Phytocompounds and Regulation of Flavonoids in In Vitro-Grown Safflower Plant Tissue by Abiotic Elicitor CdCl2.

In this study, a Gas chromatography-mass spectrometry (GC-MS) investigation of embryogenic callus and somatic embryo regenerated shoots of Carthamus tinctorius revealed the presence of a variety of sugars, sugar acids, sugar alcohols, fatty acids, organic acids, and amino acids of broad therapeutic value. The in vitro developed inflorescence contained a wide range of active compounds. In embryogenic calluses, important flavonoids like naringenin, myricetin, kaempferol, epicatechin gallate, rutin, pelargonidin, peonidin, and delphinidin were identified. To augment the synthesis of active compounds, the effect of cadmium chloride (CdCl2) elicitation was tested for various treatments (T1-T4) along with a control (T0). Varying concentrations of CdCl2 [0.05 mM (T1), 0.10 mM (T2), 0.15 mM (T3), and 0.20 mM (T4)] were added to the MS medium, and flavonoid accumulation was quantified through ultra-high-pressure liquid chromatography-tandem mass spectroscopy (UHPLC-MS/MS). The flavonoids naringenin, kaempferol, epicatechin gallate, pelargonidin, cyanidin, and delphinidin increased by 6.7-, 1.9-, 3.3-, 2.1-, 1.9-, and 4.4-fold, respectively, at T3, whereas quercetin, myricetin, rutin, and peonidin showed a linear increase with the increase in CdCl2 levels. The impacts of stress markers, i.e., ascorbate peroxidase (APX), catalase (CAT), and superoxide dismutase (SOD), on defense responses in triggering synthesis were also evaluated. The maximum APX and SOD activity was observed at T3, while CAT activity was at its maximum at T2. The impact of elicitor on biochemical attributes like protein, proline, sugar, and malondialdehyde (MDA) content was investigated. The maximum protein, proline, and sugar accumulation was noted at high elicitor dose T4, while the maximum MDA content was noted at T3. These elevated levels of biochemical parameters indicated stress in culture, and the amendment of CdCl2 in media thus could be a realistic approach for enhancing secondary metabolite synthesis in safflower.

Fungal Elicitation Enhances Vincristine and Vinblastine Yield in the Embryogenic Tissues of Catharanthus roseus.

Fungal elicitation could improve the secondary metabolite contents of in vitro cultures. Herein, we report the effect of Fusarium oxysporum on vinblastine and vincristine alkaloid yields in Catharanthus roseus embryos. The study revealed increased yields of vinblastine and vincristine in Catharanthus tissues. Different concentrations, i.e., 0.05% (T1), 0.15% (T2), 0.25% (T3), and 0.35% (T4), of an F. oxysporum extract were applied to a solid MS medium in addition to a control (T0). Embryogenic calli were formed from the hypocotyl explants of germinating seedlings, and the tissues were exposed to Fusarium extract elicitation. The administration of the F. oxysporum extract improved the growth of the callus biomass, which later differentiated into embryos, and the maximum induction of somatic embryos was noted T2 concentration (102.69/callus mass). A biochemical analysis revealed extra accumulations of sugar, protein, and proline in the fungus-elicitated cultivating tissues. The somatic embryos germinated into plantlets on full-strength MS medium supplemented with 2.24 µM of BA. The germination rate of the embryos and the shoot and root lengths of the embryos were high at low doses of the Fusarium treatment. The yields of vinblastine and vincristine were measured in different treated tissues via high-pressure thin-layer chromatography (HPTLC). The yield of vinblastine was high in mature (45-day old) embryos (1.229 µg g-1 dry weight), which were further enriched (1.267 µg g-1 dry weight) via the F. oxysporum-elicitated treatment, especially at the T2 concentration. Compared to vinblastine, the vincristine content was low, with a maximum of 0.307 µg g-1 dry weight following the addition of the F. oxysporum treatment. The highest and increased yields of vinblastine and vincristine, 7.88 and 15.50%, were noted in F. oxysporum-amended tissues. The maturated and germinating somatic embryos had high levels of SOD activity, and upon the addition of the fungal extracts, the enzyme's activity was further elevated, indicating that the tissues experienced cellular stress which yielded increased levels of vinblastine and vincristine following the T2/T1 treatments. The improvement in the yields of these alkaloids could augment cancer healthcare treatments, making them easy, accessible, and inexpensive.

