Development of a stable Agrobacterium-mediated transformation protocol for Sorghum bicolor Tx430.
ABSTRACT: Sorghum is a recalcitrant crop for Agrobacterium-mediated genetic transformation. Several parameters related to Agrobacterium-mediated transformation were tested to optimize sorghum transformation frequencies. In this study, we evaluated pretreatment of sorghum variety Tx430 immature embryos using Agrobacterium strain GV2260. Pretreatment of immature embryos with heat (43°C) treatment for 15 or 21?min, and centrifugation resulted in a transformation efficiency of up to 1.9% of immature embryos treated. Although further optimization to enhance transformation efficiency is required, this study contributes to the genetic validation of genes of interest and molecular breeding in sorghum plants.
Project description:Background:Sorghum (Sorghum bicolor L.) is one of the world's most important cereal crops grown for multiple applications and has been identified as a potential biofuel crop. Despite several decades of study, sorghum has been widely considered as a recalcitrant major crop for transformation due to accumulation of phenolic compounds, lack of model genotypes, low regeneration frequency and loss of regeneration potential through sub-cultures. Among different explants used for genetic transformation of sorghum, immature embryos are ideal over other explants. However, the continuous supply of quality immature embryos for transformation is labour intensive and expensive. In addition, transformation efficiencies are also influenced by environmental conditions (light and temperature). Despite these challenges, immature embryos remain the predominant choice because of their success rate and also due to non-availability of other dependable explants without compromising the transformation efficiency. Results:We report here a robust genetic transformation method for sorghum (Tx430) using differentiating embryogenic calli (DEC) with nodular structures induced from immature embryos and maintained for more than a year without losing regeneration potential on modified MS media. The addition of lipoic acid (LA) to callus induction media along with optimized growth regulators increased callus induction frequency from 61.3 ± 3.2 to 79 ± 6.5% from immature embryos (1.5-2.0 mm in length) isolated 12-15 days after pollination. Similarly, the regeneration efficiency and the number of shoots from DEC tissue was enhanced by LA. The optimized regeneration system in combination with particle bombardment resulted in an average transformation efficiency (TE) of 27.2 or 46.6% based on the selection strategy, 25% to twofold higher TE than published reports in Tx430. Up to 100% putative transgenic shoots were positive for npt-II by PCR and 48% of events had < 3 copies of transgenes as determined by digital droplet PCR. Reproducibility of this method was demonstrated by generating ~ 800 transgenic plants using 10 different gene constructs. Conclusions:This protocol demonstrates significant improvements in both efficiency and ease of use over existing sorghum transformation methods using PDS, also enables quick hypothesis testing in the production of various high value products in sorghum.
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.
Project description:Agrobacterium-mediated sorghum transformation frequency has been enhanced significantly via medium optimization using immature embryos from sorghum variety TX430 as the target tissue. The new transformation protocol includes the addition of elevated copper sulfate and 6-benzylaminopurine in the resting and selection media. Using Agrobacterium strain LBA4404, the transformation frequency reached over 10% using either of two different selection marker genes, moPAT or PMI, and any of three different vectors in large-scale transformation experiments. With Agrobacterium strain AGL1, the transformation frequencies were as high as 33%. Using quantitative PCR analyses of 1,182 T0 transgenic plants representing 675 independent transgenic events, data was collected for T-DNA copy number, intact or truncated T-DNA integration, and vector backbone integration into the sorghum genome. A comparison of the transformation frequencies and molecular data characterizing T-DNA integration patterns in the transgenic plants derived from LBA4404 versus AGL1 transformation revealed that twice as many transgenic high-quality events were generated when AGL1 was used compared to LBA4404. This is the first report providing molecular data for T-DNA integration patterns in a large number of independent transgenic plants in sorghum.
