scholarly journals Enhanced expression of recombinant miraculin, a tasting modifying protein, in Nicotiana tabacum hairy roots using 18.2 heat shock protein promoter and terminator

2021 ◽  
Vol 19 (2) ◽  
pp. 349-358
Author(s):  
La Viet Hong ◽  
Nguyen Thu Giang ◽  
Le Hoang Duc ◽  
Pham Bich Ngoc ◽  
Chu Hoang Ha
Keyword(s):  
Nicotiana Tabacum ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Hairy Root ◽  
Hairy Roots ◽  
Mass Production ◽  
Soluble Protein ◽  
Leaf Explants ◽  
Total Soluble Protein ◽  
Transgenic Hairy Root

Miraculin, a taste modifier, is a protein that was first isolated from miracle fruit (Richadella dulcifica). It can change a sour taste into a sweet taste when sour acids are consumed, although it does not elicit a sweet response. Miraculin may have the potential in industry as a substitute for sugars and as artificial sweeteners. Since the miracle plant has low fruit productivity, mass production of miraculin is limited. Transgenic hairy root culture is a potential alternative system for the mass production of miraculin. In this study, we investigated the expression of recombinant miraculin in tobacco (Nicotiana tabacum) hairy roots. To increase miraculin expression, the heat shock protein 18.2 promoter and terminator were used to drive the expression of miraculin gene in a potential host system. Synthetic miraculin gene was transformed into Nicotiana tobacum leaf explants via Agrobacterium rhizogenes. The transgenic hairy root clones that contained synthetic miraculin gene showed rapid growth and reached maximum growth after 35-day culture. When the expression of miraculin gene was regulated by heat shock protein 18.2 promoter and heat shock protein terminator, the expression of recombinant miraculin increased than the control regulated by CaMV 35S promoter and nopaline synthase terminator. The recombinant miraculin was 19.97 ng per µg of the total soluble protein and equivalently with approximately 2% of the total soluble protein. For the first time, a taste modifying miraculin was successfully expressed in tobacco hairy root. The results in this study have given a promising approach for the application of the transgenic hairy root system to produce recombinant miraculin.

scholarly journals Optimization of conditions for the induction of Tagetes patula L. hairy roots

2019 ◽  
Vol 9 (3) ◽  
pp. 415-420
Author(s):  
L. P. Khlebova ◽  
E. S. Brovko ◽  
O. V. Bychkova ◽  
N. V. Pavlova
Keyword(s):  
Hairy Root ◽  
Hairy Roots ◽  
Wild Strain ◽  
Leaf Explants ◽  
Biologically Active ◽  
Infected Leaf ◽  
Hairy Root Cultures ◽  
Root Cultures ◽  
Tagetes Patula ◽  
Time Of Infection

The hairy root cultures are promising sources of secondary metabolites of plants, including rare and endangered species. They possess genetic and biochemical stability, unlimited growth rate in free-hormone medium, short doubling times, high biosynthetic activity and ecological purity of plant raw materials. The hairy root cultures of Tagetes patula L. can be used to produce biologically active substances with biocidal activity. The study aimed to determine the virulent strain of Agrobacterium rhizogenes and the most effective period of co-cultivation of T. patula leaf explants with an agrobacterium to induce actively growing hairy root cultures. We used 3 strains (A-4b, 8196RT and 15834). The time of infection ranged from 3 to 33 hours in increments of 3 hours. We found that 24 h is the best time of infection to induce hairy roots with the highest transformation efficiency (92%). The wild strain A. rhizogenes 15834 turned out to be the most virulent when infected leaf explants of spreading marigold. This strain provided the maximum transformation effect, reaching 85.4%. We have identified 5 actively growing clones of hairy roots with intensive branching, the growth indices of which were 64-75. In the future, they will be transferred to a liquid medium for biomass accumulation and scaling.

scholarly journals Efficient Editing of CSLD2 Orthologue by CRISPR/Cas9 Affects Cell Morphogenesis of Root Hair in Spinach

