Changes in activity of antioxidant enzymes and photosynthetic machinery during acclimatization of micropropagated Cassia alata L. plantlets

The stimulatory effect of thidiazuron (TDZ) has been investigated in shoot multiplication for a simple, efficient, rapid, and commercially applicable regeneration protocol of an important medicinal plant, Cassia alata. Furthermore, the effects of an increased photosynthetic photon flux density (PPFD) on photosynthesis, the functioning of the photosynthetic apparatus, and the response of the antioxidant enzymatic system were studied during the ex vitro establishment of micropropagated plantlets. Multiple shoots were induced by culturing nodal explants excised from an aseptic seedling on Murashige and Skoog (MS) medium supplemented with various concentrations (1.0, 2.5, 5.0, 7.5, or 10.0 μM) of TDZ for different treatment durations (2, 3, 4, or 6 wk). The highest number of shoots (17.9?±?0.3) and longest shoot length (4.6?±?0.1 cm) were achieved when explants were exposed to 5.0 μM TDZ for 4 wk and subsequently subcultured on growth regulator-free MS medium for 8 wk. In vitro rooting of isolated shoots was best achieved on full-strength MS medium containing 0.5 μM indole-3-butyric acid (IBA). The micropropagated shoots with well-developed roots were successfully established in pots containing Soilrite? followed by garden soil and grown in greenhouse with an 85% survival rate. During the acclimatization period, significant changes in the activity of the antioxidant enzymatic system were observed. An increase in superoxide dismutase (SOD) activity was measured throughout the acclimatization period. Likewise an upregulation of catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) enzyme activities were also observed. Pigment (chlorophyll a and b and carotenoids) content in ex vitro-formed leaves was significantly higher compared with those grown in vitro. These observed changes reflected the ability of plants to develop an antioxidant enzymatic defense system aiding in survival against oxidative stress and in reducing release of free radicals.     

Brassinosteroids protect photosynthesis and antioxidant system of eggplant seedlings from high-temperature stress

The effects of 24-epibrassinolide under high temperature in eggplant (Solanum melongena L.) seedlings were studied by investigating the plant growth, chlorophyll content, photosynthesis and antioxidant systems. High temperature significantly inhibited the plant growth and markedly decreased the chlorophyll content, net photosynthetic rate, stomatal conductance and transpiration rate, while it increased intercellular CO₂ concentration. In a similar manner, high temperature also decreased significantly maximum quantum efficiency of PSII, potential photochemical efficiency, the quantum efficiency of PSII, photochemical quenching, the excitation capture efficiency of open centers, and increased non-photochemical quenching. Application of 0.05–0.2 μM EBR remarkably promoted the plant growth and alleviated high-temperature-induced inhibition of photosynthesis. Under high temperature, reactive oxygen species levels and lipid peroxidation were markedly increased, which were remarkably inhibited by application of 0.05–0.2 μM EBR. The activities of antioxidative enzymes such as superoxide dismutase, peroxidase, catalase and ascorbate peroxidase, and contents of ascorbic acid and reduced glutathione were significantly increased during high-temperature treatments, and these increases were more pronounced than those of EBR at 0.05–0.2 μM treatment. The EBR treatment also greatly enhanced contents of proline, soluble sugar and protein under high-temperature stress. Taken together, it can be concluded that 0.05–0.2 μM EBR could alleviate the detrimental effects of high temperatures on plant growth by increasing photosynthetic efficiency and enhancing antioxidant enzyme systems. Addition of 0.1 μM EBR had the best ameliorative effect against high temperature, while the addition of 0.4 μM EBR had no significant effects.     

