Regeneration in Jatropha curcas: Factors affecting the efficiency of in vitro regeneration

Factors influencing in vitro regeneration through direct shoot bud induction from hypocotyl explants of Jatropha curcas were studied in the present investigation. Regeneration in J. curcas was found to be genotype dependent and out of four toxic and one non-toxic genotype studied, non-toxic was least responsive. The best results irrespective of genotype were obtained on the medium containing 0.5 mg L⁻¹ TDZ (Thidiazuron) and in vitro hypocotyl explants were observed to have higher regeneration efficiency as compared to ex vitro explant in both toxic and non-toxic genotypes. Adventitious shoot buds could be induced from the distal end of explants in all the genotypes. The number of shoot buds formed and not the number of explants responding to TDZ treatment were significantly affected by the position of the explant on the seedling axis. Explants from younger seedlings (≤15 days) were still juvenile and formed callus easily, whereas the regeneration response declined with increase in age of seedlings after 30 days. Transient reduction of Ca²⁺ concentrations to 0.22 g L⁻¹ in the germination medium increased the number of responding explants.Induced shoot buds, upon transfer to MS medium containing 2 mg L⁻¹ Kn (Kinetin) and 1 mg L⁻¹ BAP (6-benzylamino purine) elongated. These elongated shoots were further proliferated on MS medium supplemented with 1.5 mg L⁻¹ IAA (indole-3-acetic acid) and 0.5 mg L⁻¹ BAP and 3.01-3.91 cm elongation was achieved after 6 weeks. No genotype specific variance in shoot elongation was observed among the toxic genotypes except the CSMCRI-JC2, which showed reduced response. And for proliferation among the toxic genotypes, CSMCRI-JC4 showed highest number of shoots formed. Among the rest, no significant differences were observed. The elongated shoot could be rooted by pulse treatment on half-strength MS medium supplemented with 2% sucrose, 3 mg L⁻¹ IBA (indole-3-butyric acid), 1 mg L⁻¹ IAA, 1 mg L⁻¹ NAA (α-naphthalene acetic acid) and subsequent transfer on 0.25 mg L⁻¹ activated charcoal medium. The rooted plants could be established in soil with more than 90% success. No significant differences were observed in rooting of shoots in the different toxic genotypes. However, rooting response was reduced in non-toxic genotype as compared to toxic genotypes.     

Highly efficient in vitro regeneration of the industrial oilseed crop Crambe abyssinica

A highly efficient regeneration protocol for oilseed crop Crambe abyssinica has been developed using hypocotyls as explants in this study. Crambe is a potential engineering oilseed crop for industrial purposes as it contains 55-60% erucic acid in its oil and, more importantly, it does not outcross with any food oil seed crops. However, the low regeneration frequency with the currently available protocols is still a limiting factor for genetic modification of Crambe. In this study, we investigated the effects of N-source, C-source, AgNO₃, cultural conditions as well as the concentration and combination of plant growth regulators (PGR) on the regeneration frequency of C. abyssinica. The results showed that all these factors, especially the N-source and PGR concentrations and combinations, played an important role in shoot regeneration. Among all the factors tested, the combination of using hypocotyls from C. abyssinica cv. galactica, the Lepiovre basal medium supplemented with 16 g l⁻¹ glucose, 0.5 g l⁻¹ AgNO₃, 2.2 mg l⁻¹ thidiazuron (TDZ), 0.5 mg l⁻¹ α-naphthaleneacetic acid (NAA), 2.5 g l⁻¹ Gelrite, seeds germinated in dark for 3 days and explants cultured in light, gave the best regeneration frequency (over 95%). The results also suggest that reducing the content of NH₄⁺ or keeping a suitable NO₃⁻/NH₄⁺ ratio in the regeneration medium would be crucial to Crambe shoot regeneration.     

