Carrot hairy roots (Daucus carota L.) characterisation and optimisation for high β‐carotene extraction

Nga Thi Phuong Mai(1*), Thi Van Anh Le(2), Bao Chau Nguyen(3), Nguyen Ha Trang Le(4), Quang Minh Do(5)

(1) Department of Life Science, University of Science and Technology of Hanoi (USTH), Vietnam Academy of Science and Technology (VAST). 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam
(2) Department of Life Science, University of Science and Technology of Hanoi (USTH), Vietnam Academy of Science and Technology (VAST). 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam
(3) Department of Life Science, University of Science and Technology of Hanoi (USTH), Vietnam Academy of Science and Technology (VAST). 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam
(4) Department of Life Science, University of Science and Technology of Hanoi (USTH), Vietnam Academy of Science and Technology (VAST). 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam
(5) Department of Life Science, University of Science and Technology of Hanoi (USTH), Vietnam Academy of Science and Technology (VAST). 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam
(*) Corresponding Author


Hairy roots are widely known as a biological system for the production of highly diverse biomolecules. β‐carotene – a precursor for vitamin A – is known to be an anti‐oxidant and anti‐gastric cancer and protection agent against cardiovascular disease, heart disease and stroke. β‐carotene has been chemically synthesised and consumed by humans. However, the chemical process often produces a by‐product that may be harmful to human health. Therefore, this study established a protocol to induce hairy roots (HRs) from a Vietnamese carrot variety and produce natural β‐carotene. The Rhizobium rhizogenes ATCC15834 harbouring Ri plasmid and a Vietnamese carrot variety were used as materials for genetic transformation and HR induction studies. The result showed that approximately 50 HR lines were obtained. Culture medium supplemented with 30 mg/L of sucrose that gave the highest biomass of HR was shown in carrot HR line 30, which had a doubling time of 6.5 days. The highest content of β‐carotene extraction, at 128 mg/100g hairy roots, was achieved with a ratio volume (v/v) of 2‐propanol and plant samples of 20:1, followed by two hours’ incubation with 2‐propanol at 60 °C. Our study reveals a highly efficient protocol for Vietnamese carrot hairy root establishment and multiplication. A very efficient protocol for β‐carotene extraction from the hairy root was established to produce natural β‐carotene that achieves the same β‐carotene quantity as that produced by normal roots. This study provides new insight into the production of high‐content and natural β‐carotene for therapeutic application.


β‐carotene extraction; Daucus carota L.; hairy root; Rhizobium rhizogenes; sucrose

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Baek S, Han JE, Ho TT, Park SY. 2022. Development of hairy root cultures for biomass and triterpenoid production in Centella asiatica. Plants 11(2):148. doi:10.3390/PLANTS11020148.

Cardoso JC. 2019. Silver nitrate enhances in vitro development and quality of shoots of Anthurium andraeanum. Sci. Hortic. 253:358–363. doi:10.1016/J.SCIENTA.2019.04.054.

Chen QH, Wu BK, Pan D, Sang LX, Chang B. 2021. Beta­carotene and its protective effect on gastric cancer. World J. Clin. Cases 9(23):6591–6607. doi:10.12998/WJCC.V9.I23.6591.

Chun JA, Lee WH, Han MO, Lee JW, Yi YB, Park GY, Chung CH. 2007. Optimization of abiotic factors for improved growth and extracellular production of recombinant fungal phytase in sesame hairy root cultures. Biotechnol. Bioprocess Eng. 12(3):242–249. doi:0.1007/BF02931099.

Chung IM, Rajakumar G, Thiruvengadam M. 2018. Effect of silver nanoparticles on phenolic compounds production and biological activities in hairy root cultures of Cucumis anguria. Acta Biol. Hung. 69(1):97–109. doi:10.1556/018.68.2018.1.8.

Danesh Y, Goltapeh EM, Alizadeh A, Sanavy MM. 2006. Optimizing carrot hairy root production for monoxenic culture of arbuscular mycorrhizal fungi in Iran. J. Biol. Sci. 6(1):87–91. doi:10.3923/jbs.2006.87.91.

Ellison S, Senalik D, Bostan H, Iorizzo M, Simon P. 2017. Fine mapping, transcriptome analysis, and marker development for Y2, the gene that conditions β­carotene accumulation in carrot (Daucus carota L.). G3: Genes, Genomes, Genetics 7(8):2665–2675. doi:10.1534/G3.117.043067.

