Herbicide Resistance Gene Research Articles

Starch Characteristics of Transgenic Wheat (Triticum aestivum L.) Overexpressing the Dx5 High Molecular Weight Glutenin Subunit are Substantially Equivalent to Those in Nonmodified Wheat

The effects of engineering higher levels of the High Molecular Weight Glutenin Dx5 subunit on starch characteristics in transgenic wheat (Triticum aestivum L.) grain were evaluated. This is important because of the interrelationship between starch and protein accumulation in grain, the strong biotechnological interest in modulating Dx5 levels and the increasing likelihood that transgenic wheat will be commercialized in the U.S. Unintended effects of Dx5 overexpression on starch could affect wheat marketability and therefore should be examined. Two controls with native levels of Dx5 were used: (i) the nontransformed Bobwhite cultivar, and (ii) a transgenic line (Bar-D) expressing a herbicide resistant (bar) gene, and they were compared with 2 transgenic lines (Dx5G and Dx5J) containing bar and additional copies of Dx5. There were few changes between Bar-D and Dx5G compared to Bobwhite. However, Dx5J, the line with the highest Dx5 protein (×3.5) accumulated 140% more hexose, 25% less starch and the starch had a higher frequency of longer amylopectin chains. These differences were not of sufficient magnitude to influence starch functionality, because granule morphology, crystallinity, amylose-to-amylopectin ratio, and the enthalpy of starch gelatinization and the amylose-lipid complex melting were similar to the control (P > 0.05). This overall similarity was borne out by Partial Least Squares-Discriminant Function Analysis, which could not distinguish among genotypes. Collectively our data imply that higher Dx5 can affect starch accumulation and some aspects of starch molecular structure but that the starches of the Dx5 transgenic wheat lines are substantially equivalent to the controls. Transgenic manipulation of biochemical pathways is an effective way to enhance food sensory quality, but it can also lead to unintended effects. These spurious changes are a concern to Government Regulatory Agencies and to those Industries that market the product. In this study we examined if making "specific" changes to the composition of gluten proteins in wheat seeds would simultaneously alter starch, as their synthesis is interrelated and the molecular structure of both determine flour functionality. This information may be used to address issues of "substantial equivalence" and to inform Industrial End-Users of possible changes in product performance.

Development of near‐isogenic lines and identification of markers linked to auxinic herbicide resistance in wild mustard (Sinapis arvensis L.)

Auxinic herbicides are widely used for selective control of many broadleaf weeds, e.g. wild mustard. An auxinic-herbicide-resistant wild mustard biotype may offer an excellent model system to elucidate the mechanism of action of these herbicides. Classical genetic analyses demonstrate that the wild mustard auxinic herbicide resistance is determined by a single dominant gene. Availability of near-isogenic lines (NILs) of wild mustard with auxinic herbicide resistance (R) and herbicide susceptibility (S) will help to study the fitness penalty as well as the precise characterization of this gene. Eight generations of backcrosses were performed, and homozygous auxinic-herbicide-resistant and auxinic-herbicide-susceptible NILs were identified from BC(8) F(3) families. S plants produced significantly more biomass and seed compared with R plants, suggesting that wild mustard auxinic herbicide resistance may result in fitness reduction. It was also found that the serrated margin of the first true leaf was closely linked to auxinic herbicide resistance. Using the introgressed progeny, molecular markers linked to auxinic herbicide resistance were identified, and a genetic map was constructed. The fitness penalty associated with the auxinic herbicide resistance gene may explain the relatively slow occurrence and spread of auxinic-herbicide-resistant weeds. The detection of the closely linked markers should hasten the identification and characterization of this gene.

Gabaculine selection using bacterial and plant marker genes (GSA-AT) in durum wheat transformation

