Organic Elicitors Research Articles

Physiological and phytochemical responses of Rosa damascena Mill. to the foliar application of different elicitors

The effects of foliar spraying of different elicitors on some physiological and phytochemical traits like the quality volatile oil of Rosa damascena Mill. at the field conditions in Southwestern Iran were investigated. The experimental treatments were the foliar applications of acetone, ethanol, and methanol (10% and 20% v/v), salicylic acid (1.5 and 3.0 mM), phenylalanine (1.0 and 2.0 g/L), and chitosan (0.25 and 0.50 g/L). Results indicated that the experimental treatments significantly influenced the flower petals yield and the amounts of pigments and the main compounds in the volatile oil. The highest values of the flower petals yield and chlorophyll content were obtained from the plants under the foliar application of 3 mM salicylic acid followed chitosan at 0.25 g/L. The effect of organic elicitors, especially, phenyl alanine on the total phenol, flavonoids, and anthocyanin contents were higher than the alcoholic ones. The highest volatile oil content (0.056% v/w) was obtained from the treated plants by 0.25 g/L chitosan. The foliar application of phenyl alanine, chitosan, and salicylic acid significantly improved the monoterpene alcohols compounds in the volatile oil. The amount of these compounds in treated plants by the organic elicitors 58.32% was higher than the alcoholic elicitors. In conclusion, the foliar spraying of some elicitors such as chitosan, salicylic acid, and phenyl alanine can be a good strategy to improve the flower petals yield, antioxidant capacity, and the quantity and quality of volatile oil from R. damascene in semiarid climate.

Effects of Organic Elicitors on the Recycled Production of Ginkgolide B in Immobilized Cell Cultures of Ginkgo biloba

Ginkgo biloba is a medicinal plant used in complementary and alternative medicines. Ginkgo biloba extracts contain many compounds with medical functions, of which the most critical is ginkgolide B (GB). The major role that GB plays is to function as an antagonist to the platelet-activating factor, which is one of the causes of thrombosis and cardiovascular diseases. Currently, GB is obtained mainly through extraction and purification from the leaves of Ginkgo biloba; however, the yield of GB is low. Alternatively, the immobilized cultivation of ginkgo calluses with biomaterial scaffolds and the addition of organic elicitors to activate the cell defense mechanisms were found to stimulate increases in GB production. The aim of this study was to use Ginkgo biloba calluses for immobilized cultures with different elicitors to find a more suitable method of ginkgolide B production via a recycling process.

Effective protocol for isolation and marked enhancement of psoralen, daidzein and genistein in the cotyledon callus cultures of Cullen corylifolium (L.) Medik

Cullen corylifolium (L.) Medik. (Babachi), a traditional herb is being utilized to cure several serious ailments such as psoriasis, leprosy, leucoderma, osteoporosis including leukemia due to the occurrence of many important furanocoumarins and flavonoids especially in the green seeds. Since these compounds occur in extremely low amounts, in vitro elicitation has been proved to be an effective, sustainable, and eco-friendly tool for enhanced production of such compounds. The current study aimed at the isolation of some marker bioactive compounds such as psoralen, daidzein and genistein from the green seed extract, their elicitation and callus biomass production employing various organic elicitors such as chitosan, proline and salicylic acid. The isolation of bioactive anticancerous compounds were done using the gradient of Toluene: Ethyl acetate (v:v) as a solvent system through column chromatography and thin layer chromatography (TLC). The results showed that a total of 101 fractions were collected through column chromatography, which were pooled down to 10 fractions (F1-F10) on the basis of their similar Rf values on TLC plates. The isolated fractions F2, F3 and F5 were characterized and identified as daidzein, psoralen and genistein, respectively, through NMR and HR-MS. For elicitation of such compounds the callus was induced from cotyledons of green seeds on Gamborg B5 medium +5 μM BA +10 μM IBA. Maximum cotyledon callus biomass was observed at 25 mg/L proline followed by chitosan at 50 mg/L and salicylic acid at 5 μM. Maximum psoralen content (14.8–fold) was achieved with 5 μM salicylic acid over control. Whereas, in case of daidzein 11.2–fold enhancement was seen at 50 mg/L chitosan over control. Incidentally, optimum genistein content (6.21–fold) was achieved with 100 mg/L chitosan over control. Thus, the organic elicitors especially the salicylic acid proved to enhance psoralen content, whereas, chitosan was found to be effective for the increment of daidzein and genistein content in the cotyledon callus cultures of Cullen corylifolium. This is a cost effective and industrially viable protocol for the production of important bioactive compounds psoralen, daidzein and genistein.

