Effects of exogenous nitric oxide on antioxidation and DNA methylation of <Emphasis Type="Italic">Dendrobium huoshanense</Emphasis> grown under drought stress

Nitric oxide (NO) is an important biological messenger in plants, which has been implicated in response to abiotic stress. To study the effects of exogenous NO on drought menace, the tube seedlings of Dendrobium huoshanense were selected and treated with 10% polyethylene glycol (PEG-6000) to simulate drought stress. After application of sodium nitroprusside (SNP), the relative water content (RWC) and antioxidant enzyme activities were determined. As a result, plant treated with 50 μmol L⁻¹ of SNP maintained high level of RWC and lower content of malondialdehyde (MDA). Furthermore, the antioxidant enzyme activities were obviously enhanced. However, the higher concentration of SNP (100 μmol L⁻¹) enhanced the effects of drought stress for plant. For further analysis of the response mechanism to exogenous NO, the methylation-sensitive amplified polymorphism (MSAP) technique was used to investigate the changes of DNA methylation. When the seedlings of Dendrobium huoshanense were treated with 50 μmol L⁻¹ SNP containing 10% PEG-6000, levels of global DNA methylation of Dendrobium huoshanense were decreased. Nevertheless, the demethylation rate of methylated sites increased, accounting for 12.5% of total methylation sites. These results implied that some expressed genes were involved in the response process to drought stress triggered by NO in Dendrobium huoshanense.     

Effects of citric acid as an important component of the responses to saline and alkaline stress in the halophyte <Emphasis Type="Italic">Leymus chinensis</Emphasis> (Trin.)

Some plants accumulate some compatible solutes and exude various organic acids when exposed to environmental stress. These compatible solutes including proline have been suggested to be involved in stress tolerance by maintaining sufficient cell turgor for growth, thereby improving plant growth, protecting enzymes, and membranes. However, less evidence exists regarding the protective roles of organic acids under stress conditions. Here, we investigate the effects of citric acid as a component of the response to stress on plant growth and antioxidant enzyme activities in two genotypes of halophyte Leymus chinensis (Trin.) genotypes, LcWT07 and LcJS0107. Data showed that both saline stress (200 mM NaCl) and alkaline stress (100 mM Na₂CO₃) reduced plant growth on the relative growth rate and CO₂ assimilation rate, but increased the citric acid concentrations in 6-week-old plants over the 72 h experimental period. When 50 mg l⁻¹ citric acid was exogenously applied under stress conditions, it significantly improved the plant growth and internal citric acid concentration, and also induced defense mechanisms by increasing the activities of antioxidant enzymes. To compare with the mitigative effects of exogenous citric acid on stress, exogenous application of proline was also performed under same conditions, and similar effects on the improvement of growth were observed. Based on these results, we suggested that citric acid is an important component of the stress response in L. chinensis, and exogenous application of 50 mg l⁻¹ citric acid might play a positive role on stress tolerance.     

Responses of Wheat Seedlings to Exogenous Selenium Supply Under Cold Stress

Dose-dependent effects of selenium on growth and physiological trait of wheat seedlings (Triticum aestivum L. cv Han NO.7086) exposed to cold stress are reported. Responses of seedlings were different depending on the Se concentration. The treatments with 0.5 and 1.0 mg Se kg⁻¹ significantly increased biomass and chlorophyll content of seedlings. However, the treatments at 2.0 and 3.0 mg Se kg⁻¹ only induced an evident increase in chlorophyll content and did not promote biomass accumulation of seedlings. Antioxidant compounds content (anthocyanins, flavonoids, and phenolic compounds) and antioxidant enzymes’ activities (peroxidase and catalase) increased by different Se treatments, while only the treatment with 1.0 mg Se kg⁻¹ induced a significant reduce in malondialdehyde content and the rate of superoxide radical production of wheat seedlings. The results of this study demonstrated that Se supply could increase antioxidant capacity of seedlings, and optimal Se supply reduced production of free radicals, membrane lipid peroxidation, and promoted biomass accumulation.     

