Variability of Bioactive Glucosinolates, Isothiocyanates and Enzyme Patterns in Horseradish Hairy Root Cultures Initiated from Different Organs

Horseradish hairy root cultures are suitable plant tissue organs to study the glucosinolate–myrosinase–isothiocyanate system and also to produce the biologically active isothiocyanates and horseradish peroxidase, widely used in molecular biology. Fifty hairy root clones were isolated after Agrobacterium rhizogenes infection of surface sterilized Armoracia rusticana petioles and leaf blades, from which 21 were viable after antibiotic treatment. Biomass properties (e.g. dry weight %, daily growth index), glucosinolate content (analyzed by liquid chromatography-electronspray ionization-mass spectrometry (LC-ESI-MS/MS)), isothiocyanate and nitrile content (analyzed by gas chromatography-mass spectrometry (GC-MS)), myrosinase (on-gel detection) and horseradish peroxidase enzyme patterns (on-gel detection and spectrophotometry), and morphological features were examined with multi-variable statistical analysis. In addition to the several positive and negative correlations, the most outstanding phenomenon was many parameters of the hairy root clones showed dependence on the organ of origin. Among others, the daily growth index, sinigrin, glucobrassicin, 3-phenylpropionitrile, indole-3-acetonitrile and horseradish peroxidase values showed significantly higher levels in horseradish hairy root cultures initiated from leaf blades.

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SYNTHESIS OF BIOLOGICALLY ACTIVE COMPOUNDS IN HAIRY ROOTS OF ASTRAGALUS PENDULIFLORUS LAM.

chemistry of plant raw material ◽

10.14258/jcprm.2020026284 ◽

2020 ◽

pp. 209-221

Author(s):

Elena Valerievna Ambros ◽

Ol'ga Viktorovna Kotsupiy ◽

Tat'yana Abdulkhailovna Kukushkina ◽

Tat'yana Vital'yevna Zheleznichenko ◽

Tat'yana Ivanovna Novikova

Keyword(s):

Hairy Root ◽

Hairy Roots ◽

Virulent Strain ◽

High Growth ◽

Growth Index ◽

Biologically Active ◽

Pcr Analysis ◽

Hairy Root Cultures ◽

Root Cultures ◽

Active Metabolites

Agrobacterium rhizogenes – mediated genetic transformation of medicinal plant A. penduliflorus Lam. using A4-RT, R-1601, 15834 SWISS strains was performed. The competences for transformation of three types of explants: hypocotyls, cotyledons, and primary shoots were tested. The virulent strain (15834 SWISS) and types of explants for transformation (primary shoots and cotyledons) with high growth index (I) were determined. The frequency of transformation of cotyledons by strain 15834 SWISS after 4 weeks of cultivation was 15.4% (I = 59.6), hypocotyls – 9.1% (I = 7.3) and primary shoots – 37.5% (I = 21.0). After 8 weeks of cultivation I increased 4.5 times for primary shoots (I = 94.5 ± 0.20) and cotyledons (I = 265.8 ± 0.35), for cultures from hypocotyls – 5.97 times (I = 43.6 ± 0.30). The roots’ transgenic status and the absence of agrobacterium contamination were confirmed by PCR analysis using rolB-, virC-specific primers. The lines of hairy roots characterized by active increases of biomass with high content of biologically active metabolites were selected, moreover, the content of metabolites in hairy root cultures exceeded their content in the roots of introduced plants. The maximum accumulation of compounds was found in hairy roots obtained from primary shoots (pectins – up 7.8%, protopectins – up 15.3%) and cotyledons (tannins – up 16.1%, triterpenic saponins – up 30.5%) after 8 weeks of cultivation. High perfor-mance liquid chromatography (HPLC) analysis demonstrated that hydrolysates of extracts of hairy roots from primary shoots contained 2 flavonol aglycones – quercetin and isorhamnetin whereas ethanol extracts were characterized by presence of quercetin and 4 flavonoid components. In hairy roots from cotyledons the maximum of phenolic compounds (PСs) content did not differ significantly at 8 and 12 weeks of cultivation (1.38 ± 0.01 and 1.49 ± 0.06% of dry weight, respectively). The content of PCs in hairy roots from primary shoots increased two-fold from 4 to 12 weeks of cultivation (up 1.24 ± 0.18%). To the best of our knowledge, this is the first efficient protocol reported for the establishment of hairy root cultures in A. penduliflorus using A. rhizogenes.

