Improvement of sugarcane for borer resistance using Agrobacterium mediated transformation of cry1Ac gene.
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ABSTRACT:
SUBMITTER: Dessoky ES
PROVIDER: S-EPMC7595610 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We developed sugarcane plants with improved resistance to the sugarcane borer, Diatraea saccharalis (F). An expression vector pGcry1Ac0229, harboring the cry1Ac gene and the selectable marker gene, bar, was constructed. This construct was introduced into the sugarcane cultivar FN15 by particle bombardment. Transformed plantlets were identified after selection with Phosphinothricin (PPT) and Basta. Plantlets were then screened by PCR based on the presence of cry1Ac and 14 cry1Ac positive plantlets were identified. Real-time quantitative PCR (RT-qPCR) revealed that the copy number of cry1Ac gene in the transgenic lines varied from 1 to 148. ELISA analysis showed that Cry1Ac protein levels in 7 transgenic lines ranged from 0.85 ?g/FWg to 70.92 ?g/FWg in leaves and 0.04 ?g/FWg to 7.22 ?g/FWg in stems, and negatively correlated to the rate of insect damage that ranged from 36.67% to 13.33%, respectively. Agronomic traits of six transgenic sugarcane lines with medium copy numbers were similar to the non-transgenic parental line. However, phenotype was poor in lines with high or low copy numbers. Compared to the non-transgenic control plants, all transgenic lines with medium copy numbers had relatively equal or lower sucrose yield and significantly improved sugarcane borer resistance, which lowered susceptibility to damage by insects. This suggests that the transgenic sugarcane lines harboring medium copy numbers of the cry1Ac gene may have significantly higher resistance to sugarcane borer but the sugarcane yield in these lines is similar to the non-transgenic control thus making them superior to the control lines.
| S-EPMC4836700 | biostudies-literature
Project description:BackgroundSugarcane (Saccharum spp. hybrids) is considered the most globally important sugar-producing crop and raw material for biofuel. Insect attack is a major issue in sugarcane cultivation, resulting in yield losses and sucrose content reductions. Stem borer (Diatraea saccharalis F.) causes serious yield losses in sugarcane worldwide. However, insect-resistant germplasms for sugarcane are not available in any collections all over the world, and the molecular mechanism of insect resistance has not been elucidated. In this study, cry1Ac transgenic sugarcane lines were obtained and the biological characteristics and transgene dosage effect were investigated and a global exploration of gene expression by transcriptome analysis was performed.ResultsThe transgene copies of foreign cry1Ac were variable and random. The correlation between the cry1Ac protein and cry1Ac gene copies differed between the transgenic lines from FN15 and ROC22. The medium copy lines from FN15 showed a significant linear relationship, while ROC22 showed no definite dosage effect. The transgenic lines with medium copies of cry1Ac showed an elite phenotype. Transcriptome analysis by RNA sequencing indicated that up/down regulated differentially expressed genes were abundant among the cry1Ac sugarcane lines and the receptor variety. Foreign cry1Ac gene and endogenous borer stress-related genes may have a synergistic effect. Three lines, namely, A1, A5, and A6, were selected for their excellent stem borer resistance and phenotypic traits and are expected to be used directly as cultivars or crossing parents for sugarcane borer resistance breeding.ConclusionsCry1Ac gene integration dramatically improved sugarcane insect resistance. The elite transgenic offspring contained medium transgene copies. Foreign cry1Ac gene integration and endogenous borer stress-related genes may have a synergistic effect on sugarcane insect resistance improvement.
| S-EPMC6288918 | biostudies-literature
Project description:Sorghum (Sorghum bicolor L.) ranks as the fifth most widely planted cereal in the world and is used for food as well as a biomass plant for ethanol production. Use of the TX430 non-tannin sorghum variety has enhanced Agrobacterium-mediated sorghum transformation. These protocols could not be applied, however, to other tannin producing sorghum varieties such as the BTx623 model cultivar for sorghum with full genome information of sorghum. Here we report an improved protocol for Agrobacterium-mediated genetic transformation of tannin-producing sorghum variety BTx623. We successfully developed modification of root regeneration condition for generation of transgenic plant of BTx623. We inoculated immature embryos with Agrobacterium tumefaciens strain EHA105 harboring pMDC32-35S-GFP to generate transgenic plants. In the root regeneration step, we found that regeneration from transformed calli was affected by tannin. For root regeneration, shoots that appeared were not transferred to agar plate, but instead transferred to vermiculite in a plastic pod. Direct planting of regenerated shoots into vermiculite prevented the toxic effect of tannin. Root regeneration efficiency from calli emerged shoots in vermiculite was 78.57%. Presence of sGFP transgene in the genome of transgenic plants was confirmed by PCR and sGFP expression was confirmed in transgenic plants. This improved protocol of Agrobacterium-mediated transformation for tannin-producing sorghum BTx623 could be a useful tool for functional genomics using this plant.
