Search

The plastid transformation is used for high level expression of certain metabolically and industrially important recombinant proteins in plants. The vector, pFaadAII, a tobacco based vector system, harbouring a chimeric gene consisting of aadA coding region from Escherichia coli with 5′ 16S rDNA promoter and 3′ untranslated transcript region (UTR) of chlamydomonas rbcL gene, located in between the intergenic regions of rp132 and trnL genes. This vector used for transformation of plastids targets the foreign sequences to the small single-copy region of the plastome. Biolistic mode of approach for chloroplast transformation in Scoparia dulcis L., was achieved by bombarding the leaf explants and spectinomycin based selection system was used for regeneration of transformed plants. Transplastomic lines have been successfully established with overall efficiency of two transgenic lines for twenty-five bombarded explants. Integration of aadA in selection based regenerants was characterized by PCR and protein accumulation analysis along with seedlings experiment obtained from selfing. The chloroplast transformation developed in this plant system will provide scope for research in plastid based metabolic engineering pathways.

In the present investigation we report stable plastid transformation in Scoparia dulcis L., a versatile medicinal herb via particle gun method. The vector KNTc, harbouring aadA as a selectable marker and egfp as a reporter gene which were under the control of synthetic promoter pNG1014a, targets inverted repeats, trnR/trnN of the plastid genome. By use of this heterologous vector, recovery of transplastomic lines with suitable selection protocol have been successfully established with overall efficiency of two transgenic lines for 25 bombarded leaf explants. PCR and Southern blot analysis demonstrated stable integration of foreign gene into the target sequences. The results represent a significant advancement of the plastid transformation technology in medicinal plants, which relevantly implements a change over in enhancing and regulating of certain metabolic pathways.

Here, we report for the first time, the optimized conditions for microprojectile bombardment-mediated genetic transformation in Vassourinha (Scoparia dulcis L.), a Plantaginaceae medicinal plant species. Transformation was achieved by bombardment of axenic leaf segments with Binary vector pBI121 harbouring β-glucuronidase gene (GUS) as a reporter and neomycin phosphotransferase II gene (npt II) as a selectable marker. The influence of physical parameters viz., acceleration pressure, flight distance, gap width & macroprojectile travel distance of particle gun on frequency of transient GUS and stable (survival of putative transformants) expressions have been investigated. Biolistic delivery of the pBI121 yielded the best (80.0 %) transient expression of GUS gene bombarded at a flight distance of 6 cm and rupture disc pressure/acceleration pressure of 650 psi. Highest stable expression of 52.0 % was noticed in putative transformants on RMBI-K medium. Integration of GUS and npt II genes in the nuclear genome was confirmed through primer specific PCR. DNA blot analysis showed more than one transgene copy in the transformed plantlet genomes. The present study may be used for metabolic engineering and production of biopharmaceuticals by transplastomic technology in this valuable medicinal plant.

An efficient and reproducible protocol is established for rapid in vitro multiplication of an endangered, valuable medicinal plant, Butea monosperma (Lam.) Taub. Var. lutea, through cotyledonary nodes of mature seeds. Among various cytokinins tested, high frequency of direct shoot regeneration was induced on Murashige and skoog (MS) medium supplemented with BAP, which found to be more effective and showed optimal response at 2 mg/L with a maximum number of 8.35±0.32 multiple shoots per explant. Proliferation of shoots was established by repeated subculturing on to same regeneration medium with 2-3 weeks of time interval. Rooting of regenerated shoots was achieved after 3 weeks of culture on MS medium containing 1 mg/L IBA. In vitro raised plantlets were transferred to pots containing sterilized soil and vermiculate mixture in 1:1 ratio and then shifted to greenhouse. Well established plantlets exhibited 75% survival rate.

