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.     

北苍术和茅苍术挥发油成分的比较

采用气相色谱-质谱联用技术分析了北苍术和茅苍术的挥发油成分,分别鉴定出47和50种化学成分,并测定了其相对含量。 采用色谱指纹图谱八强峰法和分区法,根据色谱峰的保留时间把总离子流色谱图分为5个区,比较了北苍术和茅苍术的挥发油成分。 结果表明,在总离子流色谱图Ⅳ和Ⅴ区中,从北苍术和茅苍术中均检出苍术的主要药效成分β-桉叶油醇、苍术酮和苍术素,且相对含量较高。 这表明《中国药典》将北苍术、茅苍术统称为苍术具有一定的科学性。     

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.     

Phytosynthesis of Silver Nanoparticle (AgNPs) Using Aqueous Leaf Extract of Knoxia sumatrensis (Retz.) DC. and Their Multi-Potent Biological Activity: An Eco-Friendly Approach

Green synthesis of silver nanoparticles (AgNPs) has gained greater interest among chemists and researchers in this current scenario. The present research investigates the larvicidal and anti-proliferation activity of AgNPs derived from Knoxia sumatrensis aqueous leaf extract (K. sumatrensis-ALE) as a potential capping and reducing candidate. The synthesized AgNPs were characterized through-UV-spectra absorption peak at 425 nm. The XRD and FT-IR studied displayed the crystalline nature and presence of functional groups in prepared samples. FE-SEM showed the hexagonal shape of NPs with the size of 7.73 to 32.84 nm. The synthesized AgNPs displayed superior antioxidant and anti-proliferative activity (IC₅₀ 53.29 µg/mL) of breast cancer cell line (MCF-7). Additionally, larvicidal activity against mosquito vector Culex quinquefasciatus larvae delivered (LC₅₀-0.40, mg/L, and LC₉₀-15.83) significant mortality rate post treatment with synthesized AgNPs. Overall, the present research illustrates that the synthesized AgNPs have high biological potential and present a perfect contender in the pharmacological and mosquitocidal arena.     

Biotechnological Approaches on Two High CBD and CBG Cannabis sativa L. (Cannabaceae) Varieties: In Vitro Regeneration and Phytochemical Consistency Evaluation of Micropropagated Plants Using Quantitative 1H-NMR

High cannabidiol (CBD) and cannabigerol (CBG) varieties of Cannabis sativa L., a species with medicinal properties, were regenerated in vitro. Explants of nodal segments including healthy axillary bud, after sterilization, were placed in Murashige-Skoog (MS) culture medium. The shoots formed after 30 days were subcultured in full- or half-strength MS medium supplemented with several concentrations of 6-benzyl-amino-purine (BA) or thidiazuron (TDZ). The highest average number and length of shoots was achieved when both full and half-strength MS media were supplemented with 4.0 μM BA. The presence of 4.0 μM TDZ showed also comparable results. BA and TDZ at concentrations of 4.0, 8.0 μM and 2.0, 4.0 μM respectively, displayed the maximum shooting frequency. The new shoots were transferred on the same media and were either self-rooted or after being enhanced with different concentrations of indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA). Presence of 2.0 or 4.0 μM IBA or 4.0 μM NAA resulted to the optimum rooting rates. The maximum average number and length of roots per shoot was observed when the culture media was supplemented with 4.0 μM IBA or NAA. Approximately 92% of the plantlets were successfully established and acclimatized in field. The consistency of the chemical profile of the acclimatized in vitro propagated clones was assessed using quantitative ¹H-NMR high throughput screening. In each variety, analysis of the micropropagated plant in comparison with the mother plant showed no statistically significant differences (p ≤ 0.05) in CBD+ cannabidiolic acid (CBDA) and CBG+ cannabigerolic acid (CBGA) content respectively, thus indicating stability of their chemical profile.     

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.     

滇桂艾纳香水溶性多糖BRP-B的分离纯化及止血活性

用蒸馏水溶解滇桂艾纳香浸膏,从中提取多糖,通过膜分离、三氯乙酸法脱蛋白、二乙氨基乙基(DEAE)脱色、SephadexG10、SephadexG50凝胶柱色谱纯化,获得滇桂艾纳香水溶性多糖(BRP-B)。 由凝胶渗透色谱（HPGPC）确定BRP-B为相对分子质量分布均一的多糖,其数均分子量和质均分子量分别为2654和2716 Da。 以血浆复钙时间（PRT）为指标,研究BRP-B的凝血活性,结果显示BRP-B质量浓度为6.25×10-2 g/L时,凝血时间为248.52 s,凝血时间抑制率达22.42%。 小白鼠离体子宫实验结果显示,BRP-B质量浓度为1.67×10-2、1.97×10-2及2.85×10-2 g/L时能明显增加子宫平滑肌的收缩频率。     

