Assessment of hairy roots induction in Echium khuzistanicum by different strains of Agrobacterium rhizogenes

Document Type : Research Paper

Authors

1 PhD student, Biotechnology Department, Faculty of Agriculture and Natural Resources, Imam Khomeini International University, Qazvin, Iran. Tel: +989350615355, Email: s1mir1mohammadi@gmail.com

2 Associate Professor, Biotechnology Department, Imam Khomeini International University, Qazvin, IR of Iran. POBox 34149-16818. Tel: +982833901242, Email: r.haddad@Eng.ikiu.ac.ir

3 Faculty member of National Institute of Genetic Engineering and Biotechnology, Tehran, Iran. Iran. Tel: +989136002347, Email: v.shariati@nigeb.ac.ir

Abstract

 
Objective
In this study for the first time, different strains of Agrobacterium rhizogenes, co-cultivation times, and concentrations of acetosyringon in leaf explants of Echium khuzistanicum were examined to produce hairy roots.
Materials and methods
The leaves of 21-day-old plant, different concentrations of acetosyringone (0,100,200 µM) and 3 strains of Agrobacterium (A4, ATCC15834 and 11325) were used for hairy root induction. In each explant, surficial wounds were created by a syringe contaminated with the bacterium and put for 10 minutes in bacterial suspension. After infection, the explants were transferred in the ½ MS solid medium supplemented with ascorbic acid (100 mg/L) to a light free growth chamber with 25 ° C temperature for 48 or 72 hours. PCR technique was used to confirm transformation by gene amplification with the rolBprimers. HPLC analysis was conducted on a high performance liquid chromatography system for shikonin measurement.
 
Results
The results of this study showed that, the first hairy roots emerged from wounded places after 14-21 days. The presence of A. rhizogenes T-DNA in the hairy root genome was confirmed by PCR using specific primers for rolB gene. Strain ATCC15834 had the highest rate of hairy root induction at 100 and 200 µM concentrations of acetosyringone indicating that the phenolic compound, acetosyringone, was effective in increasing hairy root induction. Generally, High concentrations of acetosyringone and more co-cultivation time together resulted in a significant increase in the hairy root induction in three strains of Agrobacterium rhizogenesis. Line 1 of hairy root produced 254.4±0.56 µg/g FW of shikonin.
 
Conclusions
For the first time the results of this research showed that transformation and hairy root induction in E.khuzistanicum is possible by Agrobacterium rhizogenes and it can be used in the gene transfer and hairy root culture in order to shikonin production.