Elicitation Induced α-Amyrin Synthesis in Tylophora indica In Vitro Cultures and Comparative Phytochemical Analyses of In Vivo and Micropropagated Plants.

Tylophora indica (Burm. f.) Merrill is an endangered medicinal plant that possesses various active agents, such as tylophorinine, kaempferol, quercetin, α-amyrin and beta-sitosterol, with multiple medicinal benefits. α-amyrin, a triterpenoid, is widely known for its antimicrobial, anti-inflammatory, gastroprotective and hepatoprotective properties. In this study, we investigated the metabolite profiling of tissues and the effects of cadmium chloride and chitosan on in vitro accumulation of alkaloids in T. indica. First, the callus was induced from the leaf in 2,4-D-, NAA- and/or BAP-fortified MS medium. Subsequent shoot formation through organogenesis and in vitro roots was later induced. Gas chromatography-mass spectrometry (GC-MS)-based phytochemical profiling of methanolic extracts of in vivo and in vitro regenerated plants was conducted, revealing the presence of the important phytocompounds α-amyrin, lupeol, beta-sitosterol, septicine, tocopherol and several others. Different in vitro grown tissues, like callus, leaf and root, were elicited with cadmium chloride (0.1-0.4 mg L-1) and chitosan (1-50 mg L-1) to evaluate the effect of elicitation on α-amyrin accumulation, measured with high-performance thin layer chromatography (HPTLC). CdCl2 and chitosan showed improved sugar (17.24 and 15.04 mg g-1 FW, respectively), protein (10.76 and 9.99 mg g-1 FW, respectively) and proline (7.46 and 7.12 mg g-1 FW), especially at T3 (0.3 and 25 mg L-1), in the leaf as compared to those of the control and other tissues. The antioxidant enzyme activities were also evaluated under an elicitated stress situation, wherein catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) displayed the highest activities in the leaf at T4 of both of the two elicitors. The α-amyrin yield was quantified with HPTLC in all tested tissues (leaf, callus and root) and had an Rf = 0.62 at 510 nm wavelength. Among all the concentrations tested, the T3 treatment (0.3 mg L-1 of cadmium chloride and 25 mg L-1 of chitosan) had the best influence on accumulation, irrespective of the tissues, with the maximum being in the leaf (2.72 and 2.64 µg g-1 DW, respectively), followed by the callus and root. Therefore, these results suggest future opportunities of elicitors in scaling up the production of important secondary metabolites to meet the requirements of the pharmaceutical industry.

Shotgun label-free proteomic and biochemical study of somatic embryos (cotyledonary and maturation stage) in Catharanthus roseus (L.) G. Don.

Somatic embryogenesis is an important and wonderful biotechnological tool used to develop whole plant from a single or a group of somatic cells. The differentiated somatic cells become totipotent stem cells by drastic reprogramming of a wide range of cellular activities, leading to the acquisition of embryogenic competence. After acquiring competence, the cells pass through globular, heart, torpedo and cotyledonary stages of embryo; however, all advanced embryos do not convert into full plant, produce adventive embryos or callus instead, thus reverses the programming. This is a big limitation in propagation of many plants. Understanding and unraveling the proteins at this 'embryo to plantlet' transition stage will help to get more numbers of plants. Thus, our study was aimed at an identification of differentially abundant proteins between two important advanced stages, i.e. cotyledonary-(T1) and maturation stage (T2) of somatic embryos in Catharanthus roseus. A total of 2949 and 3030 proteins were identified in cotyledonary and maturation stage, respectively. Of these, 1129 proteins were common to both. Several proteins were found to be differentially accumulated in two different embryo stages in which over 60 proteins were most accumulated during somatic embryo maturation time. More chlorophyll accumulation was noted at this time under the influence of gibberellic acid (GA3). Proteins like Mg-protoporphyrin IX chelatase, chlorophyll a-b-binding protein, photosystem I iron-sulfur center, photosystem II Psb, photosystem II subunit P-1, P-II domain-containing protein, RuBisCO large chain, RuBisCO small chain, RuBisCO activase, RuBisCO large subunit-binding proteins were synthesized. Some of the identified proteins are linked to chlorophyll synthesis, carbohydrate metabolism and stress. The identified proteins are categorized into different groups on the basis of their cellular location, role and other metabolic processes. Biochemical attributes like protein, sugar, proline, antioxidant enzyme (APX, SOD and CAT) activities were high in T2 as compared to T1. The proteins like peroxidases, pathogenesis-related proteins, the late-embryogenesis abundant proteins, argonaute, germin and others have been discussed in C. roseus somatic embryo maturation process.

