Mass Production of Transformed Hairy Root for Secondary Metabolites: A Case Study of Panax ginseng Hairy Roots

2017 ◽  
pp. 183-201 ◽  
Author(s):  
Gwi-Taek Jeong ◽  
Don-Hee Park
Keyword(s):  
Secondary Metabolites ◽  
Panax Ginseng ◽  
Hairy Root ◽  
Hairy Roots ◽  
Mass Production ◽  
Transformed Hairy Root

Hairy Roots

2021 ◽  
pp. 219-252
Keyword(s):  
Secondary Metabolites ◽  
Hairy Root ◽  
Hairy Roots ◽  
High Growth ◽  
Root Culture ◽  
Hairy Root Culture ◽  
High Growth Rate ◽  
Hairy Root Cultures ◽  
Root Cultures ◽  
Transformed Root Cultures

Agrobacterium rhizogenes induces hairy root disease in plants. The neoplastic (cancerous) roots produced by A. rhizogenes infection, when cultured in hormone free medium, show high growth rate and genetic stability. These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants. Several elicitation methods can be used to further enhance the production and accumulation of secondary metabolites. Thus, hairy root culture offer promise for high production and productivity of valuable secondary metabolites in many plants. Hairy roots can also produce recombinant proteins from transgenic roots, and thereby hold immense potential for pharmaceutical industry. Hairy root cultures can be used to elucidate the intermediates and key enzymes involved in the biosynthesis of secondary metabolites, and for phytoremediation due to their abundant neoplastic root proliferation property. Various applications of hairy root cultures and potential problems associated with them are discussed in this chapter.

scholarly journals Enhancement of Phenolic Compounds and Antioxidative Activities by the Combination of Culture Medium and Methyl Jasmonate Elicitation in Hairy Root Cultures of Lactuca indica L.

2019 ◽  
Vol 14 (7) ◽  
pp. 1934578X1986186 ◽  
Author(s):  
Tae Gyu Yi ◽  
Yeri Park ◽  
Jai-Eok Park ◽  
Nam Il Park
Keyword(s):  
Antioxidant Activity ◽  
Secondary Metabolites ◽  
Hairy Root ◽  
Hairy Roots ◽  
Total Phenolic Content ◽  
Phenolic Content ◽  
Time Dependent ◽  
Total Phenolic ◽  
Total Flavonoid Content ◽  
Flavonoid Content

Lactuca indica L. has been traditionally used as a wild vegetable and as a medicinal plant for centuries. The various compounds present in it and their biological activities have been extensively reported. Hairy-root culture combined with agrobacterium-meditated metabolic engineering is a useful technique to achieve stable production of biologically active plant compounds. Here, we evaluated the enhancement of secondary metabolites in L. indica L. and their bioactivities by testing culture media composition and the use of an elicitor. Hairy roots were induced and cultured in MS or SH liquid media for 2 weeks prior to treatment with various concentrations of MeJa, for different periods. The resulting phenolic contents and physiological activities were analyzed. Higher total phenolic, flavonoid, and hydroxycinnamic acids contents were attained by elicitation with MeJa. Metabolite accumulation, especially in SH media and in the presence of MeJa, was time dependent. Particularly, accumulation of chicoric acid increased markedly with time. Similarly, we observed time dependent positive and negative responses of antioxidant activity in DPPH and ABTS assays, respectively. As in previous studies, the highest correlation was found between total phenolic content and total flavonoid content. Further, 3,5-DCQA showed the highest correlation with total phenolic content, total flavonoid content, and antioxidant activities in hydroxycinnamic acids. Our data effectively identified optimal culture conditions to increase the accumulation of secondary metabolites and antioxidant activity in hairy roots cultures of L. indica L.

scholarly journals Genetic transformation of Ceratotheca triloba for the production of anthraquinones from hairy root cultures

2012 ◽  
Author(s):  
◽  
Leeann Naicker
Keyword(s):  
Secondary Metabolites ◽  
Cell Suspension ◽  
Hairy Root ◽  
Hairy Roots ◽  
Cell Suspension Cultures ◽  
Suspension Cultures ◽  
Control Culture ◽  
Hairy Root Cultures ◽  
Shoot Cultures ◽  
Root Cultures