Keywords

References

 
Andujar I, Rios JL, Giner RM et al. (2013) Shikonin promotes intestinal wound healing in vitro via induction of TGF-beta release in IEC-18 cells. Eur J Pharm Sci 49, 637-41.
Balasubramanian M, Anbumegala M, Surendran R et al. (2018) Elite hairy roots of Raphanus sativus (L.) as a source of antioxidants and flavonoids. 3 Biotech 8, 128.
Balasubramanian A, Venkatachalam R, Selvakesavan KR et al. (2011) Optimisation of methods for Agrobacterium rhizogenes mediated generation of composite plants in Eucalyptus camaldulensis. BMC Proc 5, O45.
Brijwal L, Tamta S (2015) Agrobacterium rhizogenes mediated hairy root induction in endangered Berberis aristata DC. SpringerPlus 4, 443.
Chen X, Yang L, Zhang N et al. (2003) Shikonin, a component of Chinese herbal medicine, inhibits chemokine receptor function and suppresses human immunodeficiency virus type 1. Anti Agents Chem 47, 2810-6.
Deng C, Zheng J, Wan W et al. (2013) Suppression of cell proliferation and collagen production in cultured human hypertrophic scar fibroblasts by Sp1 decoy oligodeoxynucleotide. Mol Med Rep 7, 785-90.
Dinarvand M, Hamzehee B (2017) Conservation status of Echium khuzistanicum. Iran nature 3, 100-103.
Fan C, Xie Y, Dong Y et al. (2015) Investigating the potential of Shikonin as a novel hypertrophic scar treatment. J Biomed Sci 22, 1-13.
Gwon SY, Choi WH, Lee DH et al. (2015) Shikonin protects against obesity through the modulation of adipogenesis, lipogenesis, and β-oxidation in vivo. J Funct Foods 16, 484-493.
Hanafy MS, Matter MA, Asker MS et al. (2016) Production of indole alkaloids in hairy root cultures of Catharanthus roseus L. and their antimicrobial activity. S Afric J Bot 105, 9-18.
Kim JY, Jeong HJ, Park JY et al. (2012) Selective and slow-binding inhibition of shikonin derivatives isolated from Lithospermum erythrorhizon on glycosyl hydrolase 33 and 34 sialidases. Bioorg med chem 20, 1740-8.
Kimura M and Isobe S (2018) Small-Molecule Screening to Increase Agrobacterium-Mediated Transformation Efficiency in Legumes. Plant Chem Gen 1795, 93-99.
Kontogiannopoulos KN, Assimopoulou AN, Tsivintzelis I et al. (2011) Electrospun fiber mats containing shikonin and derivatives with potential biomedical applications. Int J Pharm 409, 216-28.
Kumar V, Sharma A, Prasad BCN et al. (2006) Agrobacterium rhizogenes mediated genetic transformation resulting in hairy root formation is enhanced by ultrasonication and acetosyringone treatment. Plant Biotech 9, 349-358.
Lee H, Bae S, Kim K et al. (2011) Shikonin inhibits adipogenesis by modulation of the WNT/β-catenin pathway. Life Sci 88, 294-301.
Lee YJ, Choi SY and Yang JH (2014) NMDA receptor-mediated ERK 1/2 pathway is involved in PFHxS-induced apoptosis of PC12 cells. Life Sci 3, 491–492. 
Li F, Yin Z, Zhou B et al. (2015) Shikonin inhibits inflammatory responses in rabbit chondrocytes and shows chondroprotection in osteoarthritic rabbit knee. Int Immuno 29, 656-62.
Mohammadi S, Piri K, Dinarvand M (2019) Antioxidant and Antibacterial Effects of Some Medicinal Plants of Iran. Int J Sec Metabol 6, 62–78.
Mohammadi S and Piri K (2014) Antifungal Effects of Two Medicinal Plant Native to Iran. Int J Adv Bio Biomed Res 2, 2712–15.
Papageorgiou VP, Assimopoulou AN, Couladouros EA et al. (1999) The Chemistry and Biology of Alkannin, Shikonin, and Related Naphthazarin Natural Products. Ange Chem Int Edit 38, 270-301.
Park S, Eom S, lee C et al. (2010) Ep-methoxycinnamic acid production in hairy root cultures of scrophularia buergeriana miquel. Arch biol sci 62, 649-652.
Porter JR and Hector F (1991) Host range and implications of plant infection by Agrobacterium rhizogenes. Crit Rev Plant Sci 4, 387-421. 
