Callus-Mediated High-Frequency Plant Regeneration, Phytochemical Profiling, Antioxidant Activity and Genetic Stability in Ruta chalepensis L.

Efficient methods for callus induction and the high-frequency plant regeneration of Ruta chalepensis L. were established, and the phytochemical potential and antioxidant activity of a donor plant, ex-vitro-established micropropagated plants, and callus were also studied. Yellowish-green callus was induced with a frequency of 97.8% from internode shoot segments of the donor plant growing in soil in the botanical garden cultured on Murashige and Skoog (MS) medium containing 10 µM 2,4-D (2,4-dichlorophenoxyacetic acid) and 1 µM BA (6-benzyladenine). Adventitious shoots were regenerated from the yellowish-green callus on MS medium containing 5.0 µM (BA) and 1.0 µM 1-naphthaleneacetic acid (NAA), with a regeneration frequency of 98.4% and a maximum of 54.6 shoots with an average length of 4.5 cm after 8 weeks. The regenerated shoots were rooted in a medium containing 1.0 µM IBA (indole-3-butyric acid) and successfully transferred to ex vitro conditions in pots containing normal garden soil, with a 95% survival rate. The amounts of alkaloids, phenolics, flavonoids, tannins, and antioxidant activity of the ex-vitro-established micropropagated plants were higher than in the donor plant and callus. The highest contents of hesperidin and rutin (93.3 and 55.9 µg/mg, respectively) were found in the ex-vitro-established micropropagated plants compared to those obtained from the donor plant (91.4 and 31.0 µg/mg, respectively) and callus (59.1 and 21.6 µg/mg, respectively). The genetic uniformity of the ex-vitro-established micropropagated plants was appraised by the ISSR markers and compared with the donor plant. This is the first report describing the callus-mediated plant regeneration, as well as the production of phenolic compounds and antioxidant activities in R. chalepensis, which might be a potential alternative technique for the mass propagation and synthesis of bioactive compounds such as hesperidin and rutin.

Photosynthetic Parameters and Oxidative Stress during Acclimation of Crepe-Myrtle (Lagerstroemia speciosa (L.) Pers.) in a meta-Topolin-Based Micropropagation System and Genetic Fidelity of Regenerated Plants.

An improved and stable micropropagation system using the cytokinin, meta-Topolin (N6 (3-hydroxybenzylamino purine-mT), with nodal explants in Lagerstroemia speciosa L. was established. Among the different doses of mT, the maximum number of shoots with the highest shoot length was obtained using Murashige and Skoog's (MS) medium supplemented with 5.0 µM mT. The results were consistent throughout the proliferation period, when recorded at week 4, 8, and 12 of being cultured, with an average of 16.4 shoots per nodal explant, and having a mean length of 4.10 cm at week 8. Shoot proliferation rates could be further improved by a combination of 5.0 µM mT with 0.5 µM α-naphthalene acetic acid in MS medium; nodal explants produced an average of 24.3 shoots with a mean length of 5.74 cm after 8 weeks of being cultured. Among the five different concentrations of three auxins tested for the rooting of microshoots in MS medium, a 1.0 µM indole-3-butyric acid treatment was the best, with an average of 10.3 roots per microshoot at an average length of 3.56 cm in 93% of microshoots within 4 weeks of being transferred to this medium. A significant reduction of both chlorophyll a and b in leaves during the first week of acclimation corresponded with a high accumulation of malondialdehyde (MDH), indicating that lipid peroxidation affected chlorophyll pigments. From the second week of acclimation, photosynthetic pigment content significantly increased and MDH content decreased. The net photosynthetic rate and leaf carotenoid content showed almost linear increases throughout the acclimation period. Activity of antioxidant enzymes, namely, superoxide dismutase, catalase, and peroxidases, consistently increased throughout the acclimation period, corresponding with the accumulation of photosynthetic pigments, thus demonstrating the role of the improved antioxidant enzymatic defense system during acclimation. A comparison of parent plant DNA with that of the greenhouse acclimated plants using random amplified polymorphic DNA and inter-simple sequence repeat markers showed a monomorphic pattern indicating genetic stability and the suitability of the method for micropropagation of L. speciosa.