Selectable marker genes are widely used for the efficient transformation of crop plants. In most cases, selection is based on antibiotic or herbicide resistance genes because they tend to be most efficient. The SynechococcushemL gene has been successfully employed as a selectable marker for tobacco and alfalfa genetic transformation, by using gabaculine as the selective agent. The gene conferring gabaculine resistance is a mutant form of the hemL gene from Synechococcus PCC6301, strain GR6, encoding a gabaculine insensitive form of the glutamate1-semialdehyde aminotransferase (GSA) enzyme. In the present study we compared the transformation and selection efficiency of the common selection method based on the Streptomyces hygroscopicusbar gene conferring resistance to Bialaphos®, with both the Synechococcus hemL gene and a Medicago sativa mutated GSA gene (MsGSAgr) conferring resistance to phytotoxin gabaculine. Callus derived from immature embryos of the durum wheat cultivar Varano were simultaneously co-bombarded with bar/hemL and bar/MsGSAgr genes. After gene delivery, the marker genes were individually evaluated through all the selection phases from callus regeneration to adult plant formation, and compared for their transformation and selection efficiency. The integration of the three genes in the T0 generation was confirmed by PCR analysis with specific primers for each gene and southern blot analysis. Both Synechococcus hemL and MsGSA were more efficient than bar for biolistic transformation (2.8% vs. 1.8% and 1.1% vs. 0.5%) and selection (79% vs. 43% and 87% vs. 50%). Thus, an efficient selection method for durum wheat transformation was established that obviates the use of herbicide resistance genes.

Fertile transgenic Brachiaria ruziziensis (ruzigrass) plants by particle bombardment of tetraploidized callus

We have produced transgenic plants of the tropical forage crop Brachiaria ruziziensis (ruzigrass) by particle bombardment-mediated transformation of multiple-shoot clumps and embryogenic calli. Cultures of multiple-shoot clumps and embryogenic calli were induced on solidified MS medium supplemented with 0.5mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 2mg/L 6-benzylaminopurine (BAP) or 4mg/L 2,4-D and 0.2mg/L BAP, respectively. Both cultures were bombarded with a vector containing an herbicide resistance gene (bar) as a selectable marker and the β-glucuronidase (GUS) reporter gene. Sixteen hours after bombardment, embryogenic calli showed a significantly higher number of transient GUS expression spots per plate and callus than multiple-shoot clumps, suggesting that embryogenic callus is the more suitable target tissue. Following bombardment and selection with 10mg/L bialaphos, herbicide-resistant embryogenic calli regenerated shoots and roots in vitro, and mature transgenic plants have been raised in the greenhouse. Polymerase chain reaction (PCR) and DNA gel blot analysis verified that the GUS gene was integrated into the genome of the two regenerated lines. In SacI digests, the two transgenic lines showed two or five copies of GUS gene fragments, respectively, and integration at different sites. Histochemical analysis revealed stable expression in roots, shoots and inflorescences. Transgenic plants derived from diploid target callus turned out to be sterile, while transgenics from colchicine-tetraploidized callus were fertile.

Cotton genetic resources. A review

Since 6000 BC, cotton has been cultivated for lint fiber, which now dominates the natural textile industry worldwide. Common resources such as an integrated web database, a microsatellite database, and comparative quantitative trait loci (QTL) resources for Gossypium have accelerated the progress towards quantifying the impact of repeated human dispersals and selection regimes on various gene pools of the genus Gossypium. Out of 50 Gossypium species, four have been domesticated—two diploids and two tetraploids—for elimination of hard seed coat, improvement in lint percentage of about 40% and fiber length of 22%, larger boll size, and day-neutral reproductive habit. The major drawback of domestication is the lack of genetic diversity. This lack of genetic diversity is observed more in Gossypium hirsutum L. cultivars characterizing upland cotton than in Gossypium barbadense, typical of Pima and Egyptian cotton. Much of the genetic diversity among G. barbadense cultivars is attributed to the introgression of G. hirsutum alleles. This process highlights the importance of introgression of new alleles from accessions of all the Gossypium species into cultivated cotton species. Among the genomic resources, about 16,162 publicly available SSRs and 312 mapped cotton RFLP sequences containing simple sequence repeat (SSR), restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), and random amplified polymorphic DNA (RAPD) markers have been surveyed on numerous mapping populations, and developed about 26 linkage maps (SSR, RFLP, AFLP, and RAPD). Reports show the identification of DNA markers associated with over 29 important traits or QTLs such as fiber quality and yield, leaf and flower morphology, trichome density and their distribution, and disease resistance. In comparative mapping studies, 432 QTLs mapped on 11 different mapping populations were aligned on a high-density reference map containing 3,475 loci. In a meta-analysis study of over 1,000 QTLs obtained from backcross population and recombinant inbred line populations derived from the same parents, most consistent meta-clusters were reported for fiber color, fineness, and length. For exploring the function of genes, the targeting induced local lesions in genomes (TILLING) approach—avoiding gene transfer process was used for identifying a brassino steroid receptor gene that is involved in fiber development. Lastly, cotton genome has been enriched with genes isolated from distantly related organisms using various transformation methods. For example, Cry1Ac, Cry1Ab, and herbicide-resistant genes were transformed in cotton that covered a vast majority of cotton acreage worldwide. Here the authors discuss investigations for improving the efficacy of transformation and regeneration systems, and for searching new genes or silencing the unwanted cotton genes using RNAi technology. We suggest initiating projects on sequencing the diploid and tetraploid genomes for exploring the extent of genetic variations, developing TILLING populations, initiating nested association mapping studies, and developing third generation genetically modified cotton, collectively setting the stage for sustaining cotton production under continually changing production conditions, climates, and human needs.