Ethrel treatment enhanced isoflavonoids accumulation in cell suspension cultures of Pueraria tuberosa, a woody legume

The cell cultures of Pueraria tuberosa, a perennial leguminous lianas, were maintained in modified MS medium (KNO₃ 475 mg l⁻¹, thiamine 1 mg l⁻¹, biotin 1 mg l⁻¹, calcium pantothenate 1 mg l⁻¹) containing 0.1 mg l⁻¹ 2,4,5-trichloroacetic acid and 0.1 mg l⁻¹ kinetin. Isoflavonoids (puerarin, genistin, daidzein, genistein) accumulation in cell suspension cultures was increased by 14-fold to ~12 mg l⁻¹ after 48 h of adding 100 μM ethrel. Ethrel inhibitors (silver nitrate and silver thiosulfate) completely inhibited this effect in the presence of ethrel and isoflavonoids were not detected in the spent medium. The increase was dose dependent and can be explored to trigger high yield of isoflavonoids production.     

Effects of plant growth regulators and sucrose on post harvest physiology,membrane stability and vase life of cut spikes of gladiolus

Effects of post-harvest application of two plant growth regulators viz., gibberellic acid (GA₃) and benzyl adenine (BA) with sucrose in the vase solution on cell membrane stability and vase life of gladiolus were investigated. The vase solution treatment combinations of GA₃ and BA with sucrose significantly increased the membrane stability index and enhanced the vase life as compared to the sucrose alone treatments or the controls. Vase solution treatment of GA₃ (50 mg l⁻¹), followed by BA (50 mg l⁻¹) with sucrose (50 g l⁻¹) significantly increased solution uptake, fresh weight and dry weight of cut spikes. The same treatments also enhanced the concentration of reducing and non-reducing sugars in gladioli petals 4 days after treatment (DAT). Cut spikes in vase solution enriched with 50 mg l⁻¹ GA₃ + 50 g l⁻¹ sucrose showed higher antioxidative enzyme activities of superoxide dismutase (SOD) and glutathione reductase (GR), lower lipoxygenase (LOX) activity and lipid peroxidation (measured as TBARS). Petal membrane stability index was also highest in cut spikes 6 DAT with 50 mg l⁻¹ GA₃ + 50 g l⁻¹ sucrose vase solution. Treatment of gladiolus cut spikes with 50 mg l⁻¹ GA₃ + 50 g l⁻¹ sucrose vase solution showed two fold increase in vase life and improved flower quality with a higher number of open flower per spike at any one time. These results suggest that post-harvest application of GA₃ (50 mg l⁻¹) with sucrose (50 g l⁻¹) maintains higher spike fresh and dry weight, improves anti-oxidative defence, stabilizes membrane integrity leading to a delay in petal cell death.     

Antioxidant Responses of Wheat Seedlings to Exogenous Selenium Supply Under Enhanced Ultraviolet-B

The paper reports the effects of selenium (Se) supply on growth and antioxidant traits of wheat (Triticum aestivum L. cv Han NO.7086) seedlings exposed to enhanced ultraviolet-B (UV-B) stress. Antioxidant responses of seedlings were different depending on the Se concentration. Compared with the control, the lower amount used (0.5 mg Se kg⁻¹ soil) had no significant effect on biomass accumulation. The treatments with 1.0, 2.0, and 3.0 mg Se kg⁻¹ promoted biomass accumulation of wheat seedlings, and the increased amount in biomass was the most at 1.0 mg Se kg⁻¹ treatment. Se treatments with 1.0, 2.0, and 3.0 mg kg⁻¹ also significantly increased activities of peroxidase (POD) and superoxide dismutase (SOD) and reduced the rate of superoxide radical (O₂⁻) production and malondialdehyde (MDA) content of wheat seedlings. In addition, anthocyanins and phenolic compounds content in wheat seedlings evidently increased by the treatments with 1.0 and 2.0 mg Se kg⁻¹. The lower Se treatment had no significant effect on MDA content, although it increased activities of antioxidant enzymes (POD, SOD, and catalase activities) and reduced the rate of O₂⁻ production in wheat seedlings. These results suggest that optimal Se supply is favorable for the growth of wheat seedlings and that optimal Se supply can reduce oxidative stress of seedlings under enhanced UV-B radiation.     

