Ectopic expression of serotonin N-hydroxycinnamoyltransferase and differential production of phenylpropanoid amides in transgenic tomato tissues

Tomato contains high levels of amines such as serotonin and tyramine and is a suitable host to enhance phenylpropanoid amides (PAs), an important class of nutraceuticals with strong antioxidant activity and chemotherapeutic effects, by ectopic expression of the corresponding gene, serotonin N-hydroxycinnamoyltransferase (SHT). To assess whether ectopic overexpression of SHT cDNA under the control of the CaMV 35S promoter would enhance levels of PAs, we generated transgenic tomato plants and analyzed the levels of PAs. Transgenic tomato plants exhibited increased synthesis of PAs such as feruloylserotonin (FS), 4-coumaroylserotonin (CS), feruloyltyramine (FT), 4-coumaroyltyramine (CT), and feruloyloctopamine (FO) in 1-month-old leaves compared to the wild type. The increase and relative levels of PAs were even more apparent in 3-month-old leaves of transgenic tomato. When tomato leaves were challenged by wounding, levels of PAs in the best transgenic line increased by 3- and 10-fold for CS + FS and CT + FT, respectively. In contrast to leaves, tomato fruit only showed enhanced synthesis of CT + FT, whereas CS + FS levels were not enhanced. Regarding amine content, the levels of tyramine were much higher than those of serotonin in tomato leaves and fruits. The high levels of tyramine may contribute to the preferential production of CT + FT rather than CS + FS, although SHT enzyme shows the highest substrate affinity toward serotonin rather than tyramine.     

A SCAR marker linked to the N gene for resistance to root knot nematodes (Meloidogyne spp.) in pepper (Capsicum annuum L.)

The root-knot nematodes (Meloidogyne spp.) are important nematode pests and cause serious diseases in pepper in the world. No molecular markers linked to the nematodes resistance N gene have been reported. In this paper, ‘Carolina Wonder’ (Capsicum annuum L.), a sweet pepper line resistant to root-knot nematode with N gene, ‘20080-5-29’ (C. annuum L.), an inbred line susceptible to root-knot nematode with good horticultural characteristics, and their F₂ progeny with 320 individuals were used as materials. Evaluation of resistance and susceptibility of parental lines, F₁ and F₂ progeny inoculated with root-knot nematodes (Meloidogyne incognita) were carried out. ‘Bulked segregant analysis’ method was used to search for polymorphic markers from 512 pairs of AFLP primers. Based on the assessment of resistance and susceptibility and polymorphism of the AFLP marker in F₂ population, the genetic linkage distance between the AFLP marker and the N gene was estimated. One AFLP marker E39/M41-339 was obtained and transferred to a SCAR marker amplifying a 315 bp DNA fragment linked to the N resistant allele and a 331 bp fragment linked to the N⁺ susceptible allele. The distance between the molecular marker and the nematodes resistance N gene is 6.3 cM. This research delivered a valuable tool for the marker assisted selection of nematodes resistance in pepper.     

Genetics and distribution of fertility restoration associated RAPD markers in inbreds of pepper (Capsicum annuum L.)

Experiments were conducted to study genetics of fertility restoration and to examine distribution of RAPD markers (OPW19₈₀₀ and OPP13₁₄₀₀) linked with fertility restoration gene (Rf) in pepper (Capsicum annuum L.) inbreds. Forty-two hot and five sweet pepper inbreds were crossed on a cytoplasmic male sterile (cms) line CCA-4261 and F₁s were evaluated for fertility restoration under open field conditions. DNA of 5 plants of CCA-4261 and individual plants of 47 inbreds was isolated and PCR reaction was performed using OPW19 and OPP13 primers. The results revealed that most of the hot pepper lines posses Rf gene. The Rf gene associated two markers, viz., OPW19₈₀₀ and OPP13₁₄₀₀ were not frequently distributed in the restorer inbred lines because presence of marker bands often does not coincide with the presence of Rf gene identified in many restorer inbreds. The case specific applications of both the RAPD markers have been described.     

