Withania somnifera (L.) Dunal: Enhanced production of withanolides and phenolic acids from hairy root culture after application of elicitors.

Withania somnifera (L.) Dunal is an important indigenous medicinal plant with extensive pharmaceutical potential. The root is the main source of major bioactive compounds of this plant species including withanolides, withanine, phenolic acids, etc. Hairy root culture (HRC) is a crucial method for low-cost production of active compounds on a large scale. Four different Agrobacterium rhizogenes strains have been used for the hairy root induction. Maximum transformation efficiency (87.34 ± 2.13%) was achieved with A4 bacterial strain-mediated transformed culture. The genetic transformation was confirmed by using specific primers of seven different genes. Seven HR (Hairy root) lines were selected after screening 29 HR lines based on their fast growth rate and high accumulation of withanolides and phenolic acids content. Two biotic and three abiotic elicitors were applied to the elite root line to trigger more accumulation of withanolides and phenolic acids. While all the elicitors effectively increased withanolides and phenolic acids production, among the five different elicitors, salicylic acid (4.14 mg l-1) induced 11.49 -fold increase in withanolides (89.07 ± 2.75 mg g-1 DW) and 5.34- fold increase in phenolic acids (83.69 ± 3.11 mg g- 1 DW) after 5 days of elicitation compared to the non-elicited culture (7.75 ± 0.63 mg g-1 DW of withanolides and 15.66 ± 0.92 mg g-1 DW of phenolic acids). These results suggest that elicitors can tremendously increase the biosynthesis of active compounds in this system; thus, the HRC of W. somnifera is cost-effective and can be efficiently used for the industrial production of withanolides and phenolic acids.

Phytogenic silver nanoparticles from tissue-cultured Kaempferia angustifolia - an underutilized medicinal herb: a comparative antibacterial study on urinary pathogens.

BACKGROUND: Ethnomedicinally important Kaempferia angustifolia is a rhizomatous aromatic herb belonging to the family Zingiberaceae. The present manuscript deals with the green synthesis of silver nanoparticles through a rapid reduction process mediated by the rhizome extract of tissue culture-raised plants. The present study was conducted to evaluate the antimicrobial activity of the bio-nanoparticles, and the plant extracts themselves against seven multidrug-resistant urinary tract infecting (MDR-UTI) pathogens. RESULT: The ethanolic extracts of the rhizomes of the plant executed a very rapid synthesis of silver bio-nanoparticles, and the generation of the nanoparticles was confirmed through UV-vis spectrophotometry, dynamic light scattering (DLS), and electron dispersion spectroscopic (EDS) analysis. Finally, the precise shapes and dimensions of these nanoparticles were confirmed under the transmission electron microscope (TEM). The shapes of the nanoparticles obtained were diverse in nature and varied from rod, triangular, spherical, to oval shaped, with the size, ranging from 10-60 nm. Silver nanoparticles exhibited a maximum zone of inhibition (ZI) of 16.93 ± 0.04 mm against isolate no. 42332. The ex vitro and in vivo extracts exhibited ZI 14.03 ± 0.04 mm and 11.56 ± 0.04 mm, respectively, against the same strain, which are comparatively lower than the nanoparticles but unignorable. CONCLUSION: Although the pathogens used in the present study are resistant to at least three or more types of pharmacologically important antibiotics, nanoparticles, as well as the plant extracts, exhibited significant inhibition to all the seven MDR-UTI pathogens, which confirms that they are highly antimicrobic. Hence, this underutilized medicinal plant extracts of K. angustifolia and the bio-nanoparticles synthesized from these can be explored in pharmaceutical industries to treat multidrug-resistant human pathogenic bacteria. Furthermore, their broad-spectrum activity leads to the opportunity for the synthesis of future generation drugs.

Effect of different drying treatments on the physicochemical, functional, and antioxidant properties of Bacopa monnieri.

Bacopa monnieri (L.) Wettst is a very high-value medicinal plant that is commonly used for improving cognitive functions. However, the availability of very limited information on the drying method of B. monnieri has prompted to optimize a suitable drying method. The present study therefore aimed to evaluate the influence of the following six drying treatments on the quality of B. monnieri sample: microwave drying at 300 W and 600 W, hot air-drying at 50°C and 70°C, solar drying, and freeze-drying (FD). The quality attributes of the dried samples were comparatively analyzed in terms of color, total color difference, moisture content, water activity (a w), antioxidant activity, and bacoside A and bacopaside I content. The results of this study showed significant differences (P < 0.05) among the different drying methods in International Commission on Illumination (CIE) parameters, namely lightness index (L*), red-green index (a*), and yellow-blue index (b*), ΔE and a values. Among the samples dried with the six drying methods, freeze-dried B. monnieri samples had an attractive color with the lowest total color difference value (11.415%), a w value (0.15%), and maximum bacoside A (3.389%) and bacopaside I (0.620%) content. Moreover, Fourier transform infrared spectroscopy (FT-IR) analysis showed no major difference in the functional groups in B. monnieri samples processed by the different drying methods. Considering the retention of quality after drying, FD was found to be very effective for future large-scale production of good quality dried B. monnieri products.

