scholarly journals A Stable Agrobacterium rhizogenes-Mediated Transformation of Cotton (Gossypium hirsutum L.) and Plant Regeneration From Transformed Hairy Root via Embryogenesis

2020 ◽  
Vol 11 ◽  
Author(s):  
Min-Long Cui ◽  
Chen Liu ◽  
Chun-Lan Piao ◽  
Chuan-Liang Liu
Keyword(s):  
Gossypium Hirsutum ◽  
Agrobacterium Rhizogenes ◽  
Hairy Roots ◽  
Gene Function ◽  
Fluorescent Protein ◽  
Function Analysis ◽  
Green Fluorescent Protein Gene ◽  
Pcr Analysis ◽  
Cotton Transformation ◽  
Transformed Hairy Roots

Genetic transformation is a powerful tool to study gene function, secondary metabolism pathways, and molecular breeding in crops. Cotton (Gossypium hirsutum L.) is one of the most important economic crops in the world. Current cotton transformation methods take at least seven to culture and are labor-intensive and limited to some cultivars. In this study, we first time achieved plantlet regeneration of cotton via embryogenesis from transformed hairy roots. We inoculated the cotyledon explants of a commercial cultivar Zhongmian-24 with Agrobacterium rhizogenes strain AR1193, harboring a binary vector pBI-35S::GFP that contained the NPT II (neomycin phosphotransferase) gene and the GFP (green fluorescent protein) gene as a fluorescent marker in the T-DNA region. 82.6% explants produced adventitious roots, of which 53% showed GFP expression after transformation. 82% of transformed hairy roots produced embryonic calli, 12% of which regenerated into stable transformed cotton plants after 7 months of culture. The integration of GFP in the transformed cotton genomes were confirmed by PCR (Polymerase chain reaction) and Southern blot analysis as well as the stable expression of GFP were also detected by semi-quantitative RT-PCR analysis. The resultant transformed plantlets were phenotypically, thus avoiding Ri syndrome. Here we report a stable and reproducible method for A. rhizogenes-mediated transformation of cotton using cotyledon as explants, which provides a useful and reliable platform for gene function analysis of cotton.

scholarly journals Agrobacterium rhizogenes-mediated hairy root transformation as an efficient system for gene function analysis in Litchi chinensis

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yaqi Qin ◽  
Dan Wang ◽  
Jiaxin Fu ◽  
Zhike Zhang ◽  
Yonghua Qin ◽  
...  
Keyword(s):  
Functional Genomics ◽  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Hairy Roots ◽  
Gene Function ◽  
Function Analysis ◽  
Transformation System ◽  
Litchi Chinensis ◽  
Gene Function Analysis ◽  
Stem Segments

Abstract Background Litchi chinensis Sonn. is an economically important fruit tree in tropical and subtropical regions. However, litchi functional genomics is severely hindered due to its recalcitrance to regeneration and stable transformation. Agrobacterium rhizogenes-mediated hairy root transgenic system provide an alternative to study functional genomics in woody plants. However, the hairy root transgenic system has not been established in litchi. Results In this study, we report a rapid and highly efficient A. rhizogenes-mediated co-transformation system in L. chinensis using Green Fluorescent Protein (GFP) gene as a marker. Both leaf discs and stem segments of L. chinensis cv. ‘Fenhongguiwei’ seedlings were able to induce transgenic hairy roots. The optimal procedure involved the use of stem segments as explants, infection by A. rhizogenes strain MSU440 at optical density (OD600) of 0.7 for 10 min and co-cultivation for 3 days, with a co-transformation efficiency of 9.33%. Furthermore, the hairy root transgenic system was successfully used to validate the function of the key anthocyanin regulatory gene LcMYB1 in litchi. Over-expression of LcMYB1 produced red hairy roots, which accumulated higher contents of anthocyanins, proanthocyanins, and flavonols. Additionally, the genes involving in the flavonoid pathway were strongly activated in the red hairy roots. Conclusion We first established a rapid and efficient transformation system for the study of gene function in hairy roots of litchi using A. rhizogenes strain MSU440 by optimizing parameters. This hairy root transgenic system was effective for gene function analysis in litchi using the key anthocyanin regulator gene LcMYB1 as an example.

scholarly journals Comparative analysis and mutation effects of fpp2–fpp1 tandem genes encoding proteolytic extracellular enzymes of Flavobacterium psychrophilum

Microbiology ◽  
2011 ◽  
Vol 157 (4) ◽  
pp. 1196-1204 ◽  
Author(s):  
David Pérez-Pascual ◽  
Esther Gómez ◽  
Beatriz Álvarez ◽  
Jessica Méndez ◽  
Pilar Reimundo ◽  
...  
Keyword(s):  
Gene Function ◽  
Type Strain ◽  
Wild Type Strain ◽  
Proteolytic Enzymes ◽  
Function Analysis ◽  
Phenotypic Characterization ◽  
Pcr Analysis ◽  
Wild Type ◽  
Flavobacterium Psychrophilum ◽  
Genes Encoding

