Non-transformed Root Cultures Research Articles

Determination of lignans, phenolic acids and antioxidant capacity in transformed hairy root culture of Linum usitatissimum

Hairy root culture is a promising alternative method for the production of secondary metabolites. In this study, transformed root of Linum usitatissimum was established using Agrobacterium rhizogenes A4 strain from root cultures for lignans, phenolic acids and antioxidant capacity determination. Total lignin content (secoisolariciresinol diglucoside, secoisolariciresinol and matairesinol) was 55.5% higher in transformed root cultures than in the non-transformed root culture. Secoisolariciresinol was detected in higher concentration (2.107 μmol/g DM) in the transformed root culture than non-transformed culture (1.099 μmol/g DM). Secoisolariciresinol diglucoside and matairesinol were exclusively detected in the transformed root culture, but were not found in the non-transformed root culture. The overall production of phenolic acids in transformed roots was approximately 3.5 times higher than that of the corresponding non-transformed culture. Free radical scavenging DPPH˙ and ABTS˙+ assays showed 2.9-fold and 1.76-fold higher anti-oxidant activity in transformed root culture as compared to non-transformed.

Antimicrobial activity of the extracts from some Phyllanthus species cultivated in vitro

In subtropical regions the extracts from Phyllanthus species are used in the treatment of bacterial, fungal and parasitic diseases (e.g. P. niruri, P. amarus). Literature data showed that antimicrobial activity of Phyllanthus species results from synergistic effects of occurring secondary metabolites. The MIC and MBC values were determined to show antibacterial activity of extracts from shoot cultures of P. glaucus, P. multiflorus and P. juglandifolius and shoot culture and non-transformed root culture of P. amarus. The dry extracts dissolved in either DMSO or methanol were examined against strains of Gram (+) bacteria – Streptococcus G, S. aureus, S. epidermidis, B. subtilis, C. sporogenes, E. hirae, strains of Gram (-) bacteria – M. catarrhalis, K. pneumoniae, E. coli, P. vulgaris, P. aeruginosa, H. pylori as well as a yeast C. albicans. As controls, the alkaloid securinine, present as a dominant compound of the alkaloid fraction in the shoot culture of P. glaucus, and flavan-3-ol – catechin present in all tested species, were used. Using DMSO to dissolve dry extracts higher MIC values were observed than when dissolved in methanol. The obtained results showed the differences in bacterial susceptibility depending on the Phyllanthus species. For the methanol extracts antimicrobial activity expressed as MIC was in a range from 0.01 mg/mL to 5.0 mg/mL, while the MBC value was determined from 0.01 mg/mL to 10.0 mg/mL. Securinine and catechin were active in a range from 0.03 to 0.25 mg/mL showing the highest activity against Streptococcus G and P. aeruginosa (securinine) and H. pylori (catechin). All extracts and standards were inactive against E. hirae while the most susceptible strain was M. catarrhalis (MBC 0.01 mg/l). Acknowledgement: This Project was supported by the Ministry of Science and Higher Education of the Republic of Poland, from the quality-promoting subsidy, under the Leading National Research Centre (KNOW) programme for the years 2012 – 2017.

Enhanced trans-resveratrol production in genetically transformed root cultures of Peanut (Arachis hypogaea L.)