Influence of seaweed extracts and plant growth regulators on in vitro regeneration of Lycopersicon esculentum from leaf explant

An efficient in vitro regeneration protocol was developed for tomato from leaf explant using plant growth regulators, organic elicitors, polyamines, and seaweed extracts. Initially, excised leaf explant was cultured on medium containing different concentrations of auxins viz., 2,4-dichloropheonoxyacetic acid (2,4-D; 0.5–3.0 mg L−1), picloram (Pic; 0.5–3.0 mg L−1), 1-naphthaleneacetic acid (NAA; 0.5–3.0 mg L−1), and indole-3-acetic acid (IAA; 0.5–3.0 mg L−1). The most effective auxin was tested in combination with efficient cytokinins such as kinetin (Kin; 0.5–3.0 mg L−1), 6-benzylaminopurine (BAP; 0.5–3.0 mg L−1), and thidiazuron (TDZ; 0.5–3.0 mg L−1). To achieve the best organogenic capability, different elicitors (casein hydrolysate, yeast extract, glutamine and adenine sulfate), polyamines (putrescine, spermidine, and spermine), and seaweed (Gracilaria edulis and Sargassum wightii) extracts were tested at various concentrations. Among the plant growth regulators, additives, and seaweed extracts tested, the medium supplemented with 1.5 mg L−1 Pic + 1.0 mg L−1 TDZ + 80 mg L−1 casein hydrolysate induced maximum of 28.6 shoots per organogenic callus. Rapid elongation was achieved in the medium fortified with isopentenyladenine (1.2 mg L−1) and G. edulis extract (30%) and the seaweed S. wightii extract induced rooting of elongated shoots. The presence of plant growth regulators in seaweed extracts were further confirmed by FTIR and HPLC analysis. HPLC analysis of seaweed extracts and standard plant growth regulators revealed the presence of NAA and isopentenyladenine in S. wightii. The standard chromatogram of NAA correlated with the chromatogram of G. edulis. Hence this regeneration system provides an additional platform for generating transgenic plants in an efficient manner.

Conditioned media and organic elicitors underpin the production of potent antiplasmodial metabolites by endophytic fungi from Cameroonian medicinal plants.

Plasmodial resistance to artemisinin-based combination therapies emphasizes the need for new drug development to control malaria. This paper describes the antiplasmodial activity of metabolites produced by endophytic fungi of three Cameroonian plants. Ethyl acetate extracts of fungi cultivated on three different media were tested against Plasmodium falciparum chloroquine-sensitive (Pf3D7) and chloroquine-resistant (PfINDO) strains using the SYBR green florescence assay. Selected endophytes were further grown in potato dextrose broth supplemented with small organic elicitors and their extracts tested for activity. The effect of elicitors on de novo metabolite synthesis was assessed by reverse-phase HPLC. Activity screening of 81 extracts indicated that Aspergillus niger 58 (IC50 2.25-6.69μg/mL, Pf3D7), Fusarium sp. N240 (IC50 1.62-4.38μg/mL, Pf3D7), Phomopsis sp. N114 (IC50 0.34-7.26μg/mL, Pf3D7), and Xylaria sp. N120 (IC50 2.69-6.77μg/mL, Pf3D7) produced potent extracts when grown in all three media. Further culture of these endophytes in potato dextrose broth supplemented with each of the eight small organic elicitors and subsequent extracts screening indicated the extract of Phomopsis sp. N114 grown with 1% 1-butanol to be highly selective and extremely potent (IC50 0.20-0.33μg/mL; SI > 666). RPHPLC profiles of extracts of Phomopsis sp. N114 grown with or without 1-butanol showed some peaks of enhanced intensities in the former without any qualitative change in the chromatograms. This study showed the ability of selected endophytes to produce potent and selective antiplasmodial metabolites in varied culture conditions. It also showed how the production of desired metabolites can be enhanced by use of small molecular weight elicitors.

