The development of bridge lines for interspecific gene transfer between Lycopersicon esculentum and L. peruvianum

Summary Using a modified embryo callus culture technique, hybrids between Lycopersicon esculentum and L. peruvianum were developed and their usefulness as bridge lines for facilitating interspecific gene transfer was evaluated. Four of these lines showed a high level of sexual compatibility with several other L. peruvianum var. typicum accessions, as well as with accessions of L. peruvianum var. humifusum and L. peruvianum var. glandulosum and L. esculentum. These bridge line x L. peruvianum hybrids could be crossed with L. esculentum to introgress genes from L. peruvianum into L. esculentum.     

Alterations of the manifestations of hybrid breakdown in Lycopersicon esculentum L. pennellii F2 populations containing L. esculentum versus L. pennellii cytoplasm

Reproductive abnormalities reduced the percent stainable pollen, and fruit and seed set in interspecific F₂ populations derived from crosses of Lycopersicon esculentum and L. pennellii but were not observed in parental lines and interspecific F₁ populations. The degree to which these reproductive abnormalities were expressed in the interspecific F₂ populations was affected by cytoplasm. Reproduction was impeded in interspecific F₂ populations containing L. esculentum cytoplasm (F ₂ ^Le ) by reduction in pollen production, the lack of fruit set and a high proportion of parthenocarpic fruit among plants capable of fruit set. The F₂ populations containing L. pennellii cytoplasm (F ₂ ^Lp4 ) showed a reduced frequency of reproductive abnormalities at all stages of reproductive development, resulting in higher values for percent stainable pollen, fruit and seed set and higher proportions of the F ₂ ^Lp4 populations being capable of setting fruit or seed than F ₂ ^Le populations. The major barrier remaining in F ₂ ^Lp4 populations was reduced fruit set compared to parental lines. The barrier to fruit and seed set observed in the F ₂ ^Le populations, and to a lesser extent in the F ₂ ^Lp4 populations, occurs around the time of fertilization or early embryonic development. The effect of L. pennellii cytoplasm on barriers in the F ₂ ^Lp4 populations is proposed to be due to an interaction between cytoplasmic and nuclear genes during fertilization of the F₁ plants to produce F₂ populations and may also affect subsequent generations.     

Endophytic fungi in leaves of Lycopersicon esculentum Mill

Endophytic fungi were isolated from healthy tomato leaves from field-grown plants. Several surface-sterilization techniques were assayed for removing and killing epiphytes on tomato leaves. Surface-sterilization with an undiluted commercial solution of sodium hypochlorite was employed. To determine the spectrum of endophytes, samples were collected in February 1998 and 1999 from tomato plants (cultivar Tommy) cultivated in the experimental fields of Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, Buenos Aires, Argentina. In general, different spectra of species were isolated in 1998 and 1999.Alternaria alternata was the fungus most frequently isolated from tomato leaves in 1999, but it was the second most common species in 1998. In contrast, Colletotrichum gloeosporioides was the fungus most frequently isolated in 1998, but it was not found in 1999. Species of other genera, such as Cladosporium and Penicillium, were isolated in both years.     

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     

A genome-wide search for wild-species alleles that increase horticultural yield of processing tomatoes

To identify QTLs associated with horticultural yield it is necessary to conduct replicated plot trials of the tested genotypes. The first step in the utilization of an introgression-line (IL) population of Lycopersicon pennellii in a processing-tomato variety (M82) for mapping such QTLs was to screen 51 ILs in a non-replicated plot trial. The results of this survey were compared to those obtained in a replicated trial of the same genotypes grown as single plants at wide spacing. Fruit characteristics were similar between the two stands, but yield was generally different. Eight lines that outperformed the control in the plot survey were subjected to detailed analysis in the following year. The effects of these introgressions, measured on single plants, were reproducible relative to the previous year's results. In a replicated plot trial of these ILs and their hybrids involving two genetic backgrounds, the product of yield and total soluble solids (horticultural yield) in seven of the eight hybrids was 7–13% higher than that of their nearly isogenic controls. The results revealed a consistent trend in the interaction between introgression effects and genetic background. Combining the two introgressions with the largest contribution to horticultural yield in plots resulted in a 20% increase relative to the control in the third year. This research highlights the potential of wild germ plasm for yield improvement and the ability of nearly isogenic populations to achieve this goal.     

