An Efficient Root Transformation System for Recalcitrant Vicia sativa

Frontiers in Plant Science ◽

10.3389/fpls.2021.781014 ◽

2022 ◽

Vol 12 ◽

Author(s):

Vy Nguyen ◽

Iain R. Searle

Keyword(s):

Hairy Root ◽

Hairy Roots ◽

Functional Gene ◽

Gene Analysis ◽

Vicia Sativa ◽

Transformation System ◽

Common Vetch ◽

Root Transformation ◽

Functional Gene Analysis

Common vetch (Vicia sativa) is a multi-purpose legume widely used in pasture and crop rotation systems. Vetch seeds have desirable nutritional characteristics and are often used to feed ruminant animals. Although transcriptomes are available for vetch, problems with genetic transformation and plant regeneration hinder functional gene studies in this legume species. Therefore, the aim of this study was to develop a simple, efficient and rapid hairy root transformation system for common vetch to facilitate functional gene analysis. At first, we infected the hypocotyls of 5-day-old in vitro or in vivo, soil-grown seedlings with Rhizobium rhizogenes K599 using a stabbing method and produced transgenic hairy roots after 24 days at 19 and 50% efficiency, respectively. We later improved the hairy root transformation in vitro by infecting different explants (seedling, hypocotyl-epicotyl, and shoot) with R. rhizogenes. We observed hairy root formation at the highest efficiency in shoot and hypocotyl-epicotyl explants with 100 and 93% efficiency, respectively. In both cases, an average of four hairy roots per explant were obtained, and about 73 and 91% of hairy roots from shoot and hypocotyl-epicotyl, respectively, showed stable expression of a co-transformed marker β-glucuronidase (GUS). In summary, we developed a rapid, highly efficient, hairy root transformation method by using R. rhizogenes on vetch explants, which could facilitate functional gene analysis in common vetch.

Download Full-text

An efficient root transformation system for recalcitrant Vicia sativa

10.1101/2020.06.14.151175 ◽

2020 ◽

Author(s):

Vy Nguyen ◽

Iain R. Searle

Keyword(s):

Hairy Roots ◽

Functional Gene ◽

Gene Analysis ◽

Vicia Sativa ◽

Transformation System ◽

Common Vetch ◽

Root Transformation ◽

Functional Gene Analysis

AbstractCommon vetch (Vicia sativa) is a multi-purpose legume widely used in pasture and crop rotation systems. Vetch seeds have desirable nutritional characteristics and are often used to feed ruminant animals. Although transcriptomes are available for vetch, problems with genetic transformation and plant regeneration hinder functional gene studies in this legume species. Therefore, the aim of this study was to develop an efficient and rapid hairy root transformation system for common vetch to facilitate functional gene analysis. We infected the hypocotyls of five-day old in vitro or in vivo, soil grown, seedlings with Agrobacterium rhizogenes and produced transformed hairy roots 28 days later at 24% and 43% efficiency, respectively. Seventy-nine percent of the hairy roots from the in vitro plants showed stable expression of a co-transformed marker β-glucuronidase (GUS). In summary, transgenic hairy roots were obtained within 28 days, and are sufficient to facilitate functional gene analysis in common vetch.

Download Full-text

HAIRY ROOT TRANSFORMATION SYSTEM IN LARGE-SEEDED GRAIN LEGUMES

Israel Journal of Plant Sciences ◽

10.1080/07929978.1995.10676585 ◽

1995 ◽

Vol 43 (1) ◽

pp. 1-5 ◽

Cited By ~ 9

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.

Download Full-text

The application of CRISPR/Cas9 in hairy roots to explore the functions of AhNFR1 and AhNFR5 genes during peanut nodulation

BMC Plant Biology ◽

10.1186/s12870-020-02614-x ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Hongmei Shu ◽

Ziliang Luo ◽

Ze Peng ◽

Jianping Wang

Keyword(s):