2022 ◽  
Author(s):  
Yingping Cao ◽  
Yue Xu ◽  
Yue Zhang ◽  
Heng Zhang ◽  
Chen Bai ◽  
...  
Keyword(s):  
Genome Editing ◽  
Hairy Root ◽  
Hairy Roots ◽  
Membrane Trafficking ◽  
Root Hair ◽  
Large Scale ◽  
Root Hairs ◽  
Gene Expressions ◽  
Short Root ◽  
Transgenic Hairy Root

Abstract CRISPR/Cas9 is a valuable tool and has been extensively employed to perform gene editing in plants. However, CRISPR/Cas9 has not been successfully used in spinach, an important leafy vegetable crop. Here, we precisely edited Spo23361 and Spo10340, two cellulose synthase-like D (CSLD) genes involved in root hair formation of spinach hairy roots, using CRISPR/Cas9 system. Four mutation types (i.e., replacement, insertion, deletion, and combined mutations) were observed, among which the deletion accounted for the vast majority (about 64.1%). Mutation rate differed largely among different targets. Seven homozygous/bi-allelic and eight heterozygous/chimeric mutated lines of Spo23361 were obtained from 15 independent transgenic hairy root lines. All of the seven homozygous/bi-allelic lines displayed bulking and short root hairs, which exhibited the characteristics of Arabidopsis csld2 mutants. Thirteen heterozygous/chimeric mutated lines, but no homozygous/bi-allelic lines, of Spo10340 were obtained from 15 independent transgenic hairy root lines, all of which showed similar phenotype of root hair with normal hairy roots. The transcriptomic analysis further revealed that multiple gene expressions for cell wall modulation and membrane trafficking were disturbed, which might result in the inhibition of root hair growth in Spo23361 mutants. Our results indicate that Agrobacterium rhizogenes-mediated transformation using CRISPR/Cas9 is a simple and efficient genome editing tool in spinach. It lays a solid foundation for large-scale genome editing in spinach in future.

scholarly journals Production of Triterpenoid Sapogenins in Hairy Root Cultures of Silene vulgaris

2015 ◽  
Vol 10 (11) ◽  
pp. 1934578X1501001
Author(s):  
Yeon Bok Kim ◽  
Darwin W. Reed ◽  
Patrick S. Covello
Keyword(s):  
Hairy Root ◽  
Hairy Roots ◽  
Leaf Explants ◽  
Hairy Root Cultures ◽  
Root Cultures ◽  
Silene Vulgaris ◽  
In Vitro Production ◽  
Triterpenoid Saponins ◽  
Meja Treatment

Silene vulgaris (Moench) Garcke (Caryophyllaceae) is widely distributed in North America and contains bioactive oleanane-type saponins. In order to investigate in vitro production of triterpenoid saponins, hairy root cultures of S. vulgaris were established by infecting leaf explants with five strains of Agrobacterium rhizogenes (LBA9402, R1000, A4, 13333, and 15834). The A. rhizogenes strain LBA9402 had an infection of 100% frequency and induced the most hairy roots per plant. Methyl jasmonate (MeJA)-induced changes in triterpenoid saponins in S. vulgaris hairy roots were analyzed. Accumulation of segetalic acid and gypsogenic acid after MeJA treatment was 5-and 2-fold higher, respectively, than that of control root. We suggest that hairy root cultures of S. vulgaris could be an important alternative approach to the production of saponins.

scholarly journals High-Efficiency Agrobacterium rhizogenes-Mediated Transgenic Hairy Root Induction of Lens culinaris

Agronomy ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1170
Author(s):  
Chrysanthi Foti ◽  
Ourania Pavli
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Hairy Roots ◽  
High Efficiency ◽  
Culture Media ◽  
Abiotic Stress Tolerance ◽  
Root Induction ◽  
Media Composition ◽  
Transgenic Hairy Root ◽  
Transgenic Hairy Roots