The distribution and utilization of calcium by two tomato (Lycopersicon esculentum Mill.) lines differing in calcium efficiency when grown under low-Ca stress

Two lines of tomato (Lycopersicon esculentum Mill.) representing extremes in utilization effciency of absorbed Ca were studied to detect internal differences in Ca transport and distribution and factors responsible for strain differences in susceptibility to low Ca-stress. Differences in efficiency of Ca use were expressed as CaER (mg of dry weight produced for each mg of Ca absorbed by the plant).Ca-efficiency in line 113(E) appeared to be associated with a slow continuous movement of absorbed Ca, allowing for continued growth of the shoot apex and upper lamina under Ca-deficiency conditions. In the inefficient line 67(I), in contrast, Ca was rapidly deposited in the lower leaves with little upward movement in the plant after absorption.Fractionation of tissue Ca into various chemical forms suggested that Ca inefficiency also was associated with higher concentrations of insoluble Ca in the shoot tissue. The efficient line, although sustaining growth at lower levels of Ca, was capable of maintaining a higher ratio of soluble to insoluble Ca in all shoot tissues.Calcium was concentrated in the lower plant tissues of the inefficient strain, limiting its availability for continued shoot growth.Autoradiographs of lines fed⁴⁵Ca during the final 8 days of a 24-day experiment suggested that upward movement was sustained in line 113(E), in spite of vastly reduced transpiration rates and a root system characterized by leakage of K ions from the roots back into the solution.     

Isolation of milligram quantities of nuclear DNA from tomato (Lycopersicon esculentum), A plant containing high levels of polyphenolic compounds

We have developed a protocol for isolating milligram quantities of highly purified DNA from tomato nuclei. The protocol utilizes fresh seedlings or leaves without freezing. Tissues are treated with ethyl ether, thoroughly washed, and placed in a buffer containing the nuclear-stabilizing agent 2-methyl-1,4-pentanediol. Nuclei are liberated from tomato cells by homogenization in a Waring blender. The interaction of nuclear DNA with oxidized polyphenols is inhibited by compounds that adsorb polyphenols or prevent oxidation reactions. Chloroplasts and mitochondria are preferentially eliminated with Triton X-100. Nuclei are concentrated using a Percoll gradient and lysed with SDS. DNA is subsequently purified by RNase and protease digestions and phenol/chloroform extractions. The isolated DNA is essentially free of polyphenols and other major contaminants based upon its lack of coloration, A₂₆₀/A₂₈₀ ratio, digestibility with restriction enzymes, melting profile, and reassociation properties.     

Distinguishing among male sterile and fertile lines of tomatoes (Lycopersicon esculentum Mill.) by means of electrophoretic protein patterns

Vertical block SDS-PAAG electrophoresis of individual tomato seeds was used to examine the potential of seed proteins as gene markers for distinguishing male sterile from fertile lines and cultivars. Seeds of three genic male sterile lines and four fertile cultivars and lines were investigated. The line 6944 is carrier of the gene ms 10 ³⁵for pollen male sterility and the lines P-1a and PPS-3 carry the gene ps-2 for positional sterility. A modified extract solution which includes 1M tris — buffer pH 7.0, 7 % SDS, dimethylformamide and distilled water was applied. Qualitative variation in the A protein locus which could be used as a marker for distinguishing male sterile from fertile tomato lines was established.     

Salinity effects on growth and carbon balance in Lycopersicon esculentum and L. pennellii

The effects of salinity on growth and carbon balance in the cultivated tomato Lycopersicon esculentum (L.) Mill. cv. VF 234 and in a wild, salt-tolerant relative, L. pennellii (Correll) D'Arcy accession Atico, were compared. The level of salinity that significantly reduced growth and affected morphology was lower for the cultivated than for the wild species.
Net CO₂ fixation, stomatal conductance and the specific activity of ribulose bisphosphate carboxylase (EC 4.1.1.39) were more decreased by salinity in the wild tomato than in the cultivated. In both species, the salinity-associated decrease in assimilation was related to a decrease in net photosynthesis and to carbon reallocation favoring heterotrophic organs.     