Hairy Root ◽

Hairy Roots ◽

Root Nodules ◽

Crop Improvement ◽

Functional Study ◽

Nodule Formation ◽

Transformation System ◽

Knock Out ◽

Root Transformation ◽

Peanut Hairy Root

Abstract Background Peanut is an important legume crop growing worldwide. With the published allotetraploid genomes, further functional studies of the genes in peanut are very critical for crop improvement. CRISPR/Cas9 system is emerging as a robust tool for gene functional study and crop improvement, which haven’t been extensively utilized in peanut yet. Peanut plant forms root nodules to fix nitrogen through a symbiotic relationship with rhizobia. In model legumes, the response of plants to rhizobia is initiated by Nod factor receptors (NFRs). However, information about the function of NFRs in peanut is still limited. In this study, we applied the CRISPR/Cas9 tool in peanut hairy root transformation system to explore the function of NFR genes. Results We firstly identified four AhNFR1 genes and two AhNFR5 genes in cultivated peanut (Tifrunner). The gene expression analysis showed that the two AhNFR1 and two AhNFR5 genes had high expression levels in nodulating (Nod+) line E5 compared with non-nodulating (Nod-) line E4 during the process of nodule formation, suggesting their roles in peanut nodulation. To further explore their functions in peanut nodulation, we applied CRISPR technology to create knock-out mutants of AhNFR1 and AhNFR5 genes using hairy root transformation system. The sequencing of these genes in transgenic hairy roots showed that the selected AhNFR1 and AhNFR5 genes were successfully edited by the CRISPR system, demonstrating its efficacy for targeted mutation in allotetraploid peanut. The mutants with editing in the two AhNFR5 genes showed Nod- phenotype, whereas mutants with editing in the two selected AhNFR1 genes could still form nodules after rhizobia inoculation. Conclusions This study showed that CRISPR-Cas9 could be used in peanut hairy root transformation system for peanut functional genomic studies, specifically on the gene function in roots. By using CRISPR-Cas9 targeting peanut AhNFR genes in hairy root transformation system, we validated the function of AhNFR5 genes in nodule formation in peanut.

Download Full-text

Resveratrol Biosynthesis in Hairy Root Cultures of Tan and Purple Seed Coat Peanuts

Agronomy ◽

10.3390/agronomy11050975 ◽

2021 ◽

Vol 11 (5) ◽

pp. 975

Author(s):

Ye-Eun Park ◽

Chang-Ha Park ◽

Hyeon-Ji Yeo ◽

Yong-Suk Chung ◽

Sang-Un Park

Keyword(s):

Arachis Hypogaea ◽

Hairy Root ◽

Hairy Roots ◽

Seed Coat ◽

Biological Properties ◽

Fresh Weight ◽

Good Source ◽

Plant Parts ◽

Seedling Roots

Peanut (Arachis hypogaea) is a crop that can produce resveratrol, a compound with various biological properties, such as those that exert antioxidant, anticancer, and anti-inflammatory effects. In this study, trans-resveratrol was detected in the roots, leaves, and stems of tan and purple seed coat peanuts (Arachis hypogaea) cultivated in a growth chamber. Both cultivars showed higher levels of resveratrol in the roots than the other plant parts. Thus, both cultivars were inoculated with Agrobacterium rhizogenes, in vitro, to promote hairy root development, thereby producing enhanced levels of t-resveratrol. After 1 month of culture, hairy roots from the two cultivars showed higher levels of fresh weight than those of seedling roots. Furthermore, both cultivars contained higher t-resveratrol levels than those of their seedling roots (6.88 ± 0.21 mg/g and 28.07 ± 0.46 mg/g, respectively); however, purple seed coat peanut hairy roots contained higher t-resveratrol levels than those of tan seed coat peanut hairy roots, ranging from 70.16 to 166.76 mg/g and from 46.61 to 54.31 mg/g, respectively. The findings of this study indicate that peanut hairy roots could be a good source for t-resveratrol production due to their rapid growth, high biomass, and substantial amount of resveratrol.

Download Full-text

Clonal sector analysis and cell ablation confirm a function for DORNROESCHEN-LIKE in founder cells and the vasculature in Arabidopsis

Planta ◽

10.1007/s00425-020-03545-5 ◽

2021 ◽

Vol 253 (2) ◽

Author(s):

Dorothea Glowa ◽

Petra Comelli ◽

John W. Chandler ◽

Wolfgang Werr

Keyword(s):

Transcription Factor ◽

Functional Gene ◽

Gene Analysis ◽

Cell Ablation ◽

Sector Analysis ◽

Founder Cells ◽

Conventional Methods ◽

Functional Gene Analysis ◽

Lineage Analysis ◽

In Cells

Abstract Main conclusion Inducible lineage analysis and cell ablation via conditional toxin expression in cells expressing the DORNRÖSCHEN-LIKE transcription factor represent an effective and complementary adjunct to conventional methods of functional gene analysis. Abstract Classical methods of functional gene analysis via mutational and expression studies possess inherent limitations, and therefore, the function of a large proportion of transcription factors remains unknown. We have employed two complementary, indirect methods to obtain functional information for the AP2/ERF transcription factor DORNRÖSCHEN-LIKE (DRNL), which is dynamically expressed in flowers and marks lateral organ founder cells. An inducible, two-component Cre–Lox system was used to express beta-glucuronidase GUS in cells expressing DRNL, to perform a sector analysis that reveals lineages of cells that transiently expressed DRNL throughout plant development. In a complementary approach, an inducible system was used to ablate cells expressing DRNL using diphtheria toxin A chain, to visualise the phenotypic consequences. These complementary analyses demonstrate that DRNL functionally marks founder cells of leaves and floral organs. Clonal sectors also included the vasculature of the leaves and petals, implicating a previously unidentified role for DRNL in provasculature development, which was confirmed in cotyledons by closer analysis of drnl mutants. Our findings demonstrate that inducible gene-specific lineage analysis and cell ablation via conditional toxin expression represent an effective and informative adjunct to conventional methods of functional gene analysis.

Download Full-text