Transgenic Brassica Napus Plants Research Articles

Development of transgenic Brassica napus plants with AtATM3 gene to enhance cadmium and lead tolerance

Four popular genotypes of Brassica napus, cv. BARI Sarisha-8, BARI Sarisha-13, Binasarisha-4, and Binasarisha-8 were subjected to cadmium (Cd) and lead (Pb) stress to identify a genotype with enhanced tolerance of Cd and Pb. The goal was to use the selected genotype for genetic engineering, thereby improving traits related to heavy metal tolerance and accumulation to remediate polluted agricultural soils. The genotype, BARI Sarisha-8 with superior tolerance to both Cd and Pb stresses, was chosen for genetic engineering with Arabidopsis thaliana ABC transporter of the mitochondrion 3 (AtATM3) gene. The transformation process involved coculturing the cotyledon explants with Agrobacterium strain GV3101 carrying a binary vector containing the hygromycin phosphotransferase (HPT) gene as a selectable marker and the AtATM3 gene. Transformation efficiency was significantly enhanced with a two-day co-cultivation period on shoot induction medium composed of MS medium, α-naphthaleneacetic acid (0.5 mg/L), and 6-benzyladenine (3 mg/L), supplemented with acetosyringone (20 mg/L), along with a four-day delay in exposing the explants to the selective agent hygromycin. Hygromycin-resistant shoots were obtained by employing a three-step selection process. The refined protocol resulted in a transformation efficiency of 15.38%. Polymerase chain reaction (PCR) analysis confirmed the integration of AtATM3 and HPT genes into the host genome in all recombinant plants. Transgenic B. napus plants expressing the AtATM3 gene exhibited notable improvements in tolerance, demonstrating a 1.4- to 1.7fold increase in Cd tolerance and a 1.3- to 1.5-fold increase in Pb tolerance compared to the wild type (nontransformed) under both Cd and Pb stress conditions.

Comparison of three Agrobacterium-mediated co-transformation methods for generating marker-free transgenic Brassica napus plants

BackgroundGeneration of marker-free transgenic plants is very important to the regulatory permission and commercial release of transgenic crops. Co-transformation methods that enable the removal of selectable marker genes have been extensively used because they are simple and clean. Few comparisons are currently available between different strain/plasmid co-transformation systems, and also data are related to variation in co-transformation frequencies caused by other details of the vector design.ResultsIn this study, we constructed three vector systems for the co-transformation of allotetraploid Brassica napus (B. napus) mediated by Agrobacterium tumefaciens and compared these co-transformation methods. We tested a mixed-strain system, in which a single T-DNA is harbored in two plasmids, as well as two “double T-DNA” vector systems, in which two independent T-DNAs are harbored in one plasmid in a tandem orientation or in an inverted orientation. As confirmed by the use of PCR analysis, test strips, and Southern blot, the average co-transformation frequencies from these systems ranged from 24 to 81% in T0 plants, with the highest frequency of 81% for 1:1 treatment of the mixed-strain system. These vector systems are valuable for generating marker-free transgenic B. napus plants, and marker-free plants were successfully obtained in the T1 generation from 50 to 77% of T0 transgenic lines using these systems, with the highest frequency of 77% for “double T-DNA” vector systems of pBID RT Enhanced. We further found that marker-free B. napus plants were more frequently encountered in the progeny of transgenic lines which has only one or two marker gene copies in the T0 generation. Two types of herbicide resistant transgenic B. napus plants, Bar+ with phosphinothricin resistance and Bar+EPSPS+GOX+ with phosphinothricin and glyphosate resistance, were obtained.ConclusionWe were successful in removing selectable marker genes in transgenic B. napus plants using all three co-transformation systems developed in this study. It was proved that if a appropriate mole ratio was designed for the specific length ratio of the twin T-DNAs for the mixed-strain method, high unlinked co-insertion frequency and overall success frequency could be achieved. Our study provides useful information for the construction of efficient co-transformation system for marker-free transgenic crop production and developed transgenic B. napus with various types of herbicide resistance.

