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.     

Molecular analysis of the extent of asymmetry in two asymmetric somatic hybrids of tomato

Summary Two somatic hybrid plants generated from a single fusion event between Lycopersicon esculentum and irradiated L. pennellii protoplasts have been analyzed at the molecular level. Over 30 loci have been analyzed using isozymes and RFLPs. All loci tested on chromosomes 2–10 were heterozygous, while those loci on chromosome 12 were homozygous L. pennellii in both somatic hybrids. In one of the somatic hybrids, 2850, loci on chromosome 1 were also homozygous L. pennellii. The other somatic hybrid, 28F5, was heterozygous at all chromosome 1 loci tested, but exhibited altered stoichiometry of parental bands as compared to the sexual hybrid. Loci on chromosome 2 from both somatic hybrids have altered stoichiometry, with L. pennellii alleles being four times more abundant than expected. Both somatic hybrids contain the L. esculentum chloroplast genome, while only L. pennellii polymorphisms have been detected in the mitochondrial genome.     

Identification of the double genome donor in spontaneous triploid tomato plants by RFLP analysis

Independent spontaneous triploid tomato plants (Lycopersicon esculentum Mill.) were collected among diploid hybrids growing in commercial greenhouses. Ploidy levels were verified by counting chromosomes, and the donor of the double genome dose was determined by restriction fragment length polymorphism (RFLP) analysis. The TG101 probe, which is tightly linked to the Tm-2 ^a locus, revealed different restriction patterns between TMV-resistant and TMV-susceptible parent lines. The parent donor which provided two genomes to the triploid was identified by comparing the relative intensity of alleles in the triploid with that in the diploid. The results indicate that both parents can serve as a double genome donor.     

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.     

Morphological and molecular characterization of somatic hybrid plants between Lycopersicon esculentum and Solanum nigrum

Summary Mesophyll protoplasts of tomato (Lycopersicon esculentum Mill. var. cerasiforme) and of an atrazine-resistant biotype of black nightshade, (Solanum nigrum L.), were fused by using polyethylene glycol/dimethyl sulfoxide (PEG/DMSO) solution and three somatic hybrid plants, each derived from a separate callus, were recovered. A twostep selection system was used: (1) protoplast culture medium (modified 8E) in which only tomato protoplasts formed calluses; and (2) regeneration medium (MS2Z) on which only S. nigrum calluses produced shoots. These selective steps were augmented by early isozyme analysis of putative hybrid shoots still in vitro. Phosphoglucoisomerase (PGI) and glutamate oxaloacetate transaminase (GOT) mapped to five loci on four chromosomes in tomato confirmed the hybrid nature of the nuclei of regenerated shoots. The somatic hybrid plants had simple leaves, and intermediate flower and bud morphology, but anthesis was reduced to 5% due to premature bud abscission and the pollen grains were non-viable. Southern DNA blot hybridization using a pea 45 S ribosomal RNA gene probe reconfirmed the hybrid nature of the nuclear genome of the three plants. A ³²P-labeled probe of Oenothera chloroplast DNA (cpDNA) hybridized to cpDNA restricted with EcoRI or EcoRV indicated the presence of the tomato cpDNA pattern in all three hybrids. Likewise, the plants were all found to be atrazine sensitive. Analysis with two mitochondrial (mt)DNA-specific probes, maize cytochrome oxidase subunit II and PmtSylSa8 from Nicotiana sylvestris, showed that, in addition to typical mitochondrial rearrangements, specific bands of both parents were present or missing in each somatic hybrid plant.Michigan Agricultural Experiment Station Journal Article No. 12433     

Tissue culture-induced DNA methylation polymorphisms in repetitive DNA of tomato calli and regenerated plants

