Shoot regeneration from GUS-transformed tomato (Lycopersicon esculentum) hairy root

To study the influence of genetic background on the transformation and regeneration of cultivated tomato plants, hairy root lines of tomato (Lycopersicon esculentum) were obtained by inoculating the hypocotyl explants of three tomato cultivars with the Agrobacterium rhizogenes strain DCAR-2, which harbors the pBI-121 binary vector. The Ri-T-DNA transformation into the plant DNA was confirmed by both of mikimopine and GUS assay analyses. The regeneration efficiency from hairy root explants was assessed. The data indicated that white embryonic calli were formed within two weeks in the presence of 2 mgl(-1) 2, 4-D plus 0.25 mgl(-1) kinetin. Adventitious shoots emerged from the embryonic callus in the presence of 1 mgl(-1) GA3 along with 0.5 mgl(-1) NAA. The regeneration frequency was higher in the cultivar UC-97, followed by Momotaro and then Edkawi. Molecular confirmation of the integration of the GUS gene into the hairy root-derived plants genomes was done via PCR using GUS-specific primers and also using Southern blotting analysis. Our data shows that regeneration is possible from hairy roots of the cultivated tomato and this system could be used to produce transgenic tomato plants expressing the genes present in Agrobacterium rhizogenes binary vectors.     

Proteomic analysis of tomato (Lycopersicon esculentum) pollen

In flowering plants, pollen grains are produced in the anther and released to the external environment with the primary function of delivering sperm cells to the female gametophyte. This study was conducted to identify proteins in tomato pollen and to analyse their roles in relation to pollen function. Tomato is an important crop which is grown worldwide and is an excellent experimental system. Proteins were extracted from pollen, separated by two-dimensional gel electrophoresis (2-DE), and identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and peptide mass fingerprinting. Of the 960 spots observed on Colloidal Coomassie Blue (CCB)-stained 2-DE gels, 190 were selected for analysis. Of these, 158 spots, representing 133 distinct proteins, were identified by searching the NCBInr and Expressed Sequence Tag databases. The identified proteins were classified based on designated functions and the majority included those involved in defence mechanisms, energy conversions, protein synthesis and processing, cytoskeleton formation, Ca(2+) signalling, and as allergens. A number of proteins in tomato pollen were similar to those reported in the pollen of other species; however, several additional proteins with roles in defence mechanisms, metabolic processes, and hormone signalling were identified. The potential roles of the identified proteins in the survival strategy of the small, independent, two-celled pollen grain of tomato, and subsequently in pollen germination and tube growth are discussed.     

Factors influencing transformation frequency of tomato (Lycopersicon esculentum)

We developed an efficient procedure for transformation and regeneration of L. esculentum cv. Moneymaker from cotyledon explants. The effect of two parameters on the transformation frequency was investigated in detail. The use of feeder layers during cocultivation proved to be critical. In addition, it was found that Agrobacterium strains harbouring a L,L-succinamopine type helper plasmid yielded significantly higher transformation frequencies than those with octopine or nopaline type helper plasmids. The optimized protocol was used to obtain transformation frequencies averaging 9%. Of the plants produced approximately 80% proved to be diploid, of which 67% contained the transgene(s) on a single locus.Abbreviations 2,4D 2,4-dichlorophenoxy-acetic acid - gus ß-glucoronidase - IAA indoleacetic acid - LB Luria-Bertani - NPTII neomycin phosphotransferase II - ZR zeatin riboside     

Development and differentiation of haploid Lycopersicon esculentum (tomato)

Summary Haploid callus cultures of selected races of Lycopersicon (tomato) species can be obtained from anther culture. This is a further demonstration of a proposed general method of haploid culture developed with Arabidopsis thaliana. Differentiation of haploid callus of Lycopersicon esculentum can be controlled both in the dark and the light by hormones added to defined minimal media. Development to plantlets is achieved only in the light. Callus cells can be induced to develop into seedless pseudo-fruits. Chromosome counts on callus cells or root-tip cells establishes haploidy (n=12).Haploidy can be maintained in culture on defined minimal media for at least one year.     