Objectives: Lemon balm (Melissa officinalis) is a perennial herb belongs to Lamiaceae, which is native to South Europe, Middle East and North America. Lemon balm is used for anti-cancer, digestive problems, anxiety, gastrointestinal disorders, thyroid, chronic fatigue syndrome and Alzheimer’s disease. A successful and efficient system for in vitro seed sterilization and germination has been established in the present study. Methods/ Statistical Analysis: The seeds were soaked in distilled water for 10 hours and exposed with various concentrations of different surface sterilization agent for various intervals of time. Surface sterilized seeds were inoculated on different culture media such as Murashige and Skoog (MS) basal, half strength MS and B5 media with different sucrose concentrations. Findings: By comparison of measured characteristics results (ANOVA) we found that the best sterilizing agent for seed surface sterilization and uniform growth of seedling in M. officinalis was 2.5% Sodium Hypochlorite for 3 min. Maximum germination rate, shoot length and root length were recorded in half strength MS medium with 1% (W/V) sucrose. The analysis of data revealed the correlation between GP, GR, shoot length and root length were significant and positive. Application/Improvement: We optimized seed surface sterilization protocol and culture condition that may be useful quick regenerative, transformation and conservation method for Melissa officinalis, an important medicinal plant.

In the present investigation we report stable plastid transformation in Scoparia dulcis L., a versatile medicinal herb via particle gun method. The vector KNTc, harbouring aadA as a selectable marker and egfp as a reporter gene which were under the control of synthetic promoter pNG1014a, targets inverted repeats, trnR/t rnN of the plastid genome. By use of this heterologous vector, recovery of transplastomic lines with suitable selection protocol have been successfully established with overall efficiency of two transgenic lines for 25 bombarded leaf explants. PCR and Southern blot analysis demonstrated stable integration of foreign gene into the target sequences. The results represent a significant advancement of the plastid transformation technology in medicinal plants, which relevantly implements a change over in enhancing and regulating of certain metabolic pathways.

The activation of telomerase represents an early step in carcinogenesis. Increased telomerase expression in malignant tumors suggests that telomerase inactivation may represent a potential chemotherapeutic target. In this work, existing anticancer drugs were docked against telomerase reverse transcriptase (TERT) using a Lamarckian genetic algorithm (LGA). Autodock's scoring function was applied to each of the molecules in order to identify the inhibitor with the strongest pharmacological action. The structural insights provided by this study regarding binding poses and possible interactions, free energies of binding, and drug scores aided in the identification of potential inhibitory compounds. The ranks of the various ligands investigated were based on the final docked energy values. Among nine selected compounds, vindesine, temsirolimus, and cyclosporine were found to be more potent TERT inhibitors than the standard inhibitor, curcumin.

Efficient shoot regeneration and Agrobacterium-mediated genetic transformation systems were developed for Bacopa monnieri L. (Scrophulariaceae), a plant well known for its medicinal properties. Leaf explants were cultured on Murashige and Skoog (MS) medium with different concentrations of 6-benzylaminopurine (BAP), and in combination with either indole-3-acetic acid (IAA) or napthalene-3-acetic acid. A combination of BAP (17.80 μM) and IAA (2.28 μM) maximized shoot initiation (85.2 ± 2.43) with greatest shoot length (2.8 ± 0.22), and was obtained directly from leaf explants without an intervening callus phase. Leaf segments from in vitro grown plants were co-cultivated with Agrobacterium tumefaciens LBA4404 harboring pCAMBIA1301 with s-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes. The co-cultivated explants were transferred to selective shoot induction and elongation medium. The elongated hygromycin-resistant shoots were subsequently rooted on MS medium supplemented with 4.9 μM indole-3-butyric acid and 25 mg/l hygromycin (SSRM). Successful transformation was confirmed by monitoring histochemical GUS activity during shoot elongation and PCR analyses using uidA- and hpt-specific primers. Integration of hpt into the genome of transgenic plants was also verified by Southern blot analysis. The highest transformation efficiency achieved was 70.6%, with an average of 10.4 ± 0.15 transgenic plantlets per explant using the present transformation system. Therefore, these highly efficient and rapid regeneration and transformation systems create significant potential for engineering of B. monnieri with a view to detailed biomolecular analyses or for further enhancement of its medicinal properties.

