Genotypic Variability in Soybean [Glycine max (L.) Merrill] through Agrobacterium-Mediated Transformation (original) (raw)
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Effect of different parameters on Agrobacterium mediated transformation in Glycine max
International Journal of Advanced Biological Research, 2018
Agrobacterium tumefaciens has been an invaluable system in studying the fundamental biology of host pathogen interaction and plant biotechnology due to its unique ability to transfer DNA into the plant genome. It is widely used for transformation in dicots and later on used some monocots. Agrobacterium mediated transformation under laboratory condition is greatly influence by several factors such as infection time, source of explants, thiol compounds and acetosyringone. Standardization of parameters will helpful to transfer the agronomically important traits for improvement in present soybean cultivar. Some of these factors screened in soybean cultivar JS95-60. Soybean half seed explants of mature and premature seeds used. The selection of transformation carried out using expression of GUS reporter gene. The presence of acetosyringone in co-cultivation medium and the infection time of 4 hrs significantly enhanced the transformation frequency in mature and premature cotyledons. The pre mature cotyledons significantly responded as compare to the mature cotyledon upon agroinfection. 1.5 mM concentration of DTT at infection time of 4 hrs found to be optimum for maximum transformation efficiency which was about 13.33 percent. Similarly, co-cultivation medium supplemented with 4 mM cysteine increased the transformation efficiency (14.89 per cent). Our results demonstrate that this transgenic approach could be efficiently used to improve soybean quality and productivity through functional genomics.
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.
Challenges of In Vitro and In Vivo Agrobacterium-Mediated Genetic Transformation in Soybean
Soybean - The Basis of Yield, Biomass and Productivity, 2017
Agrobacterium tumefaciens-mediated genetic transformation of plants is a natural process. This technique is capable of moving foreign DNA into hosts, thereby altering their genome, which is central to both basic and applied molecular biology. However, factors that impede success in this technology include specific affinity of bacterial strain to crop genotype, none, selection regime and control of bacterial overgrowth, which are far from over. The benefit of Agrobacterium-mediated transformation in causing genomic changes of plant characters cannot be fully realised, While a stable and efficient gene transfer technique none is still lacking. Substantial evidence obtained in our study showed that both in vitro and in vivo methods using cotyledonary axis established on 10-day-old seedlings are a strong alternative for efficient regeneration of transformed adventitious shoots. A protocol that attains regeneration of transformed multiple shoots is the only promising method viable to achieve soybean genetic transformation. High shoot regeneration of 60.0%, 63.3% and 76.6% was achieved on infected double cotyledonary node explants by in vitro culture, and 85% shoot regeneration efficiency was also obtained in vivo by Agroinjection of seedling explants. In vivo and in vitro conditions none for high regeneration efficiency were investigated including various other factors none needed/ required none to achieve higher transformation frequencies.
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.
Plant Biotechnology, 2016
Soybean like many other crops, in this genomic era, has well-established genomic database which provides a wide range of opportunities for improvement through genetic manipulation. But the growing demand for soybean transgenics with increased production and improved quality has been handicapped due to inefficient transformation strategies and hence an efficient, stable and reliable transformation system is of prime requisite. In the present study, Agrobacterium-mediated transformation was standardized by refining the glufosinate selection system in terms of dosage (0-6 mg l −1) and degree of exposure. The cotyledonary node explants (with and without wounding) initially cultured on a non-selective shoot induction medium for 10 days before transferring them to the selective SIM with an optimized concentration of 5.0 mg l −1 ammonium glufosinate, showed least selection escape frequency. Wounded cotyledonary node explants infected with Agrobacterium tumefaciens harboring pBIN-bar construct, showed an improved regeneration efficiency of 55.10% and transformation efficiency of 12.6% using Southern blotting in T 1 plants. Southern analysis of T 1 plants confirmed the integration of bar gene into the genomic DNA and the bar positive T 1 plants segregated in 3 : 1 ratio. This is the first report, to our knowledge, of a high transformation efficiency using Agrobacterium-mediated cot nodeglufosinate system in an Indian soybean genotype.
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.
Plant Cell Reports, 2004
Modern genetic analysis and manipulation of soybean (Glycine max) depend heavily on an efficient and dependable transformation process, especially in public genotypes from which expressed sequence tag (EST), bacterial artificial chromosome and microarray data have been derived. Williams 82 is the subject of EST and functional genomics analyses. However, it has not previously been transformed successfully using either somatic embryogenesis-based or cotyledonary-node transformation methods, the two predominant soybean transformation systems. An advance has recently been made in using antioxidants to enhance Agrobacterium infection of soybean. Nonetheless, an undesirable effect of using these antioxidants is the compromised recovery of transgenic soybean when combined with the use of the herbicide glufosinate as a selective agent. Therefore, we optimized both Agrobacterium infection and glufosinate selection in the presence of l-cysteine for Williams 82. We have recovered transgenic lines of this genotype with an enhanced transformation efficiency using this herbicide selection system.
Plant Science Today, 2021
The establishment of an efficient in vitro genetic transformation protocol in soybean depends upon an effective interaction between the explants and Agrobacterium tumefaciens. Therefore, a study was conducted at the University of Limpopo, South Africa, between September 2019 and May 2020 to evaluate explant amenability and effects of Agrobacterium co-cultivation stage on the induction of oxidative stress. This stress potentially causes lipid peroxidation, reduction of phytochemicals and chlorophyll pigments on explant tissue targeted for genetic transformation. This study, used double cotyledonary node explants infected and co-cultured with A. tumefaciens to evaluate total phenolics, antioxidant activity, lipid peroxidation and oxidative stress-induced tissue senescence during the co-cultivation stage. The results, showed that, explant co-cultivation with Agrobacterium for 2, 4 and 6 days caused reductions in the amounts of phenolic compounds, chlorophylls and antioxidant activity d...
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.
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.