Exponential advances in Forest Genetics: the past 25 years and the next 25 weeks

Exponential advances in Forest Genetics: the past 25 years and the next 25 weeks

Gene flow increases the initial frequency of herbicide resistance alleles in unselected Lolium rigidum populations

In two different locations of the Western Australian “wheatbelt”, Lolium rigidum (rigid ryegrass) seeds were collected from organic fields (no herbicide use) and neighbouring conventional fields (persistent herbicide use), the latter infested with herbicide-resistant plants, to investigate the occurrence of gene flow among field populations as revealed by herbicide resistance gene transfer. Herbicides targeting acetyl-CoA carboxylase (ACCase) or acetolactate-synthase (ALS) were used to detect herbicide-resistant plants. Overall, the frequency of plants resistant to ACCase- or ALS-inhibiting herbicides was, respectively, 21% and 74% in the conventional fields and 2% and 37% in neighbouring organic fields. Mutant, herbicide-resistant ACCase and ALS alleles were detected in 16% and 38% of plants from conventional fields and in 0.53% and 3.7% of plants from organic fields. Identical mutant, herbicide-resistant ALS haplotypes were detected both in conventional and organic fields, supporting the occurrence of gene flow between L. rigidum populations in different fields. Gene flow can thus substantially increase the frequency of herbicide-resistant plants in unselected L. rigidum populations. Although gene flow cannot be prevented, it can be limited or managed. Hygiene tactics such as clean crop seed, weed seed removal at harvest and seed destruction post-harvest should be considered in order to minimize gene transfer among farms.

Seeds of a Possible Natural Hybrid between Herbicide-Resistant Brassica napus and Brassica rapa Detected on a Riverbank in Japan

Transgenic herbicide-resistant varieties of Brassica napus, or oilseed rape, from which canola oil is obtained, are imported into Japan, where this plant is not commercially cultivated to a large extent. This study aimed to examine the distribution of herbicide-resistant B. napus and transgene flow to escaped populations of its closely related species, B. rapa and B. juncea. Samples were collected from 12 areas near major ports through which oilseed rape imports into Japan passed—Kashima, Chiba, Yokohama, Shimizu, Nagoya, Yokkaichi, Sakai-Senboku, Kobe, Uno, Mizushima, Kita-Kyushu, and Hakata—and the presence of glyphosate- and/or glufosinate-resistant B. napus was confirmed in all areas except Yokohama, Sakai-Senboku, Uno, and Kita-Kyushu. The Yokkaichi area was the focus because several herbicide-resistant B. napus plants were detected not only on the roadside where oilseed rape spilled during transportation but also on the riverbanks, where escaped populations of B. rapa and B. juncea grew. Samples of B. napus that were tolerant to both herbicides were detected in four continuous years (2005–2008) in this area, suggesting the possibility of intraspecific transgene flow within the escaped B. napus populations. Moreover, in 2008, seeds of a possible natural hybrid between herbicide-tolerant B. napus (2n = 38) and B. rapa (2n = 20) were detected; some seedlings derived from the seeds collected at a Yokkaichi site showed glyphosate resistance and had 2n = 29 chromosomes. This observation strongly suggests the occurrence of hybridization between herbicide-resistant B. napus and escaped B. rapa and the probability of introgression of a herbicide-resistance gene into related escaped species.