Isolation of signaling mutants of tomato (Lycopersicon esculentum)

As a first step towards developing a genetic system for investigating signaling processes in plants, we have developed a screen for signaling mutants deficient in a wound response. We have isolated two mutants of tomato that lack detectable production of proteinase inhibitors induced systemically in leaves by wounding. The mutants are deficient in the induction of both proteinase Inhibitor I and proteinase Inhibitor II but can be induced to respond at near wild-type levels by methyl jasmonate, a known elicitor of inhibitor production in tomato. While completely deficient in systemic production of proteinase inhibitors, both mutants produce some proteinase inhibitor in wounded leaves. This evidence suggests the existence of two signaling pathways, one local and one systemic, that regulate the induction of proteinase inhibitor snythesis in response to wounding.     

Characterization of the subtilase gene family in tomato (Lycopersicon esculentum Mill.)

The gene family of subtilisin-like serine proteases (subtilases, SBTs) in tomato (Lycopersicon esculentum Mill.) comprises at least 15 members, 12 of which have been characterized in this study. Sequence comparison revealed that tomato subtilases fall into 5 distinct subfamilies. Single genes were shown to exist for LeSBT1, LeSBT2 and tmp, while 5 and 6 genes were found in the LeSBT3/4 and P69 subfamilies, respectively. With the exception of tmp, tomato subtilase genes were found to lack introns. Expression of subtilase genes was confirmed at the mRNA level by northern blot analysis and/or by primer extension experiments. For each of the 5 subtilase subfamilies, a distinctive pattern of expression was observed in tomato organs. At least one of the subtilases was found to be expressed in each organ analysed. Structural features evident from deduced amino acid sequences are discussed with reference to the related mammalian proprotein convertases.     

NMR characterization of endogenously O-acetylated oligosaccharides isolated from tomato (Lycopersicon esculentum) xyloglucan

Eight oligosaccharide subunits, generated by endoglucanase treatment of the plant polysaccharide xyloglucan isolated from the culture filtrate of suspension-cultured tomato (Lycopersicon esculentum) cells, were structurally characterized by NMR spectroscopy. These oligosaccharides, which contain up to three endogenous O-acetyl substituents, consist of a cellotetraose core with alpha-D-Xylp residues at O-6 of the two beta-D-Glcp residues at the non-reducing end of the core. Some of the alpha-D-Xylp residues themselves bear either an alpha-L-Arap or a beta-D-Galp residue at O-2. O-Acetyl substituents are located at O-6 of the unbranched (internal) beta-D-Glcp residue, O-6 of the terminal beta-D-Galp residue, and/or at O-5 of the terminal alpha-L-Arap residue. Structural assignments were facilitated by long-range scalar coupling interactions observed in the high-resolution gCOSY spectra of the oligosaccharides. The presence of five-bond scalar coupling constants in the gCOSY spectra provides a direct method of assigning O-acetylation sites, which may prove generally useful in the analysis of O-acylated glycans. Spectral assignment of these endogenously O-acetylated oligosaccharides makes it possible to deduce correlations between their structural features and the chemical shifts of diagnostic resonances in their NMR spectra.     

A major QTL introgressed from wild Lycopersicon hirsutum confers chilling tolerance to cultivated tomato (Lycopersicon esculentum)

Many plants of tropical or subtropical origin, such as tomato, suffer damage under chilling temperatures (under 10°C but above 0°C). An earlier study identified several quantitative trait loci (QTLs) for shoot turgor maintenance (stm) under root chilling in an interspecific backcross population derived from crossing chilling-susceptible cultivated tomato (Lycopersicon esculentum) and chilling-tolerant wild L. hirsutum. The QTL with the greatest phenotypic effect on stm was located in a 28 cM region on chromosome 9 (designated stm9), and enhanced chilling-tolerance was conferred by the presence of the Lycopersicon hirsutum allele at this QTL. Here, near-isogenic lines (NILs) were used to verify the effect of stm9, and recombinant sub-NILs were used to fine map its position. Replicated experiments were performed with NILs and sub-NILs in a refrigerated hydroponic tank in the greenhouse. Sub-NIL data was analyzed using least square means separations, marker-genotype mean t-tests, and composite interval mapping. A dominant QTL controlling shoot turgor maintenance under root chilling was confirmed on chromosome 9 using both NILs and sub-NILs. Furthermore, sub-NILs permitted localization of stm9 to a 2.7 cM interval within the original 28 cM QTL region. If the presence of the L. hirsutum allele at stm9 also confers chilling-tolerance in L. esculentum plants grown under field conditions, it has the potential to expand the geographic areas in which cultivated tomato can be grown for commercial production.     