Fikselová M, Šilhár S, Mareček J, Frančáková H. 2008. Extraction of carrot (Daucus carota L.) carotenes under different conditions. Czech J. Food Sci. 26(4):268–274. doi:10.17221/9/2008­CJFS.

Geetha G, Harathi K, Naidu C. 2016. Role of silver nitrate on in vitro flowering and shoot regeneration of Solanum nigrum (L.)—an important multipurpose medicinal plant. Am. J. Plant Sci. 7(7):1021–1032. doi:10.4236/AJPS.2016.77097.

Georgiev MI, Agostini E, Ludwig­Müller J, Xu J. 2012. Genetically transformed roots: from plant disease to biotechnological resource. Trends Biotechnol. 30(10):528–537. doi:10.1016/j.tibtech.2012.07.001.

Guerineau F, Mai NT, Boitel­Conti M. 2020. Arabidopsis hairy roots producing high level of active human gastric lipase. Mol. Biotechnol. 62(3):168–176. doi:10.1007/S12033­019­00233­Y.

Gutierrez­Valdes N, Häkkinen ST, Lemasson C, Guillet M, Oksman­Caldentey KM, Ritala A, Cardon F. 2020. Hairy root cultures—a versatile tool with multiple applications. Front Plant Sci. 11:33. doi:10.3389/FPLS.2020.00033.

Ha LT, Pawlicki­Jullian N, Pillon­Lequart M, BoitelConti M, Duong HX, Gontier E. 2016. Hairy root cultures of Panax vietnamensis, a promising approach for the production of ocotillol­type ginsenosides. Plant Cell Tissue Organ Cult. 126(1):93–103. doi:10.1007/S11240­016­0980­Y/FIGURES/7.

Hernández­Almanza A, Montanez JC, Aguilar­Gonzalez MA, Martínez­Ávila C, Rodríguez­Herrera R, Aguilar CN. 2014. Rhodotorula glutinis as source of pigments and metabolites for food industry. Food Biosci. 5:64–72. doi:10.1016/j.fbio.2013.11.007.

Huang J, Weinstein SJ, Yu K, Männistö S, Albanes D. 2018. Serum beta carotene and overall and cause­specific mortality: a prospective cohort study. Circ. Res. 123(12):1339–1349. doi:10.1161/CIRCRESAHA.118.313409.

Kasperczyk S, Dobrakowski M, Kasperczyk J, Ostałowska A, Zalejska­Fiolka J, Birkner E. 2014. Beta­carotene reduces oxidative stress, improves glutathione metabolism and modifies antioxidant defense systems in lead­exposed workers. Toxicol. Appl. Pharmacol. 280(1):36–41. doi:10.1016/J.TAAP.2014.07.006.

Kentsop RAD, Iobbi V, Donadio G, Ruffoni B, De Tommasi N, Bisio A. 2021. Abietane diterpenoids from the hairy roots of Salvia corrugata. Molecules 26(17):5144. doi:10.3390/MOLECULES26175144.

Kesharlal BM, Pratap SN, Milind BS, Ann NP, Harnarayan GS. 2001. Isolation and formulations of nutrient­rich carotenoids. US Patent 6,224,876.

Mahendran D, Kavi Kishor P, Geetha N, Venkatachalam P. 2018. Phycomolecule­coated silver nanoparticles and seaweed extracts induced high­frequency somatic embryogenesis and plant regeneration from Gloriosa superba L. J. Appl. Phycol. 30(2):1425– 1436. doi:10.1007/S10811­017­1293­1.

Mai NT, Boitel­Conti M, Guerineau F. 2016. Arabidopsis thaliana hairy roots for the production of heterologous proteins. Plant Cell Tissue Organ Cult. 127(2):489–496. doi:10.1007/s11240­016­1073­7.

Manuhara YSW, Kristanti AN, Utami ESW, Yachya A. 2015. Effect of sucrose and potassium nitrate on biomass and saponin content of Talinum paniculatum Gaertn. hairy root in balloon­type bubble bioreactor. Asian Pac. J. Trop. Biomed. 5(12):1027–1032. doi:10.1016/J.APJTB.2015.09.009.

Mehrotra S, Srivastava V, Rahman LU, Kukreja A. 2015. Hairy root biotechnology—indicative timeline to understand missing links and future outlook. Protoplasma 252(5):1189–1201. doi:10.1007/S00709­015­ 0761­1.