Chitosan enhances withanolides production in adventitious root cultures of Withania somnifera (L.) Dunal

Calli were obtained from leaf, cotyledon and internode explants of in vitro-grown plants of Indian cultivar of Withania somnifera in MS medium supplemented with 2, 4-D (2.0 mg l⁻¹) and Kinetin (0.2 mg l⁻¹). The brown, semi-friable callus (500 mg FW) derived from leaf explants produced higher number of primary adventitious roots (9 roots/callus) in half strength MS medium fortified with IBA (0.5 mg l⁻¹) and NAA (0.1 mg l⁻¹). The primary adventitious roots with an inoculum mass of 15 g FW were cultured for 6 weeks in the same medium for secondary adventitious root proliferation. Elicitation of abiotic elicitor, aluminium chloride at 10 mg l⁻¹ at the end of 4 weeks culture with 4 h exposure time enhanced withanolides productivity. Under similar culture conditions, the biotic elicitor, chitosan at 100 mg l⁻¹ stimulated higher production of all withanolides when compared to aluminium chloride treatment. This is the first report on the use of callus-derived adventitious root culture for the enhanced production of withanolides upon chitosan elicitation.     

Screening and quantification of an antiseptic alkylamide, spilanthol from in vitro cell and tissue cultures of Spilanthes acmella Murr.

The study revealed, for the first time, accumulation of spilanthol, an antiseptic alkylamide, in in vitro cultures of Spilanthes acmella Murr., a medicinal plant of immense commercial value. To achieve this, in vitro shoots were regenerated via direct organogenesis from leaf-disc explants of Spilanthes. Shoots were induced in the presence of N⁶-benzylaminopurine (BAP) alone or in combination with either α-naphthalene acetic acid (NAA) or Indole-3-acetic acid (IAA) in Murashige and Skoog medium. The best treatment for shoot regeneration was MS + BAP (5.0 μM) + IAA (5.0 μM), which promoted adventitious shoot proliferation in >82% cultures with an average of 5.3 shoots per explant. Regenerated shoots rooted spontaneously with a frequency of 100% on half strength MS medium (major salts reduced to half strength) containing 50 g l⁻¹ sucrose. The plantlets were acclimatized successfully with 90% survival rate. Additionally, ploidy stability of the regenerated plants was assessed by flow cytometry which showed that all investigated plants had the similar ploidy as that of the mother plant. For spilanthol identification, peaks eluted from HPLC were analyzed by mass spectrometry with its characteristic fragmentation pattern. For quantification studies, calibration curve was generated, which revealed a higher amount of spilanthol content (3294.36 ± 12.4 μg/g DW) in the leaves of in vitro plants compare to those of in vivo plants (2703.66 ± 9.6 μg/g DW of spilanthol). An efficient multiplication frequency, ploidy stability and enhanced spilanthol accumulation ensure the efficacy of the protocol developed for this industrially important medicinal plant.     

Large scale in vitro propagation of Stevia rebaudiana (bert) for commercial application: Pharmaceutically important and antidiabetic medicinal herb

Stevia rebaudiana is a valuable medicinal plant species and it is being used for the treatment of diabetes. Currently, there is a high demand for raw material of this medicinal herb due to ever increasing diabetes disorder among the population. In order to meet the increased demand an efficient in vitro propagation of S. rebaudiana was established. Nodal explants collected from the field were cultured on MS basal medium fortified with different concentrations of BAP (0.5-3.0 mg/l) and KIN (0.5-3.0 mg/l) individually for shoot bud induction. In vitro derived nodal buds were cultured on MS medium supplemented with different concentrations (0.5-3.0 mg/l) of BAP and KIN for multiple shoot bud regeneration. In the second experiment, in vitro derived buds were placed on MS medium supplemented with different concentrations of BAP (0.5-3.0 mg/l) in combination with 0.5 mg/l IAA or IBA or NAA for shoot bud multiplication. The highest frequency (94.50%) of multiple shoot regeneration with maximum number of shoots (15.69 shoots/explant) was noticed on MS medium supplemented with 1.0 mg/l BAP. For large scale plant production, in vitro derived nodal bud explants were cultured on MS medium fortified with 1.0 mg/l BAP, in which about 123 shoots/explant were obtained after three subcultures on the same media composition. Elongated shoots (>2 cm) dissected out from the in vitro proliferated shoot clumps were cultured on half-strength MS medium containing different concentrations of NAA (0.1-0.5 mg/l) and/or MS medium fortified with various concentrations (0.5-2.0 mg/l) of auxins (NAA, IAA and IBA) for root induction. Highest frequency of rooting (96%) was noticed on half-strength MS medium augmented with 0.4 mg/l NAA. The rooted plantlets were successfully transferred into plastic cups containing sand and soil in the ratio of 1:2 and subsequently established in the greenhouse. The present in vitro propagation protocol would facilitate an alternative method for rapid and large-scale production of this important antidiabetic medicinal plant.     