Transgenic tetraploid Isatis indigotica expressing Bt Cry1Ac and Pinellia ternata agglutinin showed enhanced resistance to moths and aphids

Co-expression of multiple genes encoding different kinds of insect resistant proteins has been developed to confer a broader spectrum of pest control. Tetraploid Isatis indigotica Fort was transformed with a plasmid, p3300BP, containing Bacillus thuringiensis Cry1Ac gene (Bt) and Pinellia ternata agglutinin gene (Pta) and the selectable marker herbicide resistance gene (Bar) driven by the CaMV35S promoter via Agrobacterium tumefaciens-mediated transformation. The integration and expression of introduced genes in regenerated transgenic plants were confirmed by PCR and Western blot assays. Insect bioassay test demonstrated transgenic lines had significant inhibition to diamondback moths (Plutella xylostella L.) and peach potato aphids (Myzus persicae Sulzer) simultaneously. Our study reported here would be a great motivation for field culture of tetraploid I. indigotica, also providing an efficient molecular breeding strategy to provide insect tolerant plants.

Registration of 34 Sorghum Germplasm Lines Resistant to Acetolactate Synthase–Inhibitor Herbicides

Sorghum [Sorghum bicolor (L.) Moench] germplasm lines designated KS201 through KS234 (Reg. No. GP‐684, PI 659520 to Reg. No. GP‐717, PI 659553) were developed and released by the Kansas Agricultural Experiment Station, Manhattan, KS in 2007. The donor of the herbicide resistance gene, Tailwind, was crossed to adapted‐seed and pollinator‐parent lines. The progenies were treated with the target herbicide, and resistant individuals were repeatedly backcrossed to the adapted parents. Backcrossing was performed until the undesirable wild characteristics from the donor parent were eliminated. Lines KS201 through KS218 are R lines and restore fertility in A1 cytoplasm system; lines KS219 through KS234 are B lines and do not restore fertility in A1 cytoplasm. All lines possess a unique genetic resistance to acetolactate synthase (ALS)–inhibitor herbicide families and are useful breeding materials for the development of commercial hybrids with ALS resistance. The lines are all of combine‐harvester height and reach 50% flowering 60 to 82 d after planting at Manhattan, KS. A majority of the lines are pigmented plant types and have variable grain color and seed sizes within the range common to existing public inbreds.

Enhancing transgenic pea (Pisum sativum L.) resistance against fungal diseases through stacking of two antifungal genes (Chitinase and Glucanase)

One way of enhancing and broadening resistance of plants to different biotic and abiotic stresses is to combine transgenes expressing several genes into a single line. This can be done using different strategies such as crossing, single vector with multiple genes, co-transformation, sequential transformation and IRES elements. In the present study conventional crossing method was used. Parental transgenic lines transformed via Agrobacterium tumefasciens-mediated gene transformation with pGreenII binary vector harbouring a bar gene as selectable marker in combination with the family 19 chitinase gene from Streptomyces olivaceoviridis for one line and 1,3-β-glucanase from barley (Hordeum vulgare) for the other line were used for crossing. Both chitinase and glucanase genes were cloned into pGreenII vector under the control of the constitutive double 35S-promoter from cauliflower mosaic virus. Progenies expressing the two genes were characterised at the molecular level using PCR, RT-PCR and Southern blot analysis, as well as segregation and stability studies of the respective expression levels. Leaf paint assay was used as functional test for herbicide resistant gene. Stable inheritance of the antifungal genes in the transgenic plants was demonstrated. The synergistic effect of crossed plants was tested using in vitro assay which shows higher inhibition of spore germination.

Construction of banana bunchy top nanovirus-DNA-3 encoding the coat protein gene and its introducing into banana plants cv. Williams

Banana ( Musa sp.) is considered as one of the most important fruit crops worldwide as well as in Egypt. The main goal of this study was to construct the open reading frame (ORF) of banana bunchy top nanovirus (BBTV)-DNA-3 that encodes the viral coat protein ( cp ) gene for banana transformation. The previously sequenced BBTV-G-DNA-3-ORF that cloned into plasmid pH1 was used as a template for PCR amplification using two specific primers containing Bam H1 site. A new plasmid called pRHA1 containing the amplified ORF under the control of maize polyubiquitin ( ubi ) promoter was created. The bar gene (herbicide-resistance gene as a selectable marker) cassette ( bar gene, Cauliflower mosaic caulimovirus (CaMV) 35S promoter and nos terminator) was released from plasmid pAB6 using Hind III-digestion and subcloned into the Hind III-digested plasmid pRHA1 to create the plasmid pRHA2 via the microprojectile bombardment transformation system. The plasmid pRHA2 was successfully introduced in the applied banana cultivar. Leaf painting test was conducted to confirm the expression of the bar gene in the putative transformed banana lines. The presence and expression of BBTV-G- cp gene were also detected using some molecular (polymerase chain reaction and dot blot using a cold DNA probe) and serological (ELISA and western blot) techniques, respectively, in the obtained transgenic banana lines.

