In the following protocol, we describe an improved a. This transformation method is particularly suitable for those plants, including legumes, whose transformation using agrobacterium tumefaciens has been challenging. Agrobacterium rhizogenesmediated transformation of. Transformed plants regenerated from explants inoculated with sha17 were fertile and phenotypically normal. Agrobacterium tumefaciensmediated transformation is a preferred method in many plants.
Composite plants are generated by inoculating wildtype shoots with agrobacterium rhizogenes, which subsequently induces the formation of transgenic roots. Methods and protocols, methods in molecular biology, vol. Agrobacterium rhizogenes recently revised as rhizobium rhizogenes. Fast, efficient and reproducible genetic transformation of. Soybean is one of the worlds most important oil and protein crops. In the present study we used agrobacterium rhizogenesmediated hairy roots as an alternative approach for agrobacterium tumefaciens transformation. 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. We describe an optimized protocol for the agrobacterium rhizogenes mediated transformation of soybean and the induction of hairy root development in vitro. Most of the in vitro procedures used for generating composite plants involve placing the a. Agrobacterium rhizogenesmediated root transformation in chickpea was performed following the protocol developed for phaseolus with few modifications. Agrobacterium rhizogenesmediated transformation of pisum. Induction of transgenic hairy roots in soybean genotypes by. 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.
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. Agrobacterium rhizogenes mediated transformation of soybean. In this study we carried out extensive work on enhancement of transformation efficiency in. 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 attila kereszt1,2, dongxue li1,2, arief indrasumunar1, cuc dt nguyen1, sureeporn nontachaiyapoom1, mark kinkema1. We designed and tested sgrnas to target nine different major storage protein genes and detected dna mutations in three storage protein genes in soybean. Agrobacterium rhizogenesmediated root transformation. Rhizobium rhizogenes formerly agrobacterium rhizogenes is a gramnegative soil bacterium that produces hairy root disease in dicotyledonous plants. Optimization of agrobacteriummediated transformation in. Jun 25, 2009 in the present study, we developed a highly efficient a. Agrobacterium rhizogenesmediated transformation has been widely used in. Mature soybean seeds were soaked for 24 h, the embryonic tips were collected and cultured on msb5 medium supplemented with 3. It has been employed in rna interference rnai studies in roots limpens et al. This study involved the formulation of protocol of agrobacterium rhizogenes mediated transformation for the detailed study of isoflavones ifs metabolism in soybean.
The quantity and ratio of different benzylisoquinoline alkaloids produced by opium poppy and california poppy were virtually identical in transformed and wild. The data presented here show that agrobacterium rhizogenes. Soybean hairy roots produced in vitro by agrobacterium rhizogenes. This system can be used to study gene functions on the. We developed a simple, effective and reproducible hairy root protocol in tobacco and arabidopsis. Kereszt a1, li d, indrasumunar a, nguyen cd, nontachaiyapoom s, kinkema m, gresshoff pm. This protocol is used to induce transgenic roots on soybean to study the function of genes required in biological processes of the root. In a comparative experiment, regeneration frequencies were not significantly different. Prior to transformation, the binary vectors were transferred into competent a. Genotypeindependent agrobacterium rhizogenes mediated. Induction of transgenic hairy roots in soybean genotypes. Agrobacterium rhizogenes an overview sciencedirect topics.
Agrobacterium rhizogenes or rhizobium rhizogenes is able to transform plant genomes and induce the production of hairy roots. Agrobacterium rhizogenes mediated transformation has several features desirable for the production of secondary metabolites. Developing an efficient and reproducible transformation method is imperative to expedite functional genomics studies in this crop. Merrill using primarynode explants from seedlings vitr cell dev biol plant, 43 2007, pp. Pdf this protocol is used to induce transgenic roots on soybean to study the function of genes required in biological processes of the root. Agrobacterium rhizogenesmediated transformation of soybean to study root biology by attila kereszt, dongxue li, arief indrasumunar, cuc nguyen, sureeporn. Agrobacterium rhizogenes mediated transformation of superroot.
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. Agrobacteriummediated transformation of soybean and recovery. Dna carried on a large plasmid, to the genome of a host plant cell. Hairy root transformation using agrobacterium rhizogenes as a. Here, we present an optimized and detailed procedure for agrobacterium rhizogenes mediated root transformation of chickpea.
Agrobacterium rhizogenes mediated transformation of. Dec 18, 2012 agrobacterium rhizogenesmediated transformation of soybean to study root biology. Lipm, inracnrs, bp 52627, 326 castanet tolosan cedex, france. Kereszt a, li d, indrasumunar a et al 2007 agrobacterium rhizogenesmediated transformation of soybean to study root biology. Briefly, mature seeds of shengli were surfacesterilized to germinate on ms medium fig. Agrobacteriummediated transformation of soybean and. A rapid, efficient and highperformance transformation protocol employing agrobacterium rhizogenes was developed for the common bean phaseolus vulgaris. In the present study, we developed a highly efficient a. Efficient transformation is a key factor for the improvement of soybean by genetic modification. Validation of a hairy roots system to study soybean. Agrobacterium rhizogenesmediated transformation of soybean to. Mutagenesis of seed storage protein genes in soybean using. A novel coldinducible zinc finger protein from soybean, scof1, enhances cold tolerance in transgenic plants. Agrobacterium rhizogenes is the etiological agent for hairy root disease also known as root mat disease.