A protocol is presented for regenerating plants from protoplasts of tropical mulberry. Leaves from seedling node cultures maintained in vitro were used as donor tissue. Optimal cell wall digestion was achieved with a combination of cellulase (2%) and macerozyme (1%). The plant growth regulator (PGR) combination zeatin (2.3 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D) (2.3 μM) resulted in the highest number (29%) of cell divisions. First cell divisions were observed at day 4 after plating. Only zeatin (2.3 μM) and 2-methoxy-3,6-dichlorobenzoic acid (dicamba) (13.5 μM) supplemented medium supported subsequent divisions in protoplast cultures. Microcolonies reached a cell number of approximately 50, after 40 to 42 days of culture. The cells of these colonies continued dividing, leading to formation of microcalli. Whole plants were obtained after culture of microcalli on Murashige and Skoog (MS) medium containing thidiazuron (TDZ) (4.5 μM) and indole-3-acetic acid (IAA) (17.1 μM). The regenerated shoots were rooted on MS medium supplemented with 4.9 μM indole butyric acid (IBA). With a low survival rate during acclimation, regenerated plants were established in the greenhouse.

A protocol for micropropagation of plants via axillary bud proliferation from nodal explants of Terminalia bellirica Roxb. seedlings has been established. Explants were cultured on Murashige and Skoog medium with different concentrations of 6-benzyladenine (BA; 4.4, 8.9, 13.3, 17.8, or 22.2 μM) or kinetin (Kn; 4.6, 9.3. 14.0, 18.6, or 23.2 μM). Within the range evaluated, the medium containing 13.3 μM BA showed the highest shoot length (1.9=0.2 cm) in the primary culture. When separated and transferred to fresh subculture medium with lower levels of BA (2.2. 4.4, 6.6, or 8.9 μM) or Kn (2.3, 4.6, 6.9, or 9.3 μM), the nodal segments from individual regenerants (obtained initially from seedling nodes) showed efficient shoot induction at 4.4 μM BA. Rooting of the shoots was achieved under in vitro conditions on two media tested, i.e., modified Gamborg's (B5) medium or Woody Plant Medium, both supplemented with 4.9 μM indole-3-butyric acid. Regenerated plants were established in the greenhouse.

An efficient procedure has been developed for inducing somatic embryogenesis from leaf cultures of potato cv. Jyothi. Leaf sections were initially cultured on 2,4-dichlorophenoxyacetic acid (2,4-D) + benzyladenine (BA) and α-naphthaleneacetic acid (NAA) + BA supplemented Murashige and Skoog (MS) media. Nodular embryogenic callus developed from the cut ends of explants on media containing 2,4-D and BA, whereas compact callus developed on media containing NAA and BA. The explants with primary callus were subsequently moved onto MS media containing zeatin and/or gibberellic acid (GA3) and BA. Treatment with zeatin (22.8 μM) and BA (10.0 μM) resulted in the induction of the highest number of somatic embryos directly from meristematic centres produced on the nodular tissue. Embryo induction and maturation took place on this medium. The cotyledonary stage embryos developed into complete plantlets on hormone-free MS medium. A distinct feature of this study is the induction of somatic embryogenesis in leaf cultures of potato which has not been reported previously.

The effects of atrazine on cotyledon cultures of Solanum melongena were investigated with a view to establishing a system for in vitro selection of resistant mutants. At herbicide levels producing little growth inhibition some chlorophyll loss occurred associated with the production of albino shoots. At 15 mg/l bleaching was more pronounced and was accompanied by the development of necrotic spots; however, efficient bleaching was associated with severe suppression of growth. Mutagenesis with EMS resulted in herbicide-resistant mutants based on the embryogenic ability of mutagenised explants placed on medium containing selective levels of sucrose (0.2%) and atrazine (15 mg/l). Different morphogenetic responses were observed when the levels of sucrose (0.2–5%) were altered. Somatic embryogenesis was observed at low sucrose concentrations (0.2–0.5%). Both embryogenesis and shoot regeneration occurred in 1% sucrose. Shoot regeneration was maximum in 2% sucrose and the regenerating ability decreased with a further increase in sucrose concentration (3–5%). However, lowering of sucrose concentration from 2% to 0.2% caused complete bleaching, permitting the selection of herbicide-resistant mutants.