Evaluation of combinations of paraquat plus photosystem II-inhibiting herbicides for controlling failed stands of maize (Zea mays)

Existing plants remaining in failed stands of maize must be removed prior to planting a subsequent crop, and glyphosate and glufosinate are generally ineffective because of the presence of herbicide-resistant genes in most US maize. Research was conducted at three locations in 2009 to evaluate maize control with paraquat as influenced by (1) the addition of three PSII-inhibiting herbicides, (2) maize growth stage at application with and without addition of atrazine, and (3) time of day of application with and without addition of atrazine. Delaying an application of paraquat beyond the V4 stage (four visible collars) of maize resulted in ineffective control of maize, and the addition of atrazine to paraquat improved control of older plants. A paraquat dose of at least 525 g/ha was often needed for acceptable control of maize. Of the three PSII-inhibiting herbicides evaluated, maize control was generally greater and more consistent with the addition of metribuzin or diuron to paraquat than with atrazine, even though atrazine did often improve control over paraquat alone. Applying paraquat at sunset did improve control over applications made near sunrise, but a PSII-inhibiting herbicide was still needed with paraquat to maximize control of maize. These findings demonstrate that undesirable maize plants can be effectively controlled with combinations of paraquat plus PSII-inhibiting herbicides when applied no later than the V4 stage of maize, and applications in close proximity to sunset can further improve control.

Production of marker-free disease-resistant potato using isopentenyl transferase gene as a positive selection marker

The use of antibiotic or herbicide resistant genes as selection markers for production of transgenic plants and their continuous presence in the final transgenics has been a serious problem for their public acceptance and commercialization. MAT (multi-auto-transformation) vector system has been one of the different strategies to excise the selection marker gene and produce marker-free transgenic plants. In the present study, ipt (isopentenyl transferase) gene was used as a selection marker gene. A chitinase gene, ChiC (isolated from Streptomyces griseus strain HUT 6037) was used as a gene of interest. ChiC gene was cloned from the binary vector, pEKH1 to an ipt-type MAT vector, pMAT21 by gateway cloning and transferred to Agrobacterium tumefaciens strain EHA105. The infected tuber discs of potato were cultured on hormone- and antibiotic-free MS medium. Seven of the 35 explants infected with the pMAT21/ChiC produced shoots. The same antibiotic- and hormones-free MS medium was used in subcultures of the shoots (ipt like and normal shoots). Molecular analyses of genomic DNA from transgenic plants confirmed the integration of gene of interest and excision of the selection marker in 3 of the 7 clones. Expression of ChiC gene was confirmed by Northern blot and western blot analyses. Disease-resistant assay of the marker-free transgenic, in vitro and greenhouse-grown plants exhibited enhanced resistance against Alternaria solani (early blight), Botrytis cinerea (gray mold) and Fusarium oxysporum (Fusarium wilt). From these results it could be concluded that ipt gene can be used as a selection marker to produce marker-free disease-resistant transgenic potato plants on PGR- and antibiotic-free MS medium.