Project description:Sorghum is the fifth most widely planted cereal crop in the world and is commonly cultivated in arid and semi-arid regions such as Africa. Despite its importance as a food source, sorghum genetic improvement through transgenic approaches has been limited because of an inefficient transformation system. Here, we report a ternary vector (also known as cohabitating vector) system using a recently described pVIR accessory plasmid that facilitates efficient Agrobacterium-mediated transformation of sorghum. We report regeneration frequencies ranging from 6% to 29% in Tx430 using different selectable markers and single copy, backbone free 'quality events' ranging from 45% to 66% of the total events produced. Furthermore, we successfully applied this ternary system to develop transformation protocols for popular but recalcitrant African varieties including Macia, Malisor 84-7 and Tegemeo. In addition, we report the use of this technology to develop the first stable CRISPR/Cas9-mediated gene knockouts in Tx430.
Project description:Agrobacterium overgrowth is a common problem in Agrobacterium-mediated plant transformation. To suppress the Agrobacterium overgrowth, various antibiotics have been used during plant tissue culture steps. The antibiotics are expensive and may adversely affect plant cell differentiation and reduce plant transformation efficiency. The SacB-SacR proteins are toxic to most Agrobacterium tumefaciens strains when they are grown on culture medium supplemented with sucrose. Therefore, SacB-SacR genes can be used as negative selection markers to suppress the overgrowth of A. tumefaciens in the plant tissue culture process. We generated a mutant A. tumefaciens strain GV2260 (recA-SacB/R) that has the SacB-SacR cassette inserted into the bacterial genome at the recA gene locus. The mutant Agrobacterium strain is sensitive to sucrose but maintains its ability to transform plant cells in both transient and stable transformation assays. We demonstrated that the mutant strain GV2260 (recA-SacB/R) can be inhibited by sucrose that reduces the overgrowth of Agrobacterium and therefore improves the plant transformation efficiency. We employed GV2260 (recA-SacB/R) to generate stable transgenic N. benthamiana plants expressing a CRISPR-Cas9 for knocking out a WRKY transcription factor.
Project description:Maize Agrobacterium-mediated transformation efficiency has been greatly improved in recent years. Antioxidants, such as, cysteine, can significantly improve maize transformation frequency through improving the Agrobacterium infection efficiency. However, the mechanism underlying the transformation improvement after cysteine exposure has not been elucidated. In this study, we showed that the addition of cysteine to the co-cultivation medium significantly increased the Agrobacterium infection efficiency of hybrid HiII and inbred line Z31 maize embryos. Reactive oxygen species contents were higher in embryos treated with cysteine than that without cysteine. We further investigated the mechanism behind cysteine-related infection efficiency increase using transcriptome analysis. The results showed that the cysteine treatment up-regulated 939 genes and down-regulated 549 genes in both Z31 and HiII. Additionally, more differentially expressed genes were found in HiII embryos than those in Z31 embryos, suggesting that HiII was more sensitive to the cysteine treatment than Z31. GO analysis showed that the up-regulated genes were mainly involved in the oxidation reduction process. The up-regulation of these genes could help maize embryos to cope with the oxidative stress stimulated by Agrobacterium infection. The down-regulated genes were mainly involved in the cell wall and membrane metabolism, such as, aquaporin and expansin genes. Decreased expression of these cell wall integrity genes could loosen the cell wall, thereby improving the entry of Agrobacterium into plant cells. This study offers insight into the role of cysteine in improving Agrobacterium-mediated transformation of maize immature embryos.