Herpetomonas spp. isolated from tomato fruits (Lycopersicon esculentum) in southern Spain

A flagellate of the family Trypanosomatidae was isolated from fruits of Lycopersicon esculentum (tomato) in southeastern Spain. The isolate was successfully adapted to in vitro culture in monophasic media. The morphology showed the kinetoplast to be positioned towards the middle of the body, and the typical opistomastigote form characteristic of members of the genus Herpetomonas. Amplification of the mini-exon gene was negative, whilst for the 5S ribosomal rRNA gene the result was positive. The DNA sequence was obtained and its alignment with other trypasomatids, obtained using the BLAST algorithm, suggested it was closely related to Herpetomonas samuelpessoai.     

The effect of growth regulators on tomato (Lycopersicon esculentum) fertility

The aim of the study was to determine the effect of 2,4 D (2,4dichlorophenoxyacetic acid) used separately and in a mixture with BAP (benzylaminopurine) on fertility and some features of tomato fruit. The plant flowers were immersed only once in water solutions of the growth regulators (2,4 D 0.001 %; 0.005 %; 2,4 D 0.001 % + BAP 0.001 %; 2,4 D 0.005 % + BAP 0.005 %). The mean fruit weight, fruit volume, specific weight, and the jelly weight were determined. The number of seeds collected from the fruit was used as a criterion of fertility estimation. No statistically significant differences were found in the mean fruit weight, fruit volume and specific weight. The fruits derived from the plants which were not exposed to the action of growth regulators were characterized by the smallest amount of jelly while the fruits set under the influence of 0.001 % 2,4 D + 0.001 % BAP had the largest jelly amount. The greatest differentiation was found in fertility which ranged from 7.5 seeds from the fruit derived from the plants treated with 0.005 % 2,4 D, to 75.7 seeds from the non-treated plants’ fruit. The BAP addition caused the limitation of fertility reduction.     

Effect of temperature on biomass allocation in tomato (Lycopersicon esculentum)

Temperature may influence dry matter partitioning between fruits and vegetative plant parts either directly or indirectly through its influence on development, flower and/or fruit abortion. The objective of the present work was to investigate whether there is any direct effect of temperature on dry matter partitioning between fruits and vegetative plant parts in tomato. A greenhouse experiment was conducted, with alternating 3-week periods of high (23°C) and low (18°C) temperature setpoint. Dry matter partitioning during these 3-week periods was determined from destructive plant harvests at two levels of fruit pruning (3 and 7 fruits per truss). Indirect temperature effects on dry matter partitioning were excluded by fruit pruning.
On average, the fraction of dry matter distributed to the fruits during a 12-week period, starting with the flowering of the fifth truss (28 days after planting), was 0.53 (3 fruits per truss) and 0.70 (7 fruits per truss). These ratios were also calculated for every 3-week period separately and did not depend on the average temperature (18–24°C) during that period.
It is concluded that dry matter distribution in tomato is not significantly affected by temperature directly, which means that the temperature effect (18–24°C) on the generative sink strength is not much different from the temperature effect on the vegetative sink strength.     

The histogenesis of somaclones from tomato (Lycopersicon esculentum Mill.) cotyledons

Summary A histological study ofin vitro cultured cotyledonary expiants of tomato (Lycopersicon esculentum) was performed in order to determine the site (differentiated tissue or developing callus) and the mode of plant regeneration.Results have shown that callus develops at the excision sites of cotyledonary expiants and that shoots are formed exclusively within the unorganized callus: excision areas are the only morphogenetic sites and the proximal excision is the preferred site for plant regeneration.Shoots differentiate by organogenesis within the superficial region of the callus. Few neocambial cells cooperate in the neoformation. Origin from a single cell is highly unlikely since rarely observed single activated cells never developed into shoots.Regenerated plants may be chimeras if invitro culture induces genetic diversity in the initial cells.Abbreviations IAA Indole-3-acetic acid - c callus - d vegetative dome - s shoot - ad adaxial - ab abaxial - t tracheid - p parenchyma - S sieve tube     

Isolation and characterization of endophytic Streptomyces strains from surface-sterilized tomato (Lycopersicon esculentum) roots