The propagation of plants through tissue culture can induce a variety of genetic and epigenetic changes. Variation in DNA methylation has been proposed as a mechanism that may explain at least a part of these changes. In the present study, the methylation of tomato callus DNA was compared with that of leaf DNA, from control or regenerated plants, at MspI/HpaII sites around five middle-repetitive sequences. Although the methylation of the internal cytosine in the recognition sequence CCGG varied from zero to nearly full methylation, depending on the probe used, no differences were found between callus and leaf DNA. For the external cytosine, small differences were revealed between leaf and callus DNA with two probes, but no polymorphisms were detected among DNA samples of calli or DNA samples of leaves of regenerated plants. When callus DNA cut with HindIII was studied with one of the probes, H9D9, most of the signal was found in high-molecular-weight DNA, as opposed to control leaf DNA where almost all the signal was in a fragment of 530 bp. Also, an extra fragment of 630 bp was found in the callus DNA that was not present in control leaf DNA. Among leaves of plants regenerated from tissue culture, the 630-bp fragment was found in 10 of 68 regenerated plants. This 630-bp fragment was present among progeny of only 4 of these 10 plants after selfing, i.e. it was partly inherited. In these cases, the fragment was not found in all progeny plants, indicating heterozygosity of the regenerated plants. The data are interpreted as indicating that a HindIII site becomes methylated in callus tissue, and that some of this methylation persists in regenerated plants and is partly transmitted to their progeny.     

Physiological response and yield of paclobutrazol treated tomato plants (Lycopersicon esculentum Mill.)

Experiments were conducted to study the physiologicaleffect of the plant growth retardant paclobutrazol(PBZ) and its impact on the yield of tomato plants(cv. Precador). Seedlings were treated at the time of prickingout with soil and foliar applications of PBZ atconcentrations of 1.0 and 25.0 mg l^-1respectively. The results established that:-- The reduced height and the increased thickness ofthe young plant stem, as well as the accelerated rootformation are a significant advantage of the PBZtreatment, contributing to the improvement of seedlingquality at planting.-- Soil treatment (1 mg l^-1) and foliar treatment(25 mg l^-1) with PBZ improves the photosyntheticactivity and water balance of tomato cv. Precador.-- PBZ accelerates fruit formation and increases earlyfruit yield.-- The concentrations of the retardant used and themode of its application ensure the production offruits without any residual retardant and harmless tohuman health from a phytosanitary point of view.     

Localization of proton-ATPase genes expressed in arbuscular mycorrhizal tomato plants

In arbuscular mycorrhizal symbioses, solutes such as phosphate are transferred to the plant in return for photoassimilates. The uptake mechanism is probably facilitated by a proton gradient generated by proton H⁺-ATPases. We investigated expression of Lycopersicon esculentum Mill. H⁺-ATPases in mycorrhizal and non-mycorrhizal plants to determine if any are specifically regulated in response to colonization. Tissue expression and cellular localization of H⁺-ATPases were determined by RNA gel blot analysis and in situ hybridization of mycorrhizal and non-mycorrhizal roots. LHA1, LHA2, and LHA4 had high levels of expression in roots and were expressed predominantly in epidermal cells. LHA1 and LHA4 were also expressed in cortical cells containing arbuscules. The presence of arbuscules in root sections was correlated with lower levels of expression of these two isoforms in the epidermis. These results suggest that LHA1 and LHA4 expression is decreased in epidermal cells located in regions of the root that contain arbuscules. This provides evidence of differential regulation between molecular mechanisms involved in proton-coupled nutrient transfer either from the soil or fungus to the plant.     

Structure and expression of an inhibitor of fungal polygalacturonases from tomato