Characterization of a cDNA encoding the protein moiety of a putative arabinogalactan protein from Lycopersicon esculentum

The nucleotide sequence of a cDNA prepared from poly(A)⁺ RNA from Lycopersicon esculentum fruit codes for a protein, M _r 20812, with features representative of the protein core of arabinogalactan proteins. The deduced amino acid sequence resembles that of peptides of arabinogalactan proteins isolated from carrot and rose and is most similar to the sequence of tryptic peptides from Lolium multiflorum (Gleeson et al., Biochem J 264 (1989) 857–862). The similar sequences include a number of Ala-Pro repeats, a feature considered distinctive of arabinogalactan proteins. The amino acid composition is similar to that of the peptide core of the Lolium multiflorum arabinogalactan protein; alanine, serine and proline account for 57% of the polypeptide. The mRNA corresponding to the cDNA sequence was detected in roots, leaves and fruit. The levels of mRNA are reduced in older leaves, in fruit that have commenced ripening and in leaves and fruit that have been wounded.     

Allotriploid somatic hybrids of diploid tomato (Lycopersicon esculentum Mill.) and monoploid potato (Solanum tuberosum L.)

Allotriploid somatic hybrids were obtained from fusions between protoplasts of diploid tomato and monohaploid potato. The selection of fusion products was carried out in two different ways: (1) The fusion of nitrate reductase-deficient tomato with potato gave rise only to hybrid calli if selection was performed on media lacking ammonium. Parental microcalli were rarely obtained and did not regenerate. (2) The fusion of cytoplasmic albino tomato with potato gave rise to albino and green hybrid calli and plants. Allotriploids were identified from the two somatic hybrid populations by counting chloroplast numbers in leaf guard cells and by flow cytometry of leaf tissue. Although some pollen fertility of allotriploids and pollen-tube growth of tomato, potato andLycopersicon pennellii into the allotriploid style were observed, no progeny could be obtained. The relevance of allotriploid somatic hybrids in facilitating limited gene transfer from potato to tomato is discussed.     

Salinity effects on water relations in Lycopersicon esculentum and its wild salt-tolerant relative species L. pennellii

Cultivated tomato Lycopersicon esculentum (L.) Mill. cv. P-73 and its wild salt tolerant relative L. pennellii (Correll) D'Arcy accession PE-47, were grown during spring-summer 1989 under unheated plastic greenhouse conditions. Plants were submitted to two different salt treatments using 0 and 140 mM NaCI irrigation water. In both tomato species, salinity caused a proportionally larger reduction in leaf area than in leaf weight and, in L. esculentum , a proportionally larger decrease in stem weight than in leaf weight. Daily variations in leaf water potential (Ψ₁) were fundamentally due to changes in the evaporative demand of the atmosphere. Reductions in Ψ₁ due to salinity were consistent only in L. esculentum . In all the conditions studied, leaf turgor was maintained. Leaf conductance (g₁)was higher in L. esculentum than in L. pennellii .Salinity induced a clear reduction in g₁ levels in L. esculentum whereas, in L. pennellii , this reduction was noted only in May. In both species the Ψ^o_s (leaf osmotic potential at full turgor) levels were reduced by salinity. The bulk modulus of elasticity (E) and relative water content at turgor loss point (RWC_tlp) were not affected by salinity. The RWC_tlp values in L. pennellii seem to be controlled by E values.     

Species-specific DNA sequences for identification of somatic hybrids between Lycopersicon esculentum and Solanum acaule

Summary Species-specific highly repeated DNA sequences can be used to screen the progeny of protoplast fusions combining different species. Such probes are easy to clone and can be detected by fast methods, e.g., hybridization to total genomic DNA. Furthermore, due to their high copy number, hybridization signals are strong and represent more than one locus, unlike isozymes or resistance markers. After cloning and screening for species-specific DNA sequences we characterized the highly repeated DNA sequences of the solanaceous species Solanum acaule and Lycopersicon esculentum var. gilva. DNA sequencing and hy ridization revealed a prominent, tandemly arranged satellite DNA repeat of 162 bp in Lycopersicon esculentum and a different satellite repeat of 183 bp, also tandemly organized, in Solanum acaule. Each repeat is absent in the respective other species. Therefore, we have used these DNA repeats as markers to distinguish regenerated interspecific somatic hybrids from the respective fusion partners. These hybrids were clearly identified by Southern hybridization and dot-blot assays to the respective ³²P-labelled satellite DNA.     