Samadi A, Carapetian J, Heidari R et al. (2012) Hairy root induction in linum mucronatum ssp. Mucronatum, an anti-tumor lignans producing plant. Notulae botanicae horti agrobotanici cluj-napoca 40, 125-131.
Sambrook J and Russell DW (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press
Sevon N and Oksman-Caldentey KM (2002) Agrobacterium rhizogenes-mediated transformation: root cultures as a source of alkaloids. Planta Medica 68, 859-68.
Singleton VL and Rossi JA (1965) Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents. Amer J Enol Viti 16, 144-158.
Skrzypczak A, Przystupa N, Zgadzaj A et al. (2015) Antigenotoxic, anti-photogenotoxic and antioxidant activities of natural naphthoquinone shikonin and acetylshikonin and Arnebia euchroma callus extracts evaluated by the umu-test and EPR method. Toxico in Vitro 30, 364-72.
Tabata M, Hajime M, Noboru H et al. (1974) Pigment Formation in Callus Cultures of Lithospermum Erythrorhizon. Phytochem 13, 927–32.
Thwe A, Valan Arasu M, Li X et al. (2016) Effect of Different Agrobacterium rhizogenes Strains on Hairy Root Induction and Phenylpropanoid Biosynthesis in Tartary Buckwheat (Fagopyrum tataricum Gaertn). Front microbiol 7, 318.
Yang Y, Wang J, Yang Q et al. (2014) Shikonin inhibits the lipopolysaccharide-induced release of HMGB1 in RAW264.7 cells via IFN and NF-kappaB signaling pathways. Int Immunopharm 19, 81-7.
Zarei b, kahrizi d, mousavi s et al. (2013) Agrobacterium rhizogense-mediated transformation of atropa belladonna. J agric biotechnol 5, 59-67.
Zhang FY, HuY, Que ZY et al. (2015) Shikonin Inhibits the Migration and Invasion of Human Glioblastoma Cells by Targeting Phosphorylated beta-Catenin and Phosphorylated PI3K/Akt: A Potential Mechanism for the Anti-Glioma Efficacy of a Traditional Chinese Herbal Medicine. Int J Mol Sci 16, 23823-48.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Andujar I, Rios JL, Giner RM et al. (2013) Shikonin promotes intestinal wound healing in vitro via induction of TGF-beta release in IEC-18 cells. Eur J Pharm Sci 49, 637-41.
Balasubramanian M, Anbumegala M, Surendran R et al. (2018) Elite hairy roots of Raphanus sativus (L.) as a source of antioxidants and flavonoids. 3 Biotech 8, 128.
Balasubramanian A, Venkatachalam R, Selvakesavan KR et al. (2011) Optimisation of methods for Agrobacterium rhizogenes mediated generation of composite plants in Eucalyptus camaldulensis. BMC Proc 5, O45.
Brijwal L, Tamta S (2015) Agrobacterium rhizogenes mediated hairy root induction in endangered Berberis aristata DC. SpringerPlus 4, 443.
Chen X, Yang L, Zhang N et al. (2003) Shikonin, a component of Chinese herbal medicine, inhibits chemokine receptor function and suppresses human immunodeficiency virus type 1. Anti Agents Chem 47, 2810-6.
Deng C, Zheng J, Wan W et al. (2013) Suppression of cell proliferation and collagen production in cultured human hypertrophic scar fibroblasts by Sp1 decoy oligodeoxynucleotide. Mol Med Rep 7, 785-90.
Dinarvand M, Hamzehee B (2017) Conservation status of Echium khuzistanicum. Iran nature 3, 100-103.
Fan C, Xie Y, Dong Y et al. (2015) Investigating the potential of Shikonin as a novel hypertrophic scar treatment. J Biomed Sci 22, 1-13.
Gwon SY, Choi WH, Lee DH et al. (2015) Shikonin protects against obesity through the modulation of adipogenesis, lipogenesis, and β-oxidation in vivo. J Funct Foods 16, 484-493.
Hanafy MS, Matter MA, Asker MS et al. (2016) Production of indole alkaloids in hairy root cultures of Catharanthus roseus L. and their antimicrobial activity. S Afric J Bot 105, 9-18.
Kim JY, Jeong HJ, Park JY et al. (2012) Selective and slow-binding inhibition of shikonin derivatives isolated from Lithospermum erythrorhizon on glycosyl hydrolase 33 and 34 sialidases. Bioorg med chem 20, 1740-8.