Agronomic characteristics and carotenoid content of five Bola-type paprika red pepper (Capsicum annuum L.) cultivars

The production of sweet paprika in Spain uses exclusively fruit of Bola-type Capsicum annuum L. This work describes the evaluation of the agronomic behaviour of five new cultivars of the Bola-type paprika red pepper, selected in the Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA), and grown in Extremadura for 3 years. The colour and the pigment content of the paprika elaborated following the traditional procedure of La Vera were also studied.     

Towards crop improvement in bell pepper (Capsicum annuum L.): Transgenics (uid A::hpt II) by a tissue-culture-independent Agrobacterium-mediated in planta approach

Agrobacterium tumefaciens-mediated transformation has been one of the methods used to generate transgenic plants in bell pepper. An alternate transformation method that avoids/minimizes tissue culture would be beneficial for the improvement of bell pepper due to its recalcitrant nature. In this report, transgenic bell pepper plants have been developed by a tissue-culture-independent A. tumefaciens-mediated in planta transformation procedure. In the present study, two open pollinated varieties viz., Arka Gaurav and Arka Mohini were used for transformation. Agrobacterium strain EHA105 harboring the binary vector pCAMBIA1301 that carries the genes for β-glucuronidase (uid A) and hygromycin phosphotransferase II (hpt II) was used for transformation. GUS histochemical analysis of T₀ and T₁ plants at various stages of growth followed by molecular analysis using PCR, Southern analysis and RT-PCR allowed selection of transgenics. The method resulted in 17.8% and 11.4% of the T₀ plants in Arka Gaurav and Arka Mohini being selected as chimeric and 35.0% and 29.7%, respectively, were identified as stable transformants in the T₁ generation based on PCR analysis.     

Fruits are more sensitive to salinity than leaves and stems in pepper plants (Capsicum annuum L.)

The effects of NaCl stress on plant growth, gas-exchange, activity of superoxide dismutase (SOD), rate of lipid peroxidation, and accumulation of Na⁺ ion and sugar were investigated in leaves and fruits of pepper plants (Capsicum annuum L.). Especially, the gene expression of l-galactono-1,4-lactone dehydrogenase (GalLDH), which is the last enzyme of ascorbic acid (AsA) biosynthesis, and the relationships between AsA level and Na⁺ concentration in plant tissue were investigated with increasing salinity. Plants were treated with three treatments: the control (0 mM NaCl) and two salinity levels (50 and 100 mM NaCl) for 21 days under greenhouse conditions. Plant growth was markedly restricted due to the reduction of photosynthetic rate and the increase of Na⁺ accumulation in leaves with the increasing intensity of NaCl stress. Salinity had more effect on fruit growth comparing to leaf growth, suggesting that fruits could be more sensitive to salinity than leaves. In comparison with the control, salt stress significantly increased lipid peroxidation (as measured as malondialdehyde content) but decreased SOD activity in both fruits and leaves although the effect was larger in fruits; and the rate of the decrease in SOD activity was greater than that of the increase in lipid peroxidation. The AsA concentration transiently increased first 7 days but it slightly decreased from the initial level in the end of treatment day 21. The change in GalLDH gene expression was similar to AsA concentration. The accumulation of Na⁺, the reduction of AsA level at severe salinity stress were greater in fruits than in leaves; and AsA level had a negative relationship with Na⁺ concentration in both leaves and fruits. These results suggest that the difference in salt sensitivity between fruits and leaves in pepper plants can be related to the difference in inhibition of AsA synthesis, which in turn is probably due to the toxicity of extreme accumulation of Na⁺.     