Biotechnological Advances in Pharmacognosy and In Vitro Manipulation of Pterocarpus marsupium Roxb.

Trees are vital resources for economic, environmental, and industrial growth, supporting human life directly or indirectly through a wide variety of therapeutic compounds, commodities, and ecological services. Pterocarpus marsupium Roxb. (Fabaceae) is one of the most valuable multipurpose forest trees in India and Sri Lanka, as it is cultivated for quality wood as well as pharmaceutically bioactive compounds, especially from the stem bark and heartwood. However, propagation of the tree in natural conditions is difficult due to the low percentage of seed germination coupled with overexploitation of this species for its excellent multipurpose properties. This overexploitation has ultimately led to the inclusion of P. marsupium on the list of endangered plant species. However, recent developments in plant biotechnology may offer a solution to the overuse of such valuable species if such advances are accompanied by technology transfer in the developing world. Specifically, techniques in micropropagation, genetic manipulation, DNA barcoding, drug extraction, delivery, and targeting as well as standardization, are of substantial concern. To date, there are no comprehensive and detailed reviews of P. marsupium in terms of biotechnological research developments, specifically pharmacognosy, pharmacology, tissue culture, authentication of genuine species, and basic gene transfer studies. Thus, the present review attempts to present a comprehensive overview of the biotechnological studies centered on this species and some of the recent novel approaches for its genetic improvement.

High-Frequency Plant Regeneration, Genetic Uniformity, and Flow Cytometric Analysis of Regenerants in Rutachalepensis L.

Ruta chalepensis L., an evergreen shrub in the citrus family, is well-known around the world for its essential oils and variety of bioactivities, indicating its potential medicinal applications. In this study, we investigated the effect of different culture conditions, including plant growth regulators, media types, pH of the medium, and carbon sources, on in vitro regeneration from nodal explants of R. chalepensis. Following 8 weeks of culture, the highest percentage of regeneration (96.3%) and maximum number of shoots (40.3 shoot/explant) with a length of 4.8 cm were obtained with Murashige and Skoog (MS) medium at pH 5.8, supplemented with 3.0% sucrose and 5.0 µM 6-Benzyladenine (BA) in combination with 1.0 µM 1-naphthaleneacetic acid (NAA). For rooting, individually harvested shootlets were transferred on ½ MS (half-strength) supplemented with IAA (indole-3-acetic acid), IBA (indole 3-butyric acid), or NAA, and the best response in terms of root induction (91.6%), number of roots (5.3), and root mean length (4.9 cm) was achieved with 0.5 µM IBA after 6 weeks. An average of 95.2 percent of healthy, in vitro regenerated plantlets survived after being transplanted into potting soil, indicating that they were effectively hardened. DNA assays (PCR-based markers) such as random amplification of polymorphic DNA (RAPD) and directed amplification of minisatellite-region (DAMD) were employed to assess in vitro cultivated R. chalepensis plantlets that produced a monomorphic banding pattern confirming the genetic stability. Additionally, no changes in the flow cytometric profile of ploidy between regenerated plantlets and donor plants were detected. Regeneration of this valuable medicinal plant in vitro will open up new avenues in pharmaceutical biotechnology by providing an unconventional steadfast system for mass multiplication and might be effectively used in genetic manipulation for enhanced bioactive constituents.

Antibacterial and Antifungal Activity of the Extracts of Different Parts of Avicennia marina (Forssk.) Vierh.