Photomixotrophic cultures of blueberries (<Emphasis Type="Italic">Vaccinium corymbosum</Emphasis>) accumulate or release phenylpropanoids via inductive treatments

The in vitro production of phenylpropanoids has been studied in temporary immersion bioreactors (TIBs) cultures of blueberries (Vaccinum corymbosum) supplemented with 0.4 MPa CO₂, light intensity of 150 μM/m²/s, and induced with ABA (4 mM) and H₂O₂ (5 mM). Results demonstrated differences in the accumulation and/or the release of patterns of metabolites. In TIBs plus ABA, a reddish-maroon color was consistent with the accumulation of phenolics, where shoots formed clusters without internodal segments. While in TIBs plus H₂O₂, the phenylpropanoids were released into the culture medium. In both cases, experimental treatments with sucrose-reduced medium displayed the highest phenylpropanoids values, in contrast with the controls (conventional culture). Altogether, differential expressions of genes, linked to key pathways as photosynthesis, phenylpropanoids, and oxidative burst, could corroborate for the increase in the phenolics production in parallel with an improvement of photosynthesis in photomixotrophic culture.     

Plant regeneration from different explant types of Bituminaria bituminosa and furanocoumarin content along plant regeneration stages

The first protocol for in vitro plant regeneration from different explants of Bituminaria bituminosa, a pasture and medicinal species, has been established. Three explant types (petiole, leaflet and petiole-leaflet attachment “PLA”) cultured on media with different combinations of benzylaminopurine (BA; 5.0, 10.0 or 20.0 μM) and naphthalene acetic acid (NAA) or indole acetic acid (IAA; 0.5 or 5.0 μM) were tested for calli induction, and with 5 μM BA + 0.5 μM NAA or IAA for shoot development. The average number of shoots (≥5 mm) per callus depended on the explant type and the calli induction medium. The highest average number of shoots per callus was achieved by culturing leaflet and PLA explants on 5 μM IAA + 10 μM BA for calli induction and on 0.5 μM IAA + 5 μM BA for shoot development, and by culturing petiole explants on 0.5 μM NAA + 10 μM BA followed by a second culture on 0.5 μM NAA + 5 μM BA. The highest frequency of shoot rooting was achieved with 10.0 μM NAA and 1.0 μM gibberellic acid (GA₃). Rooted plants were acclimatised in a culture chamber, reaching 96 % survival. Acclimatised plants were transferred to a greenhouse and finally to the field, reaching 100 % survival. The furanocoumarin (FC) accumulation was evaluated in organogenic calli, in vitro shoots, ex vitro plants in the greenhouse and in ex vitro plants in the field (after 1 and 4 months of acclimatisation). The content of FCs depended on the plant material evaluated, being higher in ex vitro plants in the field (up to 9,824 μg g^?1 DW total FC) and lowest in organogenic calli (up to 50 μg g^?1 DW total FC). This effect may be due to cell organization, longer exposure to environmental factors and the developmental stage.     

The aromatic cytokinin <Emphasis Type="Italic">meta-</Emphasis>topolin promotes in vitro propagation,shoot quality and micrografting in <Emphasis Type="Italic">Corylus colurna</Emphasis> L.

The role of 4.1 or 8.2 μM meta-topolin (mT) on shoot multiplication, rooting and ex vitro acclimatization of micropropagated Corylus colurna L., a promising non-suckering rootstock for hazelnut (Corylus avellana L.), was examined in comparison to N⁶-benzyladenine (BA), the most used cytokinin in tissue culture of Corylus spp. The influence of 8.2 μM mT and BA on photosynthetic pigments content and antioxidant enzymes activity, catalase (CAT) and guaiacol peroxidase (POD), in regenerated shoots, and on the preparation of the rootstock for micrografting was also evaluated. The highest shoot multiplication was recorded on medium containing 8.2 μM mT and an overall positive effect of mT on growth and quality of micropropagated shoots was found. The highest chlorophyll a content (1.236 mg g^?1 fresh weight, FW) and chlorophyll a/b ratio (2.48), and the lowest total carotenoids content (0.292 mg g^?1 FW) and CAT activity (25.8 μmol min^?1 mg^?1 protein) were detected after 8.2 μM mT application, while no significant differences were found in chlorophyll b content and POD activity between the two cytokinins. The best rhizogenesis response (98% for 4.1 μM and 100% for 8.2 μM mT) and ex vitro acclimatization competence (higher than 78%) were exhibited from shoots multiplied on mT. Furthermore, the multiplication of rootstock on mT allowed obtaining the highest (70%) response of successful micrografting. The present findings provide the first evidence of the successful applicability of mT in C. colurna tissue culture and development of micrografted plantlets.     