Flavobacterium psychrophilum is a very significant fish pathogen that secretes two biochemically characterized extracellular proteolytic enzymes, Fpp1 and Fpp2. The genes encoding these enzymes are organized as an fpp2–fpp1 tandem in the genome of strain F. psychrophilum THC02/90. Analysis of the corresponding encoded proteins showed that they belong to two different protease families. For gene function analysis, new genetic tools were developed in F. psychrophilum by constructing stable isogenic fpp1 and fpp2 mutants via single-crossover homologous recombination. RT-PCR analysis of wild-type and mutant strains suggested that both genes are transcribed as a single mRNA from the promoter located upstream of the fpp2 gene. Phenotypic characterization of the fpp2 mutant showed lack of caseinolytic activity and higher colony spreading compared with the wild-type strain. Both characteristics were recovered in the complemented strain. One objective of this work was to assess the contribution to virulence of these proteolytic enzymes. LD50 experiments using the wild-type strain and mutants showed no significant differences in virulence in a rainbow trout challenge model, suggesting instead a possible nutritional role. The gene disruption procedure developed in this work, together with the knowledge of the complete genome sequence of F. psychrophilum, open new perspectives for the study of gene function in this bacterium.

Induction of hairy roots in Arnica montana L. by Agrobacterium rhizogenes

2013 ◽  
Vol 8 (5) ◽  
pp. 470-479 ◽  
Author(s):  
Mariya Petrova ◽  
Ely Zayova ◽  
Mariana Vlahova
Keyword(s):  
Genetic Transformation ◽  
Agrobacterium Rhizogenes ◽  
Hairy Roots ◽  
Culture Media ◽  
Pcr Analysis ◽  
Transformed Roots ◽  
Fresh Weight ◽  
Transformation Techniques ◽  
Nutrient Media ◽  
Arnica Montana

AbstractThe induction of hairy roots in Arnica montana L. by Agrobacterium rhizogenes mediated system was established. The frequency of genetic transformation varied from 4.8 to 12% depended on method of infection. The cefotaxime at concentration of 200 mg/l proved to suppress effectively the growth of A. rhizogenes after co-cultivation. Among the three tested nutrient media: Murashige and Skoog (MS), Gamborg’s (B5) and Schenk and Hildebrandt (SH), MS medium was superior for growth and high biomass production of transformed roots compared to other culture media. After culturing for 40 days the fresh weight of clone T4 increased 7.6 fold over the non-transformed roots. The transfer of rol A, rol B and rol C genes into Arnica genome was confirmed by PCR analysis. Established genetic transformation techniques in A. montana efficiently provided and generated a large number of transformed roots — an excellent system for studying gene function and could be used for the production of secondary metabolites synthesized in roots.

scholarly journals Genetically transformed hairy roots producing agropine induced on Trigonella foenum-graecum L. plant by Agrobacterium rhizogenes 1601.

2013 ◽  
Vol 7 (1) ◽  
pp. 91-98
Author(s):  
M. M. Al-Mahdawe ◽  
M. K. Al-Mallah ◽  
A. O. Al-Attrakchii
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Roots ◽  
Root Formation ◽  
Paper Electrophoresis ◽  
Transformed Roots ◽  
Fresh Weight ◽  
Trigonella Foenum Graecum ◽  
Transformed Hairy Roots ◽  
Long Time ◽  
Trigonella Foenum Graecum L

Inoculation of leaves and stems explants excised from field-grown Trigonella foenum-graecum L. with engineered Agrobacterium rhizogenes 1601 inoculum of optical density 1.90, 2.06 and 1.96 led to the formation of hairy roots on these explants. The highest percent of infection with inoculum of O.D 1.90 was 20% in leaves, and was 53.3% in stems using inoculum of O.D 2.06. Infection percent was 50% in hypocotyl compared with non-inoculated explants. The results showed that inoculation of seedlings lacking roots were slow in hairy root formation and take long time compared with their formation in intact seedlings inoculated with the same inocula. Results of paper electrophoresis of these root proved the incidence of their genetic transformation. Moreover, these transformed roots grow happily in liquid WP medium with an average fresh weight 5.9g after four weeks of culture, whereas fresh weight of normal roots was 1.02g under the same conditions.