A polygalacturonase inhibitor protein (PGIP) was characterized from tomato fruit. Differential glycosylation of a single polypeptide accounted for heterogeneity in concanavalin A binding and in molecular mass. Tomato PGIP had a native molecular mass of 35 to 41 kDa, a native isoelectric point of 9.0, and a chemically deglycosylated molecular mass of 34 kDa, suggesting shared structural similarities with pear fruit PGIP. When purified PGIPs from pear and tomato were compared, tomato PGIP was approximately twenty-fold less effective an inhibitor of polygalacturonase activity isolated from cultures of Botrytis cinerea. Based on partial amino acid sequence, polymerase chain reaction products and genomic clones were isolated and used to demonstrate the presence of PGIP mRNA in both immature and ripening fruit as well as cell suspension cultures. Nucleotide sequence analysis indicates that the gene, uninterrupted by introns, encodes a predicted 36.5 kDa polypeptide containing amino acid sequences determined from the purified protein and sharing 68% and 50% amino acid sequence identity with pear and bean PGIPs, respectively. Analysis of the PGIP sequences also revealed that they belong to a class of proteins which contain leucine-rich tandem repeats. Because these sequence domains have been associated with protein-protein interactions, it is possible that they contribute to the interaction between PGIP and fungal polygalacturonases.     

Multivariate analysis applied to tomato hybrid production

Summary Twenty characters were measured on 60 tomato varieties cultivated in the open-air and in polyethylene plastic-house. Data were analyzed by means of principal components, factorial discriminant methods, Mahalanobis D² distances and principal coordinate techniques. Factorial discriminant and Mahalanobis D² distances methods, both of which require collecting data plant by plant, lead to similar conclusions as the principal components method that only requires taking data by plots. Characters that make up the principal components in both environments studied are the same, although the relative importance of each one of them varies within the principal components. By combining information supplied by multivariate analysis with the inheritance mode of characters, crossings among cultivars can be experimented with that will produce heterotic hybrids showing characters within previously established limits.     

Effect of the carmine spider mite (Acarida: Tetranychidae) infestation and mechanical injury on the level of ABA in tomato plants

The aim of this study was to determine the changes in the abscisic acid (ABA) content in tomato leaves infested by the carmine spider mite (CSM) (Tetranychus cinnabarinus Boidsuval) and in leaves that were mechanically injured. It was also investigated whether signalling from stressed to non-stressed organs occurred. Tomato plants (Lycopersicon esculentum Mill.) cvs. Romatos and Slonka (with various susceptibility to CSM) were stressed at the stage of first cluster flowering by either CSM feeding (72 hours) or by mechanical puncturing simulated feeding injury by CSM (18 hours). It was found that under control condition the level of ABA differed significantly between cultivars, being always higher in plants of the susceptible cv. Romatos. In response to CSM feeding, the content of ABA in infested organs of the more tolerant plant (cv. Slonka) increased by 95 % but in the susceptible one by 11 % only. ABA content in the organs non-stressed by CSM feeding either increased (Slonka cv.) or decreased (Romatos cv.). In response to mechanical wounding, ABA content in directly injured organs increased but to a lower degree (49 %) and only in Slonka cv.. The same was true for ABA content in non-injured organs of damaged plants of this cultivar. Observed changes in ABA level in non-stressed organs are probably the results of signalling from stressed organs. Plant response measured by changes in ABA level to the stress generated by CSM feeding, was much stronger than merely by mechanical injury.     

Photosynthetic acclimation in tomato plants grown in high CO2

The effects of prolonged CO₂ enrichment of tomato plants on photosynthetic performance and Calvin cycle enzymes, including the amount and activity of ribulose-1,5-bisphosphate carboxylase (RuBPco), were determined. Also the light-saturated rate of photosynthesis (P_max) of the 5th leaf throughout leaf development was predicted based on the amount and kinetics of RuBPco. With short-term CO₂ enrichment, i.e. only during the photosynthesis measurements, P_max of the young leaves did not increase while the leaves reaching full expansion more than doubled their net rate of CO₂ fixation. However, with longer-term CO₂ enrichment, i.e. growing the crop in high CO₂, the plants did not maintain this photosynthetic gain. Compared with leaves of plants grown in normal ambient CO₂ the high CO₂-grown leaves, when almost fully expanded, contained only about half as much RuBPco protein and P_max in 300 and 1000 vpm CO₂ was similarly reduced.The loss of RuBPco protein may be a factor associated with the accelerated fall in P_max since P_max was close to that predicted from the amount and kinetics of RuBPco assuming RuBP saturation. Acclimation to high CO₂ is fundamentally different from acclimation to high light. In contrast to acclimation to high light, acclimation to high CO₂ does not usually involve an increase in photosynthetic machinery so the synthesis and maintenance costs (as indicated by the dark respiration rate) are generally lower.     

