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

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

Efficient genetic transformation of Jatropha curcas L. by microprojectile bombardment using embryo axes

An efficient and reproducible protocol was established for genetic transformation in Jatropha curcas through microprojectile bombardment. Decotyledonated embryos from mature seeds were pre-cultured for 5 days and elongated embryonic axis was subjected to bombardment for the optimization of physical parameters. The frequency of transient gus expression and survival of putative transformants were taken into consideration for the assessment of physical parameters. Statistical analysis reveal that microcarrier size, helium pressure and target distance had significant influence on transformation efficiency. Among different variables evaluated, microcarrier size 1 μm, He pressure 1100 and 1350 psi with a target distance of 9 and 12 cm respectively were found optimum by co-relating microcarrier size, helium pressure and target distance on the frequency of gus expression and survival of putative transformants. Selection of putative transformants was done with increasing concentrations (5-7 mg L⁻¹) of hygromycin. The integration of desired gene into Jatropha genome was confirmed with PCR amplification of 0.96 and 1.28 kb bands of hptII and gus gene respectively from the T₀ transgenics and Southern blot analysis using PCR amplified DIG labeled hptII gene as a probe. A successful attempt of genetic transformation was made with optimized conditions using particle gene gun and establishing a stable transformation in J. curcas with 44.7% transformation efficiency. The procedure described will be very useful for the introgression of desired genes into J. curcas and the molecular analysis of gene function.     

Improvement of essential oil yield of oil-bearing (Rosa damascena Mill.) due to surfactant and maceration

The essential oil content of the oil-bearing rose (Rosa damascene Mill.) is relatively low, around 0.3-0.4 mL kg⁻¹ in fresh flowers. There is a need to increase essential oil yield of oil-bearing rose. The objective was to examine the effect of Tween 20 (polyoxyethylene sorbitan monolaurate) applied with, or without, maceration of flowers on oil content and composition of oil-bearing rose harvested at beginning of flowering, full bloom, and end of flowering. Addition of Tween 20 at 1000 mL L⁻¹ and 2500 mL L⁻¹ increased essential oil yield by 26% (to 0.44 mL kg⁻¹) and 54% (to 0.54 mL kg⁻¹) respectively relative to the untreated control that gave 0.35 mL kg⁻¹ yield. Maceration, in combination with the addition of Tween 20 at 1000 mL L⁻¹ and Tween 20 at 2500 mL L⁻¹, increased oil yield by 69% (to 0.59 mL kg⁻¹) and 94% (to 0.68 mL kg⁻¹) respectively. Among the three phenological phases of harvest, harvesting at the beginning of flowering gave the highest yield followed by the full bloom and then by the end of flowering phases. Since the interaction effect was not significant, the differences obtained among the treatments were regardless of the phase, and vice versa. Treatments did not significantly alter composition of the essential oil. Postharvest pre-extraction application of Tween 20 in combination with maceration could be used in the rose industry for increasing the essential oil yield.     

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

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

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

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

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

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

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

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

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

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

In vitro plant regeneration from decapitated embryonic axes of Clitoria ternatea L.—An important medicinal plant

In vitro clonal propagation of Clitoria ternatea has been achieved by employing decapitated embryonic axes (DEAs) explants. The explants induced multiple shoots on cytokinin-containing medium. Several cytokinins [6-benzylaminopurine (BAP), 6-furfuryl aminopurine (KIN) and thidiazuron (TDZ)] were assayed. The best response was achieved with 2 mg l⁻¹ BAP in which 100% of cultures produced 6.0 ± 0.14 shoots per explant. MS + 1 mg l⁻¹ gibberellic acid (GA₃) was the most suitable for shoot elongation. Regenerated shoots were rooted in half-strength Murashige and Skoog (MS) medium with 0.2 mg l⁻¹ indole-3-butyric acid (IBA). Plantlets were successfully acclimatized and established in soil, and they were morphologically indistinguishable from the source plant. The plantlets attained maturity and flowered normally. The efficient regeneration protocol reported here provides an important method of micropropagation of this plant. Furthermore, this protocol may be used for genetic transformation of this valuable medicinal plant for its further improvement.     

