The insertion of the Agrobacterium rhizogenes rolC gene in tomato (Solanum lycopersicum L.) affects plant architecture and endogenous auxin and abscisic acid levels

In the present paper we report on the effects of the insertion of the Agrobacterium rhizogenes rolC gene in the tomato (Solanum lycopersicum L., formerly Lycopersicon esculentum Mill.) cultivar Tondino. Several transgenic lines were successfully obtained, between which two clones, rolC1 and rolC3, were chosen for the analysis of morpho-productive traits as well as of the endogenous levels of auxin and abscisic acid. Consistent with the known phenotypic effect of this gene, the transformed tomato plants were significantly shorter than the corresponding controls. On the other hand, even if yield was not affected by the transformation in terms of average number of fruits produced, fruit weight was significantly lower in the transgenics with respect to the controls. Therefore, insertion of the rolC gene does not lead to an improvement in plant productivity.     

Improved protocol for Agrobacterium mediated transformation of tomato and production of transgenic plants containing carotenoid biosynthetic gene CsZCD

Improved protocol for Agrobacterium mediated transformation of tomato (Lycopersicon esculentum) Micro-Tom was developed to use in corporation of the carotenoid biosynthetic genes CsZCD (Crocus zeaxanthin 7,8-cleavage dioxygenase). From these experiment, a transformation methodology using explants from cotyledons cultured for 1 day on the medium with zeatin 2 mg/L, IAA 0.1 mg/L, carefully submerged in the Agrobacterium inoculum for 20 min, then concultured with the agrobacterium for 3 days on the same medium, followed by a transfer to the same medium with 500 mg/L cefotaxin for 3 days and then by a transfer to the same medium with 100 mg/L kanamycin and 500 mg/L carabenillin for 6–8 weeks and resulted in a greater than 20% transformation efficiency in the concentration of Agrobacterium OD600 = 0.2 tested. In this transformation method, no feeder layers were used and the subculture media was minimal. Among the Agrobacterium concentrations of OD600 = 0.2, 0.5 and 1.0, the best transformation efficiency, 20.87%, was obtained by using OD600 = 0.2, which was significantly higher than that of OD600 = 1.0. The presence of the inserted target genes was checked using a rapid and efficient PCR test. The protocol was successfully employed in the production of transgenic Micro-Tom tomato containing the carotenoid biosynthesis CsZCD under constructive promoter. This procedure represents a simple, efficient and general means of transforming tomato.     

Diversity,relationships, and genetic fingerprinting of the Listada de Gandía eggplant landrace using genomic SSRs and EST-SSRs

Listada de Gandía is one of the most renowned Spanish eggplant (Solanum melongena L.) landraces. Assessing its genetic diversity and relationships, as well as devising tools for its identification, is of great relevance for the enhancement and protection of this landrace. Forty-two eggplant accessions, which included 25 Striped accessions, of which 19 were of the Listada type (six accessions of Listada de Gandía, eight of Other Spanish Listada, and five of Non-Spanish Listada) and six of the Other Non-Spanish Striped group, and 17 Non-Striped accessions were characterized with 17 genomic SSRs and 32 EST-SSRs. Genomic SSRs had, as a mean, a greater polymorphism and polymorphic information content (PIC) than EST-SSRs. Although Listada de Gandía proved to be genetically diverse, specific and universal alleles for two SSR markers were found for this landrace. All the Listada accessions cluster together in the multivariate PCoA and UPGMA phenograms performed, and are separated from the Other Non-Spanish Striped and Non-Striped accessions. Also, Listada de Gandía accessions were clearly differentiated from the Other Spanish Listada and Non-Spanish Listada accessions in these analyses. SSR markers revealed of great utility to obtain a specific fingerprint for the Listada de Gandía eggplant as well as to establish the uniqueness and distinctness of this landrace. This information will be very helpful for the enhancement and protection from imitation of Listada de Gandía, and contributes to support its potential recognition with a Protected Designation of Origin (PDO) status.     

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.     

