Validation of a hairy roots system to study soybean. Agrobacterium rhizogenesmediated transformation of soybean to study root biology by attila kereszt, dongxue li, arief indrasumunar, cuc nguyen, sureeporn nontachaiyapoom, mark kinkema and peter gresshoff. Utilizing young seedlings with severed radicleshypocotyls, we developed a highly efficient procedure. Composite plants are generated by inoculating wildtype shoots with agrobacterium rhizogenes, which subsequently induces the formation of transgenic roots. Agrobacteriummediated transformation of soybean and recovery. Prior to transformation, the binary vectors were transferred into competent a. Genotypeindependent agrobacterium rhizogenes mediated. Apr 19, 2007 this protocol is used to induce transgenic roots on soybean to study the function of genes required in biological processes of the root. Development of a fast and efficient root transgenic system. Jun 25, 2009 in the present study, we developed a highly efficient a.
Methods and protocols, methods in molecular biology, vol. Lipm, inracnrs, bp 52627, 326 castanet tolosan cedex, france. This study seeks to understand the role of an arabidopsis arabidopsis thaliana purple apase gene atpap18 in soybean. In the following protocol, we describe an improved a. A comparison study of agrobacteriummediated transformation. For promoter studies, the specificity of the soybean root promoter could be used to detect expression in hairy roots. This transformation method is particularly suitable for those plants, including legumes, whose transformation using agrobacterium tumefaciens has been challenging. Agrobacterium rhizogenesmediated transformation of arachis. A novel coldinducible zinc finger protein from soybean, scof1, enhances cold tolerance in transgenic plants. Agrobacterium rhizogenesmediated root transformation in chickpea was performed following the protocol developed for phaseolus with few modifications. In this study, we examined competencies of various protocols to induce and explants that respond to hairy root transformation in bean plants.
The data presented here show that agrobacterium rhizogenes. Agrobacterium tumefaciens mediated gene delivery to callus and somatic embryos. We designed and tested sgrnas to target nine different major storage protein genes and detected dna mutations in three storage protein genes in soybean. The quantity and ratio of different benzylisoquinoline alkaloids produced by opium poppy and california poppy were virtually identical in transformed and wild. Agrobacterium rhizogenesmediated transformation has become a powerful tool for studying gene function and root biology due to its quick and simple methodology. Agrobacterium rhizogenes is the etiological agent for hairy root disease also known as root mat disease. Agrobacterium rhizogenes mediated transformation of soybean. Gene expression of reporters is indistinguishable in plants transformed by agrobacterium tumefaciens as compared with a. Kereszt a, li d, indrasumunar a, nguyen cdt, nontachaiyapoom s, kinkema m, et al.
Dec 18, 2012 agrobacterium rhizogenesmediated transformation of soybean to study root biology. We describe an optimized protocol for the agrobacterium rhizogenesmediated transformation of soybean and the induction of hairy root development in vitro. Agrobacterium rhizogenesmediated transformation of soybean to study root biology by attila kereszt, dongxue li, arief indrasumunar, cuc nguyen, sureeporn. Agrobacterium rhizogenes is the causal agent of hairy root diseases in plants, and has been used for the production of hairy root cultures from a multitude of species. Developing an efficient and reproducible transformation method is imperative to expedite functional genomics studies in this crop. Agrobacterium tumefaciensmediated transformation is a preferred method in many plants. Agrobacterium rhizogenes mediated transformation has several features desirable for the production of secondary metabolites. The objective of this study is to optimize agrobacterium mediated transformation in soybean by improving the infection efficiency of. Pdf soybean hairy roots produced in vitro by agrobacterium. In this study, we demonstrated the successful transformation of two pea pisum sativum l. Induction of hairy roots in chickpea using agrobacterium rhizogenes. The composite plant system allows for in root testing of transgenes in the context of a complete plant and can be analyzed in a variety of gene function analyses and plantmicrobe.
Agrobacterium rhizogenes recently revised as rhizobium rhizogenes. Agrobacterium mediated hairy root transformation is a widely applied method to generate transgenic soybean roots for studying root speci. Optimization of agrobacteriummediated transformation in. Jul 15, 2006 agrobacterium rhizogenes mediated genetic transformation resulting in hairy root formation is enhanced by ultrasonication and acetosyringone treatment the phytopathogenic bacteria agrobacterium rhizogenes genetically transforms plants by transferring a portion of the resident ri plasmid, the tdna to the plant. Inducing hairy roots by agrobacterium rhizogenesmediated.