Regulation of tissue differentiation by plant growth regulators on tTCLs of Panax ginseng adventitious roots

Differentiated tissue in Panax ginseng cultures was found to be very efficacious for saponin production. In order to increase the yield of saponins and preserve culture stability we were testing different plant growth regulators (PGR) and auxin/cytokinin combinations to regulate a level of tissue differentiation. For this purpose we used transverse thin cell layers (tTCLs) of adventitious roots of Panax ginseng. Adventitious roots were cultivated in Shenk and Hildebrand (SH) liquid medium supplemented with IBA (24.6 μM). Callus formation and root multiplication of adventitious root tTCLs was evaluated after 4 and following 12 weeks of cultivation, respectively, on SH basal medium containing various auxins (3 mg l⁻¹) or cytokinins (0.2 or 0.02 mg l⁻¹) or their combinations. We found that kinetin (Kin) in combination with auxin benzo[b]selenienyl acetic acid (BSAA), naphthalene acetic acid or indole-3-butric acidis the best for biomass production and following root multiplication. These combinations were tested in previously selected most suitable large-scale system—a temporary immersion system RITA. The best saponin production (15.94 ± 1.89 mg g⁻¹ dry weight) and growth value (5.62 ± 0.34) was reached on medium containing BSAA and Kin combination.     

Adventitious root cultures of Castilleja tenuiflora Benth. as a source of phenylethanoid glycosides

Castilleja tenuiflora is a highly valued medicinal plant that grows in pine-oak woods in Mexico. In this study, we identified for the first time verbascoside and isoverbascoside as the major phenylethanoid glycosides (PhGs) in C. tenuiflora. These compounds have proven biological activities, including anti-inflammatory, antioxidant, and cytotoxic activities, which may be related to the traditional uses of this plant. We developed a reverse-phase high-performance liquid chromatography (RP-HPLC) procedure to analyze PhGs, and determined their concentrations in various different tissues of wild plants. Verbascoside accumulated mainly in roots and inflorescences (9.23 and 7.88 mg g⁻¹ dry biomass, respectively), while isoverbascoside accumulated mainly in the roots (7.13 mg g⁻¹ dry biomass). To provide an alternative source of material for production of bioactive compounds, we established in vitro adventitious root cultures in which roots were grown in B5 medium containing either 10 μM indole 3-acetic acid (IAA) or 10 μM α-naphthaleneacetic acid (NAA). The greatest dry biomass yield (30 g L⁻¹) was achieved at 30 days after transfer of roots into IAA-containing medium. The highest specific yields of PhGs were also obtained using this auxin; the maximum level of verbascoside was 14.62 mg g⁻¹ dry root biomass (438.6 mg L⁻¹) at 30 days after root transfer, and the maximum yield of isoverbascoside was 37.32 mg g⁻¹ dry root biomass (522.48 mg L⁻¹) at 23 days after root transfer. Adventitious root cultures of C. tenuiflora are a promising system for further studies on scale-up and phenylethanoid glycosides biosynthesis.     

Synthesis of withanolide A depends on carbon source and medium pH in hairy root cultures of Withania somnifera

Withania somnifera (L.) Dunal. (Indian ginseng) is an important medicinal plant which yields pharmaceutically active compounds called withanolides. The present work deals with optimization of parameters of hairy root culture of W. somnifera for the production of biomass and withanolide A. We also investigated the effects of carbon source [sucrose, glucose, fructose, maltose, glucose + fructose (1:1), fructose + sucrose (1:1) and sucrose + glucose (1:1)], sucrose concentration (1%, 2%, 3%, 4%, 6% and 8%) and the initial medium pH (4.0, 4.5, 5.0, 5.5, 5.8, 6.0 and 6.5) on growth and production of withanolide A in hairy root cultures of W. somnifera. We found that biomass accumulation and production of withanolide A was highest when sucrose was used as the carbon source (11.92 g l⁻¹ DW and 11.96 mg g⁻¹ DW of withanolide A). Further 3% sucrose concentration was found to be optimal for biomass accumulation (11.92 g l⁻¹ DW) and 4% sucrose favoured the production of withanolide A (13.28 mg g⁻¹ DW) in the tested range of concentrations (1-8%). The biomass of hairy roots was optimal when the initial medium pH was 5.8 (12.1 g l⁻¹ DW) and the withanolide A production was highest in the medium pH set at 6.0 (13.84 mg g⁻¹ DW).     

