromycin B and expressed the uidA (GUS) gene. The growth of mother plants (R₀) was normal and seeds were produced. Southern blot analysis of R₀ and R₁ plants showed that hygromycin resistant plants contained intact hph genes that were inherited in a Mendelian fashion. A protocol for a simple, efficient, repeatable, genotype- and environment-independent Indica rice transformation system is described.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA -naphthalene acetic acid - kb kilobase - GUS ß-glucuronidase - hph hygromycin B phosphotransferase     

Modification of isoflavones in soybean seeds via expression of multiple phenolic biosynthetic genes

To modify the level and composition of isoflavones, the important bioactive constituents of soybean seeds, soybean was transformed via co-bombardment of embryogenic cultures with three DNA cassettes containing the CHS6-chalcone synthase and IFS2-isoflavone synthase genes, and a fragment of PAL5-phenylalanine ammonia-lyase gene, all in sense orientation under the lectin promoter mixed with the selectable marker gene, HPT (hygromycin phosphotransferase) under the 35S promoter. Four of six fertile lines produced integrated all four genes.Isoflavone levels were lower in T1 mature seeds of 5 of the 6 lines compared to the control. Transgene segregation was found in one selected line, with formation of additional sublines with different transgene composition found also in the homozygous plants. Decreased isoflavone concentrations (by about 70%) were found in T4 homozygous seeds of the two lines studied in detail here. The embryo axes accumulated most of the glycitein and contained a higher isoflavone concentration than the cotyledons. Expression of transgenes driven by the lectin promoter reduced the isoflavone concentration only in the cotyledons and not in embryo axes, indicating that this promoter is preferably active in cotyledons.     

Expression of CaMV35S-GUS gene in transgenic rice plants

Summary To understand the properties of the cauliflower mosaic virus (CaMV) 35S promoter in a monocotyledonous plant, rice (Oryza sativa L.), a transgenic plant and its progeny expressing the CaMV35S-GUS gene were examined by histochemical and fluorometric assays. The histochemical study showed that -glucuronidase (GUS) activity was primarily localized at or around the vascular tissue in leaf, root and flower organs. The activity was also detected in the embryo and endosperm of dormant and germinating seeds. The fluorometric assay of various organs showed that GUS activity in transgenic rice plants was comparable to the reported GUS activity in transgenic tobacco plants expressing the CaMV35S-GUS gene. The results indicate that the level of expression of the CaMV 35S promoter in rice is similar to that in tobacco, a dicotyledonous plant, suggesting that it is useful for expression of a variety of foreign genes in rice plants.     

A model for a bioconversion system with the promoter of the parAt gene,which confers a high level of expression of a transgene in hairy roots

The promoter of the protoplast auxin-regulated (parAt) gene of tobacco, which is expressed throughout the tissues of hairy roots, can be useful for developing a bioconversion system with hairy roots. The parAt gene is shown to be expressed in roots of seedlings and in those of mature tobacco plants. The 5-upstream region of parAt was fused to the coding sequence of the ß-d-glucuronidase (GUS) gene to generate the parAt-GUS fusion gene, which was introduced into the binary vector for Agrobacterium. Hairy roots that carried the fusion gene were obtained (parAt-GUS/hairy root) by infecting tobacco plants with A. rhizogenes carrying the fusion gene in the binary vector. Biochemical analysis with 4-methylumbelliferyl ß-d-glucuronide (MUG), a substrate for GUS, showed that the level of GUS activity was tenfold higher than that of hairy roots carrying the reporter GUS gene, which is linked to the cauliflower mosaic virus 35S RNA promoter (35S-GUS/hairy root). We also examined the rate of conversion of MUG to 4-methylumbel-liferone (MU) by hairy roots when MUG was added to the culture medium of the parAt-GUS/hairy roots. The hairy roots converted MUG to MU at more than ten times as high efficiency as the 35S-GUS/hairy roots. In addition to tobacco, the parAt-GUS gene was similarly expressed in hairy roots from Atropa and Arabidopsis. These results suggest that the promoter of the parAt gene is a useful tool for conversion of various metabolites by hairy root cultures. Correspondence to: Y. Machida     

Screening for Arabidopsis mutants affected in the Nii gene expression using the Gus reporter gene

A mutant screen was developed to isolate Arabidopsis thaliana mutants affected in the regulation of the nitrate assimilation pathway. A fusion between the tobacco Nii1 gene (that encodes a foliar nitrite reductase involved in nitrate assimilation) and the Gus reporter gene was introduced into A. thaliana , and shown to be properly regulated by nitrate. Moreover, β -glucuronidase (GUS) activity in the transgenic plants was essentially detected in the cotyledons and leaves, showing that the organ-specific expression of the tobacco Nii1 gene was retained in Arabidopsis . M2 plantlets derived from mutagenized seeds homozygous for the Nii-Gus fusion were screened by histochemical staining of whole plates for GUS activity after growth on nitrate or glutamine. About 250 progenies were screened, leading to the isolation of plants showing an enhanced or reduced staining compared to the control non-mutagenized plants. Several mutants were analyzed for the transmission of the phenotype to the M3 generation, as well as for levels of GUS or nitrite reductase activities or mRNA levels. A major problem encountered during the screening was the high background of false positives that reproducibly showed altered GUS histochemical staining compared to control plants and did not, however, display any changes in GUS activity levels. One interesting family of mutants was isolated that overexpressed GUS activity and Nii mRNA in the absence of nitrate. These mutants turned out to be cnx mutants impaired in the molybdenum cofactor biosynthesis that is necessary for nitrate reductase activity. These results may indicate that active nitrate reductase is necessary for a correct regulation of nitrate assimilation genes by nitrate.     