Elicitor-induced accumulation of stilbenes in cell suspension cultures of Cayratia trifolia (L.) Domin

Cell cultures of Cayratia trifolia (Vitaceae), a tropical lianas, were maintained in Murashige and Skoog’s medium containing 0.25 mg l⁻¹ NAA, 0.2 mg l⁻¹ kinetin and casein hydrolysate 250 mg l⁻¹. Cell suspension cultures of C. trifolia accumulate stilbenes (piceid, resveratrol, viniferin, ampelopsin), which on elicitation by any of 500 μM salicylic acid, 100 μM methyl jasmonate, 500 μM ethrel and 500 mg l⁻¹ yeast extract, added on the 7th day, were enhanced by 3- to 6-fold (5–11 mg l⁻¹) by the 15th day.     

Effects of exogenous B supply on growth, B accumulation and distribution of two navel orange cultivars

To investigate the effects of boron (B) on growth, B concentration and distribution of two navel orange cultivars, ‘Newhall’ (Citrus sinensis Osbeck) and ‘Skagg’s Bonanza’ (Citrus sinensis Osbeck) grafted on the rootstock trifoliate orange [Poncirus trifoliata (L.) Raf.], B at five levels was exogenously supplied to 1-year-old grafted plants of both cultivars under greenhouse conditions. Plants were grown in sand:perlite (1:1, v/v) medium and were irrigated every 2 days with half-strength Hoagland’s No. 2 nutrient solutions containing different B, 0.01, 0.05, 0.10, 0.25 and 2.50 mg l⁻¹ (0.25 and 2.50 mg l⁻¹ were considered as control and excess B treatment, respectively, and the other three B levels were considered as low B treatments). After treatments for 183 days, leaves (from basal, middle, upper parts of the shoots), stem of scion, stem of rootstock and root were separately sampled. Our results showed that plant growth (plant height, root volume and dry weights of various parts) was inhibited in response to low or excess B supplies in both cultivars. It was found that B concentrations in the upper leaves of both cultivars were substantially higher than those in the basal leaves when low concentrations (≤0.05 mg l⁻¹) of exogenous B were applied, suggesting that B was preferentially translocated to the upper-younger leaves to support their growth. Analysis of B distribution in different parts indicated that translocation of B from the root to the scion’s shoots (stems and leaves of scion) may be restricted upon exposure to low B conditions. When B was inadequately supplied, growth of ‘Skagg’s Bonanza’ was better than ‘Newhall’, implying that the former cultivar was more tolerant to low B status, which may be due to the higher efficiency of B translocation from the root to the scion’s shoots. However, when the plants were treated with excess B (2.50 mg l⁻¹), both cultivars showed a similar degree of B toxicity. The probability of scion–rootstock interactions in relation to the differential responses of growth and different efficiency of B translocation involved in the two orange cultivars following the long-term low B stress were discussed.     

An Enhancing Effect of Exogenous Mannitol on the Antioxidant Enzyme Activities in Roots of Wheat Under Salt Stress

The role of mannitol as an osmoprotectant, a radical scavenger, a stabilizer of protein and membrane structure, and protector of photosynthesis under abiotic stress has already been well described. In this article we show that mannitol applied exogenously to salt-stressed wheat, which normally cannot synthesize mannitol, improved their salt tolerance by enhancing activities of antioxidant enzymes. Wheat seedlings (3 days old) grown in 100 mM mannitol (corresponding to −0.224 MPa) for 24 h were subjected to 100 mM NaCl treatment for 5 days. The effect of exogenously applied mannitol on the salt tolerance of plants in view of growth, lipid peroxidation levels, and activities of antioxidant enzymes in the roots of salt-sensitive wheat (Triticum aestivum L. cv. Kızıltan-91) plants with or without mannitol was studied. Although root growth decreased under salt stress, this effect could be alleviated by mannitol pretreatment. Peroxidase (POX) and ascorbate peroxidase (APX) activities increased, whereas superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) activities decreased in Kızıltan-91 under salt stress. However, activities of antioxidant enzymes such as SOD, POX, CAT, APX, and GR increased with mannitol pretreatment under salt stress. Although root tissue extracts of salt-stressed wheat plants exhibited only nine different SOD isozyme bands of which two were identified as Cu/Zn-SOD and Mn-SOD, mannitol treatment caused the appearance of 11 different SOD activity bands. On the other hand, five different POX isozyme bands were determined in all treatments. Enhanced peroxidation of lipid membranes under salt stress conditions was reduced by pretreatment with mannitol. We suggest that exogenous application of mannitol could alleviate salt-induced oxidative damage by enhancing antioxidant enzyme activities in the roots of salt-sensitive Kızıltan-91.     