Previous efforts to transform lentil have been considerably hampered by the crop’s recalcitrant nature, giving rise to particularly low transformation and regeneration frequencies. This study aimed at optimizing an Agrobacterium rhizogenes-mediated transformation protocol for the generation of composite lentil plantlets, comprised of transgenic hairy roots and wild-type shoots. Transformation was performed by inoculating the cut hypocotyl of young lentil seedlings, while optimization involved the use of different bacterial strains, namely R1000, K599 and Arqua, and protocols differing in media composition with respect to the presence of acetosyringone and MES. Composite plantlets had a transgenic hairy root system characterized by an increased number of hairy roots at the hypocotyl proximal region, occasionally showing plagiotropic growth. Overall findings underline that transformation frequencies are subject to the bacterial strain, media composition as well as their combined effect. Among strains tested, R1000 proved to be the most capable of hairy root formation, while the presence of both acetosyringone and MES in inoculation and culture media yielded considerably higher transformation rates. The transgenic nature of hairy roots was demonstrated by the Ri T-DNA-mediated transfer of the rolB2 gene and the simultaneous absence of the virCD sequence of A. rhizogenes. Our findings provide strong evidence that A. rhizogenes-mediated transformation may be employed as a suitable approach for generating composite seedlings in lentil, a species whose recalcitrance severely hampers all efforts addressed to transformation and whole plant regeneration procedures. To the best of our knowledge, this is the first report on the development of a non-laborious and time-efficient protocol for the generation of transgenic hairy roots in lentil, thus providing an amenable platform for root biology and gene expression studies in the context of improving traits related to biotic and abiotic stress tolerance.

scholarly journals Hairy Root Induction on Justicia gendarussa by Various Density of Agrobacterium rhizogenes strain LB 510

2017 ◽  
Vol 9 (1) ◽  
pp. 26
Author(s):  
Dwi Kusuma Wahyuni ◽  
Siti Nur Hafida ◽  
Tri Muji Ermayanti ◽  
Bambang Prajoga Eko Wardoyo ◽  
Hery Purnobasuki ◽  
...  
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Hairy Roots ◽  
Solid Medium ◽  
Leaf Explants ◽  
Root Culture ◽  
Hairy Root Culture ◽  
Root Induction ◽  
Hairy Root Induction ◽  
Industrial Needs

<p>Gandarusa (<em>Justicia gendarussa</em> Burm.f.) is an Indonesian medicinal plant that has many benefits as drug and male contracetive. For industrial needs, Gandarusa must be  available in large quantity. Hairy root culture is one of methode to produce phytochemistry compound. The objective of the study was to examine the effect of various density of <em>Agrobacterium rhizogenes</em> strain LB510 on hairy roots induction of gandarusa (<em>Justicia gendarussa </em>Burm.f.) leaf plant. Leaf explants were inoculated in MS liquid medium with various density of OD<sub>600</sub> = 0.1; 0.2; 0.3; 0.4; and 0.5. Explants were co-cultivated for 2 days on MS solid medium without any hormone then sub-cultured on MS solid medium containing antibiotic cefotaxim 300 ppm, in dark condition. The data were analyzed descriptively and statistically. The results showed that various density of <em>Agrobacterium rhizogenes</em> strain LB510 was affected the lenght of hairy roots induction of <em>J</em><em>.</em><em> gendarussa</em> Burm.f., but these was not effected toward lenght formation time and number of hairy root. The treatment of OD<sub>600</sub> 0.2 was the best treatment for hairy root induction on <em>Justicia gendarussa </em>Burm. f. This data could be used for optimized the quality of methode of hairy root induction. </p>

scholarly journals Effect of Different Strains of Agrobacterium rhizogenes and Nature of Explants on Plumbago indica Hairy Root Culture with Special Emphasis on Root Biomass and Plumbagin Production

2010 ◽  
Vol 5 (12) ◽  
pp. 1934578X1000501
Author(s):  
Moumita Gangopadhyay ◽  
Saikat Dewanjee ◽  
Somnath Bhattacharyya ◽  
Sabita Bhattacharya
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Hairy Roots ◽  
Root Biomass ◽  
Leaf Explants ◽  
Pcr Analysis ◽  
Stem Explants ◽  
Root Induction ◽  
Rolb Gene ◽  
Plumbago Indica

