scholarly journals Hairy CRISPR: Genome Editing in Plants Using Hairy Root Transformation

Plants ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 51
Author(s):  
Alexey S. Kiryushkin ◽  
Elena L. Ilina ◽  
Elizaveta D. Guseva ◽  
Katharina Pawlowski ◽  
Kirill N. Demchenko
Keyword(s):  
Genome Editing ◽  
Hairy Root ◽  
Fluorescent Protein ◽  
Effective Means ◽  
Gene Encoding ◽  
Transgenic Roots ◽  
Translational Enhancer ◽  
Current State ◽  
Root Transformation ◽  
Agrobacterium Rhizogenes Strains

CRISPR/Cas-mediated genome editing is a powerful tool of plant functional genomics. Hairy root transformation is a rapid and convenient approach for obtaining transgenic roots. When combined, these techniques represent a fast and effective means of studying gene function. In this review, we outline the current state of the art reached by the combination of these approaches over seven years. Additionally, we discuss the origins of different Agrobacterium rhizogenes strains that are widely used for hairy root transformation; the components of CRISPR/Cas vectors, such as the promoters that drive Cas or gRNA expression, the types of Cas nuclease, and selectable and screenable markers; and the application of CRISPR/Cas genome editing in hairy roots. The modification of the already known vector pKSE401 with the addition of the rice translational enhancer OsMac3 and the gene encoding the fluorescent protein DsRed1 is also described.

scholarly journals Use of the rhizobial type III effector gene nopP to improve Agrobacterium rhizogenes-mediated transformation of Lotus japonicus

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yan Wang ◽  
Feng Yang ◽  
Peng-Fei Zhu ◽  
Asaf Khan ◽  
Zhi-Ping Xie ◽  
...  
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Fluorescent Protein ◽  
Transformation Efficiency ◽  
Lotus Japonicus ◽  
Nodule Bacterium ◽  
Transgenic Roots ◽  
Model Legume ◽  
Effector Gene ◽  
Root Transformation

Abstract Background Protocols for Agrobacterium rhizogenes-mediated hairy root transformation of the model legume Lotus japonicus have been established previously. However, little efforts were made in the past to quantify and improve the transformation efficiency. Here, we asked whether effectors (nodulation outer proteins) of the nodule bacterium Sinorhizobium sp. NGR234 can promote hairy root transformation of L. japonicus. The co-expressed red fluorescent protein DsRed1 was used for visualization of transformed roots and for estimation of the transformation efficiency. Results Strong induction of hairy root formation was observed when A. rhizogenes strain LBA9402 was used for L. japonicus transformation. Expression of the effector gene nopP in L. japonicus roots resulted in a significantly increased transformation efficiency while nopL, nopM, and nopT did not show such an effect. In nopP expressing plants, more than 65% of the formed hairy roots were transgenic as analyzed by red fluorescence emitted by co-transformed DsRed1. A nodulation experiment indicated that nopP expression did not obviously affect the symbiosis between L. japonicus and Mesorhizobium loti. Conclusion We have established a novel protocol for hairy root transformation of L. japonicus. The use of A. rhizogenes LBA9402 carrying a binary vector containing DsRed1 and nopP allowed efficient formation and identification of transgenic roots.

scholarly journals First‐generation genome editing in potato using hairy root transformation

2020 ◽  
Vol 18 (11) ◽  
pp. 2201-2209 ◽  
Author(s):  
Nathaniel M. Butler ◽  
Shelley H. Jansky ◽  
Jiming Jiang
Keyword(s):  
Genome Editing ◽  
Hairy Root ◽  
First Generation ◽  
Root Transformation

scholarly journals Agrobacterium rhizogenes Transformation of the Phaseolus spp.: A Tool for Functional Genomics

2006 ◽  
Vol 19 (12) ◽  
pp. 1385-1393 ◽  
Author(s):  
Georgina Estrada-Navarrete ◽  
Xochitl Alvarado-Affantranger ◽  
Juan-Elías Olivares ◽  
Claudia Díaz-Camino ◽  
Olivia Santana ◽  
...  
Keyword(s):  
Functional Genomics ◽  
Agrobacterium Rhizogenes ◽  
Fluorescent Protein ◽  
Phaseolus Vulgaris L ◽  
Rhizobium Tropici ◽  
Transgenic Roots ◽  
Root Transformation ◽  
Microbe Interactions ◽  
Wild Accessions ◽  
Green Fluorescent