Increased problems associated with side effects and bacterial resistance of chemical drugs has prompted the research focus on herbal medicines in the past few decades. In the present investigation, the antimicrobial activity of the various parts of Avicennia marina (AM), a mangrove plant, has been evaluated. The plants were collected from the Jazan area of the Kingdom of Saudi Arabia. Primary extracts of roots, stem, leaves, fruits, and seeds were made in ethanol and fractioned in ethanol, ethyl acetate, petroleum ether, chloroform, and water. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the extracts were determined against Bacillussubtilis, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. It has been observed that the chloroform extract of roots of the AM exhibited inhibitory effects against both S. aureus (MIC = 1.5 ± 0.03 mg/mL) and E. coli (MIC = 1.7 ± 0.01 mg/mL). The ethanolic extract of the AM roots has shown antibacterial activity against Pseudomonas aeruginosa (MIC = 10.8 ± 0.78 mg/mL), Bacillussubtilis (MIC = 6.1 ± 0.27 mg/mL), Staphylococcus aureus (MIC = 2.3 ± 0.08 mg/mL), and Escherichia coli (MIC = 6.3 ± 0.28 mg/mL). The leaf extract of the AM in ethyl acetate showed antibacterial activity against S. aureus and E. coli. Antifungal activity of these extracts was also investigated against Aspergillus fumigatus and Candida albicans. Ethanolic extract of roots and seeds of the AM has shown antifungal activity against Aspergillus fumigatus when applied individually. Ethanolic extract of the AM fruits has shown an inhibitory effect on the growth of Aspergillus fumigatus and Candida albicans. It is suggested that the plant extracts of AM have tremendous antimicrobial activity against a group of microbes, and this effect depends on both the plant part and the solvent used for extraction. Therefore, this plant can be considered to treat various diseases caused by antibiotic-resistant bacteria.

Nitrogen availability prevents oxidative effects of salinity on wheat growth and photosynthesis by up-regulating the antioxidants and osmolytes metabolism, and secondary metabolite accumulation.

BACKGROUND: Salinity is one of the damaging abiotic stress factor. Proper management techniques have been proposed to considerably lower the intensity of salinity on crop growth and productivity. Therefore experiments were conducted to assess the role of improved nitrogen (N) supplementation on the growth and salinity stress tolerance in wheat by analyzing the antioxidants, osmolytes and secondary metabolites. RESULTS: Salinity (100 mM NaCl) stress imparted deleterious effects on the chlorophyll and carotenoid synthesis as well as the photosynthetic efficiency. N supplementation resulted in increased photosynthetic rate, stomatal conductance and internal CO2 concentration with effects being much obvious in seedlings treated with higher N dose. Under non-saline conditions at both N levels, protease and lipoxygenase activity reduced significantly reflecting in reduced oxidative damage. Such effects were accompanied by reduced generation of toxic radicals like hydrogen peroxide and superoxide, and lipid peroxidation in N supplemented seedlings. Antioxidant defence system was up-regulated under saline and non-saline growth conditions due to N supplementation leading to protection of major cellular processes like photosynthesis, membrane structure and function, and mineral assimilation. Increased osmolyte and secondary metabolite accumulation, and redox components in N supplemented plants regulated the ROS metabolism and NaCl tolerance by further strengthening the antioxidant mechanisms. CONCLUSIONS: Findings of present study suggest that N availability regulated the salinity tolerance by reducing Na uptake and strengthening the key tolerance mechanisms.

Antioxidant, Hypoglycemic, and Neurobehavioral Effects of a Leaf Extract of Avicennia marina on Autoimmune Diabetic Mice.

Diabetes mellitus (DM) is a metabolic disease that can affect the central nervous system and behavioral traits in animals. Streptozotocin-induced diabetes is considered an autoimmune disease. The aim of the current study was to determine whether supplementation with the alcoholic extract of Avicennia marina leaves could improve diabetes-associated pathological changes. The animals were divided into four groups: a control group (A), an A. marina receiving nondiabetic group (B), a diabetic group (C), and a DM group orally supplemented with A. marina alcoholic leaf extract (D). The DM group of animals receiving the alcoholic extract of A. marina leaves had reduced blood glucose levels, improved blood picture, and organ functions. This group also showed improvement in locomotory behavior. The results of this study showed that supplementation with the alcoholic extract of A. marina leaves reduced oxidative stress and blood sugar levels, protected the liver, and improved the neurobehavioral changes associated with diabetes in mice. Introducing alcoholic leaf extract of A. marina to diabetic mice decreased inflammatory cells aggregation, vacuolation, and hemorrhage. Additionally, a positive effect of the alcoholic leaf extract on the histopathological changes was observed in the testicular tissue of treated mice.