A smoke-derived butenolide improves early growth of tomato seedlings

3-Methyl-2H-furo[2,3-c]pyran-2-one is an active compound isolated from plant-derived smoke water. It has a stimulatory role during seed germination similar to that of smoke or aqueous extracts of smoke. The present study was undertaken to gain insight into the physiological events involved in seed germination and seedling development and which are affected by butenolide using tomato (Lycopersicon esculentum Mill.) cultivar “Heinz 1370” seeds. No stimulatory role on the seed germination of tomato was recorded following the use of the butenolide, however, post-germinative growth of tomato seedlings was significantly improved over the control (P ≤ 0.05). The emergence of the radicle and elongation of the hypocotyls and radicles were accelerated in seeds imbibed with butenolide at 10⁻⁷ M. Flow cytometry studies showed that in butenolide-treated seeds the ratio of cells with replicated DNA was increased. Seedling vigour and weight were significantly increased by the butenolide (P ≤ 0.05). An inverse correlation was observed between the weight of cotyledons and the weight of the hypocotyls and radicle during seedling development. This is an indication that the butenolide is implicated in mobilization and utilization of stored reserve materials in developing tomato seedlings.     

Effects of IAA and four IAA conjugates on morphogenesis and callus growth from tomato leaf discs

The effects of indole-3-acetic acid (IAA) and four IAA conjugates, indoleacetylalanine (IAAla), indoleacetylaspartic acid (IAAsp), indoleacetylglycine (IAGly), and indoleacetylphenylalanine (IAPhe), on growth and morphogenesis in tomato leaf discs in vitro were examined. Free IAA stimulated root initiation in the absence of cytokinin and stimulated callus growth in the presence of 0.89 M benzylaminopurine (BAP). Free IAA also inhibited shoot initiation obtained with 8.9 M BAP. The activities of the IAA conjugates depended on the conjugating amino acid, the concentration of the conjugate, and the response being measured. IAAsp had little or no activity in promoting root initiation or callus growth or in inhibiting shoots, while IAPhe was similarly inactive except at the highest concentration tested (100 M). IAAla and IAGly were both very active in inhibiting shoots and promoting callus growth, but were much less active in stimulating rooting, except at 100 M, at which concentration they were as effective as free IAA. Thin-layer chromatography of the IAA conjugates revealed that IAAla, IAGly and IAPhe were largely stable to autoclaving, but that IAAsp underwent some hydrolysis to products identical with free IAA and aspartic acid. Pretreatment of seedlings with IAA, IAAla or IAGly altered the subsequent shoot initiation response from leaf discs on media with and without IAA.     

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.     

Manganese nutrition effects on tomato growth, chlorophyll concentration, and superoxide dismutase activity

The effects of Mn nutrition of tomato (Lycopersicon esculentum Mill.) seedlings on Mn-, Fe- and CuZn-superoxide dismutase (SOD, EC 1.15.1.1) enzymatic activities, metal translocation, chlorophyll concentration, and plant growth were tested using a bioassay system consisting of chelator-buffered nutrient culture with Mn2+ activities set to pMn (-log activity of Mn2+) of 6.6, 7.6, 8.6, and 9.6. The two middle levels resulted in optimal plant growth, whereas the two extreme levels resulted in a gradual decrease in chlorophyll concentration and slower plant growth. At the end of the experiment, 26 days after transfer to the Mn treatments, significant differences in shoot Mn concentration were manifested, from 10.5 mg kg(-1) in plants grown in pMn 9.6 to 207.4 mg kg(-1) in plants grown in pMn 6.6. Other element concentrations in the leaf suggest that growth inhibition and chlorophyll synthesis were affected primarily by manganese deficiency and excess. Twenty days after transfer of plants to the Mn treatments Mn-, Fe- and CuZn-SOD activities were assayed in young expanded leaf tissues by electrophoresis running gel. Whereas chloroplastic CuZn-SOD activity did not differ among Mn treatments, the cytosolic CuZn-SOD and mitochondrial Mn-SOD activities increased in both Mn-excess and Mn-deficient plants.     