Effects of root and foliar applications of exogenous NO on alleviating cadmium toxicity in lettuce seedlings

The effects of sodium nitroprusside (SNP, a donor of NO) on cadmium (Cd) toxicity in lettuce seedlings were studied. SNP was added into hydroponic systems or sprayed directly on the leaves of plants grown with and without Cd. Excess supply of Cd (100 μM) caused growth inhibition, dramatically increased Cd accumulation in both leaves and roots, and inhibited the absorption of Ca, Mg, Fe and Cu. Excess Cd also decreased activities of superoxide dismutase peroxidase and catalase in leaves and roots, and increased the accumulation of superoxide anion (O ₂ ^·? ), hydrogen peroxide (H₂O₂) and malondialdehyde (MDA). Root or foliar applications of exogenous NO alleviated Cd-induced growth suppression, especially root application of 250 μM SNP and foliar addition of 500 μM SNP. Addition of SNP promoted the chlorophyll synthesis suggesting that the photosynthesis was up-regulated. Exogenous NO increased Cd-decreased activities of antioxidant enzymes and markedly diminished Cd-induced reactive oxygen species (ROS) and MDA accumulation. Moreover, the absorption of Ca, Mg, Fe and Cu was increased, indicating that exogenous NO stimulated H⁺-ATPase activity to promote sequestration or uptake of ions. In addition, exogenous NO also inhibited Cd transfer from roots to shoots, which may indicate that Cd retention in roots induced by NO plays a significant role in Cd tolerance in lettuce seedlings. These data suggest that under Cd stress, exogenous NO improves photosynthesis by increasing chlorophyll synthesis, protects lettuce seedlings against oxidative damage by scavenging ROS, helps to maintain the uptake of nutrient elements, and inhibits Cd transferred to shoots effectively.     

Alleviation of exogenous 6-benzyladenine on two genotypes of eggplant (Solanum melongena Mill.) growth under salt stress

Cytokinins were recently shown to control plant adaptation to environmental stresses. To characterize the roles of cytokinins in the tolerance of eggplant (Solanum melongena Mill.) to salt stress, the protective effects of 6-benzyladenine (6-BA) on the growth, photosynthesis, and antioxidant capacity in the leaves of two eggplant cultivars Huqie12 (salt-sensitive) and Huqie4 (salt-tolerant) were investigated. Under 90 mM NaCl stress, Huqie4 showed higher biomass accumulation and less oxidative damage compared to the Huqie12. Application of exogenous 10 μM 6-BA significantly alleviated the growth suppression caused by salt stress in two eggplant genotypes. In parallel with the growth, 6-BA application in salt-stressed plants resulted in enhanced chlorophyll contents, as well as photosynthetic parameters such as net CO₂ assimilation rate (P _n), stomatal conductance (g _s), transpiration rate (E), and intercellular CO₂ concentration (C _i). Furthermore, exogenous 6-BA also significantly reduced the O₂ ^? production rate and malondialdehyde content and markedly increased the antioxidant enzymes superoxide dismutase and peroxidase, the antioxidant metabolites ascorbate and reduced glutathione (GSH), and proline in both genotypes under salt stress. The results indicate that exogenous 6-BA is useful to improve the salt resistance of eggplant, which is most likely related to the increase in photosynthesis and antioxidant capacity.     