Regeneration of intergeneric somatic hybrid plants between Lycopersicon esculentum and Solanum muricatum

Summary Mesophyll protoplasts of tomato (Lycopersicon esculentum) and pepino (Solanum muricatum) were fused by using an electrofusion method and cultured in modified MS medium supplemented with naphthaleneacetic acid and kinetin, in which only pepino and somatic hybrid protoplasts could divide. Somatic hybrid plants showing intermediate characteristics in morphology were regenerated from the calli exhibiting vigorous growth in contrast with those of pepino. The hybrid nature of these plants was confirmed by cytological observation and biochemical analyses of phosphoglucomutase isozymes and the fraction-1-protein. The regenerated somatic hybrids grew to flowering stage and set fruits.     

Production and characterization of intergeneric somatic hybrids through protoplast electrofusion between potato (Solanum tuberosum) and Lycopersicon pennellii

Mesophyll protoplasts of Lycopersicon pennelli Corr., a wild relative of tomato, were electrofused with those from a dihaploid potato clone, cv Nicola, with the objectives of transferring saline tolerance from L. pennellii to cultivated potato. 150 calli were selected from the fusion experiments, finally giving 2 hybrid shoots. Their hybrid nature was verified by examining isoenzyme patterns for esterases (EST), peroxidase (PRX), phosphogluconate dehydrogenase (6-PGD), and glutamate oxaloacetate transaminase (GOT). The hybrid plants had an intermediate morphology, and grew vigorously in vitro. When transplanted to soil, they were less vigorous, due to difficulties in rooting, but were still capable of flowering, and forming short stolons and mishaped tubers, probably resulting from the effects of gene dosage due to the novel association of two genomes from a tuberizing (potato) and a non tuberizing species (L. pennellii). The characteristics of such mishaped tubers provided strong evidence of a hybrid nature for the selected plants. The hybrid plants were highly sterile, producing only 3–7% viable pollen. Tests for salt tolerance showed that the growth of the somatic hybrid plants was reduced by 50% as for L. pennellii, whilst potato did not grow at all under saline conditions.Abbreviations MS Murashige and Skoog basal medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - BAP 6-benzylaminopurine - PEG polyethylen glycol 6,000 - MES 2-(N-morpholino)ethanesulfonic acid - AC alternating current - EST esterases - PRX peroxidase - 6-PGD phosphogluconate dehydrogenase - GOT glutamate oxaloacetate transaminase - FDA fluorescein diacetate     

Comparison of the response to aluminum toxicity in gametophyte and sporophyte of four tomato (Lycopersicon esculentum Mill.) cultivars

Summary We tested pollen from four tomato cultivars differing in sensitivity to aluminum in the sporophyte to determine if Al sensitivity was also expressed in pollen. Pollen sensitivity to Al was measured by the ability to germinate and grow in a control solution after a short period in a high concentration of Al. The response was ranked and compared to the Al sensitivity ranking of the four cultivars based on top growth in Al toxic soil. In addition, seedlings from the most and least sensitive cultivars, based on pollen germination, were compared for Al sensitivity in nutrient solutions. Treatment with Al significantly reduced pollen germination in the two more sensitive cultivars, but not in the two more resistant cultivars. However, the ranking was not the same as that based on the shoot growth of the sporophyte. Root growth as a criterion of sporophytic Al sensitivity produced results similar to pollen germination. The study suggests that although the correspondence is better for some phenotypic responses of the sporophyte than others, Al tolerance appears to be another character expressed in both pollen and sporophyte.     

Functional male sterility in tomato (Lycopersicon esculentum Mill.) and its application in hybrid seed production

Advantages and disadvantages in using functional male sterility (positional sterile — ps, positional sterile 2 — ps 2, and excerted stigma — ex) in tomato hybrid seed production and attempts to elaborate systems for their more efficacious use in breeding were discussed in this review. It was concluded that the application of one of these types of sterility, (ps 2) in practice, although in a limited number of countries, showed the functional male sterility in tomato was a potential not to be underestimated in developing approaches that aimed at reducting the time and cost associated with hybrid seed production.