Root restriction-induced limitation to photosynthesis in tomato (Lycopersicon esculentum Mill.) leaves

Root restriction often depresses photosynthetic capacity and the mechanism for this reduction, however, remains unclear. To identify the mechanism by which root restriction affects the photosynthetic characteristics, tomato (Lycopersicon esculentum Mill.) seedlings were subjected to root restriction stress with or without supplemental aeration to the nutrient solution. With the development of the root restriction stress, CO₂ assimilation rate was decreased only in confined plants without supplemental aeration. There were also significant decreases in leaf water potential, stomatal conductance (g_s), intercellular CO₂ concentration (C_i), and increases in the stomatal limitation (l) and the xylem sap ABA concentration. Meanwhile, the maximum carboxylation rate of Rubisco (V_cmax) and the capacity for ribulose-1,5-bisphosphate regeneration (J_max) also decreased, followed by substantial reductions in the quantum yield of PSII electron transport (Φ_PSII). Additionally, root restriction resulted in accumulation of carbohydrates in various plant tissues irrespective of aeration conditions. It is likely that root restriction-induced depression of photosynthesis was mimicked by water stress.     

Response of Vitis vinifera L. plants inoculated with Phaeoacremonium angustius and Phaeomoniella chlamydospora to thiabendazole,resveratrol and sodium arsenite

The effect of some pesticides (sodium arsenite, thiabendazole and ziram) and the natural phytoalexin (resveratrol) on mycelial growth of Phaeomoniella chlamydospora and Phaeoacremonium angustius was studied. Several strains of these species were grown on malt extract agar (MEA) plates containing different concentrations of inhibitory compounds and colony diameters were measured. While sodium arsenite and ziram had little effect on the growth of both species, thiabendazole inhibited colony growth, at minimum concentrations of 6 μM for Ph. chlamydospora and 15 μM for P. angustius. Resveratrol at concentrations equal to or grater than 867 μM also inhibited colony growth of both species. To assay the effect of these substances on plant response to infection, in vitro grapevines were inoculated with Ph. chlamydospora or P. angustius spores and were grown in the presence of sodium arsenite (0–30 μM), thiabendazole (0–30 μM) or resveratrol (0–876 μM). Infected untreated plants and sodium arsenite-treated plants developed symptoms of senescence (reduced growth, increased membrane lipid peroxidation, and decreased chlorophyll content and fluorescence). In contrast, infected plants treated with thiabendazole (30 μM) or with resveratrol (876 μM) showed similar characteristics of fresh weight, malondialdehyde accumulation and chlorophyll fluorescence to those of uninfected plants. These results are promising with respect to the use of thiabendazole-containing pesticides as alternatives to currently used pesticides for control of esca in vines. Results also suggest that the presence of resveratrol in grapevines may be useful to induce resistance to these fungi.     

Application of chlorophyll fluorescence for the diagnosis of salt stress in tomato “Solanum lycopersicum (variety Rio Grande)”

To study the response of tomato (Solanum lycopersicum cv. Rio Grande) to salinity, the effect on plant growth, water relations, stomatal conductance and Chlorophyll fluorescence was investigated. Tomato plants were grown in peat culture under controlled conditions and submitted during 28 days to saline stress ranging from 0 to 25, 50, 100, 150 and 200 mM of NaCl. At the end of the experiment period, plant growth was significantly decreased with increasing salinity.     

The effect of sucrose on the expression of the VvTFL1 and VFL genes during flower development in the “Xiangfei” grapevine

VFL and VvTFL1 genes expression patterns and the effects of sucrose on the expression of VFL and VvTFL1 genes in different organs of the “Xiangfei” grapevine (Vitis vinifera L.) were investigated. VvTFL1 gene expression was detected in the meristem of the apical bud and lateral bud, but was not detected during inflorescence differentiation and flower organ development. After sucrose treatment, VvTFL1 gene expression increased in the apical bud, but decreased in the lateral bud. These results suggested that the VvTFL1 gene might be mainly involved in the apical growth process of shoots, and exogenous sucrose had an effect on the VvTFL1 gene by increasing shoot apical meristem initiation of apical buds. The VFL gene was expressed primarily during inflorescence differentiation and early flower organ development, but it gradually reduced in later flower development. After sucrose treatment, VFL gene expression increased in the inflorescence and small or middle flower, but a little change was seen in the large flower. These results suggested that the VFL gene plays important roles in the initiation of inflorescence meristems and the morphological formation of flower organs. Exogenous sucrose had an effect on VFL gene expression at the early stage of flower development.     