Essential oil yield estimation by Gas chromatography-mass spectrometry (GC-MS) after Methyl jasmonate (MeJA) elicitation in in vitro cultivated tissues of Coriandrum sativum L.

Coriandrum sativum is an important spice plant known for its unique fragrance. Coriander oil is also one of the major essential oils in world global market. The oil yield varies with different coriander varieties; and the content and quality of oil is governed by several factors. In recent times, a variety of technologies have been exploited to improve phyto-compounds including essential oils. In this present study, Methyl jasmonate (MeJA) was amended in medium and the yield of essential oil was measured and compared in different cultivating tissues. The cultured tissues were nonembryogenic callus and embryogenic tissues (induction, proliferation and maturation stages of embryos). MeJA acts as a signaling molecule in accumulating secondary metabolites. Four different MeJA treatments i.e. T1 = 50, T2 = 100, T3 = 150 and T4 = 200 µM, along with a control (T0) were used and the yield of coriander essential oil was estimated in different in vitro cultivating tissues by using Gas chromatography-mass spectrometry (GC-MS). The addition of MEJA enriched essential oil yield, maximum oil being in maturation stage of embryos at T3 (150 µM). Other added treatments also had varied stimulatory role. The addition of MeJA induced stress as the stress marker enzymes like superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) content were high compared to non treated tissue (T0). In T4, the CAT activity was maximum i.e. 5.83 and 6.28 mg-1 protein min-1 in Co-1 and RS respectively in matured somatic embryos. The SOD activity was also high at maturation stage of embryos at T4 (5.3 mg-1 protein min-1 in RS). The APX activity on the other, was high (3.32 mg-1 protein min-1) in induction stage of embryogenesis at T3. The comparative biochemical (sugar, protein and proline) analyses of tissues were performed and presented that had high and low essential oil. MeJA induced stress may help in accumulating essential oils in C. sativum.

In vitro micropropagation of Dracaena sanderiana Sander ex Mast: An important indoor ornamental plant.

A protocol has been developed for in vitro plant regeneration from a nodal explant of Dracaena sanderiana Sander ex Mast. Nodal explant showed high callus induction potentiality on MS medium supplemented with 6.78 µM 2,4-dichlorophenoxyacetic acid (2,4-D) followed by 46.5 µM chlorophenoxy acetic acid (CPA). The highest frequency of shoot regeneration (85%) and number of shoots per explant (5.6) were obtained on medium supplemented with 7.84 µM N(6)-benzylaminopurine (BA). Rooting was high on MS solid compared to liquid medium when added with 7.38 µM indole-3-butyric acid (IBA). Fifty percent of the roots were also directly rooted as microcuttings on soil rite, sand and peat mixture (1:1:1). In vitro and ex vitro raised plantlets were used for acclimatization. More than 90% of the plantlets was successfully acclimatized and established in plastic pots. Ex vitro transferred plantlets were normal without any phenotypic aberrations.

Alliin obtained from leaf extract of garlic grown under in situ conditions possess higher therapeutic potency as analyzed in alloxan-induced diabetic rats.