In the present study, 30 Ri-transformed root lines of peanut (Arachis hypogaea) cv. JL-24, a popular Indian cultivar, obtained following infection with Agrobacterium rhizogenes strains LBA9402, A4 and R1000 were selected on the basis of growth index and maintained in vitro for 3 years. The root lines showed high degree of branching and rapid, plagiotropic growth on phytohormone free solid N/5 medium but were devoid of root hairs. Trans-resveratrol was isolated by preparative HPLC from Ri-transformed roots and identified by ESI–MS/MS. Strain independent variability was observed among 30 Ri-transformed root lines on the basis of lateral root density per cm (7.60 ± 0.30 to 4.5 ± 0.5), relative thickness (0.54 ± 0.07 to 1.54 ± 0.1 mm), growth index (9.16 ± 1.1 to 17.79 ± 1.35 FW basis and 10.77 ± 0.95 to 19.46 ± 1.78 DW basis) and trans-resveratrol content 0.27 ± 0.03 (root line R1000-1) to 0.969 ± 0.141 mg g DW−1 (root line RIX-19) in solid N/5 medium, which was 4.1–14 fold greater than in excised non-transformed root cultures (0.06 ± 0.01 mg g DW−1). Optimum growth and productivity in liquid culture was achieved in N/5 medium supplemented with 0.01 % activated charcoal. Root line RIX-19 showed maximum trans-resveratrol accumulation (1.21 ± 0.09 mg g DW−1) and productivity (0.37 ± 0.08 mg per flask), which was 19 fold higher than non-transformed root cultures. This optimized protocol can be utilized for large scale cultivation of transformed root cultures in industrial bioreactors for mass synthesis of trans-resveratrol.

Cytotoxic Lignan from the Non-Transformed Root Culture of Phyllanthus amarus.

A new lignan from the non-transformed root in vitro cultures of Phyllanthus amarus was isolated. The structure of the compound was established on the basis of one- and two-dimensional NMR, as well as mass spectrometry data, as 7'-oxocubebin dimethylether (1,4-bis(benzo[d][1,3]dioxol-5-yl)-2,3-bis(methoxymethyl)butan-1-on). The non-transformed root cultures of P. amarus showed to be a selective source of this compound. The lignan revealed strong cytotoxic activity against HeLa cell line with an IC50 value of 3.8 µg/mL.

In vitro Root Culture : An Alternative Source of Bioactives in the Rare Aphrodisiac Herb Chlorophytum borivilianum Sant et Fern

Chlorophytum borivilianum is a rare, near-endemic high value root crop. The fasciculated roots of the plant have the potential to synthesize valuable bioactives having aphrodisiac, anti stress and immuno modulatory properties. The present investigation describes an alternative protocol for the development of a stable, fast growing, non-transformed root culture system in C. borivilianum and the simultaneous quantitative analysis of total steroidal saponins, amino acids and sugars. Liquid MS fortified with various concentrations and combinations of BA (0.44 - 8.8 µM), Kn (0.23 µM) and NAA (0.27 - 5.4 µM) was effectively used for the development of root culture system. High performance thin layer chromatography (HPTLC) was used for quantification and comparative analysis of bioactives from in vitro and in vivo grown tuberous roots. D. O. I.  http://dx.doi.org/10.3329/ptcb.v23i2.17505 Plant Tissue Cult. & Biotech. 23(2): 133-146, 2013  (December)

Induction and establishment of adventitious and hairy root cultures of Plumbago zeylanica L.

This study was investigated to develop efficient protocols for adventitious and hairy root cultures of Plumbago zeylanica. Adventitious roots were initiated from leaf and stem explants cultured on MS medium with different concentrations and combinations of auxins. The highest number of roots was obtained when the explants was cultured on MS medium with 1.0 mg·L-1 IBA and 0.5 mg·L-1 NAA. Hairy root culture of P. zeylanica was obtained by infecting leaf explants cultured in vitro with Agrobacterium rhizogenes MTCC 532. The highest frequency of explant transformation was about 93%. The developed culture exhibited fast growth and high lateral branching on growth regulator free MS medium. The root cultures obtained were inoculated into B5, MS and SH media supplemented with different carbon sources with or without auxins and were placed on a rotary shaker 80 rpm for 35 days under dark or light conditions. Of the three media tested, MS medium sustained better root growth than others and sucrose proved to be the best carbon source. The biomass in hairy root culture was higher than in nontransformed root culture.

Isolation and identification of littorine from hairy roots of Atropa belladonna.