A fast, reproducible, and efficient transformation procedure employing Agrobacterium rhizogenes was developed for Phaseolus vulgaris L. wild accessions, landraces, and cultivars and for three other species belonging to the genus Phaseolus: P. coccineus, P. lunatus, and P. acutifolius. Induced hairy roots are robust and grow quickly. The transformation frequency is between 75 and 90% based on the 35-S promoter-driven green fluorescent protein and β-glu-curonidase expression reporter constructs. When inoculated with Rhizobium tropici, transgenic roots induce normal determinate nodules that fix nitrogen as efficiently as inoculated standard roots. The A. rhizogenes-induced hairy root transformation in the genus Phaseolus sets the foundation for functional genomics programs focused on root physiology, root metabolism, and root–microbe interactions.

scholarly journals CRISPR/Cas9 genome editing system confirms centriolin’s role in cytokinesis

2022 ◽  
Vol 15 (1) ◽  
Author(s):  
Eric Seronick ◽  
Jae Son ◽  
Cameron Michael ◽  
Hannah Fogg ◽  
Zeynep Gromley ◽  
...  
Keyword(s):  
Cell Division ◽  
Genome Editing ◽  
Cell Cycle Progression ◽  
Primary Cilia ◽  
Effective Means ◽  
Mrna Level ◽  
Microtubule Organizing Center ◽  
Gene Encoding ◽  
Protein Levels ◽  
Organizing Center

Abstract Objective In addition to its function as the microtubule organizing center of the cell, the centrosome has functions in many other cellular processes including primary cilia formation, DNA damage checkpoints, and cell cycle progression. But the role of individual components of the centrosome in these processes remains unclear. Previous studies used siRNA (small interfering RNA) to “knock down” protein levels of the centrosome component centriolin, resulting in failed cytokinesis. Since this approach was transient, only targeting centriolin at the mRNA level, we sought to confirm these findings by permanently disrupting the gene encoding centriolin using the CRISPR/Cas9 system of genome editing. Results This study provides evidence that the CRISPR/Cas9 system is capable of effectively reducing centriolin protein levels in the cell. Furthermore, this disruption leads to a failure of cytokinesis that is reminiscent of the phenotype previously reported for the siRNA-mediated disruption of centriolin. Furthermore, no additional defects in cell division were observed, consistent with results seen with previous siRNA studies. We conclude that the CRISPR/Cas9 system is an effective means of permanently removing the cellular pools of centriolin and that the disruption of centriolin at both the mRNA level and genomic level lead to similar cell division defects.

scholarly journals Agrobacterium rhizogenes—mediated transformation ofPisum sativumL. roots as a tool for studying the mycorrhizal and root nodule symbioses

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6552 ◽  
Author(s):  
Irina V. Leppyanen ◽  
Anna N. Kirienko ◽  
Elena A. Dolgikh
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Rhizobium Leguminosarum ◽  
Fluorescent Protein ◽  
Arbuscular Mycorrhizal ◽  
Am Fungi ◽  
Symbiotic Association ◽  
Gene Encoding ◽  
Transgenic Roots ◽  
Pea Seedlings ◽  
Green Fluorescent

In this study, we demonstrated the successful transformation of two pea (Pisum sativumL.) cultivars usingAgrobacterium rhizogenes, whereby transgenic roots in the resulting composite plants showed expression of the gene encoding the green fluorescent protein. Subsequent to infection withA. rhizogenes, approximately 70%–80% of pea seedlings developed transgenic hairy roots. We found out that the transgenic roots can be efficiently nodulated byRhizobium leguminosarumbv.viciaeand infected by the arbuscular mycorrhizal (AM) fungusRhizophagus irregularis. The morphology of nodules in the transgenic roots was found to be identical to that of nodules observed in wild-type roots, and we also observed the effective induction of markers typical of the symbiotic association with AM fungi. The convenient protocol for highly efficientA. rhizogenes-mediated transformation developed in this study would be a rapid and effective tool for investigating those genes involved in the development of the two types of symbioses found in pea plants.

scholarly journals Multi-Task Optimization and Multi-Task Evolutionary Computation in the Past Five Years: A Brief Review

Mathematics ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 864
Author(s):  
Qingzheng Xu ◽  
Na Wang ◽  
Lei Wang ◽  
Wei Li ◽  
Qian Sun
Keyword(s):  
Evolutionary Computation ◽  
Explicit Knowledge ◽  
Optimization Problems ◽  
Effective Means ◽  
Typical Application ◽  
The Past ◽  
Current State ◽  
Evolution Algorithms ◽  
Application Fields