Analysis of the effect of plant growth regulators and organic elicitors on antibacterial activity of Eucomis autumnalis and Drimia robusta ex vitro-grown biomass

The effects of plant growth regulators (PGRs) and organic elicitors (OEs) on in vitro propagation of Eucomis autumnalis was established. Three-year-old ex vitro grown plants from organogenesis of E. autumnalis and somatic embryogenesis (previously reported protocol) of Drimia robusta were investigated for antibacterial activity. In vitro propagation from leaf explants of E. autumnalis was established using different PGRs and OE treatments for mass propagation, biomass production and bioactivity analysis to supplement the use of wild plant material. Prolific shoots (16.0 ± 0.94 shoots per explant) were obtained with MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium containing 100 mg l−1 haemoglobin (HB), 10 µM benzyladenine (BA) and 2 µM naphthaleneacetic acid (NAA). The shoots were rooted effectively with a combination of 2.5 µM indole-3-acetic acid and 5.0 µM indole-3-butyric acid. The plantlets were successfully acclimatized in a vermiculite-soil mixture (1:1 v/v) in the greenhouse. Three-year-old ex vitro-grown E. autumnalis and D. robusta plants derived via organogenesis and somatic embryogenesis respectively exhibited antibacterial activity and varied with PGR and OE treatments, plant parts and bacteria. The leaves of E. autumnalis ex vitro-derived from a combination of HB, BA and NAA followed by the individual treatments of BA and HB gave the best antibacterial activities (< 1 mg ml−1: minimum inhibitory concentration from 0.098 to 0.78 mg ml−1) against all tested pathogenic bacteria (Bacillus subtilis, Enterococcus faecalis, Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa). The bulbs of D. robusta ex vitro-derived from solid culture with 10 µM picloram, 1 µM thidiazuron and 20 µM glutamine exhibited good antibacterial activity against E. faecalis, M. luteus and S. aureus when compared with other treatments and mother plants. The ex vitro-grown E. autumnalis and D. robusta biomass produced with PGRs along with OE treatments confirmed a good potent bioresource and can be used as antibacterial agents. The in vitro plant regeneration of E. autumnalis and D. robusta protocols and ex vitro plants could be used for conservation strategies, bioactivity and traditional medicinal use.

Marked enhancement of sennoside bioactive compounds through precursor feeding in Cassia angustifolia Vahl and cloning of isochorismate synthase gene involved in its biosynthesis

