In Vitro Plant Propagation of Catharanthus roseus and Assessment of Genetic Fidelity of Micropropagated Plants by RAPD Marker Assay

An investigation was carried out to develop an efficient micropropagation protocol for Catharanthus roseus. Experiments were conducted to optimize suitable media for in vitro shoot multiplication and root induction. Out of the different media compared for in vitro shoot multiplication, Murashige and Skoog (MS) medium supplemented with 1 mg/l of 6-benzylaminopurine and 0.2 mg/l α-naphthaleneacetic acid showed better response in terms of the emergence of shoots from axillary buds as well as proliferation and multiplication of shoots. The shoots when placed on half strength of MS medium having 1 mg/l indole 3-butyric acid and 0.25 % charcoal showed cent percent root induction with maximum number of roots per shoot (4.2) as well as maximum root length (1.72 cm). Further, clonal fidelity of the in vitro-raised plants was carried out using randomly amplified polymorphic DNA marker and results indicated that all the tissue culture-derived plants are true-to-type and there were no somaclonal variations among these plants.     

Ex Situ Conservation of Phyllanthus fraternus Webster and Evaluation of Genetic Fidelity in Regenerates Using DNA-Based Molecular Marker

Germplasm storage of Phyllanthus fraternus by using synseed technology has been optimized. Synseeds were prepared from nodal segments taken from in vitro-grown plantlets. An encapsulation matrix of 3 % sodium alginate and 100 mM calcium chloride with polymerization duration up to 15 min was found most suitable for synseed formation. Maximum plantlet conversion (92.5?±?2.5 %) was obtained on a growth regulator-free ½-strength solid Murashige and Skoog (MS) medium. Multiple shoot proliferation was optimum on a ½ MS medium containing 0.5 mg/l 6-benzylaminopurine (BAP). Shoots were subjected to rooting on MS media containing 1 mg/l α-naphthaleneacetic acid (NAA) and acclimatized successfully. Encapsulated nodal segments can be stored for up to 90 days with a survival frequency of 47.33 %. The clonal fidelity of synseed-derived plantlets was also assessed and compared with that of the mother plant using rapid amplified polymorphic DNA and inter-simple sequence repeat analysis. No changes in molecular profiles were observed among the synseed-derived plantlets and mother plant, which confirms the genetic stability of regenerates. This synseed production protocol could be useful for in vitro multiplication, short-term storage, and exchange of germplasm of this important antiviral and hepatoprotective plant.     

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 ??-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???M BA, 2.5???M NAA, and 30?g/l sucrose at pH?5.8. Well-developed shoots, 4?C5?cm in length, were successfully rooted ex vitro at 90?% by a 30-min pulse treatment with 150???M 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 Stability in Traditional Ginger Cultivated in Manipur,India Based on Molecular and Chemical Markers

Randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to analyze the genetic stability of ten local cultivars collected from nine districts of Manipur, India with the released ginger variety Nadia. A total of 15 RAPD and 8 ISSR primers resulted in 107 and 53 distinct and reproducible bands, respectively. A lack of polymorphism revealed the genetic stability among the local cultivars. Unlike molecular markers, analysis of essential oil composition by gas chromatography–mass spectrometry (GC-MS) revealed variation among 11 clones. Among eight major constituents obtained by GC-MS technique, cinnamyl acetate was found only in IBSD/Z-41d cultivars, whereas, in IBSD/Z-41o no trace of trans-geraniol was observed. Moreover, concentration of 6-gingerol determined by high-performance liquid chromatographic (HPLC) method shows that IBSD/Z-41r contains the highest and IBSD/Z-41i contains the lowest gingerol percentage.     

In Vitro Propagation and Assessment of the Genetic Fidelity of Musa acuminata (AAA) cv. Vaibalhla Derived from Immature Male Flowers

An efficient in vitro propagation method has been developed for the first time for Musa acuminata (AAA) cv. Vaibalhla, an economically important banana cultivar of Mizoram, India. Immature male flowers were used as explants. Murashige and Skoog’s (MS) medium supplemented with plant growth regulators (PGRs) were used for the regeneration process. Out of different PGR combinations, MS medium supplemented with 2 mg L^?1 6-benzylaminopurine (BAP) + 0.5 mg L^?1 α-naphthalene acetic acid (NAA) was optimal for production of white bud-like structures (WBLS). On this medium, explants produced the highest number of buds per explant (4.30). The highest percentage (77.77) and number (3.51) of shoot formation from each explants was observed in MS medium supplemented with 2 mg L^?1 kinetin + 0.5 mg L^?1 NAA. While MS medium supplemented with a combination of 2 mg L^?1 BAP + 0.5 mg L^?1 NAA showed the maximum shoot length (14.44 cm). Rooting efficiency of the shoots was highest in the MS basal medium without any PGRs. The plantlets were hardened successfully in the greenhouse with 96 % survival rate. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were employed to assess the genetic stability of in vitro regenerated plantlets of M. acuminata (AAA) cv. Vaibalhla. Eight RAPD and 8 ISSR primers were successfully used for the analysis from the 40 RAPD and 30 ISSR primers screened initially. The amplified products were monomorphic across all the regenerated plants and were similar to the mother plant. The present standardised protocol will find application in mass production, conservation and genetic transformation studies of this commercially important banana.     

