an-efficient-reproducible-method-for-producing-composite-plants

An Efficient and Reproducible Method for Producing Composite Plants by Agrobacterium rhizogenes-Based Hairy Root Transformation

Producing composite plants with transgenic roots and nontransgenic stems and buds using Agrobacterium rhizogenes-mediated hairy root transformation is a ...

genetic-transformation-mediated-agrobacterium-tumefaciens-solanum

Genetic Transformation Mediated by Agrobacterium tumefaciens in Solanum nigrum

Solanum nigrumis an orphan solanaceous relative of potato and tomato, and its fresh fruit is a nutritious and delicious berry. The whole plant of S. ...

Liaocheng University

 is a fast-growing rhizobial species that can establish a nitrogen-fixing symbiosis with a wide range of legume species including soybeans (). In soybeans, this interaction shows a high level of specificity such that particular  strains nodulate only a limited set of plant genotypes. Here we report the identification of a dominant gene in soybeans that restricts nodulation with  USDA193. Genetic mapping in an F2 population revealed co-segregation of the underlying locus with the previously cloned  gene. The  allele encodes a member of the Toll-interleukin receptor/nucleotide-binding site/leucine-rich repeat class of plant resistance proteins that restricts nodulation by  strains USDA257 and USDA205, and an allelic variant of this gene also restricts nodulation by  USDA122. By means of complementation tests and CRISPR/Cas9-mediated gene knockouts, we demonstrate that the  allele also is responsible for resistance to nodulation by  USDA193. Therefore, the  allele likely provides broad-spectrum resistance to nodulation by many  and  strains in soybeans.

The Soybean  Gene Restricts Nodulation by  USDA193.

Agrobacterium rhizogenes-mediated (ARM) transformation is a highly efficient technique for generating composite plants composed of transgenic roots and wild-type shoot, providing a powerful tool for studying root biology. The ARM transformation has been established in many plant species, including soybean. However, traditional transformation of soybean, transformation efficiency is low. Additionally, the hairy roots were induced in a medium, and then the generated composite plants were transplanted into another medium for growth. This two-step operation is not only time-consuming, but aggravates contamination risk in the study of plant-microbe interactions.

One-step generation of composite soybean plants with transgenic roots by Agrobacterium rhizogenes-mediated transformation.

-mediated hairy root transformation provides a powerful tool for investigating the functions of plant genes involved in rhizobia-legume symbiosis. However, in the traditional identification methods of transgenic hairy roots based on reporter genes, an expensive chemical substrate or equipment is required.

Anthocyanin, a novel and user-friendly reporter for convenient, non-destructive, low cost, directly visual selection of transgenic hairy roots in the study of rhizobia-legume symbiosis.

-mediated (ARM) transformation is an efficient and powerful tool to generate transgenic roots to study root-related biology. For loss-of-function studies, transgenic-root-induced indel mutations by CRISPR/Cas9 only with homozygous/biallelic mutagenesis can exhibit mutant phenotype(s) (excluding recessive traits). However, a low frequency of homozygous mutants was produced by a constitutive promoter to drive  expression. Here, we identified a highly efficient  gamma- promoter, termed , with strong activity in the region where the root meristem will initiate and in the whole roots in broad eudicots species.  achieved higher homozygous/biallelic mutation efficiency than the most widely used  promoter in driving  expression in soybean, , and tomato roots. Using the p-Cas9 system, the average homozygous/biallelic mutation frequency is 1.7-fold and 8.3-fold higher than the p-Cas9 system for single and two target site(s) in the genome, respectively. Our results demonstrate the advantage of the p-Cas9 system used in ARM transformation, especially its great potential in diploids with multiple-copy genes targeted mutations and polyploid plants with multiplex genome editing.  is conservatively active in various eudicots species, suggesting that  might be applied in a wide range of dicots species.

 promoter-driven clustered regularly interspaced short palindromic repeats/Cas9 highly efficiently generates homozygous/biallelic mutations in the transformed roots by -mediated transformation.