국내·외 GM식물의 개발 및 산업화 현황

지난 십년까지 상업화가 승인된 GM작물들의 특성을 보면 첫 단계에는 제초제저항성, 해충저항성, 바이러스 저항성 유전자가 도입된 작물 등으로 농업적 생산성을 증가시키기 위한 단일유전자가 도입된 것들이었으나 최근 들어 지방산 조성을 변경하거나, 특정 필수아미노산의 함량을 증가시키거나, 또는 화색을 변경 시킨 제 2세대 GM식물들의 승인건수가 점차 증가하는 추세에 있다. 뿐만 아니라, 현재 국가별로 심사 중에 있거나 환경위해성 평가용 자료를 생산하기 위하여 제한된 구역에서 포장시험을 수행하고 있는 것으로 알려진 GM 작물들을 보면 가뭄, 냉해 등에 내성이 강한 무생물적 스트레스 내성 작물, 녹색 성장의 주역이 될 biomass 증가, 또는 바이오디젤연료 생산 작물, 그리고 각종 의료용 및 산용 특수물질을 생산하는 분자농업용 작물 등이 주를 이루고 있는 것으로 조사되었다. 또한 아직은 실험실 또는 연구실 수준에서 성공하여 전문 학술지 또는 다양한 언론매체를 통하여 보도된 자료들을 분석한 결과에서도 이러한 차세대 GM 작물들의 개발건수가 현저하게 증가하고 있다. 특히 분자농업용 GM작물의 개발 건수가 가장 많은 것으로 조사되었다. 국내의 경우에도 비슷한 추세로 다양한 연구과제가 진행 중에 있는 것으로 조사되었으며. 국내에서 개발되어 최초로 환경위해성 평가를 신청한 GM 작물은 제초제 저항성잔디로서 현재까지 심사가 진행 중에 있으며, 이외에도 향후 신청을 위하여 포장시험이 진행 중에 있는 GM 작물들이 벼, 고추, 배추 등 약 10여종으로 이들 중 벼에 관한 건수가 가장 많았으며 그 중에서도 흑명나방 저항성 벼가 가장 많은 연구가 진행되어 곧 환경 위해성 평가를 신청할 것으로 조사되었다. During a last decade, the introduced traits in commercialized GM crops have been diversified from a simple trait such as herbicide resistance gene or insectresistance gene which are related to the crop production into more complicated traits such as modification of fatty acid or essential amino acid composition, modified coloring pattern of flower. In addition, it was investigated that several other GM crops bearing more refined traits expected to lead next generation are also awaiting for risk assessment (RA) or under field test for the preparation of RA in the near future. These GM crops include abiotic stress resistance including drought or cold, increased biomass, production of bioethanol or diesel, production of pharmaceuticals or functional materials for industrial. In particular, in 2008 and 2009, it was reported that the highest number of GM crops for molecular farming are under developed in laboratory or green house level in all the world. Likewise, in Korea, 171 events from 49 plant species are under developed to introduce several important traits. At present, about 10 events are under field test to select elite lines for RA application. For the first time, herbicide resistance turfgrass developed by Korean research team has been submitted for RA and currently under requested for additional data. Moreover, GM rice resistant to leaf roll (folder) disease is expected as a next event to be submitted for RA application.

Characterization of the horseweed (Conyza canadensis) transcriptome using GS‐FLX 454 pyrosequencing and its application for expression analysis of candidate non‐target herbicide resistance genes

The de novo transcriptome sequencing of a weedy plant using GS-FLX 454 technologies is reported. Horseweed (Conyza canadensis L.) was the first broadleaf weed to evolve glyphosate resistance in agriculture, and also is the most widely distributed glyphosate-resistant weed in the United States and the world. However, available sequence data for this species are scant. The transcriptomic sequence should be useful for gene discovery, and to help elucidate the non-target-based glyphosate resistance mechanism and the genomic basis of weediness. Sequencing experiments yielded 411 962 raw reads, an average read length of 233 bp and a total dataset of 95.8 Mb (NCBI accession number SRA010952). After trimming and quality control, 379 152 high-quality sequences were retained and assembled into contigs. The assembly resulted in 31 783 unique transcripts, including 16 102 contigs and 15 681 singletons. The average coverage depth for each contig and each nucleotide position was 22-fold and 12-fold respectively. A total of 16 306 unique sequences were annotated by searching a custom plant protein database. The utility of the transcriptome data was demonstrated by further exploration of ABC transporters, which were previously hypothesized to play a role in non-target glyphosate resistance. Real-time RT-PCR primers were designed from the transcriptome data, which made it possible to assess expression patterns of 17 ABC transporters from resistant and susceptible horseweed accessions from Tennessee, with and without glyphosate treatment. These results show that GS-FLX 454 sequencing is a powerful and cost-effective platform for the development of functional genomic tools for a weed species.

Backcrosses to Brassica napus of hybrids between B. juncea and B. napus as a source of herbicide-resistant volunteer-like feral populations

Introgression between genetically modified (GM) crops and wild relatives is considered to potentially modify the genetic background of the wild species. The emergence of volunteer-like feral populations through backcross of hybrids to the crop is also a concern. The progeny of spontaneous hybrids between mutant herbicide-resistant oilseed rape ( Brassica napus) and wild B. juncea was obtained. Parents, F 2 and BC 1 to B. napus were planted together in the field so as to study their performance. The chromosome number of BC 1 followed a Normal distribution. Mendelian ratio of the herbicide-resistance gene was found. The F 2 produced less seeds than B. napus, and BC 1 had intermediate production. Herbicide-resistant BC 1 were not different of their susceptible counterparts for plant weight, seed weight and seed number, but most of them exhibited B. napus morphology and larger flowers than the susceptible BC 1. They displayed an important genetic variability allowing further adaptation and propagation of the herbicide-resistance gene. Pollen flow to susceptible plants within the mixed stand was observed. As a consequence, the resistant BC 1 produced with B. napus pollen could frequently occur and easily establish as a false feral crop population within fields and along roadsides.