AIMS: To isolate endophytic Streptomyces strains from tomato and examine their antimicrobial activity. METHODS: Endophytic Streptomyces strains were isolated using surface-sterilization methods and identified by morphological characteristics. Antimicrobial activities were measured by the agar plate sensitivity method. Antifungal activity in vivo was measured by seedling mortality in infested soils. RESULTS: Twenty-one per cent of endophytic streptomycete isolates produced antibacterial metabolites and 41% produced antifungal metabolites in S medium. Sixty-five per cent of the most frequently isolated strains inhibited the growth of Rhizoctonia solani by the antibiosis assay but only 32% produced metabolites active against R. solani in S medium. Growth promotion and enhanced disease resistance of seedlings inoculated with Streptomyces sp. strain S30 were observed in tomato but not in cucumber seedlings. CONCLUSIONS: Endophytic Streptomyces spp. strains were successfully isolated using stringent methods and strain S30 promoted growth and enhanced resistance to R. solani in tomato seedlings. SIGNIFICANCE AND IMPACT OF THE STUDY: Endophytic streptomycetes showing antifungal activity in vitro and in vivo may indicate the potential for their use as biocontrol agents particularly of R. solani disease of tomato seedlings.     

Cadmium toxicity in tomato (Lycopersicon esculentum) plants grown in hydroponics

The effects of Cd have been investigated in tomato (Lycopersicon esculentum) plants grown in a controlled environment in hydroponics, using Cd concentrations of 10 and 100 μM. Cadmium treatment led to major effects in shoots and roots of tomato. Plant growth was reduced in both Cd treatments, leaves showed chlorosis symptoms when grown at 10 μM Cd and necrotic spots when grown at 100 μM Cd, and root browning was observed in both treatments. An increase in the activity of phosphoenolpyruvate carboxylase, involved in anaplerotic fixation of CO₂ into organic acids, was measured in root extracts of Cd-exposed plants. Also, significant increases in the activities of several enzymes from the Krebs cycle were measured in root extracts of tomato plants grown with Cd. In leaf extracts, significant increases in citrate synthase, isocitrate dehydrogenase and malate dehydrogenase activities were also found at 100 μM Cd, whereas fumarase activity decreased. These data suggest that at low Cd supply (10 μM) tomato plants accumulate Cd in roots and this mechanism may be associated to an increased activity in the PEPC–MDH–CS metabolic pathway involved in citric acid synthesis in roots. Also, at low Cd supply some symptoms associated with a moderate Fe deficiency could be observed, whereas at high Cd supply (100 μM) effects on growth overrule any nutrient interaction caused by excess Cd. Cadmium excess also caused alterations on photosynthetic rates, photosynthetic pigment concentrations and chlorophyll fluorescence, as well as in nutrient homeostasis.     

Cloning and characterization of the gene for phytoene desaturase (Pds) from tomato (Lycopersicon esculentum)

The gene Pds encodes phytoene desaturase, a key enzyme in carotenoid biosynthesis that converts phytoene to -carotene. We have cloned and analyzed the genomic DNA sequence of Pds from tomato. In tomato Pds is comprised of 15 exons that, together with the introns occupy over 8 kb. A putative promoter sequence has been identified by comparison with the cDNA sequence of Pds. A consensus nucleotide sequence around intron splicing sites in tomato genes was determined by compiling data on 137 introns in 34 genes. This consensus sequence generally agrees with the consensus sequence of other higher plants with only minor differences that are unique to tomato.     

Mapping of ripening-related or -specific cDNA clones of tomato (Lycopersicon esculentum)

Summary Nineteen ripening-related or -specific clones from Lycopersicon esculentum were mapped via RFLP analysis using an F₂ population from the cross L. esculentum x L. pennellii and cDNA or genomic clones of known map location. The map produced using cDNA and genomic clones of known map location corresponded well with previously published maps of tomato. The number of loci detected for each ripening-related or-specific clone varied from one to seven. These loci were located on all 12 chromosomes of the tomato genome. There was no significant clustering of ripening-related or-specific genes. Regions of very low recombination were observed. The clone for polygalacturonase (TOM6) mapped to a single region on chromosome 10, the same chromosome as the nor and alc ripening mutants. To fine map this chromosome, two backcross populations were produced from the cross of L. esculentum x L. pimpenillifolium, in which the esculentum parents used were homozygous for either the alc or the nor. The coding region for polygalacturonase is functionally unlinked to either of these two ripening mutants.