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 Specific activity of thymidine nucleotide biosynthetic enzymes in hairy roots extracts of Sesamum indicum L. transformed by two strains of Agrobacteruim rhizogenes

2014 ◽  
Vol 8 (2) ◽  
pp. 64-72
Author(s):  
Nihal E. Al- Taee ◽  
Sajida A. Abood ◽  
Mozahim K. Al-Mallah
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Roots ◽  
Dihydrofolate Reductase ◽  
Specific Activity ◽  
Sesamum Indicum ◽  
Dna And Rna ◽  
Transformed Hairy Roots ◽  
Natural Vector ◽  
Activity Of Enzymes ◽  
Control Samples

The study concluded induction of transformed hairy roots from leaves and decapitated seedlings of Sesamum indicum L. using two strains of Agrobacterium rhizogenes considered as natural vector of transformation. The strain R1601 stimulated roots on leaves and seedlings during 20 days of inoculation placed on solidified Arnon and Hoagland medium. Whereas they involved 12 days when inoculated with the strain R15834. Generally strain R15834 was efficient in inducing these roots and their numbers than strain R1601 which approached 54.4% and 41.6% respectively. The results indicated an increase in the specific activity of enzymes Thymidlate synthase (TS), Dihydrofolate reductase (DHFR), Serine hydroxy methyl transfrase (SHMT) in extract of transformed hairy roots producing agropine by stain R15834 and approach 4.610, 1.057, 0.480 µmolmin mg of protein respectively compared with the activity of 1.256, 0.097, 0.125µ molmin mg in the control samples. This was coupled with an increase in amount of DNA and RNA that approached 105, 1020 µg per gram respectively compared to 44, 462 µg per gram in control samples. The transformation of these hairy roots was pointed out through the separation of agropine spots from their extracts when electrophoreted in the presence of standared agropine.

scholarly journals Induction of transgenic hairy roots in soybean genotypes by Agrobacterium rhizogenes‑mediated transformation

2011 ◽  
Vol 46 (9) ◽  
pp. 1070-1075 ◽  
Author(s):  
Ricardo Luís Mayer Weber ◽  
Maria Helena Bodanese‑Zanettini
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Hairy Roots ◽  
Fluorescent Protein ◽  
Root Culture ◽  
Transformation Frequency ◽  
Plant Production ◽  
Uniform Size ◽  
Soybean Genotypes

The objective of this work was to perform the screening of soybean genotypes as to their ability to respond to the induction of hairy roots by Agrobacterium rhizogenes‑mediated transformation. Four Brazilian soybean cultivars (BRSMG 68 Vencedora, BRS 137, Embrapa 48, and MG/BR 46 Conquista) and two North American ones adapted to Brazilian cropping conditions (Bragg and IAS‑5) were screened for their capacity to respond to A. rhizogenes in protocols for in vitro hairy root culture and ex vitro composite plant production. Four‑day‑old seedlings with uniform size were injected with A. rhizogenes harboring the plasmid p35S‑GFP. Seedlings expressing green fluorescent protein (GFP) in at least one hairy root were used to determine the transformation frequency. Using an axenic in vitro protocol, excised cotyledons from four‑day‑old seedlings were infected with A. rhizogenes harboring the pCAMBIA1301 plasmid, containing the gusA reporter gene. The transformation frequency and the number of days for hairy root emergence after bacterial infection (DAI) were evaluated. The transformation frequency and DAI varied according to the genotype. Cultivars MG/BR 46 Conquista and BRSMG 68 Vencedora are more susceptible to A. rhizogenes and can be recommended for transformation experiments.

Bio-interaction of Agrobacterium rhizogenes with Capsicum annuum L. (sweet variety) and Establishment Hairy Roots Cultures

2020 ◽  
Vol 1 (2) ◽  
pp. 30-33
Author(s):  
Qutaiba Al-Nema
Keyword(s):  
Capsicum Annuum ◽  
Agrobacterium Rhizogenes ◽  
Hairy Roots ◽  
Future Research ◽  
Ms Medium ◽  
Transformed Roots ◽  
Transformed Hairy Roots ◽  
Petri Dishes ◽  
Suitable System ◽  
Modern Technologies

Genetic transformation using Agrobacterium is one of the techniques used to transfer desired genes to plants. This protocol is considered a short – cut to get transformed plants which could be an alternative method and suitable system compared with the classical methods. This study aimed to investigate the response of C. annuum seedlings (sweet variety) to the formation transformed hairy roots induced by A. rhizogenes strain R1601. Sterilized seeds of Capsicum annuum were inoculated with the Agrobacterium rhizogenes inoculum. The samples were then transferred to the surface of solidified MS medium. Hairy roots were developed at the inoculation sites and were enucleated 1.0-1.5 cm length and placed in 9.0 cm Petri-dishes containing 15 ml of agar solidified MS medium. Agropine test was performed according to the standard method. The inoculated seedlings showed a good response 90%. Transformed hairy roots were established at the injection sites within 10 days and these roots were easily grown on agar-solidified MS medium. The results are confirmed that these roots were transformed roots it in terms of positive agropine detection. The current study concluded that the biological interaction between Agrobacterium rhizogenes strain R1601 and Capsicum anuum L. seedlings, was successful. This study encourages future research to improve this plant by continuing and applying modern technologies to obtain genetically modified plants.

Sign in / Sign up

Export Citation Format

Share Document