Somatic embryogenesis and synthetic seed production--a biotechnological approach for true-to-type propagation and in vitro conservation of an ornamental bulbaceous plant Drimiopsis kirkii Baker.

An efficient plant regeneration protocol through indirect somatic embryogenesis pathway via callus had been developed from the leaf explant of an ornamental bulbaceous plant Drimiopsis kirkii. Optimum friable calli were induced on Murashige and Skoog (MS) basal medium supplemented with 3.0 mg/l of 2,4-dichlorophenoxyacetic acid and 1.0 mg/l of α-naphthalene acetic acid (NAA). On subculturing the callus on MS medium supplemented with 2.5 mg/l of thidiazuron (TDZ), 73.3 % of the cultures responded with 20.4 ± 0.3 somatic embryos (SEs) per 500 mg callus at different stages of development after 6 weeks of culture. The highest response of 86.7 % with 28.3 ± 0.5 embryos per 500 mg callus was observed on MS medium supplemented with 2.5 mg/l TDZ and 1.0 mg/l NAA. SEs were encapsulated in calcium alginate beads for the production of synthetic seeds (SSs) and their storability was investigated. The highest SS germination (93.3 %) was observed in 1.0 % sodium alginate followed by 86.7 % germination with 2.5 % sodium alginate. The SSs were stored at three different temperatures (4, 15, and 24 ºC) up to 6 months. The SSs kept at 15 °C showed 64.4 % germinability even after 4 months of storage. Both nonencapsulated and encapsulated SE-derived plants were successfully transferred to soil with 93.3 and 88.3 % survival rate accordingly. Randomly amplified polymorphic DNA (RAPD) analysis revealed that there were no somaclonal variations among the plants produced via somatic embryogenesis and they are true-to-type to their parental plant. These results confirmed the most reliable methods, which can be further used for genetic transformation studies as well as for mass propagation of ornamental D. kirkii at a commercial level.

Spontaneous plant regeneration in transformed roots and calli from Tylophora indica: Changes in morphological phenotype and tylophorine accumulation associated with transformation by Agrobacterium rhizogenes.

We examined the effects of genetic transformation by Agrobacterium rhizogenes on the production of tylophorine, a phenanthroindolizidine alkaloid, in the Indian medicinal plant, Tylophora indica. Transformed roots induced by the bacterium grew in axenic culture and produced shoots or embryogenic calli in the absence of hormone treatments. However, hormonal treatment was required to regenerate shoots in root explants of wild type control plants. Transformed plants showed morphological features typically seen in transgenic plants produced by A. rhizogenes, which include, short internodes, small and wrinkled leaves, more branches and numerous plagiotropic roots. Plants regenerated from transformed roots showed increased biomass accumulation (350-510% in the roots and 200-320% in the whole plants) and augmented tylophorine content (20-60%) in the shoots, resulting in a 160-280% increase in tylophorine production in different clones grown in vitro.

Genetic transformation of Tylophora indica with Agrobacterium rhizogenes A4: growth and tylophorine productivity in different transformed root clones.

We have developed an efficient transformation system for Tylophora indica, an important medicinal plant in India, using Agrobacterium rhizogenes strains LBA9402 and A4 to infect excised leaf and stem explants and intact shoots at different sites. The induction of callus and transformed roots was dependent on the bacterial strain, explant type and inoculation site used. Transformed roots were induced only in explants infected with A. rhizogenes strain A4, while an optimal transformation frequency of up to 60% was obtained with intact shoots inoculated at the nodes. The presence of the left-hand transferred DNA (T(L)-DNA) in the genome of T. indica roots induced by A. rhizogenes was confirmed by PCR amplification of the rooting locus genes of A. rhizogenes. Root growth and the production of tylophorine, the major alkaloid of the plant, varied substantially among the nine root clones studied. Both parameters increased over time in liquid cultures, with maximum biomass and tylophorine accumulation occurring within 4-6 weeks of growth in fresh medium. Interestingly, in liquid culture, the culture medium also accumulated tylophorine up to concentrations of 9.78+/-0.21 mg l(-1).

Variation in content of taxol and related taxanes in Eastern Himalayan populations of Taxus wallichiana.

Extraction and analysis of paclitaxel and other taxanes in bark, needle leaves and stem segments of male and female plants of Taxus wallichiana, representing several populations, indicate that significant variation in taxane content exists within the population. Bark accumulated maximum amount of paclitaxel in almost all plants. Populations located at higher altitude tended to accumulate more paclitaxel than lower altitude plants. Seasons in which samples were collected and plant age have also been shown to affect paclitaxel accumulation. No effects of plant sex on paclitaxel content of the plants analyzed were observed. Significant differences in baccatin-III and 10-deacetylbaccatin III content were found to exist in the trees analyzed in this study.