Transformation Of Durum Wheat Research Articles

Effect of type of mature embryo explants and acetosyringone on agrobacterium-mediated transformation of moroccan durum wheat

Drought is one of the major constraints in durum wheat production in the Mediterranean Basin. In order to overcome this problem, the genetic transformation of durum wheat is one of the choices for improvement. However, the recalcitrance to Agrobacterium-mediated transformation in durum wheat (Triticum turgidum L.) is one of the factors limiting a successful genetic transformation. The aim of this study was to investigate the effect of explant type and acetosyringone concentration for the efficient Agrobacterium-mediated genetic transformation of three Moroccan durum wheat varieties (Amria, Chaoui, and Marouane). The mature embryos (intact, halved and pieces) were inoculated with Agrobacterium tumefaciens strain EHA101 harboring the binary vector pTF101.1 containing drought tolerance gene HVA1 from barley, and a selectable marker phosphinothricin (PPT) resistance (bar) gene. The explants were inoculated with A. tumefaciens (cell density OD650 at 0.7) at four different concentrations of acetosyringone (0, 100, 200, and 400 µM). The results showed that embryogenic calli from mature embryos showed higher regeneration and transformation than mature embryo halves and pieces. The integration of the transgene was confirmed by PCR amplification using primers specific to the bar gene, 2x35S promoter, and HVA1 gene. The transformation efficiency ranging from 0.33% to 2.33% was obtained in Amira variety using embryogenic calli and acetosyringone concentrations of 200 and 400 µM. The integration, as well as inheritance of the transgene, was confirmed by PCR amplification in T0 and T1 generations. This is the first report describing a genetic transformation of Moroccan durum wheat varieties via Agrobacterium tumefaciens.

Development of an efficient regeneration system for mature bombarded calli of Moroccan durum wheat varieties

This study examined the effects of various environmental and genetic factors on callus induction and plant regeneration of bombarded calli from mature embryos of durum wheat using the biolistic method. In this study, three Moroccan durum wheat varieties ('Isly', 'Amria', 'Marouane') were cultivated on two induction media (IM1 and IM2) with different nitrogen sources and contents. After that, each variety cultured on both induction media was transferred in to two regeneration media (RM1 and RM2) with different phytohormones, whereas each variety distributed through four combinations of treatments: IM1RM1, IM1RM2, IM2RM1, and IM2RM2. A completely randomized design with five replications per treatment for each genotype was used. Parameters considered in this study were phytohormones, nitrogen source and its content, plant variety, and their interactions. The study found that variety, medium and variety × medium interactions have a statistically significant effect on callus induction and plantlets regeneration. Prior to bombardment, the maximum percentage of callus induction was obtained under IM1. Conversely, the callus survival rate was not affected by the induction media once bombarded for all three varieties. The induction media had a significant effect on all regeneration parameters (p < 0.01). The variety ‘Isly’ showed the best regeneration efficiency after bombardment, with nearly 80% of plantlets regenerated under IM1 and RM2 combination. These media can be used for genetic transformation of durum wheat

Screening Auxin Response, In Vitro Culture Aptitude and Susceptibility to Agrobacterium-Mediated Transformation of Italian Commercial Durum Wheat Varieties

The development of a robust Agrobacterium-mediated transformation protocol for a recalcitrant species like durum wheat requires the identification and optimization of factors affecting T-DNA delivery and plant regeneration. The purpose of this research was to compare the behavior of diverse durum wheat genotypes during in vitro culture and Agrobacterium tumefaciens-mediated transformation, using immature embryos as explants. Apart from plant genotype, two of the main influencing factors for a successful genetic transformation have been examined here, i.e., auxin source (Dicamba and 2,4-D) and duration of the pre-culture period (one, seven and 21 days). The addition of Dicamba to the media in combination with seven days pre-cultivation resulted in a general enhancement of T-DNA delivery for most of the analyzed cultivars, as revealed by β-glucuronidase (GUS) histochemical assay. Although all genotypes were able to produce calli, significant differences were detected in regeneration and transformation efficiencies, since only two (Karalis and Neolatino) out of 14 cultivars produced fertile transgenic plants. The estimated transformation efficiencies were 6.25% and 1.66% for Karalis and Neolatino, respectively, and χ2 analysis revealed the stable integration and segregation of the gus transgene in T1 and T2 progenies. This research has demonstrated that, among the influencing factors, genotype and auxin type play the most important role in the success of durum wheat transformation.