The aim of the present study was to determine the effect of three strains of Agrobacterium rhizogenes (ATCC 15834, A4 and LBA 9402) and the nature of explants (leaf and stem) on hairy root induction, growth and plumbagin production in Plumbago indica. The first appearance of hairy roots, the transformation frequency, dry root biomass and plumbagin accumulation were found to be maximum in hairy roots induced in leaf explants infected with A. rhizogenes ATCC 15834 as compared with the other two bacterial strains. The hairy roots generated from stem explants infected with all three strains were not found to be productive in terms of the selected parameters. Finally, the insertion of the rolB gene of A. rhizogenes ATCC 15834 in hairy roots of P. indica derived from leaf explants was confirmed by PCR analysis.

scholarly journals Methyl jasmonate enhances ursolic, oleanolic and rosmarinic acid production and sucrose induced biomass accumulation, in hairy roots of Lepechinia caulescens

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11279
Author(s):  
Victor M. Vergara-Martínez ◽  
Samuel E. Estrada-Soto ◽  
Susana Valencia-Díaz ◽  
Karlina Garcia-Sosa ◽  
Luis Manuel Peña-Rodríguez ◽  
...  
Keyword(s):  
Methyl Jasmonate ◽  
Bioactive Compounds ◽  
Hairy Root ◽  
Hairy Roots ◽  
Leaf Explants ◽  
Biomass Accumulation ◽  
Wild Plants ◽  
Bioactive Metabolites ◽  
Hairy Root Cultures ◽  
Root Cultures

Background Ursolic (UA), oleanolic (OA) and rosmarinic (RA) acids are bioactive metabolites found in Lepechinia caulescens that have generated interest for their health benefits, which include antimicrobial, antioxidant, antimutagenic, gastroprotective, antidiabetic, antihypertensive and anti-inflammatory properties, among others. To date, very few attempts have been made to evaluate the potential for simultaneous production of these bioactive compounds, using a biotechnological approach. Hairy root cultures offer a biotechnology approach that can be used to study the factors affecting the biosynthesis and the production of UA, OA and RA. In the current study, we established hairy root cultures of L. caulescens and evaluated the effect of sucrose on biomass accumulation, and the effect of different concentrations and times of exposure of methyl jasmonate (MeJA), on the accumulation of UA, OA and RA. Methods Leaves from plants of L. caulescens were inoculated with Agrobacterium rhizogenes strain ATCC 15834. PCR of rolB gene confirmed the transgenic nature of hairy roots. Hairy roots were subcultured in semisolid MSB5 medium, supplemented with 15, 30, 45 or 60 g/L sucrose and after 4 weeks, dry weight was determined. The accumulation of UA, OA and RA of wild plants and hairy roots were determined by HPLC. Finally, the hairy roots were treated with 0, 100, 200 and 300 µM of MeJA and the content of bioactive compounds was analyzed, after 24, 48 and 72 h. Results High frequency transformation (75%) was achieved, using leaf explants from axenic seedlings, infected with A. rhizogenes. The hairy roots showed an enhanced linear biomass accumulation, in response to the increase in sucrose concentration. The hairy root cultures in MSB5 medium, supplemented with 45 g/L sucrose, were capable to synthesizing UA (0.29 ± 0.00 mg/g DW), OA (0.57 ± 0.00 mg/g DW) and RA (41.66 ± 0.31 mg/g DW), about two, seven and three times more, respectively, than in roots from wild plants. Elicitation time and concentration of MeJA resulted in significant enhancement in the production of UA, OA and RA, with treatments elicited for 24 h, with a concentration of 300 µM of MeJA, exhibiting greatest accumulation. Conclusion This is the first report on development of hairy root cultures of L. caulescens. Future studies should aim towards further improving triterpenes and polyphenolic compound production in hairy roots of L. caulescens, for use in the pharmaceutical and biotechnological industry.

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