Project description:Agrobacterium-mediated genetic transformation is well established in the model grass Brachypodium distachyon. However, most protocols employ immature embryos because of their better regenerative capacity. A major problem associated with the immature embryo system is that they are available only during a limited time window of growing plants. In this study, we have developed an optimized Agrobacterium-mediated genetic transformation protocol that utilizes mature embryos. We have adopted seed shearing and photoautotrophic rooting (PR) in callus induction and root regeneration, respectively, with evident significant improvement in these aspects. We have also revealed that the newly developed chemical inducer Fipexide (FPX) had the ability to induce callus, shoots, and roots. By comparison, we have demonstrated that FPX shows higher efficiency in shoot generation than other frequently used chemicals in our mature embryo-based system. In addition, we demonstrated that the age of embryogenetic callus severely affects the transformation efficiency (TE), with the seven-week-old embryogenetic callus having the highest TE reaching 52.6%, which is comparable with that in immature embryo transformation. The new methodologies reported here will advance the development and utilization of Brachypodium as a new model system for grass genomics.
Project description:Significant progress has been made on sorghum transformation in the last decades; however, the transformation process has been constrained by the availability of immature embryos because most of the researchers have utilized immature embryos as favorable explants. Although immature embryos have been proven to be optimal for tissue culture and transformation, isolation of immature embryos is time-consuming, labor-intensive, and limited by warm weather. In this study, we developed an efficient genetic transformation system using mature seeds as explants. The <i>nptII</i> and <i>gus</i> gene, used as the selective marker and report gene respectively, have been co-transformed by particle bombardment. After optimization of tissue culture, the G418 concentration, and transgenic, the average transformation frequency at 13.33% was achieved routinely. The transgenic events and transgene copy numbers were determined by PCR and RT-PCR, respectively. The geneticin selection and GUS staining on T<sub>1</sub> seedlings confirmed that the transgenic plants were heritable. Our results demonstrated that the efficient sorghum transformation system has been established using mature seeds as explants. This transformation system will promote sorghum research on genetic engineering and genome editing without seasonal weather conditions restriction and explant resources restriction.
Project description:Agrobacterium tumefaciens has been utilized for both transient and stable transformations of plants. These transformation methods have been used in fields such as breeding GM crops, protein production in plant cells, and the functional analysis of genes. However, some plants have significantly lower transient gene transfer and stable transformation rates, creating a technical barrier that needs to be resolved. In this study, Super-Agrobacterium was updated to ver. 4 by introducing both the ACC deaminase (acdS) and GABA transaminase (gabT) genes, whose resultant enzymes degrade ACC, the ethylene precursor, and GABA, respectively. A. tumefaciens strain GV2260, which is similar to other major strains (EHA105, GV3101, LBA4404, and MP90), was used in this study. The abilities of the Super-Agrobacterium ver. 4 were evaluated in Erianthus ravennae, Solanum lycopersicum "Micro-Tom," Nicotiana benthamiana, and S. torvum. Super-Agrobacterium ver. 4 showed the highest T-DNA transfer (transient transformation) frequencies in E. ravennae and S. lycopersicum, but not in N. benthamiana and S. torvum. In tomato, Super-Agrobacterium ver. 4 increased the stable transformation rate by 3.6-fold compared to the original GV2260 strain. Super-Agrobacterium ver. 4 enables reduction of the amount of time and labor required for transformations by approximately 72%, and is therefore a more effective and powerful tool for plant genetic engineering and functional analysis, than the previously developed strains. As our system has a plasmid containing the acdS and gabT genes, it could be used in combination with other major strains such as EHA105, EHA101, LBA4404, MP90, and AGL1. Super-Agrobacterium ver. 4, could thus possibly be a breakthrough application for improving basic plant science research methods.
Project description:OBJECTIVES:Sorghum is one of the most recalcitrant species for transformation. Considering the time and effort required for stable transformation in sorghum, establishing a transient system to screen the efficiency and full functionality of vector constructs is highly desirable. RESULTS:Here, we report an Agrobacterium-mediated transient transformation assay with intact sorghum leaves using green fluorescent protein as marker. It also provides a good monocot alternative to tobacco and protoplast assays with a direct, native and more reliable system for testing single guide RNA (sgRNA) expression construct efficiency. Given the simplicity and ease of transformation, high reproducibility, and ability to test large constructs, this method can be widely adopted to speed up functional genomic and genome editing studies.