Cassia angustifolia Vahl, a chief source of anthraquinone glycosides (sennosides), extensively employed as a laxative is also reported to possess significant anticancerous activity against various cancer cell lines. HPLC analysis of different in vivo plant parts viz., leaves, nodes, roots and seeds revealed that the maximum content of both sennoside A (3816.10 µg/g fresh wt.) and sennoside B (646.74 µg/g fresh wt.) occur in leaf. Elicitation in sennoside content in leaf callus therefore, was achieved employing organic elicitors (glycine, myo-inositol, glutamine, proline, yeast extract, casein hydrolysate and sucrose) as well as precursors (α-keto glutaric acid and pyruvic acid) of anthraquinone pathway. Though there was enhancement at all levels of stress, the optimum elicitation in sennoside A and B was seen at 0.1 % pyruvic acid, their respective percentages being 16 and 32 %. Overall improvement in sennoside A and B content was seen in the order: pyruvic acid > α-keto-glutaric acid > sucrose > yeast extract > glycine > myo-inositol > proline > casein hydrolysate > glutamine. Most importantly, isochorismate synthase (ics), the key enzyme gene involved in the anthraquinone biosynthetic pathway has also been cloned from leaf and sequenced which comprised of 1377 bp. Neighbor joining tree generated through MEGA6 analysis of the nucleotide sequences revealed varied degree of homology with the ics gene sequences of Morus notabilis, Medicago truncatula, Solanum lycopersicum, Cicer arietinum, Glycine max, etc. This is the first report of elicitation of sennoside A and B from leaf callus cultures and cloning of ics gene in C. angustifolia.

Transition from in vitro to an ex vitro environment: is the metabolism altered?

The polyphenolic content and antimicrobial activity of 1-year-old ex vitro-acclimatized plant samples of the endangered plant species Merwilla plumbea were evaluated. Acclimatized plants were derived from in vitro media containing different supplements. Spectrophotometric techniques and micro-dilution assays for the determination of polyphenols and antimicrobial activities, respectively, were used. Significantly higher levels of total phenols, flavonoids, and proanthocyanidins were recorded in the greenhouse-acclimatized plants compared to the in vitro-derived samples. In contrast with the untreated field-grown plants, the levels of these compounds in both in vitro-derived and ex vitro-acclimatized plants yielded multiple-fold increases. The highest levels of flavonoids were obtained in ex vitro plants treated with 200 mg l−1 yeast malt broth (YMB), 0.45 μM thidiazuron (TDZ), and 0.054 μM naphthaleneacetic acid (NAA). The same treatment also exhibited good antimicrobial activity, an attribute that might suggest the involvement of these compounds in the observed bioactivity. The fact that Murashige and Skoog (MS) medium supplemented with organic elicitors differentially affected the accumulated levels of polyphenols in vitro, and their subsequent carry-over and higher expression in an ex vitro environment over time, makes the technology an attractive system for commercial multiplication and cultivation of medicinal plants.

Quantification of asiatic acid from plant parts of Centella asiatica L. and enhancement of its synthesis through organic elicitors in in vitro

Medicinal plants are nearly unlimited source of phytochemicals. Centella asiatica L. is a tropical medicinal plant with a long history of therapeutic uses, dermal disorders, venous insufficiency micro-angiopathy, and anticancer. The present study highlights (i) in vivo evaluation of asiatic acid (as an anticancer compound) content in different plant parts of C. asiatica and (ii) in vitro elicitation of asiatic acid employing organic elicitors. Based on the HPLC analysis, the highest amount of asiatic acid (190.2 μg·g-1 fresh wt.) was found in root part in in vivo condition. Quantitative analyses were done to enhance of asiatic acid content by some organic elicitors. Results showed the highest quantity of asiatic acid content (258.3 μg·g-1 fw.) among all of the organic elicitors was found at 200 mg·L-1 yeast extract in leaf callus cultures. All these observations suggest that exogenous application of organic elicitors could have induced a subset of secondary metabolite biosynthetic genes, which may modulate expression of genes and accumulation of compounds induced by elicitors.