Micropropagation and production of arbutin in oriental strawberry tree (<Emphasis Type="Italic">Arbutus andrachne</Emphasis> L.)

A protocol for micropropagation of Arbutus andrachne from seeds was developed. Results indicated that none of the seeds cultured on Murashige and Skoog (MS) medium, with or without plant growth regulators (PGRs), germinated. Seeds soaked in 250 mg l⁻¹ gibberellic acid (GA₃) at 4°C for 3 days, then cultured on water-agar medium containing 2.0 mg l⁻¹ GA₃ exhibited 80–100% germination and developed into usable seedlings. Shoot proliferation was tested on MS or B5 medium containing different concentrations of cytokinin. No shoot proliferation was observed on PGR-free medium. Proliferation was more successful on MS than on B5 medium. On both media, the most successful proliferation was obtained using zeatin as a cytokinin type. Rooting was tested on MS medium containing different concentrations of auxin. Rooting failed on PGR-free medium and on medium containing indole-3-acetic acid (IAA), 0.25 or 0.5 mg l⁻¹ indole-3-butyric acid (IBA), or 0.25, 0.5 or 2.0 mg l⁻¹ α-naphthaleneacetic acid (NAA). Rooting (40%) was most successful with 1.0 mg l⁻¹ NAA. Rooted plantlets exhibited 80% survival in all mixtures of peatmoss and perlite, and acclimatized plants were successfully grown in the greenhouse. Quantitative analysis of arbutin performed on in vivo and in vitro leaves using high-performance liquid chromatography (HPLC) revealed that in vivo leaves contained higher arbutin content (0.3–0.81% w/w) than in vitro leaves (0.09% w/w). The highest yield of arbutin in vivo was detected in leaves collected in August, and the lowest yield in leaves collected in December.     

Morphactin and 2iP markedly enhance accumulation of stilbenes in cell cultures of Cayratia trifolia (L.) Domin

The cell cultures of Cayratia trifolia (Vitaceae) a tropical lianas, were maintained in Murashige and Skoog’s medium containing 0.25 mg l⁻¹ naphthalene acetic acid, 0.2 mg l⁻¹ kinetin and 250 mg l⁻¹ casein hydrolysate. Cell suspension cultures of C. trifolia accumulate stilbenes (piceid, resveratrol, viniferin, ampelopsin) which on addition of 0.1–0.5 mg l⁻¹ morphactin in the medium containing naphthalene acetic acid and kinetin declined. Morphactin or 2 isopentenyl adenine alone at 0.1 mg l⁻¹ concentration enhanced stilbenes which on combination markedly enhanced the yield to ~5 mg l⁻¹ at 15th day.     

Photoinhibition-induced reduction in photosynthesis is alleviated by abscisic acid,cytokinin and brassinosteroid in detached tomato leaves

Detached leaves of tomato (Lycopersicon esculentum Mill.) experienced photoinhibition associated with sharp reductions in net photosynthetic rate (Pn), quantum efficiency of PSII (Φ_PSII) and photochemical quenching (qP) even though they were exposed to mild light intensity (400 μmol m⁻² s⁻¹ PPFD) at 28°C. Photoinhibition and the reduction in Pn, Φ_PSII and qP, however, were significantly alleviated by 1 mg l⁻¹ ABA, 0.1 mg l⁻¹ N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) and 0.01 mg l⁻¹ 24-epibrassinolide (EBR). Higher concentrations, however, reduced the effects or even exacerbated the occurrence of photoinhibition. Superoxide dismutase and ascorbate peroxidase activity in leaves increased with the increases in ABA concentration within 1–100 mg l⁻¹, CPPU concentration within 0.1–10 mg l⁻¹ and EBR concentration within 0.01–1.0 mg l⁻¹. Catalase and guaiacol peroxidase activity also increased with the increase in EBR concentration but CPPU and ABA treatments at higher concentrations caused a decrease. Malondialdehyde (MDA) content decreased with the increase in CPPU concentration. ABA and EBR, however, decreased MDA concentration only at 1 and 0.01 mg l⁻¹, respectively. In conclusion, detached leaves had increased sensitivity to PSII photoinhibition. Photoinhibition-induced decrease in photosynthesis, however, was significantly alleviated by EBR, CPPU and ABA at a proper concentration.     