| S-EPMC6566004 | biostudies-literature
Project description:Maize (Zea mays L.) is a food crop sensitive to low temperatures. As one of the abiotic stress hazards, low temperatures seriously affect the yield of maize. However, the genetic basis of low-temperature adaptation in maize is still poorly understood. In this study, maize S-adenosylmethionine decarboxylase (SAMDC) was localized to the nucleus. We used Agrobacterium-mediated transformation technology to introduce the SAMDC gene into an excellent maize inbred line variety GSH9901 and produced a cold-tolerant transgenic maize line. After three years of single-field experiments, the contents of polyamines (PAs), proline (Pro), malondialdehyde (MDA), antioxidant enzymes and ascorbate peroxidases (APXs) in the leaves of the transgenic maize plants overexpressing the SAMDC gene significantly increased, and the expression of elevated CBF and cold-responsive genes effectively increased. The agronomic traits of the maize overexpressing the SAMDC gene changed, and the yield traits significantly improved. However, no significant changes were found in plant height, ear length, and shaft thickness. Therefore, SAMDC enzymes can effectively improve the cold tolerance of maize.
| S-EPMC9291676 | biostudies-literature
Project description:Asparagus stem wilt, is a significant and devastating disease, typically leading to extensive economic losses in the asparagus industry. To obtain transgenic plants resistant to stem wilt, the hevein-like gene, providing broad spectrum bacterial resistance was inserted into the asparagus genome through Agrobacterium tumefaciens-mediated transformation. The optimal genetic transformation system for asparagus was as follows: pre-culture of embryos for 2 days, inoculation using a bacterial titre of OD600 = 0.6, infection time 10 min and co-culturing for 4 days using an Acetosyringone concentration of 200 μmol/L. Highest transformation frequencies reached 21% and ten transgenic asparagus seedlings carrying the hevein-like gene were identified by polymerase chain reaction. Moreover, integration of the hevein-like gene in the T1 generation of transgenic plants was confirmed by southern blot hybridization. Analysis showed that resistance to stem wilt was enhanced significantly in the transgenic plants, in comparison to non- transgenic plants. The results provide additional data for genetic improvement and are of importance for the development of new disease-resistant asparagus varieties.
| S-EPMC6779260 | biostudies-literature
Project description:The gram pod borer Helicoverpa armigera is a major constraint to chickpea (Cicer arietinum L.) production worldwide, reducing crop yield by up to 90%. The constraint is difficult to overcome as chickpea germplasm including wild species either lacks pod borer resistance or if possessing resistance is cross-incompatible. This study describes conversion of elite but pod borer-susceptible commercial chickpea cultivars into resistant cultivars through introgression of cry1Ac using marker-assisted backcross breeding. The chickpea cultivars (PBG7 and L552) were crossed with pod borer-resistant transgenic lines (BS 100B and BS 100E) carrying cry1Ac that led to the development of BC1F1, BC1F2, BC1F3, BC2F1, BC2F2, and BC2F3 populations from three cross combinations. The foreground selection revealed that 35.38% BC1F1 and 8.4% BC1F2 plants obtained from Cross A (PBG7 × BS 100B), 50% BC1F1 and 76.5% BC1F2 plants from Cross B (L552 × BS 100E), and 12.05% BC2F2 and 82.81% (average) BC2F3 plants derived from Cross C (PBG7 × BS 100E) carried the cry1Ac gene. The bioassay of backcross populations for toxicity to H. armigera displayed up to 100% larval mortality. BC1F1 and BC1F2 populations derived from Cross B and BC2F3 population from Cross C segregated in the Mendelian ratio for cry1Ac confirmed inheritance of a single copy of transgene, whereas BC1F1 and BC1F2 populations obtained from Cross A and BC2F2 population from Cross C exhibited distorted segregation ratios. BC1F1 plants of Cross A and Cross B accumulated Cry1Ac protein ranging from 11.03 to 11.71 µgg-1 in leaf tissue. Cry1Ac-positive BC2F2 plants from Cross C demonstrated high recurrent parent genome recovery (91.3%) through background selection using SSR markers and phenome recovery of 90.94%, amongst these 30% plants, were homozygous for transgene. The performance of BC2F3 progenies derived from homozygous plants was similar to that of the recurrent parent for main agronomic traits, such as number of pods and seed yield per plant. These progenies are a valuable source for H. armigera resistance in chickpea breeding programs.