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

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

Interactions of sunflower (Helianthus annuus) and sunflower broomrape (Orobanche cumana) as affected by sowing date,resource supply and infestation level

The holoparasitic weed Orobanche cumana (sunflower broomrape) constrains sunflower (Helianthus annuus) production in many countries. The development of efficient control strategies requires an understanding of the processes underlying the complex environment–host–parasite interrelations. Growth and development of O. cumana and sunflower were quantified under field conditions in southeastern Romania. Sunflower hybrid Florom 350 was sown at two dates, in plots infested with 0, 50, 200 and 1600 viable O. cumana seeds kg⁻¹ dry soil, under low-input (rainfed, low nitrogen supply) and high-input (irrigated, high nitrogen supply) conditions. Sunflower shoot biomass reached peak values of 760–1287 g m⁻² between the end of anthesis and physiological maturity. Seed yield varied from 221 to 446 g m⁻². Sunflower biomass and yield were affected by all experimental factors. Seed yield responded positively to delaying sowing from early April to late May as well as to irrigation and fertilisation, and negatively to O. cumana infestation. Yield reductions, which were a product of reduced seed number and size, amounted to 13%, 25% and 37% at parasite seed densities of 50, 200 and 1600 viable seeds kg⁻¹ soil, respectively. Maximum O. cumana attachment numbers, recorded in late-sown high-input crops in 2004, ranged from 11 m⁻² in plots with 50 parasite seeds kg⁻¹ soil to 188 m⁻² with 1600 seeds kg⁻¹ soil. Parasite attachment number was a function of crop sowing date, water and nutrient supply, seedbank density, and sunflower biomass and root length density, via mechanisms of parasite seed stimulation, host carrying capacity and intraspecific competition. Delayed sowing and improved water and nitrogen supply were associated with increases in parasite number that neutralised yield-boosting effects of irrigation and fertilisation at the highest infestation level. Sunflower shoot biomass was significantly reduced by O. cumana infection, with reductions affecting organs in the order head > stem > leaves. Most of the discrepancy between infected and non-infected plants was accounted for by O. cumana biomass. Parasites mainly acted as an extra sink for assimilates during sunflower generative growth and impaired host photosynthesis to a much lesser degree. Results suggest that similar mechanisms govern infection level and host–parasite biomass partitioning across different Orobanche–host systems.     

Kernel number determination differs among maize hybrids in response to nitrogen

In maize, the effects of nitrogen (N) deficiencies on the determination of kernel number per plant (KNP) have been described only by changes in plant growth rate during the critical period for kernel set (PGRcp). We hypothesize that N availability affects KNP also through variations in biomass allocation to the ear, which determines a stable N concentration in this organ. Six maize hybrids of different breeding origin were evaluated in field experiments at two N levels (0 and 400 kg N ha⁻¹ applied). Traits included were KNP and per apical ear (KN_E1), and the allometric estimation of PGRcp, ear growth rate during the critical period (EGRcp), and N content and N concentration in different plant organs. We demonstrated that (i) N availability promoted differences among genotypes (G) in the response of EGRcp and KNP to PGRcp, (ii) variations in KN_E1 were explained by EGRcp (r² = 0.64) and by ear N content at silking + 12 d (r² = 0.64), and (iii) ear N concentration was a highly conservative trait (range between 10.47 and 15.98 mg N g biomass⁻¹) as compared to N concentration in vegetative tissues (range between 4.94 and 18.04 mg N g biomass⁻¹). Three response patterns were detected among hybrids, one for which the relationship between EGRcp and PGRcp did not vary between N levels and experiments, a second one for which N availability affected this relationship, and a third one for which the response was affected by the year (Y) effect. These results, together with the high correlation between EGRcp and ear N content (r² = 0.88), evidenced the importance of both photo-assimilate and N availability on EGRcp and KNP determination. Values of 1.5–2.3 g ear⁻¹ d⁻¹ during the critical period and 0.49–0.70 g of N ear⁻¹ at silking + 12 d were determined as thresholds for maximizing KN_E1, and both could be easily estimated by means of allometric models.     

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

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

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

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

Phosphorus efficiency in long-term (15 years) wheat–maize cropping systems with various soil and climate conditions