In this study we carried out extensive work on enhancement of transformation efficiency in. Kereszt a, li d, indrasumunar a et al 2007 agrobacterium rhizogenesmediated transformation of soybean to study root biology. This study involved the formulation of protocol of agrobacterium rhizogenes mediated transformation for the detailed study of isoflavones ifs metabolism in soybean. Agrobacterium rhizogenes or rhizobium rhizogenes is able to transform plant genomes and induce the production of hairy roots. Sep 19, 2007 a novel agrobacterium rhizogenes mediated transformation method using a primarynode explant from dairyland cultivar 93061 was developed for soybean using the disarmed agrobacterium strain sha17. We developed a simple, effective and reproducible hairy root protocol in tobacco and arabidopsis.
Soybean hairy roots produced in vitro by agrobacterium rhizogenes. Young seedlings with unfolded cotyledons are infected at. Thus, the gene was isolated and a final vector atpap18pk7gwg2d was built. Agrobacterium rhizogenes mediated transformation of. This system can be used to study gene functions on the. Agrobacterium rhizogenes mediated genetic transformation. Dna carried on a large plasmid, to the genome of a host plant cell. Rhizobium rhizogenes formerly agrobacterium rhizogenes is a gramnegative soil bacterium that produces hairy root disease in dicotyledonous plants. In a comparative experiment, regeneration frequencies were not significantly different. Nov 16, 2007 agrobacterium rhizogenes is the etiological agent for hairyroot disease also known as rootmat disease. Hairy roots have been used for various purposes over the last several years. A rapid, efficient and highperformance transformation protocol employing agrobacterium rhizogenes was developed for the common bean phaseolus vulgaris. Its root inducing plasmid, containing transfer dna encoding root locus rol gene loci rola, rolb, and rolc, is responsible for the stable introduction of genetic material into host.
Transformed plants regenerated from explants inoculated with sha17 were fertile and phenotypically normal. Kereszt a1, li d, indrasumunar a, nguyen cd, nontachaiyapoom s, kinkema m, gresshoff pm. Induction of transgenic hairy roots in soybean genotypes. This transformation procedure generates, with high efficiency 7090%, hairy roots in cultivars, landraces and accessions of phaseolus vulgaris common bean. Pdf agrobacterium rhizogenesmediated transformation of. Improved cotyledonary node method using an alternative explant derived from mature seed for efficient. A novel agrobacterium rhizogenesmediated transformation method of soybean glycine max l. Agrobacterium rhizogenesmediated transformation has been. In the present study, we developed a highly efficient a.
Young seedlings with unfolded cotyledons are infected at the cotyledonary node andor hypocotyl with agrobacterium rhizogenes carrying the gene construct to be tested and the infection sites are kept in an environment of high humidity. Here, we report the establishment of an efficient, in vitro, shoot organogenesis, regeneration system for soybeans glycine max l. Developed hairy roots were compared with intact roots to. Solanum lycopersicum, an economically important crop grown worldwide, has been used as a model for the study of arbuscular mycorrhizal am symbiosis in nonlegume plants for several years and several cdna array hybridization studies have revealed specific transcriptomic profiles of mycorrhizal tomato roots. Hairy root transformation using agrobacterium rhizogenes. However, a method to easily screen candidate genes which could play an important role.
Merrill using primarynode explants from seedlings vitr cell dev biol plant, 43 2007, pp. Most of the in vitro procedures used for generating composite plants involve placing the a. Generation of soybean glycine max transient transgenic. Generation of composite plants using agrobacterium. Assessment of conditions affecting agrobacterium rhizogenes. High transformation efficiency is a prerequisite for study of gene function and molecular breeding. Fast, efficient and reproducible genetic transformation of. Agrobacterium soybean 1 updated 032210 agrobacterium mediated transformation of soybean and recovery of transgenic soybean plants.