This transformation procedure generates, with high efficiency 7090%, hairy roots in cultivars, landraces and accessions of phaseolus vulgaris common bean. 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 novel agrobacterium rhizogenesmediated transformation. The rapid and efficient induction of hairy roots from explant tissues in a wide variety of plant species, including medicinal plants, has been reported. Soybean hairy roots produced in vitro by agrobacterium. 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. Agrobacterium rhizogenesmediated transformation has been. Thus, the gene was isolated and a final vector atpap18pk7gwg2d was built. Genotypeindependent agrobacterium rhizogenes mediated root. Improved cotyledonary node method using an alternative explant derived from mature seed for efficient.
However, the transformation efficiency in soybean is still low. Induction of hairy roots in chickpea using agrobacterium rhizogenes. Agrobacterium tumefaciens mediated gene delivery to callus and somatic embryos. Young seedlings with unfolded cotyledons are infected at. Till date, there are no reports available on influence of physical and chemical treatments on enhancement of transformation rates in a. For promoter studies, the specificity of the soybean root promoter could be used to detect expression in hairy roots. Agrobacterium soybean 1 updated 032210 agrobacteriummediated transformation of soybean and recovery of transgenic soybean plants. 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. Cotyledons were separated from 4dayold soybean seedlings and infected with three different a. In this study, we demonstrated the successful transformation of two pea pisum sativum l. High transformation efficiency is a prerequisite for study of gene function and molecular breeding. 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. Kereszt a, li d, indrasumunar a, nguyen cdt, nontachaiyapoom s, kinkema m, et al. Generation of soybean glycine max transient transgenic.
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. Agrobacterium rhizogenesmediated transformation has become a powerful tool for studying gene function and root biology due to its quick and simple methodology. 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. Hairy root transformation using agrobacterium rhizogenes. Development of a fast and efficient root transgenic system. The tdna containing the transgene of interest in a disarmed binary vector is generally cotransformed with the resident a. 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. 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. 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. Agrobacterium mediated hairy root transformation is a widely applied method to generate transgenic soybean roots for studying root speci. Agrobacterium rhizogenesmediated transformation of soybean to study root biology overview of attention for article published in nature protocols, april 2007 altmetric badge. 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 rhizogenes mediated genetic transformation.
Generation of composite plants using agrobacterium. Inducing hairy roots by agrobacterium rhizogenesmediated. Utilizing young seedlings with severed radicleshypocotyls, we developed a highly efficient procedure. An improved method for agrobacterium rhizogenes mediated. The objective of this study is to optimize agrobacterium mediated transformation in soybean by improving the infection efficiency of. Agrobacterium rhizogenesmediated transformation of soybean to study root biology article pdf available in nature protocol 24. 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. The arabidopsis gene kojak, involved in root hair development, was efficiently knocked down. Agrobacterium rhizogenesmediated transformation of arachis. A comparison study of agrobacteriummediated transformation. Protocol agrobacterium rhizogenesmediated transformation of soybean to study root biology attila kereszt1,2, dongxue li1,2, arief indrasumunar1, cuc dt. Assessment of conditions affecting agrobacteriumrhizogenes.
Pdf agrobacterium rhizogenesmediated transformation of. Agrobacteriummediated hairy root transformation is a widely applied method to generate transgenic soybean roots for studying rootspeci. Validation of a hairy roots system to study soybeansoybean. In this study, we examined competencies of various protocols to induce and explants that respond to hairy root transformation in bean plants. Agrobacterium soybean 1 updated 032210 agrobacterium mediated transformation of soybean and recovery of transgenic soybean plants. Assessment of conditions affecting agrobacterium rhizogenes. 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. Pdf soybean hairy roots produced in vitro by agrobacterium. Gene expression of reporters is indistinguishable in plants transformed by agrobacterium tumefaciens as compared with a. Agrobacterium rhizogenesmediated transformation of a.
This study seeks to understand the role of an arabidopsis arabidopsis thaliana purple apase gene atpap18 in soybean. Generation of soybean glycine max transient transgenic roots. Agrobacterium rhizogenes transformed soybeans with atpap18. Composite soybean plants were created using agrobacterium rhizogenesmediated transformation. This bacterium induces the neoplastic growth of plant cells that differentiate to form hairy roots. Developed hairy roots were compared with intact roots to. A novel agrobacterium rhizogenesmediated transformation method of soybean glycine max l. However, a method to easily screen candidate genes which could play an important role.
1373 1018 341 25 758 1329 698 1262 428 737 893 1288 261 916 1603 1254 536 183 1055 760 1222 416 775 1212 1396 534 45 307 692 815 235 863 1139 915 552 978 134 1147 376 1113