Plant Regeneration Through Callus Organogenesis and True-to-Type Conformity of Plants by RAPD Analysis in Desmodium gangeticum (Linn.) DC.

An efficient plant regeneration protocol was established for an endangered ethnomedicinal plant Desmodium gangeticum (Linn.) DC. Morphogenic calli were produced from 96 % of the cultures comprising the immature leaf explants on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (4.0 mg?l^?1) in combination with 6-benzylaminopurine (BA; 0.8 mg?l^?1). For callus regeneration, various concentrations of BA (1.0–5.0 mg?l^?1) or thidiazuron (TDZ; 1.0–5.0 mg?l^?1) alone or in combination with indole-3-acetic acid (IAA; 0.2–1.0 mg?l^?1) were used. Highest response of shoot regeneration was observed on MS medium fortified with TDZ (4.0 mg?l^?1) and IAA (0.5 mg?l^?1) combination. Here, 100 % cultures responded with an average number of 22.3 shoots per gram calli. Inclusion of indole-3-butyric acid in half MS medium favored rooting of recovered shoots. Out of 45 rooted plants transferred to soil, 40 survived. Total DNA was extracted from the leaves of the acclimatized plants of D. gangeticum. Analysis of random amplified polymorphic DNA using 13 arbitrary decanucleotide primers showed the genetic homogeneity in all the ten plants regenerated from callus with parental plant, suggesting that shoot regeneration from callus could be used for the true-to-type multiplication of this plant.     

Assessment of Genetic Stability and Instability of Tissue Culture-Propagated Plantlets of Aloe vera L. by RAPD and ISSR Markers

Efficient plantlet regeneration with and without intermediate callus phase was achieved for a selected genotype of Aloe vera L. which is sweet in test and used as a vegetable and source of food. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) marker assays were employed to evaluate genetic stability of plantlets and validate the most reliable method for true-to-type propagation of sweet aloe, among two regeneration systems developed so far. Despite phenotypic similarities in plantlets produced through both regeneration systems, the differences in genomic constituents of plantlets produced through intermediate callus phase using soft base of inflorescence have been effectively distinguished by RAPD and ISSR markers. No polymorphism was observed in regenerants produced following direct regeneration of axillary buds, whereas 80% and 73.3% of polymorphism were observed in RAPD and ISSR, respectively, in the regenerants produced indirectly from base of the inflorescence axis via an intermediate callus phase. Overall, 86.6% of variations were observed in the plantlets produced via an intermediate callus phase. The occurrence of genetic polymorphism is associated with choice of explants and method used for plantlet regeneration. This confirms that clonal propagation of sweet aloe using axillary shoot buds can be used for commercial exploitation of the selected genotype where a high degree of fidelity is an essential prerequisite. On the other hand, a high degree of variations were observed in plantlets obtained through indirect regeneration and thus cannot be used for the mass multiplication of the genotype; however, it can be used for crop improvement through induction of somaclonal variations and genetic manipulations.     

Antimicrobial activity of Blumea balsamifera (Lin.) DC. extracts and essential oil

Leaves of Blumea balsamifera (Lin.) DC. are used in traditional Thai and Chinese medicine for the treatment of septic wounds and other infections. In this study, the essential oil, hexane, dichloromethane and methanol extracts of these leaves were evaluated for antibacterial and antifungal activities using the disc diffusion assay and agar microdilution method. The essential oil was the most potent, with a minimum inhibitory concentration (MIC) of 150?μg?mL?1 against Bacillus cereus and an MIC of 1.2?mg?mL?1 against Staphylococcus aureus and Candida albicans. Activity was also detected from the hexane extract against Enterobacter cloacae and S. aureus. Minimum bactericidal and fungicidal concentrations were typically equal to or two-fold higher than the MICs for both extracts, indicating microbicidal activity. The present data show that B. balsamifera extracts have activity against various infectious and toxin-producing microorganisms. This plant's active constituents could potentially be developed for use in the treatment and/or prevention of microbial disease.     

Evaluation of Genetic Homogeneity in Tissue Culture Regenerates of Jatropha curcas L. using Flow Cytometer and DNA-based Molecular Markers

The present investigation aimed to evaluate the reliability of in vitro propagation methods for elite genotypes of Jatropha curcas L., that maintain genetic integrity of tissue culture (TC) regenerates among two regeneration systems developed through direct shoot bud regeneration using nodal/apical shoot segments (protocol-A) and in vitro-derived leaves (protocol-B) as explants. Random amplified polymorphic DNA (RAPD), intersimple sequence repeat (ISSR), simple sequence repeat (SSR) molecular markers, and flow cytometery (FCM) were employed to evaluate genetic homogeneity in TC-regenerates at different passages of subcultures. RAPD markers showed genetic homogeneity in fifth-generation TC-regenerates of both protocols. ISSR markers showed genetic stability of leaf regenerates (protocol-B) at 10th generation. FCM analysis of TC-regenerates at 10th generation in protocol-B and at 20th generation in both protocols, showed stability of ploidy level. SSR assessment of TC-regenerates at 20th generation in both protocols confirmed genetic homogeneity. The results confirmed the genetic stability of the TC-regenerates and demonstrated the reliability of the regeneration systems developed so far using explants of two different origins, for large-scale multiplication of elite genotypes of Jatropha.     