A fructose-1,6-biphosphate aldolase gene from Camellia oleifera: molecular characterization and impact on salt stress tolerance

Fructose 1,6-bisphosphate aldolase (FBA) catalyzes the reverse cleavage of fructose-1,6-bisphosphate into dihydroxyacetone phosphate (DHAP) and 3-phosphate glyceraldehyde (G-3-P). DHAP acts as a precursor for the synthesis of glycerol, an important osmotically compatible solute under salt stress. Using rapid amplification of cDNA ends, the complete sequence of FBA1 cDNA was obtained from Camellia oleifera, a tree originated from China and notable as an important source of edible oil obtained from its seeds. Camellia oleifera FBA1 (CoFBA1) cDNA has a total length of 1,697-bp with 1,179-bp ORF, flanked by 250-bp 5′-UTR and 246-bp 3′-UTR. It was predicted to encode a 392 amino acid protein with a calculated molecular mass of 42.72 kDa and theoretical pI of 8.39. Real-time PCR analysis indicated that the CoFBA1 gene was strongly expressed in stems, weakly in roots, and induced under salt stress to a threshold before dropping at high salt concentrations (12 g L−1 NaCl). Over-expression of recombinant CoFBA1 resulted in increased tolerance to salinity in transgenic Brassica napus plants, which grew better at low salt concentrations than on media without NaCl. At all salt concentrations, the transgenic plants showed improved growth parameters (stem and root lengths and germination rates) in comparison with wild-type plants. These findings suggest that CoFBA1 plays very important roles in salt stress response, improving the survival ability of C. oleifera under salt stress conditions.

Altered seed oil and glucosinolate levels in transgenic plants overexpressing the Brassica napus SHOOTMERISTEMLESS gene

SHOOTMERISTEMLESS (STM) is a homeobox gene conserved among plant species which is required for the formation and maintenance of the shoot meristem by suppressing differentiation and maintaining an undetermined cell fate within the apical pole. To assess further the role of this gene during seed storage accumulation, transgenic Brassica napus (Bn) plants overexpressing or down-regulating BnSTM under the control of the 35S promoter were generated. Overexpression of BnSTM increased seed oil content without affecting the protein and sucrose level. These changes were accompanied by the induction of genes encoding several transcription factors promoting fatty acid (FA) synthesis: LEAFY COTYLEDON1 (BnLEC1), BnLEC2, and WRINKLE1 (BnWRI1). In addition, expression of key representative enzymes involved in sucrose metabolism, glycolysis, and FA biosynthesis was up-regulated in developing seeds ectopically expressing BnSTM. These distinctive expression patterns support the view of an increased carbon flux to the FA biosynthetic pathway in developing transformed seeds. The overexpression of BnSTM also resulted in a desirable reduction of seed glucosinolate (GLS) levels ascribed to a transcriptional repression of key enzymes participating in the GLS biosynthetic pathway, and possibly to the differential utilization of common precursors for GLS and indole-3-acetic acid synthesis. No changes in oil and GLS levels were observed in lines down-regulating BnSTM. Taken together, these findings provide evidence for a novel function for BnSTM in promoting desirable changes in seed oil and GLS levels when overexpressed in B. napus plants, and demonstrate that this gene can be used as a target for genetic improvement of oilseed species.

Expression of anti-sclerotinia scFv in transgenic Brassica napus enhances tolerance against stem rot

Canola is an important agricultural crop imparting a significant contribution to global oilseed production. As such, optimizing yield and quality is of paramount importance and canola production can be significantly affected by sclerotinia stem rot. The utility of recombinant antibody technology in plant protection has been explored by many researchers and shows promise for the generation of new lines of agriculturally significant crops with greater resistance to diseases. The objective of the current study was to generate recombinant pathogen specific antibody (scFv)-expressing transgenic Brassica napus plants with increased tolerance to the phytopathogenic fungus, Sclerotinia sclerotiorum. Transgenic canola (B. napus) lines expressing S. sclerotiorum-specific scFv antibody showed a significant level of tolerance towards S. sclerotiorum as compared to their non-transformed counterparts. Both incidence and progression of S. sclerotiorum-induced disease symptoms were reduced in plants expressing the recombinant scFv.

A Simplified Seed Transformation Method for Obtaining Transgenic Brassica napus Plants

We report here a seed transformation of sonication-assisted, no-tissue culture to rapidly produce transgenic Brassica napus plants. This method comprises the steps of treating seeds by ultrasonic wave, inoculating Agrobacterium tumefaciens with a recombinant ChIFN- a gene and germinating directly of treatment seed on wet filter papers. The obtained transformants were verified by GUS histochemical assay and nested PCR amplification. It suggests that seed transformation has a potential use in genetic transformation of rape.