Improvement of growth and gymnemic acid production by altering the macro elements concentration and nitrogen source supply in cell suspension cultures of Gymnema sylvestre R. Br

Gymnema sylvestre is an important medicinal plant which bears bioactive compound namely gymnemic acids. The present work deals with optimization of cell suspension culture system of G. sylvestre for the production of biomass and gymnemic acid and we investigated effects of macro elements (NH₄NO₃, KNO₃, CaCl₂, MgSO₄ and KH₂PO₄ - 0.0, 0.5, 1.0, 1.5 and 2.0× strength) and nitrogen source [NH₄⁺/NO₃⁻ ratio of: 0.00/18.80, 7.19/18.80, 14.38/18.80, 21.57/18.80, 28.75/18.80, 14.38/0.00, 14.38/9.40, 14.38/18.80, 14.38/28.20 and 14.38/37.60 (mM)] of Murashige and Skoog medium on accumulation of biomass and gymnemic acid content. The highest accumulation of biomass (165.00 g l⁻¹ FW and 15.42 g l⁻¹ DW) was recorded in the medium with 0.5× concentration of NH₄NO₃ and the highest production of gymnemic acid content was recorded in the medium with 2.0× KH₂PO₄ (11.32 mg g⁻¹ DW). The NH₄⁺/NO₃⁻ ratio also influenced cell growth and gymnemic acid production; both parameters were greater when the NO₃⁻ concentration was higher than that of NH₄⁺. Maximum biomass growth (159.72 g l⁻¹ of FW and 14.95 g l⁻¹ of DW) was achieved at an NH₄⁺/NO₃⁻ ratio of 7.19/18.80, and gymnemic acid production was also greatest at the same concentration of NH₄⁺/NO₃⁻ ratio (11.35 mg g⁻¹ DW).     

Growth and accumulation of caffeic acid derivatives in Echinacea angustifolia DC. var. angustifolia grown in hydroponic culture

In separate experiments conducted in 2007 and 2008, growth and accumulation of selected caffeic acid derivatives (CADs; i.e., caftaric acid, chlorogenic acid, echinacoside, caffeic acid, cynarin, p-coumaric acid, ferulic acid and cichoric acid) were determined in Echinacea angustifolia DC. var. angustifolia seedlings grown in hydroponic culture (floating raft system) at a density of 122 plant m⁻² (at planting). Plants were harvested 11 (2007) or 16 (2008) weeks after transplanting (i.e., 15 or 20 weeks after sowing). In both years, plants grew vigorously and at harvest approximately half of the plants under observation had developed one to three inflorescences. In 2008, the root yield (2940 kg ha⁻¹) harvested in nearly eight months from two consecutive hydroponic cultures was within the yield reported in the literature for field cultivations lasting two to four years. None of the selected CADs was found in the leaves, while the inflorescences (stem and capitulum) contained only caftaric acid and echinacoside at concentrations higher than the detection limits (0.05 mg g⁻¹ dry weight). Echinacoside, cynarin and chlorogenic acid were found in root tissues at concentrations ranging from 0.36 to 5.25 mg g⁻¹ dry weight. In all plant samples, echinacoside, which is the marker compound for E. angustifolia material, did not reach the minimum quality standard (10 mg g⁻¹ dry weight) for the production of standardized extract. We concluded that short-cycle, high-density greenhouse hydroponic culture stimulates plant growth and root production in E. angustifolia, but it does not ensure sufficient CADs accumulation in dried roots.     

In vitro regeneration from petiole explants of non-toxic Jatropha curcas

Jatropha curcas, a multipurpose shrub has acquired significant economic potential as biodiesel plant. The seeds or pressed cake is toxic due to the presence of toxic substances and is not useful as food/fodder despite having the best protein composition. A simple, efficient, and reproducible method for plant regeneration through direct organogenesis from petiole explants of non-toxic J. curcas was developed using Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (57.61%), and number of shoot buds (4.98) per explant were obtained when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 μM TDZ. The Induced shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM 6-benzyl aminopurine (BA), and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation and subsequent elongation was achieved on MS medium supplemented with 2.25 μM BA and 8.5 μM IAA. The elongated shoots could be rooted on half-strength MS medium with 15 μM IBA, 11.4 μM IAA and 5.5 μM NAA with more than 90% survival rate.     