Investigation of the coumarins and furocoumarins ofHippomarathrum caspium (DC.) Grossh.

Summary 1. Osthole, oxypeucedanin, heraclenin, and oxypeucedanin hydrate have been isolated from the roots ofHippomarathrum caspium (DC) Grossh. and identified; the presence of traces of isoimperatorin, imperatorin, and umbelliferone has been established chromatographically. A substance with mp 149–150° apparently belonging to the sitosterol group has been obtained.2. Xanthotoxin has been isolated from the leaves and stems; the presence of a small amount of bergaptene and umbelliferone has been detected chromatographically.Khimiya Prirodnykh Soedinenii, Vol. 1, No. 5, pp. 356–359, 1965     

Circadian variation and in vitro cytotoxic activity evaluation of volatile compounds from leaves of Piper regnellii (Miq) C. DC. var. regnellii (C. DC.) Yunck (Piperaceae)

Aiming detection of circadian variation in the chemical composition of volatiles from Piper regnellii, the leaves were collected during four different periods (8, 12, 16 and 20 h) in the same day. After extraction by hydrodistillation and GC/MS analysis, no significant variation was observed for the main compounds: germacrene D (45.6 ± 1.5–51.4 ± 3.1%), α–chamigrene (8.9 ± 1.3–11.3 ± 2.7%) and β–caryophyllene (8.2 ± 0.9–9.5 ± 0.3%). Evaluation of in vitro cytotoxicity against several cancer and non-tumourigenic cells indicated promising activity, especially to HeLa (human cervical carcinoma) with IC₅₀ ranging from 11 ± 3 to 17 ± 3 μg/mL. The obtained volatile oils were pooled and subjected to fractionation to afford pure β-caryophyllene, α-chamigrene and germacrene D, being this last compound the more active against HeLa cells with IC₅₀ of 7 ± 1 μg/mL (34 ± 5 μM). Therefore, the predominance of germacrene D in all analysed oils could justify, at least in part, the activity observed for the volatile compounds from P. regnellii leaves.     

A Sensitive HPTLC Method for Estimation of Amentoflavone,a Bioactive Principle from Biophytum sensitivum (Linn.) DC. and Putranjiva roxburghii Wall.

JPC – Journal of Planar Chromatography – Modern TLC - A sensitive high-performance thin-layer chromatographic (HPTLC) method has been developed for estimation of amentoflavone, an...     

A Tandem Olefin Migration and Prins Cyclization Using Cu(OTf)(2)-Bisphosphine Complexes: An Improved Synthesis of Functionalized Tetrahydropyrans

In situ-generated (bis-DPPMB)-Cu(OTf)₂ complex has been examined to catalyze a tandem olefin migration and Prins cyclization of an alkenol with various aldehydes. The reaction proceeded with electron-rich aromatic aldehydes at room temperature and provided functionalized tetrahydropyrans in good yields. An efficient synthesis of the bis-DPPMB ligand has also been described.     

Biogenic Synthesis of Rod Shaped ZnO Nanoparticles Using Red Paprika (Capsicum annuum L. var. grossum (L.) Sendt) and Their in Vitro Evaluation

Journal of Cluster Science - Metal oxide nanoparticles (NPs) have gained attention in biomedicine due to their broad spectrum of applications, such as targeted drug delivery, their use as...     

Molecular structure of orthosilicic acid and importance of (p-d)π bonding. An ab initio molecular orbital study

OH and SiO bond lengths, the SiOH bond angle, favoured conformations of the protons and rotational barriers are obtained from 6-31G* calculations (assuming a tetrahedral SiO₄ structure). The role of d orbitals on silicon and/or oxygen atoms is assessed and the origin of the rotational barriers for silanol protons is discussed.     

Comparison of the Electrochemical Behavior of the High Molecular Mass Cadmium Proteins in Arabidopsis thaliana and in Vegetable Plants on Using Preparative Native Continuous Polyacrylamide Gel Electrophoresis (PNC‐PAGE)

In Arabidopsis cytosol (supernatant) and in supernatants of vegetable plants high molecular mass cadmium proteins with molecular mass 200 kDa were isolated by using p reparative n ative c ontinuous p olya crylamide g el e lectrophoresis (PNC‐PAGE). Because of a different electrochemical behavior of the Cd proteins in Arabidopsis and endive supernatants on using the same PAGE method, it is concluded that the high molecular mass cadmium proteins of Arabidopsis and endive possess different isoelectric points. Consequently, different chemical structures of the Cd proteins with molecular mass 200 kDa are present in Arabidopsis thaliana and in endive. During the electrophoretic separation of vegetable metalloproteins by using the Model 491 Prep Cell from BioRad, electroanalytical processes like electrode reactions may play an important role.