Centranthus ruber (L.) DC. and Tropaeolum majus L.: Phytochemical Profile,In Vitro Anti-Denaturation Effects and Lipase Inhibitory Activity of Two Ornamental Plants Traditionally Used as Herbal Remedies

Ornamental plants often gain relevance not only for their decorative use, but also as a source of phytochemicals with interesting healing properties. Herein, spontaneous Centranthus ruber (L.) DC. and Tropaeolum majus L., mainly used as ornamental species but also traditionally consumed and used in popular medicine, were investigated. The aerial parts were extracted with methanol trough maceration, and resultant crude extracts were partitioned using solvents with increasing polarity. As previous studies mostly dealt with the phenolic content of these species, the phytochemical investigation mainly focused on nonpolar constituents, detected with GC-MS. The total phenolic and flavonoid content was also verified, and HPTLC analyses were performed. In order to explore the potential antiarthritic and anti-obesity properties, extracts and their fractions were evaluated for their anti-denaturation effects, with the use of the BSA assay, and for their ability to inhibit pancreatic lipase. The antioxidant properties and the inhibitory activity on the NO production were verified, as well. Almost all the extracts and fractions demonstrated good inhibitory effects on NO production. The n-hexane and dichloromethane fractions from T. majus, as well as the n-hexane fraction from C. ruber, were effective in protecting the protein from heat-induced denaturation (IC₅₀ = 154.0 ± 1.9, 270.8 ± 2.3 and 450.1 ± 15.5 μg/mL, respectively). The dichloromethane fractions from both raw extracts were also effective in inhibiting pancreatic lipase, with IC₅₀ values equal to 2.23 ± 0.02 mg/mL (for C. ruber sample), and 2.05 ± 0.02 mg/mL (T. majus). Obtained results support the traditional use of these species for their beneficial health properties and suggest that investigated plant species could be potential sources of novel antiarthritic and anti-obesity agents.     

Antioxidant,Hypoglycemic and Molecular Docking Studies of Methanolic Extract,Fractions and Isolated Compounds from Aerial Parts of Cymbopogon citratus (DC.) Stapf

Traditionally, Cymbopogon citratus is used to treat a variety of ailments, including cough, indigestion, fever, and diabetes. The previous chemical and bioactive research on C. citratus mainly focused on its volatile oil. In this study, 20 non-volatile known compounds were isolated from the dried aerial part of C. citratus, and their structures were elucidated by MS, NMR spectroscopy, and comparison with the published spectroscopic data. Among them, 16 compounds were reported for the first time from this plant. The screening results for antioxidant and α-glucosidase inhibitory activities indicated that compounds caffeic acid (5), 1-O-p-coumaroyl-3-O-caffeoylglycerol (8), 1,3-O-dicaffeoylglycerol (9) and luteolin-7-O-β-D-glucopyranoside (12) had potent antioxidant capacities, with IC₅₀ values from 7.28 to 14.81 μM, 1.70 to 2.15 mol Trolox/mol and 1.31 to 2.42 mol Trolox/mol for DPPH, ABTS, and FRAP, respectively. Meanwhile, compounds 8 and 9 also exhibited significant inhibitory activities against α-glucosidase, with IC₅₀ values of 11.45 ± 1.82 μM and 5.46 ± 0.25 μM, respectively, which were reported for the first time for their α-glucosidase inhibitory activities. The molecular docking result provided a molecular comprehension of the interaction between compounds (8 and 9) and α-glucosidase. The significant antioxidant and α-glucosidase inhibitory activities of compounds 8 and 9 suggested that they could be developed into antidiabetic drugs because of their potential regulatory roles on oxidative stress and digestive enzyme.     

Flavanone Glycosides,Triterpenes, Volatile Compounds and Antimicrobial Activity of Miconia minutiflora (Bonpl.) DC. (Melastomataceae)

Chemical composition of the essential oils and extracts and the antimicrobial activity of Miconia minutiflora were investigated. The flavanone glycosides, pinocembroside and pinocembrin-7-O-[4″,6″-HHDP]-β-D-glucose, were identified, along with other compounds that belong mainly to the triterpene class, besides the phenolics, gallic acid and methyl gallate. Sesquiterpenes and monoterpenes were the major compounds identified from the essential oils. Screening for antimicrobial activity from the methanolic extract of the leaves showed that the MIC and MMC values against the tested microorganisms ranged from 0.625 to 5 mg·mL⁻¹ and that the extract was active against microorganisms, Staphyloccocus aureus, Escherichia coli, and Bacillus cereus.