An alginate-derived oligosaccharide enhanced wheat tolerance to cadmium stress

Hydroponic experiments were carried out to study the role of alginate-derived oligosaccharides (ADO) in enhancing wheat (Triticum aestivum L.) tolerance to cadmium stress. Data were collected on plant biomass, chlorophyll content, photosynthetic rate, antioxidant enzyme activity and malondialdehyde (MDA) content. Under 100 μM Cd stress, plant growth was significantly inhibited. Shoot length, root length, fresh and dry weight were sharply reduced by 24.21, 34.59, 22.1 and 14.7%, respectively of the control after 10 day of Cd exposure. Superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities were increased and MDA content increased. Wheat seeds were soaked for 5 h in 1,000 mg L⁻¹ ADO solution before cadmium stress. ADO pretreatment alleviated cadmium toxicity symptoms, which were reflected by increasing root and shoot lengths, fresh and dry weight, chlorophyll content and photosynthetic rate (P _n ). Furthermore, ADO pretreatment significantly increased antioxidant enzyme (SOD, CAT and POD) activities and reduced MDA content in leaves and roots. The results indicated that ADO pretreatment partially protected the seedlings from cadmium toxicity during the following growth period.     

Efficient regeneration and antioxidant potential in regenerated tissues of <Emphasis Type="Italic">Piper nigrum</Emphasis> L.

The organogenic potential and antioxidant potential (1, 1-diphenyl-2-picrylhydrazyl-scavenging activity) of the medicinal plant Piper nigrum L. (black pepper) were investigated. Callus induction and shoot regeneration were induced from leaf explants of potted plants cultured on MS medium supplemented with different plant growth regulators. The best callogenic response was observed on explants cultured for 30 days on MS medium supplemented with either 0.5 or 1.5 mg l⁻¹ 6-benzyladenine (BA) + 1.0 mg l⁻¹ α-naphthaleneacetic acid. Subsequent transfer of the callogenic explants onto MS medium supplemented with 1.5 mg l⁻¹ BA + 1.0 mg l⁻¹ gibberellic acid (GA₃) achieved 85% shoot organogenesis after 30 days of culture. The maximum number (7.2) of shoots/explant was recorded for explants cultured in MS medium supplemented with 1.0 mg l⁻¹ BA. Following the transfer of shoots to an elongation medium, the longest shoots (5.4 cm) were observed on MS medium supplemented with 1.0 mg l⁻¹ BA + 1.0 mg l⁻¹ GA₃. The elongated shoots were rooted on MS medium supplemented with different concentrations of indole butyric acid. An assay of the antioxidant potential of the in vitro-grown tissues revealed that the antioxidant activity of the regenerated shoots was significantly higher than that of callus and the regenerated plantlets.     

Plant regeneration via somatic embryogenesis of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> in temporary immersion system (TIS)