Plant regeneration via somatic embryogenesis in Drimia robusta

A simple efficient in vitro plant regeneration system was developed by direct and indirect somatic embryogenesis of Drimia robusta, a medicinal plant extensively used in South African traditional medicine. Different developmental stages of somatic embryos (SEs: globular embryos, partial pear-shaped embryos and club-shaped embryos), club-shaped cotyledon initiation, plumule initiation and plantlets were directly obtained from leaf explants on Murashige and Skoog (MS) medium containing 3.5 % (w/v) sucrose and different plant growth regulators (PGRs). In MS medium containing 3.5 % (w/v) sucrose and supplemented with 10 μM picloram, 1 μM thidiazuron (TDZ) and 20 μM glutamine, a higher number of SEs and plantlets were achieved. These were established onto half-strength MS medium followed by successful acclimatization (100 %) in the greenhouse. Liquid somatic embryo medium (SEML) containing 500 mg of friable embryogenic callus on MS medium supplemented with different concentrations and combinations of PGRs and organic elicitors produced different stages of SEs. Somatic embryo production was enhanced by 0.5 μM picloram, 1 μM TDZ and mebendazole treatment. The highest number of plantlets (9.0 ± 0.70) was obtained in SEML containing 0.5 μM picloram, 1 μM TDZ and 25 mg l−1 haemoglobin. All the cotyledon and plumule embryos germinated on half-strength MS medium, however 90 % of SEs germinated on half-strength MS medium containing 0.5 μM naphthaleneacetic acid. All plantlets were successfully acclimatized in the greenhouse. This first report of D. robusta somatic embryogenesis provides an opportunity to control extinction threats, ensure germplasm conservation and provides a system for analysis of bioactive compounds and bioactivity.

Increased Ginsenosides Production by Elicitation of In vitro Cultivated Panax Ginseng Adventitious Roots

Ginsenosides, which belong to group of triterpenoid saponins, are considered to be main constituents responsible for the biological effects of ginseng drug. Effect of organic and inorganic elicitors and other additives on the ginsenoside biosynthesis and biomass production by the in vitro cultivated adventitious root cultures of Korean ginseng was investigated in present study. Elicitation potential of natural mixtures (coconut water, pineapple extract, casein hydrolyzate, yeast extract, malt extract), organic elicitors (putrescine, spermidine, spermine, jasmonic acid), inorganic ions (calcium) and their complexes with organic acids (titanium ascorbate, titanium citrate) was tested. Changes in concentration of 9 main ginsenosides (Re, Rg1 , Rf, Rg2 , Rb1 , Rb2 , Rc, Rg3 and Rd) were monitored using HPLC-PDA and LC/MS/MS techniques. Jasmonic acid was the most effective elicitor of ginsenoside production (about 3.5 mg/g of DW in comparison with 1.4 mg/g of DW in control); however, its application (similar to spermine) was followed with significant reduction of the biomass growth. Among tested additives, casein hydrolyzate showed the highest increase of the ginsenoside content (2.2 mg/g DW) together with no effect on the growth of the culture. Both tested titanium complexes showed slight inhibition of the saponin accumulation in comparison with untreated control.

In vitro plant regeneration, phenolic compound production and pharmacological activities of Coleonema pulchellum

Effects of plant growth regulators (PGRs) and organic elicitors (OEs) on Coleonema pulchellum in vitro micropropagation, secondary product production and pharmacological activities were evaluated. In vitro, ex vitro and parental plants of C. pulchellum were investigated for their potential to produce phenolic and pharmacological compounds. Different morphogenic characteristics of shoots were obtained with PGRs- and OEs-containing media. A higher number of normal shoots were achieved with a low concentration of thidiazuron (TDZ: 4.5μM). Lesser numbers were found with combinations of TDZ (13.6μM)+indole-3-acetic acid (IAA: 2.9μM); haemoglobin (HB: 300mgl−1) or glutamine (GM: 40μM)+benzyladenine (BA: 8.8μM). Shoots were rooted in vitro and successfully acclimatized. Plant growth regulators and OEs had a significant effect on the synthesis and accumulation of phenolic compounds and flavonoids. In particular, casein hydrolysate (CH) as well as a combination of GM and BA induced high levels of total phenolics and flavonoids during in vitro culture. Cytokinins and OEs had a significant effect on DPPH radical scavenging and antibacterial activities of C. pulchellum extracts. Acclimatized C. pulchellum plants can be used as substitute alternative to natural populations.