Long-term (over 15 years) winter wheat (Triticum aestivum L.)–maize (Zea mays L.) crop rotation experiments were conducted to investigate phosphorus (P) fertilizer utilization efficiency, including the physiological efficiency, recovery efficiency and the mass (the input–output) balance, at five sites across different soil types and climate zones in China. The five treatments used were control, N, NP, NK and NPK, representing various combinations of N, P and K fertilizer applications. Phosphorus fertilization increased average crop yield over 15 years and the increases were greater with wheat (206%) than maize (85%) across all five sites. The wheat yield also significantly increased over time for the NPK treatments at two sites (Xinjiang and Shanxi), but decreased at one site (Hunan). The P content in wheat was less than 3.00 g kg⁻¹ (and 2.10 g kg⁻¹ for maize) for the N and NK treatments with higher values for the Control, NP and NPK treatments. To produce 1 t of grain, crops require 4.2 kg P for wheat and 3.1 kg P for maize. The P physiological use efficiency was 214 kg grain kg⁻¹ P for wheat and 240 kg grain kg⁻¹ P for maize with over 62% of the P from P fertilizer. Applying P fertilizer at 60–80 kg P ha⁻¹ year⁻¹ could maintain 3–4 t ha⁻¹ yields for wheat and 5–6 t ha⁻¹ yields for maize for the five study sites across China. The P recovery efficiency and fertilizer use efficiency averaged 47% and 29%, respectively. For every 100 kg P ha⁻¹ year⁻¹ P surplus (amount of fertilizer applied in excess of crop removal), Olsen-P in soil was increased by 3.4 mg P kg⁻¹. Our study suggests that in order to achieve higher crop yields, the long-term P input–output balance, soil P supplying capacity and yield targets should be considered when making P fertilizer recommendations and developing strategies for intensively managed wheat–maize cropping systems.     

Postharvest harpin or Bacillus thuringiensis treatments suppress citrus black spot in ‘Valencia’ oranges

Citrus black spot (CBS) caused by Guignardia citricarpa represents an important threat to citriculture in Brazil. Limited information is available regarding potential biological control agents and new alternative compounds that may provide protection of orange fruits against the disease. In this study, the effects of commercial products based on Bacillus thuringiensis var. kurstaki (Bt) bacterium, Bt pure isolates and Harpin protein (Messenger^®) on the postharvest control of CBS, were evaluated in ‘Valencia’ sweet orange fruits harvested for three consecutive years in a citrus grove. The fruits were sprayed with the following products: DiPel^® WP (Bt, subspecies, kurstaki strain HD-1, 16,000 International Units mg⁻¹, 32 g active ingredient kg⁻¹) (1, 20 and 50 mg ml⁻¹), Dimy Pel^® WP (Bt, subspecies, kurstaki, strain HD-1, 17,600 IU mg⁻¹, 26 g active ingredient l⁻¹) (2, 20 and 50 mg ml⁻¹), Messenger^® (3% harpin protein) (1 and 2 mg ml⁻¹) and fungicide Tecto^® Flowable SC (thiabendazole, 485 g l⁻¹) (0.8 g active ingredient l⁻¹), besides the Bt isolates, Bt- HD-567, Bt- DiPel and Bt- Dimy (9 × 10⁸ CFU ml⁻¹). Ten days after treatment, the number of newly developed CBS lesions and pycnidia produced were evaluated using fifty fruits per treatment. The Dimy Pel^® and Messenger^® reduced the number of new developed CBS lesions on fruits in up to 67% and 62%, respectively. All applied treatments drastically decreased the number of pycnidia produced in the CBS lesions on orange fruits with 85% to 96% reductions compared to the untreated control. Volatile compounds produced by the isolates Bt- HD-567, Bt- Dimy and Bt- DiPel, reduced the number of lesions on treated fruits by 70%, 65% and 71% compared to the control, respectively. In addition, the survival of Bt isolates on orange fruit surfaces were evaluated by recovering and quantifying the number of CFU every seven days for up to 28 days. The declines in survival rates on orange fruit surfaces were drastic for the three strains of Bt in the first week. The CFU numbers of all applied isolates declined by 4 to 5 orders of magnitude after storage at room temperature for 28 days. In vitro assays revealed that the Bt isolates significantly reduced the mycelial growth of the pathogen, ranging from 32% to 51%, compared to the control, whereas no inhibitory effect was observed in the presence of Messenger^®.     

Accumulation of cadmium by flax and linseed cultivars in field-simulated conditions: A potential for phytoremediation of Cd-contaminated soils