Effect of Ticarcillin, Cefotaxime and Carbenicillin doses on Efficacy of Tissue Culture Parameters Associated with Durum wheat Transformation

Agrobacterium-mediated genetic trans-formation is one of the most efficient and widely used method for plant transformation. In this study three types of β-lactam antibiotics (ticarcillin, cefotaxime and carbenicillin) individu-ally and in different concentrations and combi-nations, were used to suppress post co-cultivation growth of Agrobacterium tumefa-ciens (strain EHA105, harboring binary vector PBI 121) during plant transformation. High concen-tration of antibiotics used for eliminating Agro-bacterium has negative effect on callus growth, plant regeneration and embryo germination. Therefore the optimal concentration of antibi-oticthat efficiently prevents growth of Agrobac-terium without impairing the above parameters needs to be identified. Different concentrations of ticarcillin, cefotaxime, and carbenicillin (50–250 mg/l) individually and in combination (25–100 mg/l) were added to callusing, regeneration and germination media. Individual antibiotics at 200 mg/l and in combination at 50 mg/l and above were effective for control of agrobacte-rium. In the medium containing combination of ticarcillin, cefotaxime and carbenicillin (50 mg/l each), relative callus weight gain (21.73 mg), percentage plant regeneration (80%) and percentage germination rate (66.12%) was high-er than media containing otherwise effective doses of single antibiotic (200 and 250 mg/l) or combinations of three antibiotics at the rate of 75 mg/l and 100 mg/l of each.

Overexpression of Puroindoline a gene in transgenic durum wheat (Triticum turgidum ssp. durum) leads to a medium–hard kernel texture

Durum wheat is the second-most widely grown wheat species, and is primarily used in the production of pasta and couscous. The grain utilization of durum wheat is partly related to its very hard kernel texture because of the lack of the D genome and consequentially the Puroindoline genes. Our previous study reported the transformation of durum wheat with the Puroindoline a (Pina) gene. Here, we characterized the transgenic durum wheat lines expressing the Pina gene, and studied the effects of PINA on grain texture and other kernel characteristics. SDS-PAGE and Western blotting results demonstrated that starch-bound PINA levels of Pina-overexpressing lines were lower than that of Pina-positive control, common wheat cv. Chinese Spring, suggesting a weak association of PINA protein with starch granules in the absence of Pinb. Grain hardness analysis and flour milling tests indicated that the overexpression of PINA resulted in decreased grain hardness and increased flour yield in transgenic durum wheat lines. The agronomic performance of the transgenic and control lines was also examined and it was found that no significant differences in measured traits were observed between Pina-overexpressing and control lines in the 2-year field trials. Since grain hardness strongly affects milling and end-use qualities, the development of medium–hard-textured durum wheat lines is not only of significance for our knowledge of grain hardness and Puroindolines, but also has practical implications for plant breeders and food technologists for the expansion of utilization of durum wheat.

Efficient callus induction and plantlets regeneration in durum wheat using mature embryos

The use of mature embryos as explants to initiate cultures is a best alternative to save time and costs, especially for producing somatic embryos for genetic transformation of durum wheat. However, plantlets regeneration from cultures derived from matured embryos is usually low. In this study, we tested matured embryos as explants from eight Moroccan durum wheat varieties (‘Irden’, ‘Marzak’, ‘Kyperounda’, ‘Isly’, ‘Amria’, ‘Karim’, ‘Marouane’ and ‘Tomouh’) to define suitable culture media for obtaining high frequencies of somatic embryogenesis and in vitro plantlets regeneration. For this purpose, we tested five induction and maintenance media (M1 to M5) based on MS media (macro and oligo-elements) which differed with respect to concentrations of plant hormones (2,4-D and BA), vitamins, sucrose, maltose, L-asparagine, and solidifying agents. All tested media induced embryogenic callus for the varieties and regenerate plantlets. However, a significant effect of variety, medium and variety × medium interaction were observed for callus induction and regeneration. Average callus growth as measured by relative fresh weight growth rate (RFWGR) across different media was the highest for ‘Amria’ (7215.4%) and the lowest for ‘Tomouh’ (2088.2%). M1 (2 mg/L 2,4-D) and M5 (3 mg/L 2,4-D) media gave highest RFWGR(6892.1% and 6332%, respectively) and M3 (1 mg/L 2,4-D) was the lowest (3708.8%), across different varieties. However, the embryogenic callus from M3 media regenerated the highest percentage of plantlet, upon transfer to regeneration medium, for most of the varieties. For the varieties ‘Marouane’, ‘Kyperounda’, ‘Marzak’, ‘Karim’, and ‘Tomouh’, the favourable medium was M3, whereas, for ‘Isly’, ‘Irden’ and ‘Amria’, both M2 (2.5 mg/L BA and 2.5 mg/L 2,4-D) and M3 were the favourable media for embryogenic callus induction. In this study, for the first time, favourable media for induction and regeneration from mature embryo of Moroccan durum wheat varieties were identified. These media will be used for callus induction and genetic transformation.

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.

The Medicago Sativa HemL gene as an innovative plant-derived selectable marker for durum wheat transformation

The Medicago Sativa HemL gene as an innovative plant-derived selectable marker for durum wheat transformation

Agrobacterium-mediated transformation of durum wheat (Triticum turgidum L. var. durum cv Stewart) with improved efficiency.