Evaluation of crucial factors for implementing shed-microspore culture of Indonesian hot pepper (Capsicum annuum L.) cultivars

A shed-microspore culture protocol was developed in Wageningen for producing doubled haploid plants in several genotypes of Indonesian hot pepper (Capsicum annuum L.). For transfer of technology to Indonesia, three factors were studied that appeared crucial for successful implementation in practice. First, application in the culture medium of a combination of the antibiotics timentin and rifampicin at the concentrations of 200 and 10 mg/l, respectively, prevented bacterial contamination from the donor explants. Second, in vitro application of colchicine (100 μM) during the first week of culture was highly effective in increasing the percentage of doubled haploid plants. Third, a comparative analysis of the ploidy level of plants regenerated from shed-microspore-derived embryos using chloroplast counts in guard cells of leaf stomata and flow cytometric measurement of leaf nuclear DNA content, revealed that the first procedure is a reliable and an easy to use method for ploidy determination with hot pepper.     

Defence responses in leaves of resistant and susceptible pepper (Capsicum annuum L.) cultivars infected with different inoculum concentrations of Phytophthora capsici Leon

Three pepper cultivars (PM-702: resistant, Demre-8 and KM-hot: susceptible) with different resistances to Phytophthora capsici-22 (P. capsici-22) were inoculated with different concentrations of zoospores to analyze the time course of phenylalanine ammonia lyase (PAL) activity, malondialdehyde (MDA), hydrogen peroxide (H₂O₂), proline and total protein.     

Refinement of shed-microspore culture protocol to increase normal embryos production in hot pepper (Capsicum annuum L.)

A low percentage of normal-looking embryos (20%) is still a problem in an efficient shed-microspore culture of hot pepper (Capsicum annuum L.) and it is more a serious problem in other androgenesic culture systems of pepper. Therefore, several factors were investigated to refine the protocol in order to increase the percentage of normal-looking embryos. The most important factors which improved the protocol and resulted in a significantly higher percentage of normal-looking embryos produces (>50%), were delayed enrichment of the liquid upper layer medium with 2.5 μM Zeatin and 5 μM IAA, and reduced incubation temperature from 28 °C to 21 °C, both after 3 weeks of culture. Addition of 1% activated charcoal in the solid lower layer of the medium enhanced the total embryo yield only, while an application of abscisic acid and increased osmolality medium had a detrimental effect on embryo production. The use of doubled haploid lines as anther donor plants has clearly decreased the variability and improved the statistical analysis of treatment effects. A higher percentage of normal embryos are produced with this refined protocol, and is therefore more suitable for implementation in the breeding programs of hot pepper.     

The genetics of non-virus vein-banding in aromatic pepper Capsicum annuum L.

Vein-banding is a genetically inherited trait controlled by one locus, Vb, which is expressed as a wave-like banding of the main and side veins of pepper leaves. It was studied as a morphological defect always present in the ‘Nsukka Yellow’ pepper, Capsicum annuum L., and was shown to be inherited as an incompletely dominant characteristic with an F₂ segregation ratio of 1:2:1. The F₁ population was uniformly mild in expression of the vein-handing characteristic of the parent ‘Nsukka Yellow’ pepper. VbVb was designated as the genetic constitution of severe vein-banding, and vbvb as a normal genotype, the F₁ being Vbvb. A selection of normal plants from the back-cross with the normal-leafed parent yield a stable normal-leafed strain of ‘Nsukka Yellow’ christened ‘UN-Yellow’.     

Evaluating variability of root size system and its constitutive traits in hot pepper (Capsicum annum L.) under water stress