Anticancer effect of Tamarix gallica extracts on human colon cancer cells involves Erk1/2 and p38 action on G2/M cell cycle arrest

Taking into account that oxidative stress is among the factors causing cancer-related death; chemoprevention which consists in using antioxidant substances such as phenolics could prevent cancer formation and progression. In the present study, phenolic contents and antioxidant activities of methanolic extracts from the halophyte Tamarix gallica shoots were determined. Moreover, the anticancer effect of this species on human colon cancer cells and the likely underlying mechanisms were also investigated. Shoot extracts showed an appreciable total phenolic content (85 mg GAE/g DW) and a high antioxidant activity (IC₅₀ = 3.3 μg/ml for DPPH test). At 50 and 100 μg/ml, shoot, leaf, and flower extracts significantly inhibited Caco-2 cell growth. For instance, almost all plant part extracts inhibited cell growth by 62 % at the concentration 100 μg/ml. DAPI staining results revealed that these extracts decrease DNA synthesis and confirm their effect on Caco-2 cells proliferation, principally at 100 μg/ml. More importantly, cell mitosis was arrested at G₂/M phase. The changes in the cell-cycle-associated proteins (cyclin B1, p38, Erk1/2, Chk1, and Chk2) are correlated with the changes in cell cycle distribution. Taken together, our data suggest that T. gallica is a promising candidate species to be used as a source of anticancer biomolecules.     

High-Frequency Regeneration by Abscisic Acid (ABA) from Petiole Callus of Jatropha curcas

Jatropha curcas L. is attaining worldwide interest as an important biofuel crop. Experiments were conducted to improve the prevailing micropropagation technique as well as to develop a new ex vitro rooting method for J. curcas plant regeneration. Regeneration and ex vitro rooting efficiency was enhanced by augmenting the culture medium with abscisic acid (ABA). Different concentrations of 6-benzylaminopurine (BAP) and indole-3-butyric acid (IBA) were tested for callus generation from both in vitro and in vivo explants (leaf and petiole) on Murashige and Skoog (MS) medium. The best regenerative callus was achieved on MS medium supplemented with BAP (4.44 μM) and IBA (2.45 μM) from in vitro-cultured petioles. Highest regeneration (91%) was achieved by culturing petiole callus on MS medium supplemented with BAP (8.88 μM), IBA (0.49 μM), and ABA (1.9 μM), whereas 61% regeneration was obtained from in vitro leaf callus. Shoot proliferation and elongation was achieved on BAP (2.22 μM) and IAA (8.56 μM) with 10–13 shoots per explants. Highest rooting (65%) was achieved from M1 shoots (BAP, IAA, and ABA) on MS medium supplemented with IBA (2.45 μM), naphthaleneacetic acid NAA (0.54 μM), and 0.02% activated charcoal. Ex vitro rooting of 1-mo-old M1 shoots obtained from the charcoal-containing medium resulted optimum rooting (>72%) when transferred to polybags containing sterile sand. The plantlets were successfully acclimatized in soil with more than 98% survival rate in the greenhouse.     

In vitro propagation and the acclimatization effect on the synthesis of 2-hydroxy-4-methoxy benzaldehyde in Decalepis hamiltonii Wight and Arn.

The present study reports a high frequency in vitro propagation protocol through apical bud sprouting and basal organogenic nodule formation in shoot tip explants of Decalepis hamiltonii, an endemic and endangered medicinal liana. Among different combinations of plant growth regulators (PGRs) and growth additives, maximum of 8.20 shoots per explant with mean shoot length of 6.54 cm were induced on Murashige and Skoog’s medium (MS) supplemented with 5.0 µM 6-benzyladenine (BA) + 0.5 µM indole-3-acetic acid (IAA) + 30.0 µM adenine sulphate (ADS) through apical bud sprouting. On single cytokinin treatment explants did not exhibit good multiplication but showed nodulation (N₁) from the basal cut end similar to cytokinin–auxin combination (N₂). Between two types of nodular tissues, N₂ was proved to be better for maximum shoot regeneration (15.40 shoots per explant) and shoot length (4.56 cm) when cultured on MS medium supplemented with 5.0 µM BA, 0.5 µM IAA, 30.0 µM ADS and 1.0 µM gibberellic acid (GA₃). Microshoots were efficiently rooted on half-strength MS medium supplemented with 2.5 μM α-naphthalene acetic acid (NAA). After successful acclimatization in Soilrite, 95.10 % plantlets were survived in field conditions. Histological investigation proved useful in ascertaining the callogenic nature of the regenerating nodular tissue formed at the basal cut end of shoot tip explant. Acclimatized plantlets were studied for the estimation of chlorophyll and carotenoid content as well as the net photosynthetic rate (PN) during subsequent days of transfer to ex vitro condition. Moreover, acclimatization had a significant effect on biomass production and the synthesis of 2-hydroxy-4-methoxy benzaldehyde (2HMB). Maximum fresh weight (3.78 gm/plant), dry weight (0.39 gm/plant) of roots and 2HMB content (15.94 µg/ml of extract) were noticed after 8 weeks of acclimatization.     