Transfer of Lysine-rich Protein Gene into Rice and Production of Fertile Transgenic Plants

Lysine-rich protein gene (lys) was cloned from Psophocarpus tetragonolobus (L.) DC. A plant expression plasmid was constructed and lys gene was under the control of maize ubiquitin promoter which is the highest efficient monocotyledon promoter. The plasmid was introduced into rice embryogenic calli by microprojectile bombardment. The regenerated fertile plants were obtained by effective selection for hygromycin B resistance. Genomic PCR and Southern blotting analyses showed that the lys gene has been integrated into rice genome. Simultaneously, the results of GUS histochemical assay demonstrated the transgenic rice plants. Data analysis showed that lysine content in most of the 11 transgenic plants is differently improved, and in one of them increased by 16.04%.     

Use of 4-methylindole or 7-methyl-DL-tryptophan in a transformant selection system based on the feedback-insensitive anthranilate synthase α-subunit of tobacco (ASA2)

Effective selectable markers are needed for basic research and commercial applications that do not involve antibiotic or herbicide resistance. A novel selection system based on a feedback-insensitive anthranilate synthase α-subunit of tobacco (ASA2) as selectable marker using either 4-methylindole (4MI) or 7-methyl-DL-tryptophan (7MT) as the selection agent was developed. We found that these two components were able to discriminate better between ASA2 expressing and untransformed lines than the most commonly used analog 5-methyltryptopan (5MT) in the seedling growth inhibition test. We successfully integrated an expression cassette containing an ASA2 cDNA driven by a cauliflower mosaic virus 35S promoter into tobacco leaf discs by A. tumefaciens and selected transgenic plants on medium supplemented with 300 μM of 7MT or 4MI. Due to the expression of the feedback-insensitive ASA2, the transgenic lines produced showed higher free tryptophan (Trp) concentrations than the untransformed WT control. These results demonstrate the feasibility of the selection system with the ASA2 gene in combination with the use of Trp or indole analogs as selective agent.     

Microprojectile-DNA delivery in conifer species: factors affecting assessment of transient gene expression using the β-glucuronidase reporter gene

The Biolistic^® microprojectile DNA-delivery method was used to test the usefulness in conifers of eight gene constructs based on the 35S promoter, the AMV translational enhancer, and gene fusion between the P-glucuronidase and the neomycin phosphotransferase II genes. The evaluation was done with embryogenic cells of Picea glauca, where the relative strengths of the promoters were 35S-35S-AMVE>35S-AMVE>35S-35S>35S as evaluated by transient gene expression. The fusion gene of GUS and NPT II gave lower levels of transient gene expression than the unfused GUS gene as detected by X-GLU histochemical assays. Experiments comparing the EM promoter of wheat and the 35S-35S-AMVE promoter (with and without fusion between GUS and NPT II) were done in Picea rubens, P. mariana, P. glauca, and Larix x eurolepis. The unfused gene with the 35S-35S-AMVE promoter gave higher levels of transient gene expression than the fused GUS-NPT II gene. The fluorescent MUG assay was more sensitive than the histochemical X-GLU assay to detect the activity of the -glucuronidase gene.Abbreviations AMV alfalfa mosaic virus - AMVE alfalfa mosaic virus translational enhancer - EM protein of mature wheat embryo - GUS P-glucuronidase gene - MUG 4-methylumbelliferyl -D-glucuronide - NPT II neomycin phosphotransferase - X-GLU 5-bromo-4-chloro-3-indolyl -D-glucuronic acid     

高赖氨酸蛋白基因导入水稻及可育转基因植株的获得

构建了一个植物高效表达质粒,使来源于四棱豆（Psophocarpus tetragonolobus(L.)DC)的高赖氨酸蛋白基因（lys）受控于单子叶植物ubiqutin强启动子下表达。用基因枪法将其导入水稻(Oryza sativa L.)幼胚诱导的愈伤组织,经潮霉素抗性筛选,得到可育的再生植株。经PCR和Southem blotting检测,表明该基因已整合到水稻的基因组织。GUS组织化学染色表明转基因水稻植株的叶、茎和根中均有gus基因的表达。测定112株转基因水稻叶片中赖氨酸叶量,大部分植株有不同程度的提高,最高幅度为16．04％。     

Tissue-specific and ABA-regulated Maize GIN gene expression in transgenic tobacco

Summary To study the regulatory functions of the ON promoter region, a ppG1b1GUS construct, consisting of 1402 bp 5 flanking sequence ofGlbl, 1919 by GUS coding sequence, and 283 by 3 NOS terminator, was cloned into a binary vector and introduced into tobacco plants byAgrobacterium-mediated transformation. Histochemical GUS assays of T_o tobacco mature seeds indicate that theGlbl promoter drives GUS expression in ABA treated seeds. Further GUS assays of the T, seeds at different developmental stages revealed that without ABA treatment, theGibl promoter drives GUS expression in immature seeds. The results from both T_o and T₁ tobacco plants indicated thatGlbl-driven GUS expression in tobacco is embryo specific.