Alteration of tomato fruit quality by root inoculation with plant growth-promoting rhizobacteria (PGPR): Bacillus subtilis BEB-13bs

In this study the effect of inoculation of tomato (Lycopersicon esculentum Mill.) roots with plant growth-promoting rhizobacteria (PGPR) on yield and fruit quality was evaluated. The control treatment was non-inoculated (CTL) and the PGPR treatment was inoculated with Bacillus subtilis BEB-lSbs (BS13). Yield per plant and marketable yield, as well as fruit weight and length were increased by the BS13 treatment when compared to the CTL treatment. Texture of red fruits was also enhanced by the BS13 treatment compared to that in the CTL treatment. These results demonstrated that PGPR have positive effects on tomato fruit quality attributes, particularly on size and texture.     

Chitinase and β-1,3-glucanase enzymatic activities in response to infection by Alternaria alternata evaluated in two stages of development in different tomato fruit varieties

Chitinase (ChA) and β-1,3-glucanase (GA) activity had been related with plant defense mechanism against pathogen attack in vegetative tissues. Scarce information is available about the behavior of these enzymes in response to different stages of development and fungi infection in fruits. The changes in ChA and GA activities in response to Alternaria alternata infection were evaluated in mature green (MG) and red ripe (RR) developmental stages of Sunpride, Geronimo and Charleston varieties of tomato fruit. Tomato fruits were inoculated with a conidial suspension of A. alternata and stored for 10 days at 25 °C and 90–92 H.R.%. The degree of fruit infection was measured by a hedonic scale every 2 days. ChA activity was determined fluorometrically by quantifying the release of 4-methylumbelliferyll (4-MU) from 4-methylumbelliferyll β-d-N,N′N″-triacetylchitotrioside, and GA activity was measured quantifying the release of glucose from β-1,3-glucan (laminarin) by HPLC. Tomato fruit in RR stage was more susceptible to fungi infection than MG stages. Geronimo was the most resistant variety, whereas Sunpride was the most susceptible for both stages of development (MG and RR). Higher levels of ChA and GA activities were observed for mature green stage in Charleston variety at the end of the storage period. An induction in ChA and GA in response to infection by A. alternata was observed in all varieties. Particularly high levels of ChA were found for inoculated Geronimo in RR stage and inoculated Charleston in MG stage which correlated with low levels of fungi infection. Higher levels of GA induction in response to fungi infection were recorded for Sunpride variety in RR stage, whereas no substantial induction was observed for Geronimo and Charleston varieties at the same stage of development. This GA induction correlated negatively with the resistance showed by the different varieties to fungi infection. We concluded that chitinase and β-1,3-glucanase induction are part of the tomato fruit defense mechanism against A. alternata infection with a different behavior depending upon stage of development and variety.     

Translocation of heavy metals to tomato (Solanum lycopersicom L.) fruit irrigated with treated wastewater

A field experiment was conducted to investigate the extent of translocation of heavy metals to tomato (Solanum lycopersicom L. cvs. “GS₁₂” and “RS₅₈₉₉₅₆”) fruit produced in an open field near to Abu-Nusiar Wastewater Treatment Plant, Amman-Jordan. Seedlings were planted during the seasons of 1999 and 2000 and furrow irrigated with different mixtures of potable water to treated wastewater {100%:0% (1:0, control); 25%:75% (1:3); 50%:50% (1:1); 0%:100% (0:1)}. Tomato fruit, soil and water were examine for heavy metals concentrations, and changes in the pH and electrical conductivity (EC) of the soil were tested. The obtained results revealed an increase in the concentrations of Cu, Mn and Fe, in the soil correlated with high concentrations found in the wastewater. Additionally, an increase in the pH and EC in the soil was observed with increasing the proportions of wastewater. Results of tomato fruit analysis showed an increased concentration of Fe, Cu, Ni, Mn and Zn in the cultivar “RS₅₈₉₉₅₆”, whereas, an increased concentration of Mn and Zn were detected in the cultivar “GS₁₂”, but no accumulation of Cd and Pb in both cultivars. The accumulation of heavy metals in fruit was below the Jordanian standard limits, thus, the use of treated wastewater in irrigation might be feasible.     