The importance of root size system has long been recognized as crucial to cope with drought conditions. This investigation was conducted to: (i) evaluate the variability in root size system of hot pepper at maturity; (ii) estimate the effect of root size system on yield under drought conditions; and (iii) effect of water stress on xylem vessel development and total xylem cross-sectional area in roots of hot pepper cultivars. Twelve diverse hot pepper cultivars were grown in wooden boxes with two different water treatments, normal and in 50% water application as water deficit condition. Mean primary root length (PRL) showed a significant positive correlation with final fruit yield at normal as well as stressed condition. Total dry mass of fruit was reduced by 34.7% in drought treatments (DI) compared to full watered treatment (FI). At harvest, water-stressed plants had 21% lower root dry weight mass but higher root:shoot ratio other than FI. PRL, lateral root density, total xylem area per root cross-section showed a significant positive relationship with fruit yield. Also, lateral root density was higher in cultivars with higher xylem density, particularly in tolerant cultivars. Lateral root density (r = 0.847, P < 0.001) and total xylem cross-sectional area in root (r = 0.926, P < 0.001) were tightly related with total biomass production. The importance of root traits contributing to withstand drought in hot pepper is discussed.     

Manganese nutrition of pepper (Capsicum annuum L.): Growth,Mn uptake and fruit disorder incidence

This research included two independent yet build up objectives: (i) to examine the effects of Mn concentration in the irrigation water on the yield and fruit quality of greenhouse-grown bell pepper exposed to heat stress; and (ii) to investigate in a laboratory experiment the role of Mn addition on oxidative and anti-oxidative components of fruit apoplast. The field experiment included four concentrations of Mn in the irrigation water (0, 0.2, 0.6 and 1.0 mg L⁻¹). The effect of Mn application rate on vegetative growth and total fruit yield was insignificant and despite low Mn concentrations in zero-Mn-fed plants, no visible foliar Mn symptoms were observed. A quadratic regression was obtained between high-quality fruit yield and Mn concentration in fruit. Based on the quadratic equation, maximum high-quality yield was obtained when fruit-Mn concentration approached 44 mg kg⁻¹ DW. The correlation also indicates that variations in high-quality yield could be explained by differences in fruit-Mn concentration. The combination of low-Mn nutrition and high air temperature which prevailed during the experiment (summer season) induced severe pale spots and blemished fruits; approximately 50% of fruits were infected in zero-Mn-fed plants. In a laboratory experiment with detached-fruit under high temperature the H₂O₂ concentration increased whereas ascorbic acid concentration decreased in the apoplast. In Mn-fed fruit the H₂O₂ concentration was attenuated and the activity of ascorbate oxidase was suppressed, with the result that apoplastic ascorbic acid concentration increased. In light of the known crucial role of Mn in enzyme activities and in detoxification of oxygen free-radicals, the pale spots might be related to Mn effect on oxidative stress-related apoplastic activities. The relationships between incidence of pale spots and plant-Mn concentration may support this hypothesis, but further research is needed to validate it.     

Responses of some different pepper (Capsicum annuum L.) genotypes to inoculation with two different arbuscular mycorrhizal fungi

Eight different pepper genotypes inoculated by two different arbuscular mycorrhizal fungi (AMF) [Glomus intraradices (Gi) and Gigaspora margarita (Gm)] in a growth chamber experiment under normal seedling growing conditions were evaluated for seedling traits, colonization and relative mycorrhizal dependency (RMD). In general, inoculated plants had greater dry weights compared to non-inoculated plants. Five cultivars responded positively to inoculation with AM fungi and three responded negatively. A great variation in mycorrhizal colonization dependency was observed among the pepper genotypes, with the N52 genotype showing the highest RMD and the Karaisali genotype the lowest. RMD and dry weights of pepper genotypes were inversely correlated.     

Induced synthesis of caffeoylserotonin in pepper fruits upon infection by the anthracnose fungus, Colletotrichum gloeosporioides

Caffeoylserotonin (CaS) is a serotonin derivative that belongs to the family of phenylpropanoid amides, but has not previously been identified in plants. In this study, pepper fruits challenged with the anthracnose fungus, Colletotrichum gloeosporioides, were found to synthesize CaS, which accumulated at concentrations of up to 2.3 μg/g fresh weight. The induction of CaS was accompanied by the upregulation of both 4-coumarate-CoA ligase (4CL) and serotonin N-hydroxycinnamoyl transferase (SHT) in unripe pepper fruits. However, no induction of SHT and 4CL was observed in ripe pepper fruits consistent with the lower level of CaS production.     