The present study describes a protocol for plant regeneration via somatic embryogenesis in temporary immersion system (TIS) for Camptotheca acuminata. Somatic embryos were induced by culturing hypocotyl segments from 14-day-old in vitro grown C. acuminata seedlings in TIS. Hypocotyl segments were placed in culture vessels modified with a mechanical device to support the fixation of explants. Cultures were maintained under a 16 h photoperiod with a light intensity of 60 μmol m⁻² s⁻¹ PPF at 25 ± 1°C. After 16 weeks of incubation embryogenic calli were formed above the edge of the mechanical device in the basal Murashige and Skoog (MS) medium containing 35 g l⁻¹ sucrose and without hormonal supplementation. For plantlet regeneration, somatic embryos at cotyledonary stage were cultured in three different concentrations of 6-benzylamino-purine (0.5, 1.0 and 1.5 mg l⁻¹ BAP) and in plant growth regulator (PGR) free medium. In general, 0.5 mg l⁻¹ BAP was found to be the most effective concentration for growth and development of Camptotheca embryos in TIS. Conversion of somatic embryos into plantlets was also successfully achieved on sterile substrates moistened with 0.5 mg l⁻¹ BAP. Plantlets derived from cotyledonary embryos were rooted in vitro with 0.5 mg l⁻¹ indole-3-butyric acid (IBA) before transfer to ex vitro conditions.     

Decolorization of 1-aminoanthraquinone-2-sulfonic acid by Sphingomonas xenophaga

Sphingomonas xenophaga QYY from sludge samples could effectively decolorize 1-aminoanthraquinone-2-sulfonic acid (ASA-2), one kind of anthraquinone dye intermediate, under aerobic conditions. More than 98% of ASA-2 could be removed within 120 h at the dye concentration from 200 mg l⁻¹ to 1,000 mg l⁻¹ due to oxidative degradation. The strain converted ASA-2 to 2-(2′-hydroxy-3′-amino-4′-sulfo-benzoyl)-benzoic acid, 2-(2′-amino-3′-sulfo-6′-hydroxy-benzoyl)-benzoic acid, o-phthalic acid and 2-amino-3-hydroxy-benzenesulfonic acid, which were identified using HPLC-MS and NMR. A possible initial decolorization pathway was proposed according to these metabolites. The decolorization of ASA-2 by cells in the basal salt medium was induced by ASA-2, and was due to soluble cytosolic enzymes. Combined initial decolorization pathway and the analysis of decolorization enzyme(s), the major enzyme responsible for ASA-2 decolorization was a NADH-dependent oxygenase.     

The effect of thidiazuron level on in vitro regeneration type and peroxidase profile in <Emphasis Type="Italic">Eucalyptus microtheca</Emphasis> F. Muell

The green twigs of 1-year-old Eucalyptus microtheca F. Muell seedlings were cultured on modified MS medium, supplemented with α-naphthalene acetic acid (NAA) and kinetin (Kin) hormones at 12 different concentrations. After 4 weeks, the combination of 1 mg l⁻¹ NAA + 1 mg l⁻¹ Kin induced the highest number of axillary shoots. Meanwhile, embryogenic calli were observed in media containing 4 mg l⁻¹ NAA + 0.5 mg l⁻¹ Kin, without any regeneration. The hormone treatments were followed by subculturing the twigs in different levels of thidiazuron (TDZ). The combination of 1 mg l⁻¹ NAA + 1 mg l⁻¹ Kin together with 0.01 mg l⁻¹ TDZ resulted in an increase of direct shoot, while higher amounts of TDZ led to adventitious shoot induction. Somatic embryogenesis was observed in the treatment containing 0.01 mg l⁻¹ TDZ + 4 mg l⁻¹ NAA + 0.5 mg l⁻¹Kin. The peroxidase (POD) band patterns in regenerated plantlets were investigated in order to determine the effect of different levels of TDZ on loci synthesis. A dimer locus, a tetramer locus and two epigenetic bands (a new band for NAA + Kin and the other for TDZ) were observed in the POD profiles. In case of low (0.01 mg l⁻¹ and 0.1 mg l⁻¹) levels of TDZ, one heterozygote allele was disappeared from dimer locus, while at higher TDZ levels, the dimer locus lost its stability and tetramer locus showed a high activity. Thus, POD allele patterns seems to be a feasible marker for different types of regeneration.     