Traditional evolution algorithms tend to start the search from scratch. However, real-world problems seldom exist in isolation and humans effectively manage and execute multiple tasks at the same time. Inspired by this concept, the paradigm of multi-task evolutionary computation (MTEC) has recently emerged as an effective means of facilitating implicit or explicit knowledge transfer across optimization tasks, thereby potentially accelerating convergence and improving the quality of solutions for multi-task optimization problems. An increasing number of works have thus been proposed since 2016. The authors collect the abundant specialized literature related to this novel optimization paradigm that was published in the past five years. The quantity of papers, the nationality of authors, and the important professional publications are analyzed by a statistical method. As a survey on state-of-the-art of research on this topic, this review article covers basic concepts, theoretical foundation, basic implementation approaches of MTEC, related extension issues of MTEC, and typical application fields in science and engineering. In particular, several approaches of chromosome encoding and decoding, intro-population reproduction, inter-population reproduction, and evaluation and selection are reviewed when developing an effective MTEC algorithm. A number of open challenges to date, along with promising directions that can be undertaken to help move it forward in the future, are also discussed according to the current state. The principal purpose is to provide a comprehensive review and examination of MTEC for researchers in this community, as well as promote more practitioners working in the related fields to be involved in this fascinating territory.

scholarly journals Versatile CRISPR/Cas9 Systems for Genome Editing in Ustilago maydis

2021 ◽  
Vol 7 (2) ◽  
pp. 149
Author(s):  
Sarah-Maria Wege ◽  
Katharina Gejer ◽  
Fabienne Becker ◽  
Michael Bölker ◽  
Johannes Freitag ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cell Biology ◽  
Gene Deletion ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Model Organism ◽  
Ustilago Maydis ◽  
Genomic Locus ◽  
Targeted Gene Deletion ◽  
Molecular Cell Biology

The phytopathogenic smut fungus Ustilago maydis is a versatile model organism to study plant pathology, fungal genetics, and molecular cell biology. Here, we report several strategies to manipulate the genome of U. maydis by the CRISPR/Cas9 technology. These include targeted gene deletion via homologous recombination of short double-stranded oligonucleotides, introduction of point mutations, heterologous complementation at the genomic locus, and endogenous N-terminal tagging with the fluorescent protein mCherry. All applications are independent of a permanent selectable marker and only require transient expression of the endonuclease Cas9hf and sgRNA. The techniques presented here are likely to accelerate research in the U. maydis community but can also act as a template for genome editing in other important fungi.

scholarly journals CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yoshihiko Nanasato ◽  
Masafumi Mikami ◽  
Norihiro Futamura ◽  
Masaki Endo ◽  
Mitsuru Nishiguchi ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cryptomeria Japonica ◽  
Fluorescent Protein ◽  
Japanese Cedar ◽  
Targeted Mutagenesis ◽  
Embryogenic Tissue ◽  
Breeding Period ◽  
Single Mutation ◽  
Knock Out ◽  
Guide Rna

AbstractCryptomeria japonica (Japanese cedar or sugi) is one of the most important coniferous tree species in Japan and breeding programs for this species have been launched since 1950s. Genome editing technology can be used to shorten the breeding period. In this study, we performed targeted mutagenesis using the CRISPR/Cas9 system in C. japonica. First, the CRISPR/Cas9 system was tested using green fluorescent protein (GFP)-expressing transgenic embryogenic tissue lines. Knock-out efficiency of GFP ranged from 3.1 to 41.4% depending on U6 promoters and target sequences. The GFP knock-out region was mottled in many lines, indicating genome editing in individual cells. However, in 101 of 102 mutated individuals (> 99%) from 6 GFP knock-out lines, embryos had a single mutation pattern. Next, we knocked out the endogenous C. japonica magnesium chelatase subunit I (CjChlI) gene using two guide RNA targets. Green, pale green, and albino phenotypes were obtained in the gene-edited cell lines. Sequence analysis revealed random deletions, insertions, and replacements in the target region. Thus, targeted mutagenesis using the CRISPR/Cas9 system can be used to modify the C. japonica genome.

HAIRY ROOT TRANSFORMATION SYSTEM IN LARGE-SEEDED GRAIN LEGUMES

1995 ◽  
Vol 43 (1) ◽  
pp. 1-5 ◽  
Author(s):  
H.J. Siefkes-Boer ◽  
M.J. Noonan ◽  
D.W. Bullock ◽  
A.J. Conner
Keyword(s):  
Vicia Faba ◽  
Hairy Root ◽  
Hairy Roots ◽  
Faba Bean ◽  
Grain Legumes ◽  
Transformation System ◽  
Root Transformation ◽  
Specific Strain ◽  
Size And Morphology ◽  
Root Size

Hairy roots were produced on faba bean (Vicia faba L.) and chickpea (Cicer arietinum L.) plants by inoculation with Agrobacterium root-inducing strains. Examination of 14 plant genotypes and eight Agrobacterium strains in all possible combinations revealed specific strain/genotype interactions. Hairy root size and morphology differed substantially between faba bean and chickpea hairy roots. Sixty percent of chickpea hairy roots were 10–15 mm in length and forty percent, 15–25 mm. All were <1.0 mm in thickness. Sixty-three percent of faba bean hairy roots were 15–25 mm long and thirty-seven percent, 25–40 mm. All faba bean hairy roots were between 1.0 and 1.5 mm in thickness.

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