Biochemical and physiological changes produced by Azotobacter chroococcum (INIFAT5 strain) on pineapple in vitro-plantlets during acclimatization

This study highlights some of the effects of the application of Azotobacter chroococcum (INIFAT5 strain) on in vitro-pineapple plantlets during acclimatization. The bacteria were sprayed immediately after transplanting to the ex vitro environment; the plants were then sprayed every 4 week. Subsequently (4 months) the evaluated variables included plantlet fresh and dry weights, leaf and root lengths, and composition of minerals, amino-acids, carbohydrates and proteins. Photosynthesis indicators were also evaluated. Significant effects of the application of Azotobacter over pineapple plantlets during acclimatization were observed in the mineral, amino-acid, carbohydrate and protein levels, as well as, in the photosynthesis indicators. Contrastingly, plant growth parameters showed modest increases caused by the bacteria, although they were statistically significant. Looking into specific minerals, the following significant effects of Azotobacter should be highlighted: increased levels of nitrogen, phosphorous, potassium, magnesium, copper and zinc. Moreover, contents of all amino-acids recorded showed significant increases in their levels in sprayed plantlets. Carbohydrates were also increased in leaves of plantlets bio-fertilized with the bacteria, mainly sucrose and fructose. Chlorophyll b levels were also significantly increased by Azotobacter. The biofertilizer did not modify levels of calcium, iron or manganese.     

Micropropagation,antioxidant properties and phytochemical assessment of Swertia corymbosa (Griseb.) Wight ex C. B. Clarke: a medicinal plant

Swertia corymbosa (Griseb.) Wight ex C. B. Clarke, a valuable medicinal plant, has been investigated for its regeneration potential using nodal explants. Out of a range of concentrations of cytokinins [6-benzyl adenine (BA), 6-furfurylaminopurine (Kn), 2-isopentenyl adenine (2iP), thidiazuron (TDZ), and zeatin (Z)] used as supplements with MS, BA at 4.40 μM concentration proved best for multiple shoot induction yielding 26.50 ± 0.26 shoots after 12 weeks of culture. Addition of low concentration of NAA (1.3 μM) in MS medium supplemented with the cytokinin BA (4.40 μM) favoured shoot multiplication. A mean number of 35.78 ± 0.81 shoots were produced per explant. Additive effect of BA (4.40 μM) in combination with Kn (4.64 μM) produced highest number of shoots (83.20 ± 4.29). Addition of GA₃ (1.4 μM) to the above medium not only favored shoot elongation but also enhanced the number of shoots (113.98 ± 3.80). The microshoots were rooted successfully on half-strength MS medium supplemented with 9.8 μM of IBA. The plantlets were successfully transferred to hardening medium containing vermiculite with 87 % survival rate. Screening of the antibacterial, antioxidant activity and estimation of total phenolic and flavonoid content of methanolic extracts of micropropagated plants were also carried out and compared with that of the wild-grown plants. In all the tests, methanolic extract from wild-grown plants showed higher antioxidant, antimicrobial activity, total phenolic and flavonoid content than in vitro propagated plants. The content of secondary metabolites in wild-grown plants and in vitro propagated plants was determined by HPLC coupled with ESI-MS and the presence of loganic acid, swertiamarin, sweroside, gentiopicroside, isovitexin, amoroswertin, amarogentin, gentiacaulein, decussatin, and swertianin in the samples were confirmed. Gentiopicroside (40.726 mg/g) and swertianin (29.598 mg/g) were found to be the major compounds which may be responsible for the antimicrobial and antioxidant activities. The results of the present study confirmed the therapeutic potency of S. corymbosa used in the traditional medicine; in addition, the protocol for in vitro production developed in the present study could be applied for mass multiplication and for the conservation of germplasm.     