With the advent of multiple omics and Genome-Wide Association Studies (GWAS) technology, genome-scale functional analysis of candidate genes is to be conducted in diverse plant species. Construction of plant binary expression vectors is the prerequisite for gene function analysis. Therefore, it is of significance to develop a set of plant binary expression vectors with highly efficient, inexpensive, and convenient cloning method, and easy-to-use in screening of positive recombinant in . In this study, we developed a set of plant binary expression vectors, termed pBTR vectors, based on Golden Gate cloning using I restriction site. Foreign DNA fragment of interest (FDI) can be cloned into the destination pBTR by one-step digestion-ligation reaction in a single tube, and even the FDI contains internal I site(s). Markedly, in one digestion-ligation reaction, multiple FDIs (exemplified by cloning four soybean , , , and  genes) can be cloned into the pBTR vector to generate multiple corresponding expression constructs (each expression vector carrying an FDI). In addition, the pBTR vectors carry the visual marker, a brightness monomeric red fluorescent protein mScarlet-I, that can be observed with the unaided eye in screening of positive recombinants without the use of additional reagents/equipment. The reliability of the pBTR vectors was validated in plants by overexpression of / in tomato and  in soybean roots -mediated transformation, promoter activity analysis of  in -mediated transformation, and protein subcellular localization of the  VvCEB1 in tobacco, respectively. These results demonstrated that the pBTR vectors can be used in analysis of gene (over)expression, promoter activity, and protein subcellular localization. These vectors will contribute to speeding up gene function analysis and the process of plant molecular breeding.

Development of a set of novel binary expression vectors for plant gene function analysis and genetic transformation.

Composite plants containing transgenic hairy roots produced with Agrobacterium rhizogenes-mediated transformation have become an important method to study the interaction between plants and arbuscular mycorrhizal fungi (AMF). Not all hairy roots induced by A. rhizogenes are transgenic, however, which leads to requirement of a binary vector to carry a reporter gene to distinguish transgenic roots from non-transformed hairy roots. The beta-glucuronidase gene (GUS) and fluorescent protein gene often are used as reporter markers in the process of hairy root transformation, but they require expensive chemical reagents or imaging equipment. Alternatively, AtMYB75, an R2R3 MYB transcription factor from Arabidopsis thaliana, recently has been used as a reporter gene in hairy root transformation in some leguminous plants and can cause anthocyanin accumulation in transgenic hairy roots. Whether AtMYB75 can be used as a reporter gene in the hairy roots of tomato and if the anthocyanins accumulating in the roots will affect AMF colonization, however, are still unknown. In this study, the one-step cutting method was used for tomato hairy root transformation by A.rhizogenes. It is faster and has a higher transformation efficiency than the conventional method. AtMYB75 was used as a reporter gene in tomato hairy root transformation. The results showed that the overexpression of AtMYB75 caused anthocyanin accumulation in the transformed hairy roots. Anthocyanin accumulation in the transgenic hairy roots did not affect their colonization by the arbuscular mycorrhizal fungus, Funneliformis mosseae strain BGC NM04A, and there was no difference in the expression of the AMF colonization marker gene SlPT4 in AtMYB75 transgenic roots and wild-type roots. Hence, AtMYB75 can be used as a reporter gene in tomato hairy root transformation and in the study of symbiosis between tomato and AMF.

Application of AtMYB75 as a reporter gene in the study of symbiosis between tomato and Funneliformis mosseae.