An efficient and reproducible method for in vitro clonal multiplication of Rauvolfia tetraphylla L. and evaluation of genetic stability using DNA-based markers.

An efficient protocol is described for the rapid in vitro clonal propagation of an endangered medicinal plant, Rauvolfia tetraphylla L., through high frequency shoot induction from nodal explants collected from young shoots of a field grown plant. Effects of growth regulators [6-benzyladenine (BA), kinetin (Kin) 2iP, or α-naphthalene acetic acid (NAA)], carbohydrates, different medium [Murashige and Skoog (MS), Woody Plant Medium (WPM), Gamborg medium (B5), Linsmier and Skoog medium (LS)], and various pH levels on in vitro morphogenesis were investigated. The highest frequency of shoot regeneration (90 %) and maximum number of shoot (35.4 ± 2.3) per explant were observed on WPM medium supplemented with 7.5 µM BA, 2.5 µM NAA, and 30 g/l sucrose at pH 5.8. Well-developed shoots, 4-5 cm in length, were successfully rooted ex vitro at 90 % by a 30-min pulse treatment with 150 µM IBA prior to their transfer in planting substrates. The survival rate of transplantation reached 90 % when transferred to field condition. Genetic stability of micropropagated plantlets was assessed and compared with mother plant using Random Amplified Polymorphic DNA and Inter Simple Sequence Repeats markers. No variation was observed in DNA fingerprinting patterns among the micropropagated plants, which were similar to that of the donor plant illustrating their genetic uniformity and clonal fidelity. This confirms that clonal propagation of this plant using axillary shoot buds can be used for commercial exploitation of the selected genotype where a high degree of fidelity is an essential prerequisite. The work contributed to a better in vitro regeneration and clonal mass multiplication of R. tetraphylla and to develop a strategy for the germplasm conservation of this endangered medicinal plant.

Assessment of genetic fidelity in Rauvolfia serpentina plantlets grown from synthetic (encapsulated) seeds following in vitro storage at 4 °C.

An efficient method was developed for plant regeneration and establishment from alginate encapsulated synthetic seeds of Rauvolfia serpentina. Synthetic seeds were produced using in vitro proliferated microshoots upon complexation of 3% sodium alginate prepared in Llyod and McCown woody plant medium (WPM) and 100 mM calcium chloride. Re-growth ability of encapsulated nodal segments was evaluated after storage at 4 °C for 0, 1, 2, 4, 6 and 8 weeks and compared with non-encapsulated buds. Effects of different media viz; Murashige and Skoog medium; Lloyd and McCown woody Plant medium, Gamborg's B5 medium and Schenk and Hildebrandt medium was also investigated for conversion into plantlets. The maximum frequency of conversion into plantlets from encapsulated nodal segments stored at 4 °C for 4 weeks was achieved on woody plant medium supplement with 5.0 µM BA and 1.0 µM NAA. Rooting in plantlets was achieved in half-strength Murashige and Skoog liquid medium containing 0.5 µM indole-3-acetic acid (IAA) on filter paper bridges. Plantlets obtained from stored synseeds were hardened, established successfully ex vitro and were morphologically similar to each other as well as their mother plant. The genetic fidelity of Rauvolfia clones raised from synthetic seeds following four weeks of storage at 4 °C were assessed by using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. All the RAPD and ISSR profiles from generated plantlets were monomorphic and comparable to the mother plant, which confirms the genetic stability among the clones. This synseed protocol could be useful for establishing a particular system for conservation, short-term storage and production of genetically identical and stable plants before it is released for commercial purposes.