Many secondary metabolites that have been extracted from medicinal plants have been used as source of clinical drugs. However, the concentration of the active metabolites in plants is generally low. An attractive alternative for producing these important secondary metabolites is via plant tissue culture technology. More particularly, the genetic transformation of a plant tissue by Agrobaterium rhizogenes has been employed for producing high yields of secondary metabolites. In a previous study, three structurally similar anthraquinones: 9,10-Anthracenedione, 1-Hydroxy-4-methylanthraquinone and 5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, and one steroid; Androst-5-ene-3, 17, 19-triol were isolated from the root extracts of C. triloba. The anthraquinones have shown to exhibit the anticancer mechanism which involves the inhibition of the activity of the human topoisomerase II enzyme that transforms supercoiled DNA to linear DNA. However, these anthraquinones were found in very low concentrations. Therefore, in this study we used plant cell and tissue culture systems (cell suspension, shoot and hairy root cultures) of C. triloba to increase the production of anthraquinones. Since the establishment of C. triloba in vitro plant systems required a source sterile explants, a protocol that involved the use of NaCIO was optimized for the sterilization and subsequent germination of C. triloba seeds which were micro-propagated into shoot cultures. These cultures provided a source explants for the induction of callus and hairy root cultures. The biomass of these plant cell and tissue cultures were subsequently bulked up for the extraction for anthraquinones and the yields were compared followed by fractionation and identification of the major compounds. The bioactivity of the fractions was evaluated by testing their cytotoxicity on cancer cells and anti-topoisomerase activity. The sterilization protocol that provided sterile seeds was found to be a solution of 30% NaCIO at an exposure time of 10 minutes. From the sterilized seeds shoot cultures were established on MS medium. The leaf explants of the shoot cultures were then used to induce callus cultures which subsequently were transferred to liquid medium whereby the total biomass of suspension cultures increased from 4 g to 134.18 g (wet weight). Also hairy roots cultures were established from stem explants with a low cell density inoculum of A. rhizogenes at a transformation efficiency of 73%. The growth of these hairy roots was slow in hormone free medium. This was overcomed with the use NAA and IAA which increased the xvii biomass from 1.03 g in the control culture (without hormone) to 23.91 g and 46.13 g respectively. An evaluation of the anthraquinones in the field root and hairy root, cell suspension and shoot culture extracts was carried out by using their Thin Layer Chromatography profiles and the High Performance Liquid Chromatography profiles as well as the standards, 9,10-Anthracenedione and 1-Hydroxy-4-methylanthaquinone. TLC analysis showed that the RF values of the fractions CT01 and CT02 matched the RF values of anthraquinones standards while HPLC analysis revealed that hairy root cultures supplemented with IAA (125.03 μg.mg-1) or NAA (98.25 μg. mg-1) produced a higher concentration of anthraquinones than the control culture (without hormone) (13.33 μg.mg-1), the field roots (33.51 μg. mg-1) and the shoot (3.23 μg.mg-1) and cell suspension cultures (13.17 μg.mg-1). Due to co-elution of the compounds in HPLC analysis, six fractions were isolated by Preparative Thin Layer Chromatography from the hairy root extract (obtained from the culture supplemented with NAA) and were coded as CT01, CT02, CT03, CT04, CT05 and CT06. The compounds in these fractions were identified by Electron Ionization-Liquid chromatography-Mass Spectroscopy and it was found that the hairy roots produced one acridone derivative; 5-Methoxy-2-nitro-10H-acridin-9-one, one naphthoquinone derivative; 2H-Naphto[2,3-b]pyran-5,10-dione,3,4-dihydro-2,2-dimethyl- and seven anthracenedione derivatives. These were: i) 5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, ii) 9,10-Anthracenedione, 2-methyl-, iii) 1-Hydroxy-4-methylanthraquinone, iv) 9,10-Anthracenedione, 2-ethyl-, v) 1,5-Diaminoanthraquinone, vi) Phenanthrene, 3,6-dimethoxy-9-methyl-, vii) 9,10-Anthracenedione, 1,4-dimethyl-. Fractions CT01 (5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, 9,10-Anthracenedione, 2-methyl- and 1-Hydroxy-4-methylanthraquinone) and CT02 (9,10- Anthracenedione, 2-ethyl-) were cytotoxic to the DU-145 cancer cell line at concentrations of 125 μg.mg-1 to 1000 μg.mg-1. These fractions also showed anti-topoisomerase activity as they inhibited the conversion of supercoiled DNA into linear DNA. In conclusion this is the first study that describes the transformation of C. triloba by A. rhizogenes mediated transformation and compares the production of anthraquinones in C. triloba hairy roots to the field roots, shoot and cell suspension cultures. This study has xviii indicated that hairy root cultures is a high-yielding production system for anthraquinones (5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, 1-Hydroxy-4-methylanthraquinone, 9,10-Anthracenedione, 2-methyl- and 9,10- Anthracenedione, 2-ethyl-) which could have the potential to be used in cancer therapy. In addition the discovery of C. triloba hairy roots having the biosynthetic capacity to synthesize five valuable anthraquinone derivatives that are not found the field roots has also been revealed.