Effectiveness of water pillow irrigation method on yield and water use efficiency on hot pepper (Capsicum annuum L.)

A study was conducted to elucidate the effect of water pillow (WP) irrigation method, a new alternative method to furrow irrigation, on the yield and water use efficiency (WUE) of hot pepper in a semi-arid climatic condition. In this research, treatments used were: (i) WP method and its 7-day irrigation interval (WP₇), (ii) WP method and its 9-day irrigation interval (WP₉), (iii) WP method and its 11-day irrigation interval (WP₁₁) and (iv) furrow irrigation (FI) method and its 5-day irrigation interval (control) were employed. Although the plants were grown under different irrigation methods and interval conditions, there were no statistical differences in yield and biomass of hot pepper plants between FI and WP treatments (P < 0.05). Water use efficiency (WUE) and irrigation water use efficiency (IWUE) values significantly increased with the application of WP irrigation method (P < 0.05). The highest WUE and IWUE values obtained from WP₁₁ treatment in both years. As a result, we conclude that WP method is a way to save water and increase the yield in semi-arid areas where climatic conditions require repeated irrigation in the hot pepper production area.     

Influence of growing season and donor plant age on anther culture response of some pepper cultivars (Capsicum annuum L.)

The effects of growing season and donor plant age on anther culture in Capsicum were investigated. Pepper cultivars Kekova and Sera Demre 8 were grown under unheated greenhouse conditions in winter season and field conditions in summer season. Flower buds were collected and cultured in weekly intervals from November to May in winter and from April to December in summer. The results of this study indicated that anthers from these two pepper genotypes gave different embryogenic response to seasonal effects. Kekova cultivar gave the highest embryogenic yield in summer season while this was occurred in winter season for Sera Demre 8 cultivar. The results of the study revealed that the effect of donor plant age was also an important factor in anther culture of pepper. Since 4-month-old plants gave the highest embryo yield in both cultivars in each seasons, it is possible to say that anthers collected from old plants have sufficient embryogenic response when the optimum developmental stage is selected.     

Sensitive stages of fruit and seed development of chili pepper (Capsicum annuum L. var. Shishito) exposed to high-temperature stress

The sensitivity of developmental stages to high temperature was investigated in chili pepper (Capsicum annuum L. var. Shishito). Plants were subjected to heat stress (38/30 °C day/night) immediately after anthesis for 5 or 10 days, or from 10 to 30 days after anthesis (DAA), from 30 DAA until harvest of the seeds, or immediately after anthesis until harvest of the seeds. Control plants were grown at 30/22 °C (day/night). Exposure to high temperature (heat stress) during different periods of development after anthesis adversely affected fruit growth, seed yield, and seed quality in chili pepper. Heat stress for the whole period after anthesis, and from 30 DAA until harvest reduced the growth period of chili fruits by 15 and 10 days, respectively. Heat stress from 10 to 30 DAA reduced fruit width and fruit weight. The early stage of seed development from anthesis until 10 DAA was sensitive to high temperature, which affected fruit length, fruit weight and seed set. Applying high temperatures to plants for 10 DAA increased the proportion of abnormal seeds per fruit. High temperatures from 10 DAA until 30 DAA inhibited carbohydrate accumulation and adversely affected seed germinability and vigor. These results suggest that the stage of development at which chili peppers are exposed to high temperatures is an important factor in fruit and seed growth and in seed quality.     