| S-EPMC9039336 | biostudies-literature
Project description:Genetic transformation is one of the most important technologies for revealing or modulating gene function. It is used widely in both functional genomics and molecular breeding of rice. Demands on its use in wild Oryza species is increasing because of their high genetic diversity. Given the difficulties in genetic crosses between distantly related species, genetic transformation offers a way to alter or transfer genetic traits in wild rice accessions. However, transformation of wild Oryza accessions by conventional methods using calli induced from scutellum tissue of embryos in mature seeds often fails. Here, we report methods using immature embryos for the genetic transformation of a broad range of Oryza species. First, we investigated the ability of callus induction and regeneration from immature embryos of 192 accessions in 20 species under several culture conditions. We regenerated plants from immature embryos of 90 accessions in 16 species. Next, we optimized the conditions of Agrobacterium infection using a vector carrying the GFP gene driven by the maize ubiquitin promoter. GFP signals were observed in 51 accessions in 11 species. We analyzed the growth and seed set of transgenic plants of O. barthii, O. glumaepatula, O. rufipogon, and O. brachyantha. The plants grew to maturity and set seeds normally. Southern blot analyses using DNA from T0 plants showed that all GFP plants were derived from independent transformation events. We confirmed that the T-DNAs were transmitted to the next generation through the segregation of GFP signals in the T1 generation. These results show that many Oryza species can be transformed by using modified immature-embryo methods. This will accelerate the use of wild Oryza accessions in molecular genetic analyses and molecular breeding.
| S-EPMC7270233 | biostudies-literature
Project description:BackgroundPlant transformation is an invaluable tool for basic plant research, as well as a useful technique for the direct improvement of commercial crops. Barley (Hordeum vulgare) is the fourth most abundant cereal crop in the world. It also provides a useful model for the study of wheat, which has a larger and more complex genome. Most existing barley transformation methodologies are either complex or have low (<10%) transformation efficiencies.ResultsA robust, simple and reproducible barley transformation protocol has been developed that yields average transformation efficiencies of 25%. This protocol is based on the infection of immature barley embryos with Agrobacterium strain AGL1, carrying vectors from the pBract series that contain the hpt gene (conferring hygromycin resistance) as a selectable marker. Results of large scale experiments utilising the luc (firefly luciferase) gene as a reporter are described. The method presented here has been used to produce hundreds of independent, transgenic plant lines and we show that a large proportion of these lines contain single copies of the luc gene.ConclusionThis protocol demonstrates significant improvements in both efficiency and ease of use over existing barley transformation methods. This opens up opportunities for the development of functional genomics resources in barley.
| S-EPMC2562381 | biostudies-literature
Project description:Agrobacterium tumefaciens is a plant pathogenic bacterium that causes neoplastic growths, called 'crown gall', via the transfer and integration of transferred DNA (T-DNA) from the bacterium into the plant genome. We characterized an acetosyringone (AS)-induced tumour-inducing (Ti) plasmid gene, tzs (trans-zeatin synthesizing), that is responsible for the synthesis of the plant hormone cytokinin in nopaline-type A. tumefaciens strains. The loss of Tzs protein expression and trans-zeatin secretions by the tzs frameshift (tzs-fs) mutant is associated with reduced tumorigenesis efficiency on white radish stems and reduced transformation efficiencies on Arabidopsis roots. Complementation of the tzs-fs mutant with a wild-type tzs gene restored wild-type levels of trans-zeatin secretions and transformation efficiencies. Exogenous application of cytokinin during infection increased the transient transformation efficiency of Arabidopsis roots infected by strains lacking Tzs, which suggests that the lower transformation efficiency resulted from the lack of Agrobacterium-produced cytokinin. Interestingly, although the tzs-fs mutant displayed reduced tumorigenesis efficiency on several tested plants, the loss of Tzs enhanced tumorigenesis efficiencies on green pepper and cowpea. These data strongly suggest that Tzs, by synthesizing trans-zeatin at early stage(s) of the infection process, modulates plant transformation efficiency by A. tumefaciens.
| S-EPMC6640272 | biostudies-literature
Project description:Development of resistance in target pests is a major threat to long-term use of transgenic crops expressing Bacillus thuringiensis (Bt) Cry toxins. To manage and/or delay the evolution of resistance in target insects through the implementation of effective strategies, it is essential to understand the basis of resistance. One of the most important mechanisms of insect resistance to Bt crops is the alteration of the interactions between Cry toxins and their receptors in the midgut. A Cry1Ac-selected strain of Asian corn borer (ACB), Ostrinia furnacalis, a key pest of maize in China, evolved three mutant alleles of a cadherin-like protein (OfCAD) (MPR-r1, MPR-r2 and MPR-r3), which mapped within the toxin-binding region (TBR). Each of the three mutant alleles possessed two or three amino acid substitutions in this region, especially Thr1457→Ser. In highly resistant larvae (ACB-Ac200), MPR-r2 had a 26-amino acid residue deletion in the TBR, which resulted in reduced binding of Cry1Ac compared to the MPR from the susceptible strain, suggesting that the number of amino acid deletions influences the level of resistance. Furthermore, downregulation of OfCAD gene (ofcad) transcription was observed in the Cry1Ac resistant strain, ACB-Ac24, suggesting that Cry1Ac resistance in ACB is associated with the downregulation of the transcript levels of the cadherin-like protein gene. The OfCAD identified from ACB exhibited a high degree of similarity to other members of the cadherin super-family in lepidopteran species.
| S-EPMC4179154 | biostudies-literature