An efficient protocol for micropropagation of lemon (<Emphasis Type="Italic">Citrus limon</Emphasis>) from mature nodal segments

The influence of the basal medium and different plant growth regulators on micropropagation of nodal explants from mature trees of lemon cultivars was investigated. Although the basal medium did not affect any of the variables, explants on DKW medium were greener. Several combinations of 6-benzyladenine (BA) and gibberellic acid (GA) were used to optimise the proliferation phase. The number of shoots was dependent on the BA and GA concentrations and the best results were obtained with 2 mg l⁻¹ BA and 1 or 2 mg l⁻¹ GA. Explants length was shorter with the higher BA concentrations and, in all genotypes, shoot length was greater with 2 mg l⁻¹ GA. The best results for productivity (number of shoots × the average shoot length) were obtained with 2 mg l⁻¹ BA and 2 mg l⁻¹ GA, although explants with chlorosis and narrow leaves were observed. The presence of BA and GA in the proliferation medium was essential for the explant multiplication but GA had a greater influence. The transfer of in vitro shoots to rooting media, containing different concentrations of indole butyric acid (IBA) and indole acetic acid (IAA) produced complete plantlets. Lemon shoots rooted well in all rooting combinations. The highest rooting percentages were obtained on media containing 3 mg l⁻¹ IBA alone or IBA in combination with 1 mg l⁻¹ IAA and on these media the highest numbers of roots were produced. The average root length was affected significantly by the IBA and IAA concentrations. Root length was greater when only 3 mg l⁻¹ IBA was used, and in this rooting medium explants had a better appearance, with greener and larger leaves. The success during the acclimatisation was close to 100% and the plantlets exhibited normal growth in soil under greenhouse conditions.     

Synergistic effect of morphactin on cytokinin-induced production of isoflavonoids in cell cultures of Pueraria tuberosa (Roxb. ex. Willd.) DC

Isoflavonoids, the functional molecules of Fabaceae, are under clinical trials against cancer, osteoporosis and cardiovascular diseases. In this study, the efficacy of different plant growth regulators was evaluated for optimizing the production of isoflavonoids in Pueraria tuberosa. The cultures were maintained in Murashige and Skoog’s medium containing 0.1 mg l⁻¹ 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 0.1 mg l⁻¹ kinetin. The addition of 5.0 mg l⁻¹ N⁶-(2-Isopentenyl) adenine (2iP) resulted in about ∼32-folds increase in production of isoflavonoids, while about ∼23-folds increase was recorded in the absence of kinetin in the maintenance medium. A maximum yield of isoflavonoids (∼80 mg l⁻¹; 82-folds increase) was obtained in cultures grown at 0.1 mg l⁻¹ morphactin and 5.0 mg l⁻¹ of 2iP. However, 2,4,5-T in combination with 2iP was ineffective for their production. Among different plant growth regulators tested, maximum yields of puerarin, genistin, daidzein and genistein were 17.4, 15.9, 69.0 and 0.04 mg l⁻¹, respectively. The study suggested that the presence of two cytokinins or 2iP with morphactin in the culture medium markedly enhanced the production of isoflavonoids in P. tuberosa.     

Exogenous salicylic acid alleviates effects of long term drought stress and delays leaf rolling by inducing antioxidant system

Salicylic acid (SA) is one of the important signal molecules modulating plant responses to environmental stress. In this study, the effects of exogenous SA on leaf rolling, one of drought avoidance mechanisms, and antioxidant system were investigated in Ctenanthe setosa during long term drought stress. The plants were subjected to 38-day drought period and they were treated with or without SA (10⁻⁶ M) on the 25th, 27th and 29th days of the period. Leaf samples were harvested on the 30th, 34th and 38th days. Some antioxidant enzyme activities (superoxide dismutase, catalase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, glutathione reductase), reactive oxygen species (hydrogen peroxide and superoxide) and lipid peroxidation were determined during the drought period. Treatment with SA prevented water loss and delayed leaf rolling in comparison with control leaves. Exogenous SA induced all antioxidant enzyme activities more than control leaves during the drought. Ascorbate and glutathione, α-tocopherol, carotenoid and endogenous SA level were induced by the SA treatment. Levels of reactive oxygen species were higher in SA treated plants than control ones on the 34th day. Their levels on the 38th day, however, fastly decreased in SA treated plants. SA treatment prevented lipid peroxidation while the peroxidation increased in control plants. The results showed that exogenous SA can alleviate the damaging effect of long term drought stress by decreasing water loss and inducing the antioxidant system in the plant having leaf rolling, alternative protection mechanism to drought.