Increase of the tocochromanol content in transgenic Brassica napus seeds by overexpression of key enzymes involved in prenylquinone biosynthesis

Lipid soluble tocochromanols, only synthesised in photosynthetic organisms, are industrially interesting compounds because of their antioxidative properties and their essential function in nutrition. In order to increase the tocochromanol content in the seed oil of transgenic plants, approaches were undertaken to engineer the flux of substrates and intermediates through the pathway. To this end, we overexpressed genes encoding hydroxyphenylpyruvate dioxygenases, alone or in combination with chimeric homogentisate phytyltransferase and tocopherol cyclase genes, in seeds of transgenic Brassica napus plants and analysed total tocochromanol content and composition. Overexpression of chimeric hydroxyphenylpyruvate dioxygenase genes, both in the cytosol or in the plastids of developing seeds, yielded a slight although significant increase in total tocochromanol level. Coexpression of a hydroxyphenylpyruvate dioxygenase gene with both a homogentisate phytyltransferase gene and a tocopherol cyclase gene elevated this effect with maximum increases of up to two-fold in individual lines and this phenotype was found to be stably inherited. These data showed that the three enzymes are critical in determining the total tocochromanol content in the seed oil of Brassica napus plants, while the tocopherol cyclase, unlike hydroxyphenylpyruvate dioxygenase and homogentisate phytyltransferase, had additionally an effect on the relative abundance of individual tocochromanol species and resulted in an increase of δ-tocopherol and plastochromanol-8 in the seeds.

Development of a novel Agrobacterium-mediated transformation method to recover transgenic Brassica napus plants.

We report here an in planta method to produce transgenic Brassica napus plants. The procedure included Agrobacterium-mediated inoculation of plants at various development stages along with a vacuum infiltration step. The flowering stage appeared to be the most receptive stage for transformation and production of transgenic plants. In some cases, the flowering stage was induced either by cold treatment or by high density planting. Molecular and genetic analysis revealed that single and multiple copy events were generated and that the transgenes were transmitted to the T1 and T2 progeny in a Mendelian fashion.

Transgenic canola lines expressing pea defense gene DRR206 have resistance to aggressive blackleg isolates and to Rhizoctonia solani

We have previously demonstrated that transgenic Brassica napus plants expressing pea DRR206 constitutively are resistant to the hemibiotrophic blackleg fungus, Leptosphaeria maculans isolate PG2. The present work seeks to determine whether DRR206 is effective against a wider range of fungi. Transgenic plants expressing DRR206 exhibit decreased severity of stem canker in adult plants inoculated with aggressive L. maculans isolates PG3 and PG4. Decreased seedling mortality with the biotrophic root pathogen Rhizoctonia solani is also seen. Finally, leaves of DRR206 transgenic plants inoculated with the necrotroph Sclerotinia sclerotiorum show smaller lesions at 48 h after inoculation, leading to a delay, but not a prevention, of disease development. These results demonstrate the effectiveness of DRR206 against several fungal species with three distinct modes of pathogenicity. Although its precise function remains to be determined, a recent report shows that pea DRR206 shares strong amino acid sequence similarity with `dirigent proteins' which couple monolignol radicals to form the lignan (+) pinoresinol.

Phenotypic alterations and component analysis of seed yield in transgenic Brassica napus plants expressing the tzs gene

Cytokinins play an important role in plant development. We investigated the possibility that the nopaline Ti plasmid gene (tzs) from Agrobacterium tumefaciens could encode a protein able to participate in plant cytokinin production and lead to alterations in plant phenotype as a result of the expression of endogenous tzs. tzs was placed under the control of a heat‐inducible promoter from the Zea mays hsp70 gene. The expression of this fused gene was examined in transgenic Brassica napus plants. The tzs gene, which encodes the enzyme dimethylallyl transferase, was used as a cytokinin biosynthetic gene. The expression of the tzs gene was monitored by RNA hybridization and analysis of cytokinin content. Overproduction of cytokinin was observed even when the plants had not been heat‐shocked, and the plants displayed a reduced root system, increased height and branching, and delayed flowering. In addition, a significant increase in seed yield was observed in the transgenic plants, accounted for by increased number of seeds per silique and seed weight. The results suggest that increased levels of cytokinins, through the expression of tzs, are correlated with growth rather than with differentiation processes.

Effect of sulphur levels on transgenic double‐low Brassica napus plants expressing a seed‐specific gene encoding a methionine‐rich 2S albumin

AbstractThe effects of supplying varying levels of sulphur to transgenic Brassica napus plants expressing the Brazil nut 2S albumin in their seeds were studied in the field. The aim was to determine if transgenic plants responded differently to such stress, and whether expression of the transgene would be effected. The transgenic plants were similar to normal plants in their response to sulphur stress. Increases in sulphur concentration were correlated with an increase in fresh and dry matter quantities as well as an increase in the carbon, nitrogen and sulphur levels in the vegetative parts of the plants. No effect on yield parameters was observed. Increasing sulphur levels resulted in an increase in the glucosinolate content, and shifts in fatty acid composition were observed. None of these changes could be correlated with the response of the transgene. The expression of the latter was altered at neither the mRNA nor The protein levels, presumably because the promoter used does not respond directly to sulphur level changes. The procedures used to create the transgenic plants in this work appear not to result in plants that respond to sulphur stress differently from normal plants in the ranges studied.