Increases in leaf artemisinin concentration in Artemisia annua in response to the application of phosphorus and boron

Malaria resurgence particularly in the third world is considerable and exacerbated by the development of multi-drug resistances to chemicals such as chloroquinone. Drug therapies, as recommended by WHO include the use of antimalarial compounds derived from Artemisia annua L., i.e. artemisinin-based therapies. This work aims to determine how A. annua plant dry matter can be enhanced while maximising artemisinin concentration from understanding the plant's mineral requirements for P and B. Experiments with differing of P, from 5 to 120 mg L⁻¹ and B from 0.1 to 0.9 mg L⁻¹ were undertaken. Mineral nutrients were supplied in irrigation water to potted plants and after a period of growth, dry matter production and leaf artemisinin concentration were determined. Increases in P application enhanced plant growth and total dry matter production. An optimal application rate, with respect to dry matter, was apparent around 30 mg P L⁻¹. Despite increases in P application having no influence on leaf artemisinin concentration, optimal yields of artemisinin, on a per plant basis, were again achieved at supply rate around 30-60 mg L⁻¹. Increasing B application rate had little influence on dry matter production despite increases in B leaf tissue concentration promoting the total amount of B per plant. Leaf artemisinin concentration significantly increased with B application rate up to 0.6 mg B L⁻¹. The higher artemisinin concentrations when multiplied by total leaf dry matter at the higher B application rates produced an increase in total artemisinin production per plant. There was however no further significant effect on leaf artemisinin concentration when B supply concentrations increased further (0.9 mg L⁻¹). Artemisinin production varied between the two experiments to a greater extent than plant dry matter production and the reasons for this are discussed in relation to growing environments and their possible impacts on artemisinin biosynthesis.     

Role of exogenous phytohormones on growth and plumbagin accumulation in Plumbago indica hairy roots and conservation of elite root clones via synthetic seeds

The present study describes the role of different exogenous hormones on morphology and plumbagin production in Plumbago indica hairy roots. It was also aimed to conserve elite root clones via synthetic seed technology. Insertion of rolB gene in transformed roots was confirmed by polymerase chain reaction followed by southern blot analysis. Hairy roots were treated with single or in combination of different phytohormones viz. IAA, IBA, 2, 4-D, NAA, BAP, GA₃ and ABA. Cultures incubated with GA₃ (0.5 mg l⁻¹) yielded highest root growth due to formation of profuse lateral branching while NAA (0.5 mg l⁻¹) treatment caused highest plumbagin accumulation. Cultures incubated with 2, 4-D exhibited highest inhibitory effect in terms of both root growth and plumbagin production. All phytohormones were found to be effective at lower concentration. In combinatorial study, GA₃ + NAA (0.5 mg l⁻¹, each) was found optimum for root biomass and plumbagin production at earlier stage of culture. Different combinations of auxins and BAP induced different morphologies ranging from reduction of lateral branching to rapid disorganization of root matrix. The combinations of ABA and selected auxins were not found promising at any of selected concentration. Based on the effect of exogenous hormones on hairy root culture, elite root clones were selected and encapsulated with sodium alginate matrix. Uniform shaped alginate coated synthetic seeds were conserved up to 6 months exhibited high regeneration potential without disturbing plumbagin content.     

An improved and efficient micropropagation of Eclipta alba through transverse thin cell layer culture and assessment of clonal fidelity using RAPD analysis

An improved and efficient in vitro regeneration system has been developed for Eclipta alba, a medicinally important plant, through transverse thin cell layer culture (tTCL). The transverse section of the nodal segment of field grown plants was used as tTCL explants for plant regeneration. Shoot multiplication from tTCL nodal explants was influenced by BAP and their interaction with Kin or NAA. MS medium containing 13.2 μM BAP and 4.6 μM Kin was most effective for shoot multiplication from tTCL nodal explants. Upon this medium, percent response for shoot proliferation was 100% with an average of 32.6 shoot buds per tTCL nodal explant. Regenerated shoots from tTCL nodal explants were rooted on the growth regulator free MS medium. The rooted plantlets were successfully acclimatized and established in soil with a survival frequency of 90-100%. Random amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic fidelity of the micropropagated plants. RAPD profile analysis indicated that micropropagated plants were genetically similar to mother plant.     