Mohammed AA, Al­Mallah MK. 2013. Determination of β­carotene in Carrot (Daucus carota L.) plants regenerated from stems callus. Rafidain J. Sci. 24(5):27– 36. doi:10.33899/RJS.2013.74507.

Parr AJ. 2017. Secondary products from plant cell cultures: Early experiences with­transformed hairy roots. Springer. doi:10.1007/978­3­319­28669­3_20.

Pedreño MA, Almagro L. 2020. Carrot hairy roots: Factories for secondary metabolite production. J. Exp. Bot. 71(22):6861–6864. doi:10.1093/jxb/eraa435.

Rachamallu RR. 2016. Hairy roots production through Agrobacterium rhizogenes genetic transformation from Daucus carota explants. Int. J. Adv. Res. Biol. Sci. 3(8):23–27. doi:­o­­2016­3­8­5.

Rekha K, Thiruvengadam M. 2017. Secondary metabolite production in transgenic hairy root cultures of cucurbits. Transgenes Second. Metab. p. 267. doi:10.1007/978­3­319­28669­3_6.

Ruslan K, Selfitri AD, Bulan SA, Rukayadi Y, et al. 2012. Effect of Agrobacterium rhizogenes and elicitation on the asiaticoside production in cell cultures of Centella asiatica. Pharmacogn. Mag. 8(30):111. doi:10.4103/0973­1296.96552.

Syahid SF, Wahyuni S. 2019. Effect of silver nitrate on shoot multiplication, rooting induction and plantlet characteristics of St. John’ s wort (Hypericum perforatum L.) in vitro culture. Afr. J. Agric. Res. 14(27):1149–1153. doi:10.5897/AJAR2019.14203.

Tahoori F, Majd A, Nejadsattari T, Ofoghi H, Iranbakhsh A. 2018. Effects of silver nitrate (AgNO3) on growth and anatomical structure of vegetative organs of liquorice (Glycyrrhiza glabra L.) under in vitro condition. Plant Omics 11(3):153–160. doi:10.3316/informit.011073689743957.

Tepfer D. 2017. DNA transfer to plants by Agrobacterium rhizogenes: A model for genetic communication between species and biospheres. Transgenes Second. Metab. p. 3–43. doi:10.1007/978­3­319­28669­3_19.

Thiruvengadam M, Praveen N, Maria John K, Yang YS, Kim SH, Chung IM. 2014. Establishment of Momordica charantia hairy root cultures for the production of phenolic compounds and determination of their biological activities. Plant Cell Tissue Organ Cult. 118(3):545–557. doi:10.1007/S11240­014­ 0506­4.

Tisserant LP, Aziz A, Jullian N, Jeandet P, Clément C, Courot E, Boitel­Conti M. 2016. Enhanced stilbene production and excretion in Vitis vinifera cv Pinot Noir hairy root cultures. Molecules 21(12):1703. doi:10.3390/MOLECULES21121703.

Vishwakarma K, Upadhyay N, Singh J, Liu S, Singh VP, Prasad SM, Chauhan DK, Tripathi DK, Sharma S. 2017. Differential phytotoxic impact of plant mediated silver nanoparticles (AgNPs) and silver nitrate (AgNO3) on Brassica sp. Front. Plant Sci. 8:1501. doi:10.3389/FPLS.2017.01501.

Yang DC, Choi YE. 2000. Production of transgenic plants via Agrobacterium rhizogenes­mediated transformation of Panax ginseng. Plant Cell Rep. 19(5):491– 496. doi:10.1007/S002990050761.

Yang J, Guo L. 2014. Biosynthesis of β­carotene in engineered E. coli using the MEP and MVA pathways. Microb. Cell factories 13(1):1–11. doi:10.1186/S12934­ 014­0160­X.

Yoon JY, Chung IM, Thiruvengadam M. 2015. Evaluation of phenolic compounds, antioxidant and antimicrobial activities from transgenic hairy root cultures of gherkin (Cucumis anguria L.). S. Afr. J. Bot. 100:80– 86. doi:10.1016/J.SAJB.2015.05.008.

Zhao JL, Zou L, Zhang CQ, Li YY, Peng LX, Xiang DB, Zhao G. 2014. Efficient production of flavonoids in Fagopyrum tataricum hairy root cultures with yeast polysaccharide elicitation and medium renewal process. Pharmacogn. Mag. 10(39):234–240. doi:10.4103/0973­1296.137362.


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