The culture of isolated microspores of ornamental kale (Brassica oleracea var. acephala) and the importance of genotype to embryo regeneration

The culture of isolated microspores of kale (Brassica oleracea var. acephala) was studied including the importance of genotype to embryo regeneration, medium composition chiefly the sucrose concentration and the use of colchicine, simultaneously medium renovation. It was initiated using 29 different genotypes as donor plants. Embryos were induced from six of the kale genotypes and these corresponded to the more out-bred genotypes. Embryogenesis was achieved using four different combinations of culture media: (a) microspores initially cultured in NLN medium supplemented with 13% (w/v) sucrose (NLN-13) for 48 h, followed by transfer to fresh NLN-13 medium; (b) microspores cultured for 48 h in NLN-13 medium supplemented with colchicines (50 mg/L) followed by transfer to unsupplemented NLN-13 medium; (c) microspores cultured for 48 h in NLN-16 medium supplemented with colchicines (50 mg/L) followed by transfer to unsupplemented NLN-16 medium; (d) microspores cultured for 48 h in NLN-16 medium supplemented with colchicines (50 mg/L) followed by transfer to unsupplemented NLN-13 medium. The embryos obtained from four of the genotypes developed into plantlets and these regenerated plants have been successfully transplanted to soil.     

Production of transgenic sweetpotato plants resistant to stem nematodes using oryzacystatin-I gene

Stem nematode (Ditylenchus destructor Thorne) is one of most serious diseases limiting sweetpotato (Ipomoea batatas (L.) Lam.) production, and it is urgent to develop sweetpotato varieties resistant to this disease. In present study, we have developed transgenic sweetpotato (cv. Xushu 18) plants resistant to stem nematodes using oryzacystatin-I (OCI) gene with Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harbors a binary vector pCAMBIA1301 with OCI gene, uidA gene and hptII gene. Selection culture was conducted using 7 mg/l hygromycin. A total of 2119 plants were produced from the inoculated 1710 cell aggregates of Xushu 18 via somatic embryogenesis. GUS assay and PCR analysis of the regenerated plants randomly sampled showed that 92.8% of the regenerated plants were transgenic plants. Transgenic plants exhibited enhanced resistance to stem nematodes compared to the untransformed control plants by the field evaluation and the inoculation test with stem nematodes and stem nematode-resistant plants were selected from the transgenic plants. Stable integration of the OCI gene into the genome of resistant transgenic plants was confirmed by Southern blot analysis, and the copy number of integrated OCI gene ranged from 1 to 3. High level of OCI gene expression in the resistant transgenic plants was demonstrated by real-time quantitative PCR analysis. This study provides a way for improving stem nematode resistance of sweetpotato.     

Isolation and expression analysis of a cobalamin-independent methionine synthase gene from the parasitic plant Orobanche ramosa

Broomrapes (Orobanche spp) are holoparasitic weeds that cause devastating losses in many economically important crops. The branched broomrape (Orobanche ramosa) represents a real threat for many vegetable crops including tobacco, tomato and potato.     

DNA content,morphometric and molecular marker analyses of Citrus limonimedica, C. limon and C. medica for the determination of their variability and genetic relationships within the genus Citrus

This work investigated the fingerprinting and phenotyping of Citrus germplasm; species selected were of historical importance belonging to Citrus limonimedica Lush. and its supposed ancestors, along with some other species of the Citrus genus. An integrated approach based on the exploitation of nuclear DNA content, morphological traits and molecular markers, such as RAPD fingerprints and ITS-based SNPs, was employed. We studied a core collection of 54 distinct accessions, including 43 genotypes of the Citrus species (18 species or supposed species) and 11 genotypes of the Poncirus genus, which was used as the reference outgroup. Morphological trait analysis and statistical analysis of DNA content and markers were useful for reconstructing a Citrus phylogeny. In particular, our experiments aimed at estimating the genetic variation within and the genetic relatedness among C limon (L.) Burm., C. limonimedica and C. medica L. to shed light on the hybrid origin hypothesis of C. limonimedica. The results of the multidisciplinary analyses allowed us to confirm a remarkable differentiation between Poncirus and Citrus genera and to highlight a close relationship among the three investigated Citrus species but a distinct difference between these three species and other species in the Citrus genus. RAPD fingerprints and ITS polymorphisms enabled us to point out a variation gradient between lemon and citron, with C. limonimedica as a possible intermediate species. Some accessions of C. medica and C. limonimedica that deviate from such a trend suggest recurrent introgression and/or hybridisation with other species of Citrus.     