Tocochromanol content and composition in Jatropha curcas seeds

Physic nut (Jatropha curcas L.) is a promising seed oil source for biodiesel production. Natural antioxidants play a major role in maintaining oxidative stability of oils and they also have important food and industrial applications. Among them, tocochromanols are the most abundant in seeds. The objective of this research was to evaluate the variation for tocochromanol content and profile in a germplasm collection of 52 accessions of J. curcas. Seeds collected in two different periods, August and November of 2009, were analysed for tocochromanol content. Additionally, the dynamics of tocochromanol accumulation in developing seeds was studied. Total seed tocochromanol content averaged 307.2 mg kg⁻¹ in August and 303.7 mg kg⁻¹ in November, whereas total oil tocochromanol content averaged 507.4 mg kg⁻¹ in August and 500.8 mg kg⁻¹ in November. The tocochromanol fraction was made up of 15.4% gamma-tocopherol, 83.8% gamma-tocotrienol, and 0.8% delta-tocotrienol in August and 18.0% gamma-tocopherol, 80.4% gamma-tocotrienol, and 1.6% delta-tocotrienol in November. Genotype × environment effects were identified for tocochromanol content but not for the proportion of major tocochromanol homologues, which showed a high positive correlation between both environments. Developing seeds contained primarily alpha-tocopherol and gamma-tocopherol at early stages of development, with gamma-tocotrienol and delta-tocotrienol being practically undetectable. Gamma-tocotrienol content remained practically undetectable till 66 DAP and then increased pronouncedly to final levels of 177.1 mg kg⁻¹ (74.8% of the total tocochromanol content). The powerful antioxidant and health-promoting properties of gamma-tocotrienol encourages further studies on selection for the tocopherol/tocotrienol ratio in Jatropha and on the potential of tocochromanols as high added-value products derived from Jatropha seed oil production.     

Foliar application of chitosan activates artemisinin biosynthesis in Artemisia annua L.

There has been much interest in artemisinin owing to its excellent activity against malaria, an infectious disease threatening the tropical world. However, the low artemisinin content (0.01-0.8%, DW) in Artemisia annua, which is the only commercial source of artemisinin, makes artemisinin expensive to produce and not yet available on a global scale. Here we show that foliar application of 100 mg l⁻¹ chitosan improved artemisinin biosynthesis in A. annua. The content of dihydroartemisinic acid and artemisinin in chitosan-treated leaves increased by 72% and 53% compared with control values, respectively. Chitosan induced the expression of ADS and DBR2, which could explain the increase in level of artemisinic metabolites. After chitosan treatment, the amounts of hydrogen peroxide (H₂O₂) and superoxide anion (O₂⁻) in leaves of A. annua were 1.4 and 3.0 times higher than those of the control, respectively. Accumulation of reactive oxygen species (ROS) probably accelerated the conversion of dihydroartemisinic acid to artemisinin. Foliar application of 100 mg l⁻¹ chitosan had no harmful effect on A. annua growth. The simple method described here could be an effective method to improve artemisinin production in A. annua field cultivation.     

Effect of boron on the development of brown rot (Monilinia laxa) on peaches

Requirements of consumers for products with low residues of pesticides have increased the need for alternative disease management practices. The concentration of boron in fruit affects its quality, shelf life and the development of physiological disorders. However, the effect of boron on the susceptibility of peach to fruit rots has not been reported. This study investigated the effect of boron (Power B and Borax) on the development of Monilinia laxa on peaches (cv Andross). Mycelial growth of M. laxa was inhibited on potato dextrose agar supplemented with 750 μg ml⁻¹ of Borax or 1000 μg ml⁻¹ of Power B. The EC 50 values were 107.9 and 522.4 for Borax and Power B respectively. Field investigations showed that the incidence of peach infections by M. laxa was negatively correlated with the content of Boron in the leaves. Post-harvest dipping of peaches in Power B or Borax solution, at concentrations recommended by manufacturer (2 μg ml⁻¹ for Power B and 1 mg ml⁻¹ for Borax), significantly reduced the development of M. laxa. Power B, at rates of 6 μg ml⁻¹, and Borax at rates of 3 mg ml⁻¹ were the most effective in reducing infections by M. laxa. Finally, post-harvest dipping of fruit in Power B or Borax reduced losses of fruit weight and improved fruit firmness one month after storage, showing that boron increased the maintainability of peaches in cold storage. Peaches treated with 6 μg ml⁻¹ Power B or 3 mg ml⁻¹ Borax had the highest flesh firmness and the lowest water losses, while untreated control peaches were the least firm. Generally, Borax was significantly less effective than Power B, but better than the control treatment.     

