Detection of Target Sites Edited in Upland Cotton By The CRISPR/Cas9 System Mediated By Agrobacterium Rhizogenes

2021 ◽  
Author(s):  
Lili Zhou ◽  
Yali Wang ◽  
Peilin Wang ◽  
Jiamin Wang ◽  
Hongmei Cheng
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Hairy Roots ◽  
Upland Cotton ◽  
Gene Editing ◽  
Target Site ◽  
Shoot Meristem ◽  
Multiple Target ◽  
Root Transformation ◽  
Target Sites

Abstract Background CRIPSR/Cas9 gene editing has the ability to effectively modify plant genomes. Multiple target sites usually were designed and the effective target sites were selected for editing. However, upland cotton is allotetraploid and is commonly considered as difficult and inefficient to transform. Therefore, it’s important to quickly identify feasibility of the target site. In this study, we use Agrobacterium rhizogenes K599 strain to infect cotton shoot meristem and induce them to grow hairy roots to detect the feasibility of a selected target designed in GhMYB25-like gene. Results We designed a sgRNA within the second exons of GhMYB25-likeA and GhMYB25-likeD and constructed the CRISPR vector. Transient hairy root transformation using A. rhizogenes K599 with four OD600s (0.4, 0.6,0.8, 1.0) was performed in Coker 312 (R15). The results show that A. rhizogenes at OD600 = 0.6–0.8 is the best concentration range for inducing cotton hairy roots. The other three cultivars (TM-1, Lumian 21, Zhongmian 49) were injected using A. rhizogenes K599 with OD600 = 0.6-0.8 and all produced hairy roots. We characterized ten R15 plants with hairy roots and detected different degrees of base deletions and insert at the target site in five R15 plants. Conclusion Overall, our data show A. rhizogenes-mediated transient hairy root transformation offers a rapid and efficient method to detect sgRNA feasibility in cotton.

A reliable and efficient protocol for induction of hairy roots in Agastache foeniculum

Biologia ◽  
2014 ◽  
Vol 69 (7) ◽  
Author(s):  
Elnaz Nourozi ◽  
Bahman Hosseini ◽  
Abbas Hassani
Keyword(s):  
Salicylic Acid ◽  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Hairy Roots ◽  
High Performance ◽  
Root Culture ◽  
Hairy Root Culture ◽  
Biochemical Properties ◽  
Separate Experiment ◽  
Transformed Roots

AbstractHairy root culture system is a valuable tool to study the characteristics of gene expression, gene function, root biology, biochemical properties and biosynthesis pathways of secondary metabolites. In the present study, hairy roots were established in Anise hyssop (Agastache foeniculum) via Agrobacterium rhizogenes. Three strains of Agrobacterium rhizogenes (A4, A7 and 9435), were used for induction of hairy roots in four various explants (hypocotyl, cotyledon, one-month-old leaf and five-month-old leaf) of Anise hyssop. The highest frequency of transformation was achieved using A4 strain in one-month-old leaves (51.1%). The transgenic states of hairy root lines were confirmed by PCR (Polymerase chain reaction) method. High performance liquid chromatography analysis revealed that the production of rosmarinic acid (RA) in transformed roots of A. foeniculum was almost 4-fold higher than that of the non-transformed roots. In a separate experiment, hairy roots obtained from one-month-old leaves inoculated with A4 strain, were grown in liquid medium and the effects of different concentrations of salicylic acid (0.0, 0.01, 0.1 and 1 mM) and chitosan (0, 50, 100 and 150 mg L−1) (as elicitor) and sucrose (20, 30, 40 and 50 g L−1) on the growth of hairy roots were evaluated. The results showed that, 30 g L−1 sucrose and 100 mg L−1 chitosan increased the biomass of hairy root cultures and application of salicylic acid reduced the growth of hairy roots compared with control roots.

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.