Asr genes belong to a gene family comprising at least three closely linked loci on chromosome 4 in tomato

Asr1, Asr2 andAsr3 are three homologous clones isolated from tomato whose expression is believed to be regulated by abscisic acid (ABA); the corresponding genes thus participate in physiological and developmental processes such as responses of leaf and root to water stress, and fruit ripening. In this report, results obtained with Near Isogenic Lines reveal thatAsr1, Asr2 andAsr3 represent three different loci. In addition, we map these genes on the restriction fragment length polymorphism (RFLP) map of the tomato genome by using an F₂ population derived from an interspecific hybrid crossL. esculentum × L. penelli. RFLP data allow us to map these genes on chromosome 4, suggesting that they belong to a gene family. The elucidation of the genomic organization of theAsr gene family may help in understanding the role of its members in the response to osmotic stress, as well as in fruit ripening, at the molecular level.     

Ac transposition in transgenic tomato plants

Summary As an initial step towards developing a transposon mutagenesis system in tomato, the maize transposable element Ac was transformed into tomato plants via Agrobacterium tumefaciens. Southern analysis of leaf tissue indicated that in nine out of eleven transgenic plants, Ac excised from the T-DNA and reintegrated into new chromosomal locations. The comparison of Ac banding pattern in different leaves of the same primary transformant provided evidnece for transposition during later stages of transgenic plant development. There was no evidence of Ds mobilization in tomato transformants.     

Photocontrol of anthocyanin biosynthesis in tomato

Juvenile anthocyanin biosynthesis has been studied in dark-grown seedlings of tomato (Lycopersicon esculentum Mill.) wild types (WTs) and photomorphogenic mutants. During a subsequent 24-hr period of monochromatic irradiation at different fluence rates of red light (R) the fluence-rate response relationships for induction of anthocyanin in all the WTs are similar, yet complex, showing a response at low fluence rates (LFRR) followed by a fluence rate-dependent high irradiance response (HIR). In the hypocotyl this response is restricted to the sub-epidermal layer of cells. The high-pigment-1 (hp-1) mutant exhibits a strong amplification of both response components. Theatroviolacea (atv) mutant shows strongest amplification of the HIR component. In contrast, a transgenic line overexpressing an oat phytochrome A gene (PHYA3 ⁺) shows a most dramatic amplification of the LFRR component. The far-red light (FR)-insensitive (fri) mutant, deficient in phytochrome A (phyA), lacks the LFRR component whilst retaining a normal HIR. The temporarily R-insensitive (tri) mutant, deficient in phytochrome B1 (phyB1) retains the LFRR, but lacks the HIR. Thehp-1,fri andhp-1,tri double mutant, exhibit amplified, yet qualitatively similar responses to the monogenicfri andtri mutants. Thefri,tri double mutant lacks both response components in R, but a residual response to blue light (B) remains. Similarly, theaurea (au) mutant deficient in phytochrome chromophore biosynthesis and presumably all phytochromes, lacks both response components in the R and FR regions of the spectrum. Experiments at other wavelengths demonstrate that while there is only a small response in the FR spectral region (729 nm) in tomato, there is an appreciable HIR response in the near FR at 704 nm, which is retained in thetri mutant. This suggests that the labile phyA pool participates in the HIR at this wavelength. The intense pigmentation (Ip) mutant appears to be specifically deficient in the B1 induced anthocyanin biosynthesis. Adult plants, grown under fluorescent light/dark cycles, show a reduction of anthocyanin content of young developing leaves upon application of supplemtary or end-of-day FR. The involvement of different phytochrome species in anthocyanin biosynthesis based on micro-injection studies into theau mutant and studies using type specific phytochrome mutants is discussed.