Kimura M and Isobe S (2018) Small-Molecule Screening to Increase Agrobacterium-Mediated Transformation Efficiency in Legumes. Plant Chem Gen 1795, 93-99.
Kontogiannopoulos KN, Assimopoulou AN, Tsivintzelis I et al. (2011) Electrospun fiber mats containing shikonin and derivatives with potential biomedical applications. Int J Pharm 409, 216-28.
Kumar V, Sharma A, Prasad BCN et al. (2006) Agrobacterium rhizogenes mediated genetic transformation resulting in hairy root formation is enhanced by ultrasonication and acetosyringone treatment. Plant Biotech 9, 349-358.
Lee H, Bae S, Kim K et al. (2011) Shikonin inhibits adipogenesis by modulation of the WNT/β-catenin pathway. Life Sci 88, 294-301.
Lee YJ, Choi SY and Yang JH (2014) NMDA receptor-mediated ERK 1/2 pathway is involved in PFHxS-induced apoptosis of PC12 cells. Life Sci 3, 491–492. 
Li F, Yin Z, Zhou B et al. (2015) Shikonin inhibits inflammatory responses in rabbit chondrocytes and shows chondroprotection in osteoarthritic rabbit knee. Int Immuno 29, 656-62.
Mohammadi S, Piri K, Dinarvand M (2019) Antioxidant and Antibacterial Effects of Some Medicinal Plants of Iran. Int J Sec Metabol 6, 62–78.
Mohammadi S and Piri K (2014) Antifungal Effects of Two Medicinal Plant Native to Iran. Int J Adv Bio Biomed Res 2, 2712–15.
Papageorgiou VP, Assimopoulou AN, Couladouros EA et al. (1999) The Chemistry and Biology of Alkannin, Shikonin, and Related Naphthazarin Natural Products. Ange Chem Int Edit 38, 270-301.
Park S, Eom S, lee C et al. (2010) Ep-methoxycinnamic acid production in hairy root cultures of scrophularia buergeriana miquel. Arch biol sci 62, 649-652.
Porter JR and Hector F (1991) Host range and implications of plant infection by Agrobacterium rhizogenes. Crit Rev Plant Sci 4, 387-421. 
Samadi A, Carapetian J, Heidari R et al. (2012) Hairy root induction in linum mucronatum ssp. Mucronatum, an anti-tumor lignans producing plant. Notulae botanicae horti agrobotanici cluj-napoca 40, 125-131.
Sambrook J and Russell DW (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press
Sevon N and Oksman-Caldentey KM (2002) Agrobacterium rhizogenes-mediated transformation: root cultures as a source of alkaloids. Planta Medica 68, 859-68.
Singleton VL and Rossi JA (1965) Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents. Amer J Enol Viti 16, 144-158.
Skrzypczak A, Przystupa N, Zgadzaj A et al. (2015) Antigenotoxic, anti-photogenotoxic and antioxidant activities of natural naphthoquinone shikonin and acetylshikonin and Arnebia euchroma callus extracts evaluated by the umu-test and EPR method. Toxico in Vitro 30, 364-72.
Tabata M, Hajime M, Noboru H et al. (1974) Pigment Formation in Callus Cultures of Lithospermum Erythrorhizon. Phytochem 13, 927–32.
Thwe A, Valan Arasu M, Li X et al. (2016) Effect of Different Agrobacterium rhizogenes Strains on Hairy Root Induction and Phenylpropanoid Biosynthesis in Tartary Buckwheat (Fagopyrum tataricum Gaertn). Front microbiol 7, 318.
Yang Y, Wang J, Yang Q et al. (2014) Shikonin inhibits the lipopolysaccharide-induced release of HMGB1 in RAW264.7 cells via IFN and NF-kappaB signaling pathways. Int Immunopharm 19, 81-7.
Zarei b, kahrizi d, mousavi s et al. (2013) Agrobacterium rhizogense-mediated transformation of atropa belladonna. J agric biotechnol 5, 59-67.
Zhang FY, HuY, Que ZY et al. (2015) Shikonin Inhibits the Migration and Invasion of Human Glioblastoma Cells by Targeting Phosphorylated beta-Catenin and Phosphorylated PI3K/Akt: A Potential Mechanism for the Anti-Glioma Efficacy of a Traditional Chinese Herbal Medicine. Int J Mol Sci 16, 23823-48.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Agricultural Biotechnology Journal
Volume 12, Issue 1
April 2020
Pages 63-80

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