Challenges of In Vitro and In Vivo Agrobacterium-Mediated Genetic Transformation in Soybean (original) (raw)
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Transgenic Crops - Emerging Trends and Future Perspectives, 2018
An efficient and reproducible plant regeneration protocol is essential for genetic manipulation of important crops in vitro through Agrobacterium-mediated genetic transformation. However, the establishment of such a procedure for recalcitrant legumes like soybean is still a major challenge. Genotype specificity, culture conditions and inefficient recovery of transgenic microshoots are some of the most important factors which requires optimisation before an efficient system of regeneration can be developed. The purpose of this chapter was to provide a review, and report on the varied responses obtained during the assessment of factors that cause recalcitrance during genetic transformation of soybean. Agrobacterium infected double cotyledonary-node explants were tested on MS basal culture medium containing combinations of cytokinins-auxins, as well the different concentrations of antibiotics for callus and shoot proliferation. The study showed that, the efficiency of microshoots and callus induction varied widely between cultures and among the genotypes. About 1.0-20.0 and 5.0-20.0% of callus and shoot induction frequency were obtained on cotyledonary explants transformed with Agrobacterium compared to more than 60% efficiency obtained in the controls, respectively. This study revealed that, there are some neglected factors playing a crucial role in genetic manipulation, which require optimisation before genetic transformation and in vitro regeneration of transgenic plants could be achieved.
TAG Theoretical and Applied Genetics, 2003
An efficient transformation system was developed for multiple soybean [Glycine max (L.) Merrill.] cultivars using Agrobacterium-mediated gene transfer. A significantly high number of hygromycin-resistant somatic embryos (SEs) was obtained when immature zygotic cotyledons were inoculated with Agrobacterium tumefaciens strain KYRT1 and when the abaxial side of explants was oriented upwards (i.e., the adaxial side of explants was in contact with the medium). Most hygromycinresistant SEs on selective medium were induced along the periphery of the abaxial side of cotyledonary explants. Extended periods of selection (up to 10 weeks postcocultivation) increased the frequency of somatic embryogenesis, and more than 50% of selected SEs tested positive for b-glucuronidase (GUS). Following maturation and regeneration of selected SEs, ten independent transgenic soybean plants of cv Jack were obtained, and the overall transformation frequency ranged from 1.1 to 1.7%. Six and two transgenic plantlets were obtained from cvs Dwight and Williams, respectively. In addition, transgenic suspension lines were established from cvs Jack, Williams, Dwight, Rend and Ina. Molecular analysis of embryogenic lines and/or transgenic plants, established from different cultivars, confirmed stable integration, expression, and/or inheritance of transgenes in both T 0 and T 1 plants.
Euphytica, 2004
Conditions affecting Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merr.], including seed vigor of explant source, selection system, and cocultivation conditions, were investigated. A negative correlation between seed sterilization duration and seed vigor, and a positive correlation between seed vigor and regenerability of explants were observed in the study, suggesting that use of high vigor seed and minimum seed sterilization duration can further improve transformation efficiency. Selection schemes using glufosinate or bialaphos as selective agents in vitro were assessed. Glufosinate selection enhanced soybean transformation as compared to bialaphos. The use of 6 mg L −1 glufosinate during shoot induction and shoot elongation stages yielded higher final transformation efficiency ranging from 2.0% to 6.3% while bialaphos at 4 to 8 mg L −1 gave 0% to 2.1% efficiency. Including cysteine and DTT during cocultivation increased the transformation efficiency from 0.2-0.9% to 0.6-2.9%. This treatment also improved T-DNA transfer as indicated by enhanced transient GUS expression. Shoot regeneration and Agrobacterium infection were attained in twelve soybean cultivars belonging to maturity groups I-VI. These cultivars may be amenable to genetic transformation and may provide a valuable tool in soybean improvement programs.
A rapid method for optimization of Agrobacterium-mediated transformation of Indian soybean genotypes
An Agrobacterium-mediated transformation method optimized for two high yielding/popular Indian soybean genotypes JS335 and DS 9712, using the cotyledonary node (CN) and hypocotyl segment systems. The explants were co-cultivated with Agrobacterium tumefaciens harboring the binary vector with bar (phosphinothricin acetyltransferase gene, a selectable marker gene) and the gus reporter gene. The results revealed a higher regeneration frequency using CN as explants in the both cultivars JS335 (75%) and DS 9712 (50%), as compared to regeneration frequency of 44% (JS335) and 27% (DS 9712) using hypocotyl segments as explants. An addition of dithiothreitol (DTT) and sodium thiosulfate along with L-cysteine during co-cultivation of JS-335 showed an increase (33%) in the transformation efficiency. Shoot induction on the non-selective shoot induction media (SIM) for 10 days before transferring the explants to SIM containing 5 mg/L of glufosinate greatly stimulated the regeneration of the transformed shoots. The transformed shoots elongated on selective shoot elongation media (SEM) (2×14 days) were successfully rooted and transplanted to pot mix and grown to maturity under greenhouse conditions. Stable integration confirmed by PCR and Southern analysis of the T 1 progeny revealed a transformation efficiency of 3.1% and 2.4% in JS 335 and DS 9712, respectively. The protocol resulted in stable transformants of soybean, with T 1 seeds developing to maturity within six months after the initiation of this protocol. The study indicated a better efficiency of the CN node transformation system over the hypocotyl segment system in cultivar JS335 and may be beneficial for versatile gene function analysis in popular Indian genotypes.