Technology and Application of Hairy Root Culture in Monocotyledons

2021 ◽  
Vol 07 ◽  
Author(s):  
Chang-Qi Hao ◽  
Shuai-Run Wang ◽  
Yi Wang ◽  
Xin-Yi Hou ◽  
Ya-Xuan Jiang ◽  
...  
Keyword(s):  
Medicinal Plants ◽  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Hairy Roots ◽  
Root Culture ◽  
Hairy Root Culture ◽  
Food Crops ◽  
Root Induction ◽  
Hairy Root Induction ◽  
Selection Of

Background: Hairy root culture has been widely used in the production of metabolites in dicotyledons, and a large number of food crops and medicinal plants in monocotyledons need to be developed, but there are many difficulties in the induction of hairy roots in monocotyledons. The purpose of this paper is to introduce the inducing methods, influencing factors and application of hairy roots in monocotyledons, and to promote the development of hairy root system in monocotyledons. Methods: The mechanism of action of Agrobacterium rhizogenes and the current situation of hairy root induction, induction methods and influencing factors of monocotyledons were summarized so as to provide convenience for efficient acquisition of hairy root of monocotyledons. Results: Monocotyledons are not easy to produce phenols, cells are prone to lignification, adverse differentiation and selective response to Agrobacterium rhizogenes strains. It is proposed that before induction, plant varieties and explants should be selected, and different infection strains should be screened. In the process of hairy root induction, exogenous inducers such as acetosyringone can be added. Although these factors can provide some help for the induction of hairy roots in monocotyledons, we still need to pay attention to the disadvantages of monocotyledons from dicotyledons at the cellular level. Conclusion: A large number of food crops and medicinal plants are monocotyledons. Hairy root culture can be used to help the breeding and production of medicinal substances. Therefore, it is necessary to pay attention to the selection of varieties and explants, the selection of Agrobacterium rhizogenes and the addition of acetosyringone in the process of hairy root induction so as to improve the production efficiency and facilitate the development and utilization of monocotyledons.

scholarly journals Evaluating α-glucosidase inhibitory activity of hairy roots cultured from some plants in malvaceae family induced by Agrobacterium rhizogenes ATCC 15834

2020 ◽  
Vol 2 (6) ◽  
pp. 165-174
Author(s):  
Vu Thi Bach Phuong ◽  
Pham Thi Anh Hong ◽  
Quach Ngo Diem Phuong
Keyword(s):  
Type 2 Diabetes ◽  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Hairy Roots ◽  
Inhibitory Activity ◽  
Root Culture ◽  
Root Induction ◽  
Hibiscus Sabdariffa ◽  
Sida Acuta

One of the most effective methods for type 2 diabetes treatments is inhibition of enzyme α-glucosidase in the intestines to slow down the release of glucose from carbohydrates in the diet, reduce plasma glucose levels and prevent hyperglycemia after meals. Therefore, seeking α-glucosidase inhibitors used in the treatment of diabetes from plant is the attention of many scientists. Based on the potential of the hairy root culture technology in increasing valuable chemical compounds accumulating, this study aimed to induce hairy roots from six plants of the Malvaceae family including Urena lobata, Abutilon indicum, Hibiscus Sabdariffa, Hibiscus rosa-sinensis, Sida acuta, Sida rhombifolia, and screening which materials has the highest in α-glucosidase inhibitory activity. We have successfully induced hairy roots from six plant species by using the Agrobacterium rhizogenes ATCC 15834 strain. The highest rates of hairy root induction were observed in Hibiscus Sabdariffa and Urena lobata. The stable introduction of rolB and rolC genes to plant genomes was confirmed by PCR. Under liquid-shake culture conditions on MS medium, hairy roots of Hibiscus sabdariffa, Urena lobata and Sida acuta showed better development than other species, and therefore, they are selected for the study of α-glucosidase inhibitory activity. This study proved that Urena lobata was stronger in inhibiting α-glucosidase activity than other studied plants, with the IC50 value of 7.65 μg/ml. The results of this study demonstrated Urena lobata hairy root might be considered as a potential supply of medicinal plants for the treatment of type 2 diabetes.  

scholarly journals Hairy root culture of white mulberry (Morus alba L.) for a source of tyrosinase inhibitors

2020 ◽  
Vol 93 (1) ◽  
Author(s):  
Hanh Thi My Nguyen ◽  
Tan Quoc Tran ◽  
Anh Lan Bui ◽  
Phuong Ngo Diem Quach
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Hairy Roots ◽  
Root Culture ◽  
Hairy Root Culture ◽  
Morus Alba ◽  
Root Induction ◽  
Skin Whitening ◽  
Tyrosinase Inhibitors ◽  
White Mulberry