Hairy Roots

2022 ◽  
pp. 735-759
Author(s):  
Pradip Chandra Deka
Keyword(s):  
Secondary Metabolites ◽  
Hairy Root ◽  
Hairy Roots ◽  
High Growth ◽  
Root Culture ◽  
Hairy Root Culture ◽  
High Growth Rate ◽  
Hairy Root Cultures ◽  
Root Cultures ◽  
Transformed Root Cultures

Agrobacterium rhizogenes induces hairy root disease in plants. The neoplastic (cancerous) roots produced by A. rhizogenes infection, when cultured in hormone free medium, show high growth rate and genetic stability. These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants. Several elicitation methods can be used to further enhance the production and accumulation of secondary metabolites. Thus, hairy root culture offer promise for high production and productivity of valuable secondary metabolites in many plants. Hairy roots can also produce recombinant proteins from transgenic roots, and thereby hold immense potential for pharmaceutical industry. Hairy root cultures can be used to elucidate the intermediates and key enzymes involved in the biosynthesis of secondary metabolites, and for phytoremediation due to their abundant neoplastic root proliferation property. Various applications of hairy root cultures and potential problems associated with them are discussed in this chapter.

scholarly journals Transformation of Amorphadiene Synthase and Antisilencing P19 Genes into Artemisia annua L. and its Effect on Antimalarial Artemisinin Production

2020 ◽  
Vol 10 (3) ◽  
pp. 464-471
Author(s):  
Elfahmi Elfahmi ◽  
Fany Mutia Cahyani ◽  
Tati Kristianti ◽  
Sony Suhandono
Keyword(s):  
Hairy Root ◽  
Hairy Roots ◽  
Artemisia Annua ◽  
Pcr Analysis ◽  
Sequencing Analysis ◽  
Pcr Products ◽  
Transformed Hairy Root ◽  
Polymerase Chain ◽  
Infiltration Method

Purpose : The low content of artemisinin related to the biosynthetic pathway is influenced by the role of certain enzymes in the formation of artemisinin. The regulation of genes involved in artemisinin biosynthesis through genetic engineering is a choice to enhance the content. This research aims to transform ads and p19 gene as an antisilencing into Artemisia annua and to see their effects on artemisinin production. Methods: The presence of p19 and ads genes was confirmed through polymerase chain reaction (PCR) products and sequencing analysis. The plasmids, which contain ads and/or p19 genes, were transformed into Agrobacterium tumefaciens, and then inserted into leaves and hairy roots of A. annua by vacuum and syringe infiltration methods. The successful transformation was checked through the GUS histochemical test and the PCR analysis. Artemisinin levels were measured using HPLC. Results: The percentages of the blue area on leaves by using vacuum and syringe infiltration method and on hairy roots were up to 98, 92.55%, and 99.00% respectively. The ads-p19 sample contained a higher level of artemisinin (0.18%) compared to other samples. Transformed hairy root with co-transformation of ads-p19 contained 0.095% artemisinin, where no artemisinin was found in the control hairy root. The transformation of ads and p19 genes into A. annua plant has been successfully done and could enhance the artemisinin content on the transformed leaves with ads-p19 up to 2.57 folds compared to the untransformed leaves, while for p19, cotransformed and ads were up to 2.25, 1.29, and 1.14 folds respectively. Conclusion: Antisilencing p19 gene could enhance the transformation efficiency of ads and artemisinin level in A. annua.

scholarly journals (286) Hairy Root Culture as a Source of Unique Secondary Metabolites from Echinacea Species

HortScience ◽  
2005 ◽  
Vol 40 (4) ◽  
pp. 1080C-1080
Author(s):  
Fredy R. Romero ◽  
David J. Hannapel ◽  
Kathleen Delate
Keyword(s):  
Growth Rate ◽  
Secondary Metabolites ◽  
Hairy Root ◽  
Hairy Roots ◽  
The United States ◽  
Molecular Techniques ◽  
Wild Populations ◽  
Transformed Roots ◽  
Degree Of Branching ◽  
Medicinal Properties