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Development of Hairy Root Cultures for Biomass and Triterpenoid Production in Centella asiatica

Plants ◽

10.3390/plants11020148 ◽

2022 ◽

Vol 11 (2) ◽

pp. 148

Author(s):

Seungeun Baek ◽

Jong-Eun Han ◽

Thanh-Tam Ho ◽

So-Young Park

Keyword(s):

Hairy Root ◽

Mass Production ◽

Adventitious Roots ◽

Transformation Efficiency ◽

Growth Index ◽

Centella Asiatica ◽

Asiatic Acid ◽

Hairy Root Cultures ◽

Root Cultures ◽

Petiole Explants

Centella asiatica (Apiaceae) is a tropical/subtropical medicinal plant, which contains a variety of triterpenoids, including madecassoside, asiaticoside, madecassic acid, and asiatic acid. In this study, we tested the efficiency of hairy root (HR) induction in C. asiatica from leaf and petiole explants. Leaves and petioles collected from C. asiatica plants were suspended in agro-stock for 30 min and co-cultured with Agrobacterium rhizogenes for 3 days to induce HR formation. The transformation efficiency of leaf and petiole explants was approximately 27% and 12%, respectively. A total of 36 HR lines were identified by PCR-based amplification of rol genes, and eight of these lines were selected for further analysis. Among all eight HR lines, the petiole-derived lines HP4 and HP2 displayed the highest growth index (37.8) and the highest triterpenoids concentration (46.57 mg∙g−1), respectively. Although triterpenoid concentration was >2-fold higher in leaves than in petioles of C. asiatica plants, the accumulation of triterpenoids in petiole-derived HR cultures was 1.4-fold higher than that in leaf-derived HR cultures. Additionally, in both leaf- and petiole-derived HR cultures, terpenoid production was higher in HRs than in adventitious roots. These results demonstrate that the triterpenoid content in the explant does not affect the triterpenoid content in the resultant HRs. The HR culture of C. asiatica could be scaled up to enable the mass production of triterpenoids in bioreactors for the pharmaceutical and cosmetic industries.

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

Ukrainian Journal of Ecology ◽

10.15421/2019_119 ◽

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.

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Panax quinquefolium L. Ginsenosides from Hairy Root Cultures and Their Clones Exert Cytotoxic, Genotoxic and Pro-Apoptotic Activity towards Human Colon Adenocarcinoma Cell Line Caco-2

Molecules ◽

10.3390/molecules25092262 ◽

2020 ◽

Vol 25 (9) ◽

pp. 2262 ◽

Cited By ~ 1

Author(s):

Ewa Kochan ◽

Adriana Nowak ◽

Małgorzata Zakłos-Szyda ◽

Daria Szczuka ◽

Grażyna Szymańska ◽

...

Keyword(s):

Cell Line ◽

Hairy Root ◽

Biologically Active ◽

Hairy Root Cultures ◽

Dependent Manner ◽

Root Cultures ◽

Panax Quinquefolium ◽

Adenocarcinoma Cell Line ◽

Concentration Dependent Manner ◽

Adenocarcinoma Cell

American ginseng, Panax quinquefolium (L.), is traditionally used in folk medicine. It exhibits a range of anti-inflammatory, hepatoprotective, anti-diabetic, anti-obesity, anti-hyperlipidemic and anti-carcinogenic effects. Its main components are ginsenosides, also known as panaxosides or triterpene saponins. In order to obtain high yields of ginsenosides, different methods of controlled production are involved, i.e., with hairy root cultures. However, they are still employed under in vitro conditions. Our studies revealed that hairy root cultures subjected to an elicitation process can be considered as a potent source of ginsenosides. The present study examines the biological activity of ginseng hairy root cultures against the Caco-2 human adenocarcinoma cell line. Among our six different clones of P. quinquefolium hairy roots, extracts B and Be (treated with elicitor) were the strongest inhibitors of the cellular metabolic activity. While all extracts induced DNA damage, B and Be also generated reactive oxygen species (ROS) in a concentration-dependent manner, which was correlated with the depletion of the mitochondrial membrane potential and induction of apoptosis. These findings indicate that further research concerning P. quinquefolium hairy root cultures should focus on the activity of rare ginsenosides and other biologically active compound profiles (i.e., phenolic compounds).

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Alkaloid Spectrum in Diploid and Tetraploid Hairy Root Cultures of Datura stramonium

Zeitschrift für Naturforschung C ◽

10.1515/znc-2003-1-207 ◽

2003 ◽

Vol 58 (1-2) ◽

pp. 42-46 ◽

Cited By ~ 36

Author(s):

Strahil Berkov ◽

Atanas Pavlov ◽

Petia Kovatcheva ◽

Pepa Stanimirova ◽

Stefan Philipov

Keyword(s):

Mass Spectrometry ◽

Gas Chromatography ◽

Hairy Root ◽

Hairy Roots ◽

Datura Stramonium ◽

Gas Chromatography Mass Spectrometry ◽

Hairy Root Cultures ◽

Root Cultures ◽

Mass Spectral Data ◽

Mass Spectral

Hairy root cultures were obtained from diploid and induced tetraploid plants of Datura stramonium and analyzed by gas chromatography/mass spectrometry. Twenty alkaloids (19 for diploid and 9 for tetraploid hairy root cultures) were identified. A new tropane ester 3-tigloyloxy-6-propionyloxy-7-hydroxytropane was identified on the basis of mass spectral data. Hyoscyamine was the main alkaloid in both diploid and tetraploid cultures. In contrast to diploid hairy roots, the percentage contributions of the alkaloids, with exceptions for hyoscyamine and apoatropine, were higher in the total alkaloid mixture of tetraploid hairy roots

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