In vitro propagation, proscillaridin A production and antibacterial activity in Drimia robusta

Drimia robusta is a threatened traditional medicinal plant extensively used in South Africa. Rapid in vitro mass propagation of the species was developed for commercial cultivation from leaf explants using various concentrations and combinations of plant growth regulators and organic elicitors. The highest number of regenerated shoots per explant (14.6 ± 0.54) was obtained on Murashige and Skoog (MS) medium supplemented with a combination of 2.27 μM thidiazuron (TDZ), 2.22 μM benzyladenine (BA) and 20 μM glutamine. Adventitious shoots were rooted and the plantlets were successfully acclimatized (100 %) in a vermiculite-soil mixture (1:1 v/v) in the greenhouse. Proscillaridin A (PsA) content and the antibacterial activity of in vitro and ex vitro regenerated plants were evaluated in different tissues in comparison to naturally-grown plants. The highest content of PsA (19.68 μg mg−1 DW) was recorded in roots of ex vitro plants which were grown on MS medium containing 2.27 μM TDZ, 2.22 μM BA and 100 mg l−1 casein hydrolysate. In vitro regenerated plants grown on MS medium containing 2.27 μM TDZ, 2.22 μM BA and 50.8 μM MBZ gave high antibacterial activity (MIC of 0.156 mg ml−1) against both Gram-positive and Gram-negative bacteria. Using this protocol the regenerated plants can be used in traditional medicine as an alternative to naturally collected plants.

In vitro propagation and secondary product production by Merwilla plumbea (Lindl.) Speta

Merwilla plumbea is a popular but threatened traditional medicinal plant sold at herbal markets in South Africa. It produces bioactive compounds and contributes to Traditional African Medical systems for the treatment of various diseases. Due to high demand, the plant is recommended for commercial cultivation. We investigated in vitro propagation and secondary compound production in in vitro cultures of M. plumbea, cultivated on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium supplemented with various plant growth regulators [PGRs: adenine sulphate (Ads), benzyladenine (BA), 2-isopentenyladenine (2iP), meta-topolin (mT), meta-topolin riboside (mTR), thidiazuron (TDZ), kinetin (Kin) and naphthaleneacetic acid (NAA)] and organic elicitors [casein hydrolysate (CH), glutamine (GM), haemoglobin (HB), mebendazole (MBZ), trimethoprim (TMP), yeast extract (YE) and yeast malt broth (YMB)]. A combination of 20 μM GM, 0.45 μM TDZ and 0.054 μM NAA produced the highest number of adventitious shoots per explant (30.6 shoots/explant) after 12 weeks of culture. The regenerated shoots were rooted and the plantlets successfully acclimatized. The total phenolic, flavonoid, gallotannin and condensed tannin contents were determined. The highest levels of flavonoids (50.97 μg CTE/g in shoots) and gallotannins (99.55 μg GAE/g in shoots) were recorded in combinations of 200 mg l−1 YE, 0.45 μM TDZ and 0.054 μM NAA and 100 mg l−1 YMB, 0.45 μM TDZ and 0.054 μM NAA. These increases were around 3- to 16-fold higher than in naturally-grown plants. This system offers the possibility to use in vitro culture techniques for mass propagation, secondary metabolite and pharmacological studies.

Quantification of Psoralen in Plant Parts of Psoralea Corylifolia growing in vivo and in vitro and Enhancement of Psoralen by Organic Elicitors