Chemical compositions and insecticidal effects of essential oils isolated from Achillea gypsicola, Satureja hortensis, Origanum acutidens and Hypericum scabrum against broadbean weevil (Bruchus dentipes)

The essential oils of aerial parts of Achillea gypsicola Hub.-Mor., Hypericum scabrum L., Satureja hortensis L., and Origanum acutidens (Hand.-Mazz.) Letswaart were analyzed in this study by GC and GC–MS and their oils were tested for toxicity against broadbean weevil (Bruchus dentipes). A. gypsicola oil contained camphor (40.17%), 1,8-cineole (22.01%), piperitone (11.29%), borneol (9.50%) and α-terpineol (1.56%) as major components. A total of 74 components were identified by GC–MS in H. scabrum oil, including α-pinene (9.26%), terpinen-4-ol (5.12%), camphor (5.94%), δ-cadinene (4.52%), pulegone (4.45%), γ-muurolene (4.12%), pinocarvone (3.97%) and β-caryophyllene (3.42%) as predominant components. The essential oils of O. acutidens and S. hortensis were characterized by high contents of carvacrol (86.99% and 55.74%), γ-terpinene (0.71% and 20.94%), p-cymene (1.95% and 12.30%), α-terpinene (0.13% and 2.04%) and β-caryophyllene (1.30% and 1.08%). All of the essential oils were toxic to adults of B. dentipes and insect mortality increased with increasing concentration of each oil. The oils (20 μl dose) brought about 100% mortality in 36 h. Although desirable insecticidal activities against the pest were achieved with the oils from all four plant species, S. hortensis and O. acutidens oils were more effective, particularly after 6 h of treatment. The current results concluded that the essential oils, in particular O. acutidens and S. hortensis oils, may be used as potential botanical insecticides against B. dentipes.     

Micropropagation of Capsicum frutescens L. using axillary shoot explants

A novel micropropagation protocol was established for Capsicum frutescens L. cv. ‘Uchithi’, a pungent chilli cultivar, through induction of axillary shoot proliferation of in vitro raised plantlets by decapitation and using the axillary shoots as explants for multiple shoot bud induction. About 2–6 axillary shoots were induced within 2 weeks when 4-week-old in vitro raised plantlets were decapitated. The axillary shoot-tip explants produced multiple shoot buds when cultured on Murashige and Skoog's (MS) medium containing 8.8–44.4 μM 6-benzylaminopurine (BAP) or 9.3–46.7 μM kinetin alone or 8.8–44.4 μM BAP with 4.6 μM kinetin or 5.7 and 28.5 μM indole-3-acetic acid (IAA). Maximum number of shoots (5.6) were induced on medium containing 22.2 μM BAP in combination with 4.65 μM kinetin. The separated shoots rooted and elongated on medium containing 2.8 μM IAA or 2.4–4.9 μM indole-3-butyric acid (IBA). Rooted plantlets were successfully established in the soil. Efficient mass multiplication of this important food crop was achieved.     

Anthocyanins from flowers of Hippeastrum cultivars

The anthocyanins, cyanidin 3-O-(6″-O-α-rhamnopyranosyl-β-glucopyranoside) (1) and pelargonidin 3-O-(6″-O-α-rhamnopyranosyl-β-glucopyranoside) (2), were isolated from the ornamental flowers of a Ugandan Hippeastrum cultivar by a combination of chromatographic techniques, and their structures were elucidated mainly by the use of homo- and heteronuclear nuclear magnetic resonance spectroscopy and electrospray mass spectrometry. The same anthocyanins were found in six different Hippeastrum cultivars purchased in Norway. However, the absolute amount of the anthocyanins (0.08–1.79 mg/g, fresh weight) and the relative proportions of the individual anthocyanins varied from cultivar to cultivar (13.2–96.5% of 1). The colours of the fresh petals of the three cultivars ‘Red Lion’, ‘Royal Velvet’ and ‘Liberty’ were described by the CIELab coordinates L^* (lightness), C^* (chroma) and h_ab (hue angles). All the cultivars showed hue angles corresponding to scarlet nuances (h_ab = 22–35°), with the highest value in ‘Red Lion’. The most reddish petals (in ‘Royal Velvet’) contained the highest relative proportion of 1. Thus, the in vivo colours of these cultivars seem to be correlated with the relative proportions of individual anthocyanin in the petals.