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 (SEM_L) 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 SEM_L 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.     

Micropropagation of Hohenbergia penduliflora (A. Rich.) Mez. for sustainable production of plant proteases

Hohenbergia penduliflora (A. Rich.) Mez. inhabits the protected ecological area of Cunagua Highland, Ciego de Ávila, Cuba. The availability of this plant for experimental purposes is exceedingly limited. Tissue cultured plants of this specie, would be useful for propagation purposes. Experiments were carried out to optimize the micropropagation process from the disinfection of fruits to ex vitro hardening of regenerated plantlets. The best results were obtained when seeds were disinfected with 2 % (v:v) sodium hypochlorite for 20 min and placed in vitro for 45 days for seed germination. Tissue cultured shoots (1 cm) with vertical wounds in the basal region (5 mm long) were placed in a medium containing Murashige and Skoog (MS) salts, 100 mg l^?1 myo-inositol, 0.1 mg l^?1 thiamine-HCl, 30 g l^?1 sucrose, 8.8 μM 6-benzyladenine (BA) and 1.6 μM naphthalene acetic acid (NAA). Shoots were proliferated for 45 days to obtain 8.21 new shoots per explant; they were subsequently divided and rooted on a medium containing 1.6 μM NAA for 30 days. For ex vitro hardening, plastic trays containing 82 cm³ of filter-cake-sugarcane ashes were used; 100 % survival rate was recorded. After 6 months of hardening, plants were established ex vitro and ready for protease extraction. Comparisons between protein contents, proteolytic activities and specific proteolytic activities of extracts from stems of macro- and micropropagated plants were acquired. Tissue cultured stems showed statistically lower figures which is why Ethrel was tested here to increase proteolytic activity in micropropagated plant stems. After Ethrel applications, protein contents, proteolytic activities and specific proteolytic activities of extracts from stems were the three main indicators recorded. However, other biochemical effects of Ethrel were also evaluated, such as, levels of chlorophyll pigments, malondialdehyde and other aldehydes; and superoxide dismutase, and guaiacol peroxidase activities. Rising concentrations of Ethrel (0, 1.5, 3.0, 4.5 and 6.0 mg l^?1) decreased protein contents at 72 h but increased proteolytic and specific proteolytic activities of stem extracts. Ethrel was effective in increasing proteolytic activity in in vitro culture-derived plant stems, at a level higher than in field-grown plant stems. Moreover, Ethrel increased superoxide dismutase and guaiacol peroxidase-specific activities in leaves; and decreased chlorophyll pigments. Ethrel did not affect levels of malondialdehyde and other aldehydes.     

The influence of ABA on water relation, photosynthesis parameters, and chlorophyll fluorescence under drought conditions in two maize hybrids with contrasting drought resistance

Drought is a major limitation of maize cultivation in Brazil. Agronomic and physiological practices have been considered to overcome this stress and consequently, increase grain production. The present study investigated the role of abscisic acid (ABA) application in some physiological parameters, in two hybrids with contrasting drought resistance (DKB 390 and BRS 1030 resistant and sensitive, respectively). Contrasting resistance to drought in these genotypes was determined in previous studies. Water deficit was imposed for 10 days at flowering stage, in association with the application of 100 μM abscisic acid on plant canopy. Evaluations of gas exchange, chlorophyll fluorescence, relative water content (RWC), and endogenous ABA content were performed during stress period and also at water recovery (recovery irrigation). A significant functional relationship was observed between RWC and the parameters of gas exchange and fluorescence. During water recovery, no differences were observed among the treatments. DKB 390 presented higher photosynthesis rate (P _n) and electron transport rate (ETR) under water stress, while BRS 1030 presented higher intercellular CO₂ concentration (C _i) and lower photochemical quenching (qP), non-photochemical quenching (NPQ), and lower F _v/F _m ratio. DBK 390 was more responsive to ABA application than BRS 1030, presenting higher endogenous ABA content in the first day of stress. DBK 390 with ABA application reduced the effect of water stress through maintenance of water status, an increase of photosynthetic parameters, and a decrease of decline in the functions of photosystem II during stress.     