Identification and antimicrobial activity of two alkaloids from traditional Chinese medicinal plant Tinospora capillipes

Two antimicrobial alkaloids, palmatine and jatrorrhizine, were isolated from tubers of traditional Chinese medicinal plant Tinospora capillipes using activity-guided isolation method and chromatography. Their antimicrobial activity was determined in vitro. The results showed that palmatine and jatrorrhizine had inhibitory activity against plant pathogens Colletotrichum gloeosporioides, Fusarium oxysporum f. sp. niveum, Mycosphaerella sentina, Pestalotia mangiferae, Cercospora kaki, Gymnosporangium haraeanum, Rhizoctonia solani and Colletotrichum graminicola, with the EC₅₀ values of 0.0348-0.8356 g L⁻¹ and 0.0240-0.8649 g L⁻¹, respectively. Palmatine and jatrorrhizine also exhibited inhibition against animal pathogens Bacillus cereus, Bacillus megaterium, Bacillus subtilis, Staphyloccocus aureus, Staphylococcus epidermidi, Micrococcus lysodeikticus, Proteus vulgaris, Salmonella typhi and Escherichia coli, with the MIC values of 0.1-0.8 g L⁻¹ and 0.1-0.6 g L⁻¹, respectively. These results suggested that palmatine and jatrorrhizine showed relatively broad spectrum antimicrobial activity against plant and animal pathogens.     

In vitro propagation and regeneration of an industrial plant kenaf (Hibiscus cannabinus L.)

The present study report a protocol for the efficient in vitro propagation of kenaf (Hibiscus cannabinus L., an industrial crop having high cellulosic fiber content) on hormone free MS medium using the shoot apex and nodal explants. Shoot tips and nodes were isolated from 15 days old seedlings cultivated on MS medium. Different combinations and concentrations of auxin/cytokinin were used and added to the MS medium to assess the shoot and root induction of theses explants. Several subcultures were drived in order to enhance the multiplication rate. Healthy and well developed in vitro propagated shoots were transferred for acclimatization under greenhouse conditions in pots filled with different substrates (sand + compost or perlite). Our results showed that shoots could elongate and root within 4-6 weeks on MS basal medium without any callus formation. However, addition of growth regulators to the MS medium leaded to a decrease in shoot and root induction rates. Indeed, the highest shoot regeneration frequency (90.5%) was obtained on MS control medium. Elongated shoots were transferred onto the same hormone free MS medium using five subcultures where the multiplication rate reached the highest value (3.66) at the fifth and last step. The in vitro rooted plantlets were acclimatized in greenhouse and successfully transplanted to natural conditions with 70% survival.     

Impact of organic manure and chemical fertilizers on artemisinin content and yield in Artemisia annua L

Artemisinin isolated from the aerial parts of Artemisia annua L. is a promising and potent antimalarial drug. It posses remarkable activity against both chloroquinine resistant as well as chloroquinine sensitive strains of Plasmodium falciparum. It is also useful in the treatment of cerebral malaria. The relatively low content of artemisinin in A. annua and unavailability of cost effective and viable synthetic protocol however, are major obstacles to the commercial production of the drug. The enhanced production of artemisinin is hence, highly desirable, which can be achieved by adequate and judicious supply of plant nutrients. The present experiment was therefore, designed to study the effect of organic manure (15 tonnes ha⁻¹) and chemical fertilizers (N₄₀₊₄₀, P₄₀, K₄₀, S₁₅₊₁₅ kg ha⁻¹; nitrogen, phosphorus, potassium and sulphur) on the accumulation of artemisinin and biomass in various plant parts through the developmental stages of A. annua L. Artemisinin yield (kg ha⁻¹) was also determined through the developmental stages of A. annua L. Artemisinin content and artemisinin yield of dried leaves were increased significantly at pre-flowering stage in the plants treated with NPKS (27.3% and 53.6%) and NPK (18.2% and 33.5%), respectively, when compared with control. Maximum dry yield of leaf ranging from 2596 to 3141 kg ha⁻¹ was observed at pre-flowering stage with various treatments.