The possibility of use of two technological types of Linum usitatissimum L., namely flax (grown for fibre) and linseed (grown for seed), for phytoextraction of cadmium (Cd) from Cd-contaminated soil was studied. A four-year field-simulated experiment was carried out with 6 flax and 4 linseed cultivars in order to study organ accumulation of Cd by flax and linseed plants at artificial concentration range 10-1000 mg Cd kg⁻¹ soil. The most Cd was accumulated by roots, followed by shoots, while reproductive parts (capsules and seeds) played comparably smaller role. The increasing soil Cd concentration resulted in increasing Cd accumulation by roots, while transport to above-ground plant parts was progresivelly inhibited. Even high soil Cd concentrations (1000 mg Cd kg⁻¹ soil) had not dramatic negative effect on plant growth and development. Cultivar differences as well as the differences between both technological Linum types have been found in Cd accumulation (flax being better Cd accumulator than linseed). Nevertheless, the recorded variation between technological types and within cultivars is in multiples of Cd values (units of mg Cd kg⁻¹ DW), not in orders of magnitude as needed for practical phytoextraction. A significant year-to-year effect on plant growth/development resulting in high variation in Cd accumulation was observed. Flax cv. Jitka exhibited good transport of Cd from roots to above-ground parts, while flax cv. Merkur showed high retention of Cd in roots. Further, the contrasting cultivars in total Cd accumulation (high accumulating flax cv. Jitka versus low accumulating linseed cv. Jupiter) were selected for future experiments. The uptake of Cd by flax/ linseed from ha per season was calculated and the strategy for flax/linseed growing on heavy metal polluted soils with subsequent utilization of heavy metal-contaminated biomass is discussed.     

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

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

Enhanced selenium content in wheat grain by co-inoculation of selenobacteria and arbuscular mycorrhizal fungi: A preliminary study as a potential Se biofortification strategy

Cereal crops grown in southern Chilean Andisol provide suboptimal levels of this metalloid for human diet. Certain rhizosphere microorganisms, such as rhizobacteria and arbuscular mycorrhizal fungi can increase the selenium uptake in plants. The purpose of this study was to evaluate selenium acquisition by wheat plants through the co-inoculation of native selenobacteria strains (Stenotrophomonas sp. B19, Enterobacter sp. B16, Bacillus sp. R12 and Pseudomonas sp. R8), both individually and in mixture, as selenonanosphere source with one arbuscular mycorrhizal fungus (Glomus claroideum). Total selenium content in plant tissues and substrate was analyzed. According to our results, significant higher selenium content was found in inoculated plants in comparison to uninoculated controls (P ≤ 0.05). Independently of fungal presence, selenium content in grain from plants inoculated with Enterobacter sp. B16 (236 mg kg⁻¹) was higher than the rest of the strains (116–164 mg kg⁻¹). However, when plants were co-inoculated with a mixture of selenobacteria strains and G. claroideum, selenium content in grain was 23.5% higher (725 mg kg⁻¹) than non-mycorrhizal plants (587 mg kg⁻¹). The results suggest a synergistic effect between the selenobacteria mixture and G. claroideum associated to major biodiversity and demonstrate a great potential of these rhizosphere microorganisms for biofortification of cereals and its derivates.     

Impact of intercropping of medicinal and aromatic plants with organic farming approach on resource use efficiency in arecanut (Areca catechu L.) plantation in India

The present investigation was conducted at Vittal, Karnataka, India during 2004-2007 to study the feasibility of intercropping of medicinal and aromatic plants (MAPs) in arecanut plantation. The results revealed that MAPs can be successfully grown as intercrops in arecanut plantation with increased productivity and net income per unit area. Kernel equivalent yield of MAPs varied between 272 kg ha⁻¹ in case of Piper longum to 1218 kg ha⁻¹ in Cymbopogon flexuosus. Pooled data indicated that Asparagus racemosus produced fresh root yield of 10,666 kg ha⁻¹ of arecanut plantation and contributed to maximum kernel equivalent yield of 1524 kg ha⁻¹ among all medicinal and aromatic plants. Intercropping of MAPs in arecanut was found economical. The net return per rupee investment was highest in C. flexuosus (4.25) followed by Bacopa monnieri (3.64), Ocimum basilicum (3.46) and Artemisia pallens (3.12). The total system productivity of arecanut + MAPs intercropping system varied from 2990 to 4144 kg ha⁻¹. Arecanut + O. basilicum intercropping system registered significantly higher production efficiency 8.2 kg ha⁻¹ day⁻¹ than other systems. Intercropping of MAPs had more positive effect on soil pH in arecanut based cropping system. The soil pH was 5.6 in 2004 and it was 0.3-0.9 units higher in 2007. Soil organic carbon (SOC) content varied significantly due to intercropping of MAPs at the end of experiment. The SOC content increased in Aloe vera, A. pallens, P. longum and B. monnieri, while it depleted in grasses and rhizomatic MAPs. Based on demand and marketing opportunities for MAPs, farmers are advised to grow aromatic plants in large areas on a community basis to meet huge industrial demand and variety of medicinal crops in small areas to meet the requirement of traditional systems of medicine.