Oilseed Rape Transformation by Electroporation: Effects of Nurse Culture on Transformation.

Transgenic Brassica napus plants have been obtained after electroporation of hypocotyl protoplasts. We have tested electroporation parameters based on transient expression of β-glucuronidase (GUS) gene in the protoplasts. The number of GUS positive cells 4 days after electroporation was affected by use of tobacco suspension cells as nurse culture as well as the amplitude of the electric pulse. The defined conditions for the electroporation was used for the stable transformation. To obtain transgenic plants, neomycin phosphotransferase II (npt II) or hygromycin phosphotransferase (hph) genes with a GUS gene were co-transformed. Five and six transgenic plants out of 140 kanamycin and 33 hygromycin resistant calli, respectively, were obtained. Presence of the npt II and hph genes in the plants was confirmed by PCR and Southern blot analysis of genomic DNA. GUS activity was detected in one plant out of three hygromycin resistant transgenic plants analyzed.

In‐situ enzyme histochemistry on plastic‐embedded plant material. The development of an artefact‐free β‐glucuronidase assay

SummaryAn in‐situ enzyme histochemical method is described that preserves the tissue and cell structure as well as the enzyme activities. Flower buds at different developmental stages from wild‐type and transgenic Brassica napus plants, the latter containing the GUS gene under the control of a tapetum‐specific promoter, were used as starting material. The method is based on the following principles: processing of the tissue on crushed ice, no fixation but a pretreatment with spermidine, partial dehydration with acetone, and a final embedding in a water‐miscible glycol methacrylate resin at 5°C. This method was used to set up a sensitive p‐glucuronidase histochemical assay with a high resolution. A succinate dehydrogenase assay was included as a control for tissue and cell viability as well as a standard for the relative metabolic activities between different tissues and cell types.

Arrest of embryo development in Brassica napus mediated by modified Pseudomonas aeruginosa exotoxin A

Intracellularly expressed cytotoxins are useful tools both to study the action of plant regulatory sequences in transgenic plants and to modify plant phenotype. We have engineered a low mammalian toxicity derivative of Pseudomonas aeruginosa exotoxin A for intracellular expression in plant cells by fusing the ADP ribosylating domain of the exotoxin gene to plant regulatory sequences. The efficacy of exotoxin A on plant cells was demonstrated by transient expression of the modified exotoxin gene in tobacco protoplasts: the exotoxin gene inhibited the expression of a co-electroporated beta-glucuronidase gene. An exotoxin with an introduced frameshift mutation was also effective at inhibiting beta-glucuronidase expression in the transient assay; the activity of the frameshifted gene was presumably a result of frameshifting during translation or initiation of translation at a codon other than AUG. When fused to napin regulatory sequences, the exotoxin gene specifically arrested embryo development in the seeds of transgenic Brassica napus plants concomitant with the onset of napin expression. The napin/exotoxin chimeric gene did not have the same pattern of expression in tobacco as in B. napus; in addition to exhibiting an inhibition of seed development, the transgenic tobacco plants were male-sterile.

Transgenic Brassica napus plants obtained by cocultivation of protoplasts with Agrobacterium tumefaciens

Hypocotyl protoplasts of German winter oilseed, rape (Brassica napus) lines of double-low quality were transformed using Agrobacterium tumefaciens harbouring pGV 3850∶1103 neo (dimer) containing chimaeric kanamycin resistance reporter genes. Transformed protoplasts were regenerated to fertile and phenotypically normal plants. Transformation was confirmed by kanamycin resistance, nopaline production, neomycinphosphotransferase II activity, and Southern blot hybridization. Seed progeny from self-pollinated transformants expressed the introduced kanamycin resistance as a Mendelian trait.

Transformation of Brassica napus with Agrobacterium tumefaciens based vectors

A reproducible system to produce transgenic Brassica napus plants has been developed using stem segments. Stem segments from 6-7 week old plants were inoculated with an Agrobacterium tumefaciens strain containing a disarmed tumor-inducing plasmid pTiT37-SE carrying a chimeric bacterial gene encoding kanamycin resistance (pMON200). Stem explants were cocultured for 2 days before transfer to kanamycin selection medium. Shoots regenerated directly from the explant in 3-6 weeks and were excised, dipped in Rootone®, and rooted in soil. Transformation was confirmed by opine production, kanamycin resistance, and DNA blot hybridization in the primary transformants. Final proof of transformation was demonstrated by the co-transfer of opine production and kanamycin resistance to progeny in a Mendelian fashion. Over 200 transgenic Brassica napus plants have been produced using this system.