White mulberry (Morus alba L.) root was reported to contain potential tyrosinase inhibitors such as norartocarpetin, streppogenin, oxyresveratrol (5, 7, 2′, 4′-tetrahydroxy-flavavone) and 2-arylbenzofuran. This study aims at investigating Morus alba L. hairy root culture to create a source of tyrosinase inhibitors for skin-whitening application. Morus alba L. leaf (including mesophyll and petiole) and stem explants were randomly wounded and inoculated with Agrobacterium rhizogenes ATCC 15834 in 10-40 minutes, and coculturing for 4 days. After inoculation, Agrobacterium rhizogenes were removed by using MS medium adding 200mg/L cefotaxime as antibiotics. Then, fresh inducted hairy roots were grown in nonphytohormone liquid medium (MS, White, Gamborg B5 or WPM) on a shaker (80rpm) at 25 ±1°C. Inducted hairy roots were tested for the present of rolB by PCR analysis and its effect on tyrosinase activity using a 96-well plate reader. The suitable explants for hairy root induction are leaves, especially petioles in 10 minutes (root induction frequency: 83%). After induction, hairy roots on MS was grown more efficiently (increased 3.933 times in FW) than on WPM, B5 and White medium. Inducted hairy roots were successfully transgenes by the present of rolB in their genome and had the same anti-tyrosinase activity as the natural roots without significance differences (IC50: 3.860 and 3.597 μg/mL, respectively). Cultured Morus alba L. hairy roots are potential source of tyrosinase inhibitors for future skin-whitening ingredient.

scholarly journals Comparison of Different Strains of Agrobacterium rhizogenes for Hairy Root Induction and Betulin and Betulinic Acid Production in Morus alba

2017 ◽  
Vol 12 (4) ◽  
pp. 1934578X1701200
Author(s):  
Chang Ha Park ◽  
Shicheng Zhao ◽  
Hyeon Ji Yeo ◽  
Ye Eun Park ◽  
Thanislas Bastin Baska ◽  
...  
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Hairy Roots ◽  
Betulinic Acid ◽  
Acid Production ◽  
Leaf Tissue ◽  
Dry Weight ◽  
Morus Alba ◽  
Root Induction ◽  
Different Strains

Three Agrobacterium rhizogenes strains were tested for their ability to transform the plant Morus alba L. and to induce production of the secondary metabolites betulin and betulinic acid. All the tested strains of A. rhizogenes (R1601, LBA9402 and R1000) were able to induce hairy root formation in leaf tissue explants. Strain LBA9402 had the highest rate of infection (92.7% ± 8.8%), whereas strain R1601 had the lowest rate (87.4% ± 9.3%). The highest number of hairy roots per explant (5.6 ± 0.5) and the greatest root length (2.4 ± 0.2 mm) were obtained with strain LBA9402. We also evaluated dry weight (a measure of growth) and betulin and betulinic acid production in hairy roots and found that the highest growth (167.8 ± 14.5 mg/flask) occurred after infection with strain LBA9402. Furthermore the highest production of betulin (5.4 ± 0.4 mg/g dry weight) and betulinic acid (2.3 ± 0.2 mg/g dry weight) was noted using strain LBA9402. Among three elicitors, yeast extract showed the highest induction of betulin production (8.7 ± 0.4 mg/g) and silver nitrate induced the highest yield of betulinic acid (4.1 ± 0.2 mg/g). Our study showed that A. rhizogenes strain LBA9402 was the most effective of the three tested strains for production of transformed root cultures and betulin and betulinic acid.

scholarly journals Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation for gene functional and gene editing analysis in soybean

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yuanyuan Cheng ◽  
Xiaoli Wang ◽  
Li Cao ◽  
Jing Ji ◽  
Tengfei Liu ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Subcellular Localization ◽  
Root System ◽  
Hairy Root ◽  
Protein Interactions ◽  
Gene Editing ◽  
Transformation Frequency ◽  
Highly Efficient ◽  
Root Transformation ◽  
Gene Functional Analysis