Plant Cell Reports, 2006
The utility of transformation for soybean improvement requires an efficient system for production of stable transgenic lines. We describe here an improved cotyledonary node method using an alternative explant for Agrobacterium tumefaciens-mediated soybean transformation. We use the term “half-seed” to refer to this alternative cotyledonary explant that is derived from mature seed of soybean following an overnight imbibition and to distinguish it from cotyledonary node derived from 5–7-day-old seedlings. Transformation efficiencies using half-seed explants ranged between 1.4 and 8.7% with an overall efficiency of 3.8% based on the number of transformed events that have been confirmed in the T 1 generation by phenotypic assay using the herbicide Liberty® (active ingredient glufosinate) and by Southern analysis. This efficiency is 1.5-fold higher than the cotyledonary node method used in our laboratory. Significantly, the half-seed system is simple and does not require deliberate wounding of explants, which is a critical and technically demanding step in the cotyledonary node method.
In Vitro Cellular & Developmental Biology - Plant, 2007
A novel Agrobacterium rhizogenes-mediated transformation method using a primary-node explant from Dairyland cultivar 93061 was developed for soybean using the disarmed Agrobacterium strain SHA17. Transformed plants regenerated from explants inoculated with SHA17 were fertile and phenotypically normal. In a comparative experiment, regeneration frequencies were not significantly different between explants inoculated with A. rhizogenes strain SHA17 and Agrobacterium tumefaciens strain AGL1; however, a 3.5-fold increase in transformation efficiency [(number of Southern or TaqMan-positive independent events/ total number of explants inoculated)×100] was found for explants cocultured with SHA17 compared to AGL1 (6.6 and 1.64%, respectively). Southern analysis of 48 T 0 plants suggested that 37.5, 23, and 39.6% of the T 0 plants contained 1, 2, and 3 or more T-DNA fragments integrated into the genome, respectively. Additionally, T 1 progeny analysis of 8 independent events resulted in typical Mendelian inheritance of T-DNA genes. Of seven T 0 plants that had two or more T-DNA fragments, six contained multiple loci segregating in T 1 progenies. Further analysis of four lines confirmed the presence of PAT, GUS, and/or DsRED2 proteins in transgenic plants that were encoded on the T-DNA into the T 2 generation.
In Vitro Cellular & Developmental Biology - Plant, 2018
In the present study, Agrobacterium tumefaciens-mediated transformation of Glycine max (L.) Merr. (soybean) cv. DS-9712 using half-seed explants was optimized for eight different parameters, including seed imbibition, medium pH, infection mode (sonication and vacuum infiltration), co-cultivation conditions, concentrations of supplementary compounds, and selection. Using this improved protocol, maximum transformation of 14% and regeneration efficiencies of 45% were achieved by using explants prepared from mature seeds imbibed for 36 h, infected with A. tumefaciens strain EHA105 at an optical density (OD 600) of 0.8, suspended in pH 5.4 medium containing 0.2 mM acetosyringone and 450 mg L −1 L-cysteine, followed by sonication for 10 s, vacuum infiltration for 2 min, and co-cultivated for 3 d on 35 mg L −1 kanamycin-containing medium. Independent transgenic lines were confirmed to be transgenic after ß-glucuronidase histochemical assays, polymerase chain reaction, and southern hybridization analysis. The protocol developed in the present study showed high regeneration efficiency within a relatively short time of 76 d. This rapid and efficient protocol might overcome some hurdles associated with the genetic manipulation of soybean.
Agrobacterium mediated transformation of soybean (glycine max l.): some conditions standardization
Pakistan Journal of Botany, 2010
Present study was aimed to standardize some transformation conditions for soybean cultivar NARC-4 using Agrobacterium tumefaciens strain EHA 101 harboring pGUSintNPTII. Soybean half seed cotyledonary node method was opted. It was found that explant preparation in Agrosuspension culture resulted in highest transformation efficiency (48.3%) than in infection medium and water. One hour infection time was found optimum (55.9% transformation efficiency) in culture OD 600 1.0. Co-cultivation of soybean half seed explants with Agrobacterium for five days showed better results as compared with three and four days. Two hr washing in washing medium containing 1g/L cefotaxime controlled prevalence of Agrobacterium in further steps. It was found that varying kanamycin concentration in selection medium resulted in high survival rate of transformed shoots. NARC-4 soybean cultivar showed better transformation efficiency than NARC-7 when genotype dependency was examined using Agrobacterium strain EHA101.
A Review: Agrobacterium-mediated Gene Transformation to Increase Plant Productivity
The Journal of Phytopharmacology
In genetics and molecular biology Gene transformation is a gene alteration technique that involves the introduction and expression of a foreign gene into the host organism. There are many gene transformation methods like particle bombardment electroporation micro-injection, (PEG), for different biotechnological experiments But Plant gene transformation is a widely used procedure for obtaining transgenic plants and plant models to understand gene functions. Agrobacterium tumefaciens is a natural genetic engineer which is rod-shaped gram-negative soil-born barteri. Initially Agrobacterium was utilized to transform only dicot plants but over the year’s modification in plant transformation protocol. It was now utilized in monocot plants as well as in fruits plants too. Agrobacterium tumefaciens inserts its (DNA), (Transfer DNA-T-DNA), into the host plant. The transmitted (DNA), is randomly integrated into the host cell's genetic material inside the infected plant cell nucleus. Alter...