Psoralen were evaluated from in vivo and in vitro plant parts of Psoralea corylifolia by HighPerformance Liquid Chromatography (HPLC). The high amount of psoralen (3038 μg/g fresh weight) in invivo condition were obtained in brown seeds. Psoralen content in plant parts growing in vitro were lower thanthose growing in vivo. Quantitative analysis on psoralen in callus derived from different plant parts showedthat maximum psoralen (2601.8 μg/g fw) was recorded in callus derived from cotyledons. Estimation of psoralenin shoots derived from different callus were indicated high amount of psoralen (572.3 μg/g fw) in plant drivatedfrom node callus. Identification of psoralen in the callus of Psoralea corylifolia is reported for first time. In theother section attempts were done to enhance of psoralen by some organic elicitors including; yeast extract,proline, myzo-inositol and sucrose.in different medium. For the first time a comparative study of mature nodal,juvenile nodal explants and cotyledonary callus cultures were evaluated. These explants elicitated with differentorganic elicitor. Results revealed that maximum quantity of psoralen were present in cotyledonary callus cultures,mature and juvenile nodal cultures, respectively. Also, results indicated that there were significant variations inthe psoralen content in mature nodal, juvenile nodal and cotyledonary callus cultures, when their mediumsupplemented with different elicitors in the wide range of concentrations. Interestingly, high amount of psoralencontent (2881.9 μg/g fw.) even among all of the organic elicitors tried was found at 300 mg/l yeast extract incotyledonary callus cultures.

Management of citrus blue and green moulds through application of organic elicitors

The present studies aimed to develop control of citrus green and blue moulds caused by Penicillium digitatum and P. italicum, respectively, using elicitors as an alternate to synthetic chemicals. The dose-responsive toxicity of two organic elicitors was evaluated through a series of in vitro and in vivo experiments including pre- and postharvest application of different concentrations of salicylic acid (SA) or methyl jasmonate (MeJ) to ‘Lane Late’ sweet orange fruit for resistance induction. In in vitro experiments, SA significantly inhibited the radial growth, sporulation and spore germination of both Penicillium spp. compared to MeJ and control. SA (≥6 mM) showed direct fungitoxic effects and completely inhibited the radial growth, sporulation and conidial germination of both the pathogens. MeJ showed only a suppressive effect on fungal propagules and exhibited slightly better efficacy at concentrations ≥4 mM. A pre-harvest spray application of 8 mM SA or 3 mM MeJ to ‘Lane Late’ orange proved to be effective in reducing colony/lesion diameter, wound rotting and spore mass density of P. digitatum in contrast to postharvest treatment. We believe this is the first report demonstrating the efficacy of two organic elicitors in controlling the green mould in sweet orange. The present studies provide valuable information on standardization of minimum inhibitory concentrations (MICs) and pre- and postharvest treatment of sweet orange fruit with natural organic elicitors to induce resistance against postharvest pathogens.

In vitro isolation, elicitation of psoralen in callus cultures of Psoralea corylifolia and cloning of psoralen synthase gene

Psoralen, an important furanocoumarin occurring abundantly in seeds of Psoralea corylifolia is used as an anticancerous compound against leukemia and other cancer cell lines. Evaluation and isolation of psoralen from the calluses derived from different plant parts, viz. cotyledons, nodes, leaves and roots have been done in the present case for the first time. Amongst all, a maximum of 1934.75 μg/g f.w. of psoralen was recorded in callus derived from cotyledons, followed by 1875.50 and 1465.75 μg/g f.w. of psoralen in node and leaf derived calluses, respectively. Amount of psoralen enhanced further when cotyledonary calluses were exposed to different concentrations of organic elicitors (yeast extract, proline, inositol, casein hydrolyzate (CH), glycine, glutamine and sucrose) and precursors of psoralen (umbelliferone, cinnamic acid and NADPH). Isolation of psoralen was done using methanol as solvent through column chromatography and TLC. FT-IR and NMR further characterized and confirmed the structure of psoralen. In addition, the putative gene, psoralen synthase involved in psoralen synthesis pathway has been isolated, cloned and sequenced which comprised 1237 bp length. BLAST analysis of the gene sequence of psoralen synthase revealed that its nucleotide sequence showed 93% homology with psoralen synthase isolated from Ammi majus. This is the first report of isolation, cloning and characterization of psoralen synthase from Psoralea corylifolia.