Abstract Background Agrobacterium-mediated genetic transformation is a widely used and efficient technique for gene functional research in crop breeding and plant biology. While in some plant species, including soybean, genetic transformation is still recalcitrant and time-consuming, hampering the high-throughput functional analysis of soybean genes. Thus we pursue to develop a rapid, simple, and highly efficient hairy root system induced by Agrobacterium rhizogenes (A. rhizogenes) to analyze soybean gene function. Results In this report, a rapid, simple, and highly efficient hairy root transformation system for soybean was described. Only sixteen days were required for the whole workflow and the system was suitable for various soybean genotypes, with an average transformation frequency of 58–64%. Higher transformation frequency was observed when wounded cotyledons from 1-day-germination seeds were inoculated and co-cultivated with A. rhizogenes in 1/2 B5 (Gamborg’ B-5) medium. The addition of herbicide selection to root production medium increased the transformation frequency to 69%. To test the applicability of the hairy root system for gene functional analysis, we evaluated the protein expression and subcellular localization in transformed hairy roots. Transgenic hairy roots exhibited significantly increased GFP fluorescence and appropriate protein subcellular localization. Protein–protein interactions by BiFC (Bimolecular Fluorescent Complimentary) were also explored using the hairy root system. Fluorescence observations showed that protein interactions could be observed in the root cells. Additionally, hairy root transformation allowed soybean target sgRNA screening for CRISPR/Cas9 gene editing. Therefore, the protocol here enables high-throughput functional characterization of candidate genes in soybean. Conclusion A rapid, simple, and highly efficient A. rhizogenes-mediated hairy root transformation system was established for soybean gene functional analysis, including protein expression, subcellular localization, protein–protein interactions and gene editing system evaluation.

scholarly journals Efficient genetic transformation of Impatiens hawkerii Bull. (Balsamiaceae) using agrobacterium rhizogenes

2009 ◽  
Vol 61 (3) ◽  
pp. 467-474 ◽  
Author(s):  
Snezana Milosevic ◽  
Angelina Subotic ◽  
A. Cingel ◽  
Sladjana Jevremovic ◽  
Slavica Ninkovic
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Hairy Roots ◽  
Lateral Roots ◽  
Basal Medium ◽  
Growth Patterns ◽  
Pcr Analysis ◽  
Gus Assay ◽  
Hairy Root Clones ◽  
First Time

Transformation of Impatiens hawkerii Bull. mediated by Agrobacterium rhizogenes strain A4M70GUS was studied. Hairy roots developed 10 days after inoculation were excised from the shoot explants and transferred onto Murashige and Skoog's (MS) basal medium lacking plant growth regulators. More than 20 hairy root clones were established and eight of them were further analyzed. Each clone differed significantly from the others in growth capacity and lateral branching. Clone C2 showed the highest biomass (20.6 g L-1) as well as the highest number of lateral roots (37 ? 2.2). The transgenic nature of the established hairy root clones was confirmed by GUS assay and PCR analysis. In conclusion, hairy roots were developed for the first time in I. hawkerii Bull., and transgenic hairy root clones showed a distinct morphological nature and growth patterns.

scholarly journals Study on the growth of Agrobacterium rhizogenes-induced Catharanthus roseus hairy roots in liquid media

2019 ◽  
Vol 2 (1) ◽  
pp. 5-12
Author(s):  
Nguyen Nhu Nhut ◽  
Bui Van Le
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Hairy Roots ◽  
Catharanthus Roseus ◽  
Growth Kinetics ◽  
Exponential Phase ◽  
Liquid Media ◽  
Hairy Root Line ◽  
Initial Ph ◽  
Initial Results

Catharanthus roseus hairy roots induced by Agrobacterium rhizogenes get potential to apply to many fields. The suitable conditions for culturing depend on each hairy root line. In shaken liquid media in dark at 25 oC, VIN002-12 and VIN005-07 hairy root lines had the best growth in hemi-concentrated Gamborg’B5 media while VIN072-15 and VIN077-09 lines showed the best growth in in hemi-concentrated White media. The appropriate initial pH of medium for the lines was in range of 5.7–6.5. The hairy root lines used 2–5% sucrose as an optimal source of carbon for their growth. In selected conditions, the growth kinetics curves showed the end of exponential phase at the 28th day of culture with VIN005-07 and VIN072- 15 lines whereas at the 35th day with VIN002-12 and VIN077-09 lines. The initial results are quite possible to produce biomass for researches on four hairy root lines in the future.  

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