A novel agrobacterium rhizogenesmediated transformation. Efficient transformation is a key factor for the improvement of soybean by genetic modification. Here, we present an optimized and detailed procedure for agrobacterium rhizogenes mediated root transformation of chickpea. However, the transformation efficiency in soybean is still low. The tdna containing the transgene of interest in a disarmed binary vector is generally cotransformed with the resident a. In this study, we set out to test the efficiency of the crisprcas9 system in editing soybean storage protein genes using agrobacterium rhizogenesmediated hairy root transformation system. Agrobacterium rhizogenesmediated transformation of soybean to study root biology overview of attention for article published in nature protocols, april 2007 altmetric badge. Oct 16, 2008 agrobacterium rhizogenesmediated transformation has become a powerful tool for studying gene function and root biology due to its quick and simple methodology. This protocol is used to induce transgenic roots on soybean to study the function of genes required in biological processes of the root.
In the present study we used agrobacterium rhizogenesmediated hairy roots as an alternative approach for agrobacterium tumefaciens transformation. Soybean is one of the worlds most important oil and protein crops. The rapid and efficient induction of hairy roots from explant tissues in a wide variety of plant species, including medicinal plants, has been reported. Genotypeindependent agrobacterium rhizogenes mediated root. Soybean hairy roots produced in vitro by agrobacterium. Agrobacterium rhizogenesmediated transformation of soybean to study root biology. An improved method for agrobacterium rhizogenes mediated. Agrobacterium rhizogenes mediated transformation of superroot. Kereszt a, li d, indrasumunar a, nguyen cd, nontachaiyapoom s, kinkema m and gresshoff pm 2007 agrobacterium rhizogenesmediated transformation of soybean to study root biology. Hairy root transformation using agrobacterium rhizogenes as a. Agrobacterium rhizogenesmediated transformation of pisum. Induction of transgenic hairy roots in soybean genotypes by.
Agrobacteriummediated transformation of soybean and. Agrobacterium rhizogenesmediated transformation of soybean to. Agrobacterium rhizogenesmediated transformation of a. Agrobacterium rhizogenesmediated transformation of soybean. Cotyledons were separated from 4dayold soybean seedlings and infected with three different a. The present study involved the formulation of protocol of agrobacterium rhizogenes mediated transformation for the detailed study of isoflavones metabolism in soybean.
Agrobacterium rhizogenesmediated transformation of soybean to study root biology attila kereszt1,2, dongxue li1,2, arief indrasumunar1, cuc dt nguyen1, sureeporn nontachaiyapoom1, mark kinkema1. Till date, there are no reports available on influence of physical and chemical treatments on enhancement of transformation rates in a. Agrobacteriummediated hairy root transformation is a widely applied method to generate transgenic soybean roots for studying rootspeci. Agrobacterium rhizogenesmediated transformation of. Briefly, mature seeds of shengli were surfacesterilized to germinate on ms medium fig.
Pdf this protocol is used to induce transgenic roots on soybean to study the function of genes required in biological processes of the root. Protocol agrobacterium rhizogenesmediated transformation of soybean to study root biology attila kereszt1,2, dongxue li1,2, arief indrasumunar1, cuc dt. The arabidopsis gene kojak, involved in root hair development, was efficiently knocked down. Composite soybean plants were created using agrobacterium rhizogenesmediated transformation. It has been employed in rna interference rnai studies in roots limpens et al. Agrobacterium soybean 1 updated 032210 agrobacteriummediated transformation of soybean and recovery of transgenic soybean plants. Agrobacterium rhizogenesmediated transformation of soybean to study root biology article pdf available in nature protocol 24.
Mature soybean seeds were soaked for 24 h, the embryonic tips were collected and cultured on msb5 medium supplemented with 3. The production of axenic hairy root cultures is an interesting tool, because the transgenic root explants can be clonally propagated in studies of, for example. We describe an optimized protocol for the agrobacterium rhizogenes mediated transformation of soybean and the induction of hairy root development in vitro. Agrobacterium rhizogenes an overview sciencedirect topics. Assessment of conditions affecting agrobacteriumrhizogenes. Agrobacterium rhizogenesmediated root transformation. Mar 27, 2019 in this study, we set out to test the efficiency of the crisprcas9 system in editing soybean storage protein genes using agrobacterium rhizogenes mediated hairy root transformation system. This bacterium induces the neoplastic growth of plant cells that differentiate to form hairy roots. Rna interference rnai is a powerful reverse genetic tool to study gene function. Agrobacterium rhizogenesmediated transformation has been widely used in. Agrobacterium rhizogenes transformed soybeans with atpap18.
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