Photosynthesis of field-grown Arracacha (Arracacia xanthorriza Bancroft) cultivars in relation to root-yield

There has been limited research on measuring potential differences in leaf gas exchange of Arracacha (Peruvian parsnip, Arracacia xanthorriza Bancroft) cultivars, as affected by different environments, as well as its relation to storage root-yield. The present paper reports field measurements of leaf CO₂ assimilation rates (A) for five contrasting cultivars grown at two different high-altitude locations. Using a design of plots chosen at random with three repetitions, commercial root production was determined in the two locations at different altitude (1580 and 1930 m). Daily leaf gas exchange was repeatedly monitored with a portable open-mode infrared gas analyzer at different times in both locations during the growth cycle. Root-yield, leaf area and dry weight were measured. Significant differences in leaf photosynthetic rate and in specific leaf area (SLA) were observed among cultivars. Cultivars with high SLA, had high CO₂ assimilation. Mean (A_n) and total (A_tot) of CO₂ assimilation and SLA were significantly correlated with storage root-yield across cultivars and locations. The three cultivars with the greatest commercial root production also had the highest maximum values for A and the highest specific leaf area, indicating that these two parameters can be used to select for highly productive cultivars of A. xanthorriza.     

Effects of plastic mulches on root zone temperature and on the manifestation of tomato spotted wilt symptoms and yield of tomato

Tomato spotted wilt (TSW) disease is a serious constraint to tomato production in various regions of the world. The effect of TSW on tomato yield is largely influenced by time of infection. Early infection usually results in severe stunting of the seedling and even death of the plant. Plastic film mulches affect both the incidence of TSW, and plant growth and yield of tomato. The objective of the present study was to determine the effect of root zone temperature (RZT) as affected by plastic film mulch on the manifestation of symptoms of TSW, and growth and yield of tomato plants either artificially inoculated with tomato spotted wilt virus (TSWV) or under natural TSW infection. In artificially inoculated plants as well as in plants under natural TSW infection, vegetative top fresh weight (FW) and fruit FW both increased with the length of time after transplanting that the plants remained free from TSW symptoms. The root zone temperature was highest under black mulch (seasonal mean = 27.5 °C), followed by gray (27.0 °C), silver (25.8 °C), and white (24.8 °C) mulches. The plants grown on black mulch showed the earliest appearance of TSW symptoms, and had significantly reduced vegetative growth and fruit yields compared to plants on the other mulches. In conclusion, utilization of plastic mulches that created conditions of high RZT stress resulted in reduced plant growth and yield and predisposed the plants to earlier expression of TSW symptoms compared to plants grown at RZTs more favorable to tomato plant growth (optimal RZT = 26.1 °C [Díaz-Pérez, J.C., Batal, K.D., Granberry, D., Bertrand, D., Giddings, D., Pappu, H., 2003. Vegetative top growth and yield of tomato grown on plastic film mulches as affected by the appearance of symptoms of Tomato spotted wilt virus. HortScience 38, 395–399]). Since these plant responses to TSW under heat stress occurred in artificially inoculated plants as well as in plants under natural TSW infection, high RZTs probably affected the plants directly, independently of any possible effects on the thrips vectors.     

Interspecific variations in seed germination of Corylopsis

This study was initiated to investigate the differences in germination percentages and rates between Corylopsis coreana Uyeki and Corylopsis sinensis var. calvescens Rehder & E.H. Wilson following a warm stratification (WS) and cold stratification (CS), and to study the effect of different WS temperatures interacting with different durations of CS. Warm stratification at 10 °C, 15 °C, 20 °C, and 25 °C was given for 1 month (1 M 10 °C, 15 °C, 20 °C, and 25 °C WS) followed by 0 M, 1 M, 2 M, and 3 M of CS at 5 °C (0 M, 1 M, 2 M, 3 M CS) and seeds were germinated in an air conditioned greenhouse maintained at 18.5 °C/18 °C. On average, less than 1% of C. coreana seeds germinated when sown without any WS and CS or with 1 M 15 °C, 20 °C, and 25 °C WS without CS treatment. However, 26% C. coreana seeds germinated after 1 M 10 °C WS without any CS treatment. Germination was not affected by WS temperatures when followed by 2 M 5 °C CS. It is concluded that C. coreana exhibited low seed germination at 10 °C and that this temperature could be considered the upper limit of CS for C. coreana. Only 2 M CS was required for more than 90% seeds to germinate. However, C. sinensis var. calvescens required longer than 3 M CS for more than 29% seeds to germinate. This clearly shows that there is an interspecific variation in optimum dormancy-breaking requirements.