Modulation in growth,photosynthetic efficiency,activity of antioxidants and mineral ions by foliar application of glycinebetaine on pea (Pisum sativum L.) under salt stress

A pot experiment was carried out to explore the role of glycinebetaine (GB) as foliar spray foliar on two pea (Pisum sativum L.) varieties (Pea 09 and Meteor Fsd) under saline and non-saline conditions. Thirty-two-day-old plants were subjected to two levels 0 and 150 mM of NaCl stress. Salt treatment was applied in full strength Hoagland’s nutrient solution. Three levels 0, 5 and 10 mM of GB were applied as foliar treatment on 34-day-old pea plants. After 2 weeks of foliar treatment with GB data for various growth and physiochemical attributes were recorded. Rooting-medium applied salt (150 mM NaCl) stress decreased growth, photosynthesis, chlorophyll, chlorophyll fluorescence and soluble protein contents, while increasing the activities of enzymatic (POD and CAT) and non-enzymatic (ascorbic acid and total phenolics) antioxidant enzymes. Foliar application of GB decreased root and shoot Na⁺ under saline conditions, while increasing shoot dry matter, root length, root fresh weight, stomatal conductance (g _s), contents of seed ascorbic acid, leaf phenolics, and root and shoot Ca²⁺ contents. Of three GB (0, 5, 10 mM) levels, 10 mM proved to be more effective in mitigating the adverse effects of salinity stress. Overall, variety Pea 09 showed better performance in comparison to those of var. Meteor Fsd under both normal and salinity stress conditions. GB-induced modulation of seed ascorbic acid, leaf phenolics, g _s, and root Ca²⁺ values might have contributed to the increased plant biomass, reduction of oxidative stress, increased osmotic adjustment and better photosynthetic performance of pea plants under salt stress.     

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.     

Asymmetric effects of litter removal and litter addition on the structure and function of soil microbial communities in a managed pine forest

Aims

The effect of different MeJA doses applied prior to or simultaneously with toxic Al on biochemical and physiological properties of Vaccinium corymbosum cultivars with contrasting Al resistance was studied.

Methods

Legacy (Al-resistant) and Bluegold (Al-sensitive) plants were treated with and without toxic Al under controlled conditions: a) without Al and MeJA, b) 100 μM Al, c) 100 μM Al + 5 μM MeJA, d) 100 μM Al + 10 μM MeJA and e) 100 μM Al + 50 μM MeJA. MeJA was applied to leaves 24 h prior to or simultaneously with Al in nutrient solution. After 48 h, Al-concentration, lipid peroxidation (LP), H₂O₂, antioxidant activity, total phenols, total flavonoids, phenolic compounds and superoxide dismutase activity (SOD) of plant organs were analyzed.

Results

Al-concentrations increased with Al-treatment in both cultivars, being Al, LP and H₂O₂ concentrations reduced with low simultaneous MeJA application. Higher MeJA doses induced more oxidative damage than the lowest. Legacy increased mainly non-enzymatic compounds, whereas Bluegold increased SOD activity to counteract Al³⁺.

Conclusions

Low MeJA doses applied simultaneously with Al³⁺ increased Al-resistance in Legacy by increasing phenolic compounds, while Bluegold reduced oxidative damage through increment of SOD activity, suggesting a diminution of its Al-sensitivity. Higher MeJA doses could be potentially toxic. Studies are needed to determine the molecular mechanisms involved in the protective MeJA effect against Al-toxicity.