The application of the yeast N-acetyltransferase MPR1 gene and the proline analogue L-azetidine-2-carboxylic acid as a selectable marker system for plant transformation

The yeast N-acetyltransferase MPR1 gene has previously been shown to confer resistance to the toxic proline analogue azetidine-2-carboxylic acid (A2C) in yeast and transgenic tobacco. Here experiments were carried out to determine if MPR1 and A2C can work as a selectable marker system for plant transformation. The MPR1 gene was inserted into a binary vector under the control of the cauliflower mosaic virus 35S promoter and nopaline synthase terminator, and transformed into tobacco via the Agrobacterium tumefaciens-mediated leaf disc method. A2C was applied in the selection medium to select for putative transformants. PCR analysis showed that 28.4% and 66.7% of the plantlets selected by 250 μM and 300 μM A2C were positive for the MPR1 gene, respectively. Southern and northern blot analysis and enzyme activity assay confirmed the stable gene incorporation, transcription, and translation of the MPR1 transgene in the transgenic plants. The transgene-carrying T1 progeny could be distinguished from the recessive progeny when grown on 400, 450, or 500 μM A2C. Examination of the metabolism of 22 transgenic plants by gas chromatography–mass spectrometry profiling did not reveal any significant changes. In conclusion, the results demonstrate that MPR1/A2C is a safe and efficient selection system that does not involve microbial antibiotic or herbicide resistance genes. Recent studies showed that MPR1 can protect yeast against oxidative stresses by decreasing the accumulation of the proline catabolite Δ1-pyrroline-5-carboxylate (P5C). However, H2O2 treatment resulted in contradictory responses among the five transgenic lines tested. Further experiments are required to assess the response of MPR1 transgenic plants under oxidative stress.

AtMYB12 gene: a novel visible marker for wheat transformation

Efficiency of plant transformation is less than optimal for many important species, especially for monocots which are traditionally recalcitrant to transformation, such as wheat. And due to limited number of selectable marker genes, identification or selection of those cells that have integrated DNA into appropriate plant genome and to regenerate fully developed plants from the transformed cells, becomes even more difficult. Some of the widely used marker genes belong to the categories of herbicide or antibiotic resistance genes and flourescent protein genes. As they become an integral part of plant genome along with promoters prokaryotic or eukaryotic origin, there are certain health and environmental concerns about the use of these reporter genes. These marker genes are also inefficient with respect to time and space. In this study we have found a novel visible selection agent AtMYB12, to screen transgenic wheat, with in days after transformation. Transformed coleoptiles as well as cells regenerating from transformed cultured scutella, phenotypically exhibit purple pigmentation, making selection possible in limited and reasonable cost, time and space.

Transgenic Cucumis sativus Expressing the Hepatitis B Surface Antigen

A DNA fragment encoding the hepatitis B virus surface antigen was amplified from a positive blood (hepatitis B) sample and introduced into the pET 32c prokaryotic expression vector. The gene encoding the HBV surface protein antigen was introduced into pCAMBIA 3300, and immobilized into Agrobacterium tumefaciens strain LBA4404. Cotyledonary leaf sections of Cucumis sativus (cucumber) cv ‘Swarnamukhi’ were cocultivated with Agrobacterium harboring the binary vector pCAMBIA 3300 carrying the HBV surface antigen gene driven by the CaMV35S promoter and the herbicide resistance gene phosphinothricin. Putative transformed shoots were induced on a Murashige and Skoog (MS) medium containing phosphinothricin, and these were then rooted on MS basal medium supplemented with 1 mg/L Indole 3-butyric acid (IBA). Integration of the T-DNA into in putative transgenic plants was confirmed by PCR and Southern blot analyses. RT-PCR and Northern blot analyses were conducted to determine RNA expression. Levels of expression in transgenic plants were confirmed by Western blot analysis, and quantification of the protein was determined by enzyme linked immuno assay (ELISA). Molecular mass of the recombinant protein was measured by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) Mass Spectrometry.