 (Solanaceae family) is widely consumed as a fruit or local leafy vegetable after boiling; it also serves as a medicinal plant. Although -mediated genetic transformation has been established in . , the transformation period is long. Specifically, induction of roots takes approximately five weeks for tetraploid and hexaploid . , and 7 weeks for diploid . In this study, we developed an improved rooting-induced method that requires only about 1 week and avoids the use of tissue culture. After generating the transgenic shoots, they were directly transplanted into the soil to facilitate root formation. Remarkably, 100% of the transgenic shoots developed roots within 6 days. Our improved method is time-saving (saving more than 1 month) and simpler to operate. The improved rooting-induced step can be applied to induce roots in various plants using tissue culture, exemplified by the carnation ( L.). Furthermore, we applied the improved method to generate .  plants expressing  from kiwifruit ( spp.). This method will contribute to speeding up gene functional analysis and trait improvement in .  and might have potential in fast plant molecular breeding processes in crops and rapid rooting induction in tissue culture.

An Improved and Simplified -Mediated Genetic Transformation Protocol for  with a Shorter Growth Time.

A simplified Agrobacterium tumefaciens-mediated transformation protocol accelerates plant molecular breeding.

To clone DNA sequences quickly and precisely into plasmids is essential for molecular biology studies. Some cloning vectors have been developed for the cloning of PCR products, including blunt-end and T-A cloning. However, different plasmids are required for the cloning of PCR products with blunt ends and 3' A overhang ends. Here, a novel cloning vector, pYFRed, which is based on the pUC19 backbone, has emerged and can be applied in both blunt-end and T-A cloning. PCR products can be cloned into the pYFRed by a one-step digestion-ligation reaction in a tube. The endonuclease recognition sequences of I, 53kI, RV, I, and I for blunt-end cloning and I for T-A cloning were designed and added between the  promoter and the starting codon ATG of the  gene of pYFRed. The ligation efficiency was significantly higher because the restriction enzyme sites utilized were removed from the vector after being successfully constructed. The  gene was introduced into the pYFRed for the screening of the positive recombinants by the unaided eye without the need for additional reagents/equipment. pYFRed is easy to construct in an ordinary laboratory, which facilitates researchers to develop their cloning vector without purchasing commercial cloning vectors.

'Two in One' Cloning Vector Applied for Blunt-End and T-A Cloning with One-Step Digestion-Ligation and Screening of Positive Recombinants by Unaided Eyes.

ArticleTotal : 2	Year
Publication title Cited by 1	2023
Publication title	2025

Article	Year
The Soybean Gene Restricts Nodulation by USDA193. Frontiers in plant science\| PubMed ID: 28936222	2017
One-step generation of composite soybean plants with transgenic roots by Agrobacterium rhizogenes-mediated transformation. BMC plant biology\| PubMed ID: 32397958	2020
Anthocyanin, a novel and user-friendly reporter for convenient, non-destructive, low cost, directly visual selection of transgenic hairy roots in the study of rhizobia-legume symbiosis. Plant methods\| PubMed ID: 32647533	2020
promoter-driven clustered regularly interspaced short palindromic repeats/Cas9 highly efficiently generates homozygous/biallelic mutations in the transformed roots by -mediated transformati... Frontiers in plant science\| PubMed ID: 36330262	2022
Development of a set of novel binary expression vectors for plant gene function analysis and genetic transformation. Frontiers in plant science\| PubMed ID: 36714700	2022
Application of AtMYB75 as a reporter gene in the study of symbiosis between tomato and Funneliformis mosseae. Mycorrhiza\| PubMed ID: 37198421	2023
An Improved and Simplified -Mediated Genetic Transformation Protocol for with a Shorter Growth Time. Plants (Basel, Switzerland)\| PubMed ID: 39124132	2024
A simplified Agrobacterium tumefaciens-mediated transformation protocol accelerates plant molecular breeding. Plant physiology\| PubMed ID: 39484976	2024
'Two in One' Cloning Vector Applied for Blunt-End and T-A Cloning with One-Step Digestion-Ligation and Screening of Positive Recombinants by Unaided Eyes. Current issues in molecular biology\| PubMed ID: 39852132	2024

Shanhua Lyu

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