     

Evaluation of heme oxygenase 1 (HO 1) in Cd and Ni induced cytotoxicity and crosstalk with ROS quenching enzymes in two to four leaf stage seedlings of <Emphasis Type="Italic">Vigna radiata</Emphasis>

Research on heme oxygenase in plants has received consideration in recent years due to its several roles in development, defense, and metabolism during various environmental stresses. In the current investigation, the role of heme oxygenase (HO) 1 was evaluated in reducing heavy metal (Cd and Ni) uptake and alleviating Cd and Ni toxicity effects in the hydroponically grown seedlings of Vigna radiata var. PDM 54. Seedlings were subjected to Cd- and Ni-induced oxidative stress independently at different concentrations ranging from 10 to 100 μM. After 96 h (fourth day) of treatment, the stressed plants were harvested to study the cellular homeostasis and detoxification mechanism by examining the growth, stress parameters (LPX, H₂O₂ content), and non-enzymatic and enzymatic parameters (ascorbate peroxidase (APX), guaicol peroxidase (GPX), and catalase (CAT)) including HO 1. At 50 μM CdCl₂ and 60 μM NiSO₄, HO 1 activity was found to be highest in leaves which were 1.39 and 1.16-fold, respectively. The greatest HO 1 activity was reflected from the reduction of H₂O₂ content at these metal concentrations (50 μM CdCl₂ and 60 μM NiSO₄) which is correlated with the increasing activity of other antioxidant enzymes (CAT, APX). Thus, HO 1 works within a group that generates the defense machinery for the plant’s survival by scavenging ROS which is confirmed by a time-dependent study. Hence, it is concluded that seedlings of V. radiata were more tolerant towards metal-induced oxidative stress in which HO 1 is localized in its residential area (plastids).     

Ex vitro rescue,physiochemical evaluation,secondary metabolite production and assessment of genetic stability using DNA based molecular markers in regenerated plants of <Emphasis Type="Italic">Decalepis salicifolia</Emphasis> (Bedd. ex Hook.f.) Venter

To ensure replenishment, a refine protocol for micropropagation of Decalepis salicifolia (Bedd. ex Hook.f.) Venter a critically endangered and endemic medicinal plant was developed using mature nodal explants. A high frequency shoot regeneration system was obtained on Murashige and Skoog (MS) medium comprised of 6- benzyladenine (BA) (5.0 µM)?+?α-naphthalene acetic acid (NAA) (0.5 µM)?+?adenine sulphate (ADS) (30.0 µM) corresponds to a highest mean number of 9.97?±?0.01 shoots/explants with maximum shoot length of 6.46?±?0.1 cm. Successful rooting in microshoots was achieved on half strength MS medium supplemented with indole-3-butyric acid (IBA) (2.5 µM). A maximum of 6.10?±?0.07 roots/microshoot with average root length of 2.30?±?0.06 cm was obtained. As much as 90% plantlets survived when Soilrite^? was used as planting substrate and finally established in soil without any casualty and morphological variation. Acclimatized plantlets were screened for pigment content, net photosynthetic rate (PN), stomatal conductance (Gs) and transpiration rate (E) during subsequent days of acclimatization as well as the changes in antioxidant was also evaluated. A steady rise was observed in the activity of superoxide dismutase (SOD) for initial 21 days and then after a decrease was found showing improved acclimatization efficiency of the plant. Similarly, the activities of catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) enzyme shows reliable increase as the days of acclimatization advanced which play their precautionary role against oxidative damage to the plant. The genetic fidelity of the in vitro raised plantlets with that of mother plant was further confirmed using random amplified polymorphic DNA (RAPD) and inters simple sequence repeats (ISSR) analysis. Additionally, the effect of acclimatization on the biosynthesis of 2-hydroxy-4-methoxybenzaldehyde (2H4MB) in the root system was also evaluated in relation to their biomass production. Maximum fresh weight (4.9 g/plant), dry weight (0.65 g/plant) of roots and 2H4MB content (6.8 µg ml^?1 of root extract) was obtained after 10 weeks of acclimatization. Accelerated multiplication rate with the stability of genetic virtue, physiological and biochemical parameter assure the efficacy of the protocol developed for the propagation of this critically endangered medicinal plant.