An efficient Agrobacterium-mediated durum wheat transformation system has been developed for the production of 121 independent transgenic lines. This improved system used Agrobacterium strain AGL1 containing the superbinary pGreen/pSoup vector system and durum wheat cv Stewart as the recipient plant. Acetosyringone at 400 μM was added to both the inoculation and cultivation medium, and picloram at 10 mg l−1 and 2 mg l−1 was used in the cultivation and induction medium, respectively. Compared with 200 μM in the inoculation and cultivation media, the increased acetosyringone concentration led to significantly higher GUS (β-glucuronidase) transient expression and T-DNA delivery efficiency. However, no evident effects of acetosyringone concentration on regeneration frequency were observed. The higher acetosyringone concentration led to an improvement in average final transformation efficiency from 4.7% to 6.3%. Furthermore, the concentration of picloram in the co-cultivation medium had significant effects on callus induction and regeneration. Compared with 2 mg l−1 picloram in the co-cultivation medium, increasing the concentration to 10 mg l−1 picloram resulted in improved final transformation frequency from 2.8% to 6.3%, with the highest frequency of 12.3% reached in one particular experiment, although statistical analysis showed that this difference in final transformation efficiency had a low level of significance. Stable integration of foreign genes, their expression, and inheritance were confirmed by Southern blot analyses, GUS assay, and genetic analysis. Analysis of T1 progeny showed that, of the 31 transgenic lines randomly selected, nearly one-third had a segregation ratio of 3:1, while the remainder had ratios typical of two or three independently segregating loci.

Efficient and rapid Agrobacterium-mediated genetic transformation of durum wheat (Triticum turgidum L. var. durum) using additional virulence genes

Genetic transformation of wheat, using biolistics or Agrobacterium, underpins a range of specific research methods for identifying genes and studying their function in planta. Transgenic approaches to study and modify traits in durum wheat have lagged behind those for bread wheat. Here we report the use of Agrobacterium strain AGL1, with additional vir genes housed in a helper plasmid, to transform and regenerate the durum wheat variety Ofanto. The use of the basic pSoup helper plasmid with no additional vir genes failed to generate transformants, whereas the presence of either virG542 or the 15 kb Komari fragment containing virB, virC and virG542 produced transformation efficiencies of between 0.6 and 9.7%. Of the 42 transgenic plants made, all but one (which set very few seeds) appeared morphologically normal and produced between 100 and 300 viable seeds. The transgene copy number and the segregation ratios were found to be very similar to those previously reported for bread wheat. We believe that this is the first report describing successful genetic transformation of tetraploid durum wheat (Triticum turgidum L. var. durum) mediated by Agrobacterium tumefaciens using immature embryos as the explant.

Phosphomannose isomerase, pmi, as a selectable marker gene for durum wheat transformation

Transgene technology provides a powerful tool for developing traits that are otherwise difficult to achieve through conventional methods. The development of transgenic plants requires the use of selectable marker genes, as the efficiency of plant transformation is less than optimal for many important species, especially for monocots such as durum wheat ( Triticum turgidum var. durum). Many concerns have been expressed about the persistence of currently used marker genes in plants used for field cultivation. To sustain further progress in this area, alternative efficient selection methods are desirable. A recent development is the use of selective genes that give transformed cells a metabolic advantage (positive selection) compared to untransformed cells, which are slowly starved with a concomitant reduction in growth and viability. This selection strategy is in contrast to traditional negative selections during which the transgenic cells are able to survive on a selective medium whereas the non-transgenic cells are actively killed by the selective agent. We compared the ‘selection efficiency’ of a commonly used negative selection method that employs the Streptomyces hygroscopicus bar gene to confer resistance to the herbicide bialaphos, to a positive selection employing the Escherichia coli phosphomannose isomerase ( pmi) gene as the selectable gene and mannose as the selective agent. Calli derived from immature embryos of the durum wheat cultivar Svevo were bombarded separately with bar and pmi genes using a biolistic system. The integration and expression of the two genes in the T 0 generation were confirmed by PCR analysis with specific primers for each gene and the chlorophenol red assay, respectively. The selection efficiency, calculated as the number of expressing plants divided by the number of total regenerants, was higher when pmi was used as the selectable marker gene (90.1%) than when bar was used (26.4%). Thus, an efficient selection method for durum wheat transformation was established that obviates the use of herbicide résistance genes.

Genetic Transformation of Durum Wheat (Triticum durum Desf.) through Particle Bombardment of Scutellar Tissues.

This is the first report of the production of transgenic durum wheat. A f transformation system or common wheat through particle bombardment of scutellar tissues of immature embryos was successfully applied to emmer wheat. Three emmer wheats, Triticum durum var. reichenbachii, T. durum var. agricunum and T. aethiopicum Jakubz., were used in the transformation experiments. The scutellar tissues isolated from immature seeds were bombarded with a plasmid, pDM302 containing the bialaphos-resistant gene (bar) under control of the rice actin 1 gene (Act1) promoter. In T. durum var. agricunum, 25 bialaphos-resistant plants were independently regenerated from each green-spotted calli. On the other hand, no bialaphos-resistant plants were recovered from the other two emmer wheat, T. durum var. reichenbachii and T. aethiopicum. Most regenerated plants grew into mature and fertile plants. The integration and inheritance of the bar gene were confirmed by the PCR amplification and Southern analysis in T0 and T1 plants. All 25 plants showed the positive band of the bar gene. The frequency of transformation was 1.17 % (25 independent transformants/2144 immature embryos).