Echinacea is one of the best-selling medicinal plants in the United States. It was historically harvested from wild populations, but its demand has increased so significantly that commercial production has become a necessity to supply the increasing demand and to protect wild populations. The medicinal properties of echinacea are associated with secondary metabolites that are produced mainly in the roots. Hairy roots, induced by the Ri plasmid of Agrobacterium rhizogenes, have been produced in other crops as alternative sources of secondary metabolites that commonly are produced and synthesized in the roots of mature plants. This method of production offers some advantages over traditional agricultural systems, such as the possibility of producing novel compounds year-round. The overall goal of this project is to explore the utility of hairy root cultures (mediated by A. rhizogenes) as an efficient, alternative, and enriched source of secondary compounds with medicinal properties, such as alkamides, flavonoids, and caffeic acid derivatives. We have been successful in transforming roots from E. angustifolia, E. pallida, and E. purpurea plants, and confirming the presence of rol ABC genes in hairy roots using molecular techniques. Roots from control plants show no active growth under dark conditions, whereas transformed roots from E. pallida and E. purpurea show a low degree of branching with a slow growth rate on solid media under darkness. However, transformed E. angustifolia show a faster growth rate and higher degree of branching under the same conditions. Currently, we are working on the optimization of the growing conditions of the transformed roots and will proceed to the biochemical analysis phase of the project.

scholarly journals AGROBACTERIUM-MEDIATED TRANSFORMATION OF PANAX VIETNAMENSIS HA ET GRUSHV.

2018 ◽  
Vol 15 (4) ◽  
pp. 641-650
Author(s):  
Duong Tan Nhut ◽  
Nguyen Phuc Huy ◽  
Trinh Thi Huong ◽  
Vu Quoc Luan ◽  
Vu Thi Hien ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Genetic Transformation ◽  
Hairy Root ◽  
Hairy Roots ◽  
Large Scale ◽  
Continuous Production ◽  
Pcr Amplification ◽  
Limited Area ◽  
Root Induction

In recent years, the Agrobacterium-mediated genetic transformation system has become the most useful method widely used for the introduction of foreign genes into plant cells followed by regeneration of genetically improved plants. Panax vietnamensis Ha et Grushv. is a highly valued medicinal plant native to Vietnam with limited area of distribution. This report illustrates the possibilities of biotechnology for genetic transformation aimed at establishing an effective production of secondary metabolites in P. vietnamensis. In the present investigation, 0.5 cm2 leaf blades, 1 cm long leaf petioles and 0.5 cm3 callus clusters were used for the hairy root induction. Results indicated that hairy roots were induced on P. vietnamensis callus clusters co-cultivated with Agrobacterium rhizogenes strain ATCC15834 at OD600 of 0.5 with an infection time of 20 min and a supplementation of 100 mM acetosyringone. PCR amplification of the DNA isolated from the resulting hairy roots was used to confirm the presence of rol genes. Compared to in vitro rhizome cultures, hairy root cultures appear to be potential for continuous production of valuable secondary metabolites with similar saponin profiles. The protocol described in this study is simple and rapid and therefore, can be used for large-scale experiments for the rapid production of valuable compounds.

scholarly journals Biological activity and hairy roots induction of Hibiscus sabdariffa L.

2019 ◽  
Vol 2 (6) ◽  
pp. 66-74
Author(s):  
Vu Thi Bach Phuong ◽  
Cao Minh Dai ◽  
Pham Thi Anh Hong ◽  
Quach Ngo Diem Phuong
Keyword(s):  
Hairy Root ◽  
Hairy Roots ◽  
Reducing Power ◽  
Root Production ◽  
Pcr Analysis ◽  
Root Extract ◽  
Root Induction ◽  
Hibiscus Sabdariffa ◽  
Transformed Hairy Root ◽  
Pharmaceutical Production

Hibiscus sabdariffa L. has been used traditionally in many countries of the world as food, especially as a flavouring agent in food industry. H. Sabdariffa is used for treating heart, nerve, liver disease, high blood pressure, arteriosclerosis, sore throat, cough, hypoglycaemia, laxative, diuretic, kidney stone, scurvy... The aims of this study are evaluation of bioactivities of H. Sabdariffa and the production of transformed hairy root of H. Sabdariffa for pharmaceutical production. In this study, Yen and Duh method showed the reducing power of ethanol the root extract and leaf extract are higher than that of stem. The root extract showed the α-glucosidase inhibitory activity with IC50 at 0.2 mg/mL is higher than those of stem and leaf. These results shows that root has higher bioactivites than stem and leaf. In this study, hairy roots of H. Sabdariffa were successfully induced via Agrobacterium rhizogenes ATCC 15834 in the plant cells. The frequency of hairy root and number of hairy root induction from the wounded sites of leaves are the highest (100% and 12.89 roots). The stable introduction of rolB and rolC genes of A. rhizogenes ATCC 15834 into H. Sabdariffa plants was confirmed by PCR analysis. Besides, the absence of virG gene confirmed hairy roots as bacteria-free. Subsequently, these results demonstrated that H. Sabdariffa, particularly the roots, has great potential as pharmacological values and hairy root production can be used as pharmaceutical sources.  

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.

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