CONTEXT: Garlic, Allium sativum L. (Liliaceae), possesses high therapeutic and pharmacological properties. Hypoglycemic activity is attributed to alliin (S-allyl cysteine sulfoxide), the main active principle localized in garlic cloves. OBJECTIVE: To compare the production and therapeutic efficiency of alliin extracted from garlic leaves of plants grown under ex situ and in situ conditions. MATERIALS AND METHODS: Alliin content of leaves was quantified and aqueous leaf extracts (from ex situ and in situ grown plants) were given to normal and alloxan-induced diabetic rats for five weeks. RESULTS: Alliin production noted ~50% enhancement in leaves from plants grown under in situ conditions. Serum glucose, triglycerides, total lipids, total cholesterol, low-density lipoprotein (LDL)-, and very low-density lipoprotein (VLDL)-cholesterol in diabetic rats treated with alliin produced from in situ grown plants noted significant reduction of ~54%, 15%, 14%, 20%, 24%, and 15%, while 35%, 14%, 10%, 12%, 17% and 11% reduction was noted in diabetic rats treated with alliin produced from ex situ grown plants in comparison with those administered with distilled water. High-density lipoprotein (HDL)-cholesterol did not show any significant change. Leaf extract of plants lowered serum enzyme levels (alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase) toward the norm better than glibenclamide. The histopathological alteration in pancreas caused by alloxan was also reduced by leaf extract. DISCUSSION AND CONCLUSION: These findings demonstrate leaf extract obtained from plants grown under in situ condition possess higher therapeutic efficiency in comparison with leaf extract obtained from plants grown under ex situ condition. Studies suggest that environmental factors influence production of alliin and its therapeutic potential.

Somatic embryogenesis, scanning electron microscopy, histology and biochemical analysis at different developing stages of embryogenesis in six date palm (Phoenix dactylifera L.) cultivars.

An efficient somatic embryogenesis system has been established in six date palm (Phoenix dactylifera L.) cultivars (Barhee, Zardai, Khalasah, Muzati, Shishi and Zart). Somatic embryogenesis (SE) was growth regulators and cultivars dependent. Friable embryogenic callus was induced from excised shoot tips on MS medium supplemented with various auxins particularly 2,4-dichlorophenoxyacetic acid (2,4-D, 1.5 mg 1(-l)). Suspension culture increased embryogenesis potentiality. Only a-naphthaleneacetic acid (NAA, 0.5 mg 1(-1)) produced somatic embryos in culture. Somatic embryos germinated and converted into plantlets in N(6)-benzyladenine (BAP, 0.75 mg 1(-l)) added medium following a treatment with thidiazuron (TDZ, 1.0 mg 1(-l)) for maturation. Scanning electron microscopy showed early stages of somatic embryo particularly, globular types, and was in masses. Different developing stages of embryogenesis (heart, torpedo and cotyledonary) were observed under histological preparation of embryogenic callus. Biochemical screening at various stages of somatic embryogenesis (embryogenic callus, somatic embryos, matured, germinated embryos and converted plantlets) of date palm cultivars has been conducted and discussed in detail. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as a good source of alternative propagation. Genetic modification to the embryo precursor cell may improve the fruit quality and yield further.

Salinity stress enhances production of solasodine in Solanum nigrum L.

Various in vitro grown tissues (non-regenerative callus, regenerative callus and microshoot derived leaves) of Solanum nigrum L. were cultured under salinity stress (0-150 mM NaCl) for enhanced production of solasodine, a steroidal alkaloid and an alternative to diosgenin, which is used as a precursor for the commercial production of steroidal drugs. The role of plant growth regulators and various concentrations of NaCl during in vitro production of solasodine was studied. The in vitro yield was compared with the yield from leaves of field grown plant. Solasodine content was maximum (2.39 mg/g dry wt.) in regenerative callus when grown on medium added with 150 mM NaCl; followed by in vitro raised leaf of microshoot. Quantitative estimation of solasodine was carried out using a new HPTLC method, which is validated for its recovery and precession. The proposed HPTLC method showed a good linear relationship (r(2)=0.994) in 50-2000 ng/spot concentration ranges. The data demonstrate that the solasodine production in cultures was growth dependent.