A hairy root clone (M8) of Atropa belladonna, producing high levels of tropane alkaloids, was established by transformation with Agrobacterium rhizogenes (MAFF 03-01724). Littorine, an intermediate of tropane alkaloids, was detected by high-performance liquid chromatography and gas chromatography-mass spectrometry in the alkaloid fraction of the hairy roots and identified by nuclear magnetic resonance analysis. Littorine was also detected in the non-transformed root culture of A. belladonna.

Indole alkaloid production by transformed and non-transformed root cultures ofCatharanthus roseus

Ten transformed and two non-transformed root lines ofCatharanthus roseus were established. A systematic study of the growth kinetics and alkaloid content was performed over a culture cycle and showed significant differences between transformed and non-transformed cultures. Mean doubling times for transformed and normal root lines were 2.8 and 19.5 days, respectively. Alkaloid content in hairy roots was from two- to threefold higher than in the non-transformed tissues. The established transformed root lines produced a wide variety of indole alkaloids as can be observed from their complex thin layer chromatography patterns. A large quantity of serpentine was determined in two of the transformed root cultures. Alkaloid content, both quantitatively and qualitatively, has been stable in the hairy root cultures for more than 2 yr of subculturing.

Transformation of opium poppy (Papaver somniferum L.) with Agrobacterium rhizogenes MAFF 03-01724

Transformed cultures of opium poppy (Papaver somniferum L.) were established by infecting hypocotyl segments with Agrobacterium rhizogenes MAFF 03-01724. Undifferentiated calli formed on the infected site grew satisfactorily on phytohormone-free solid medium in the dark and produced opine, mikimopine, which could not be detected in a normal culture. Numerous adventitious shoots developed from transformed calli during subculture. The transformed shoots separated individually were cultured on phytohormone-free MS solid medium at 22 ° C under 14 h/day light. They displayed wider leaves and longer internodes than shoots established from seeds or non-transformed root culture. The content of morphinan alkaloids in the cultures and regenerated shoots were quantitatively analyzed by enzyme-linked immunosorbent assay and high performance liquid chromatography. HPLC analysis revealed that non-transformed shoots contained much more codeine (1310 gmg/g dry wt.) than morphine (50 μg/g dry wt.), while the transformed shoot cultures did not contain morphine, although the level of morphinan alkaloids in the transformed shoots (213 μg morphine equivalents/g fr. wt.) was comparable to that in non-transformed shoots (182 μg morphine equivalents/g fr. wt.) by ELISA.

Hernandulcin in hairy root cultures of Lippia dulcis

The hairy root culture of Lippia dulcis Trev., Verbenaceae, was established by transformation with Agrobacterium rhizogenes A4. The transformed roots grew well in Murashige and Skoog medium containing 2% sucrose. The roots turned light green when they were cultured under 16 h/day light. The green hairy roots produced the sweet sesquiterpene hernandulcin (ca. 0.25 mg/g dry wt) together with 20 other mono- and sesquiterpenes, while no terpenes were detected in the nontransformed root cultures. The growth and hernandulcin production in the hairy root cultures were influenced by the addition of auxins to the medium. The addition of a low concentration of chitosan (0.2 - 10.0 mg / l) enhanced the production of hernandulcin 5-fold.

Accumulation of Unusual Phenylpropanoids in Transformed and Non-Transformed Root Cultures of Coreopsis tinctoria

Using Coreopsis tinctoria liquid cultures of Agrobacterium rhizogenes transformed roots (socalled “hairy roots”) and non-transformed roots have been established. The production of different phenylpropanoid com pounds by these cultures grown in light or darkness, respectively, was compared. Whereas the com position of phenylpropanoids in the root cultures was not influenced by the culture conditions, the total production of phenylpropanoids as well as the formation of single com pounds each analyzed were higher in darkness than in light. The biomass production of the transformed root cultures exceeded that of non-transformed root cultures by a factor of 2.6. A t the same time com pound accumulation paralleled the biomass production, so that the dark grown transformed root cultures were producing maximum yields of several phenylpropanoids, especially l′-isobutyryloxy-eugenol-isobutyrate (7 mg per culture flask during a single culture passage).