As a preface for the tomato special issue, a significance of tomato for research and development was described. Tomato is a model species of Solanaceae, and its genome is currently being sequenced. In order to fully utilize the genome information, scientists have to develop resources and tools for tomato functional genomics. In this special issue, such resources and tools, mainly developed by Japanese scientists were reviewed. In addition, scientific name of tomato was discussed because, for tomato being a model plant, scientists should use a common word for tomato.

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As DNA sequence data were rapidly accumulated in the last decade, the intense debate on the position of the genus “Lycopersicon” with respect to “Solanum” was recently settled. In this review, we focus on chloroplast DNA restriction fragment length polymorphism (cpRFLP) analysis and molecular phylogenetic study of the chloroplast-encoded NADH dehydrogenase (ndhF) gene of numerous Solanaceae family members including tomato, Solanum lycopersicum (formerly Lycopersicon esculentum). Both studies revealed that S. lycopersicum is clearly included in the large genus Solanum, which includes potato (S. tuberosum) and eggplant (S. melongena). All cpRFLP and different algorithms in ndhF phylogenetic analysis classified S. lycopersicum into the same clade as S. tuberosum. Comparative genome analysis using a high-density genetic linkage map of S. lycopersicum and S. tuberosum indicated that the chromosome structure is highly conserved between the two species. These recent studies indicated that tomato and potato are phylogenetically close and share a recent common ancestor.

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Mutant-based studies have contributed to the elucidation of gene functions in plants including tomato. In this review, we introduce some outstanding research performed using spontaneous and artificially induced mutants. Tomato (Solanum lycopersicum L.) is a model species in the solanaceae family and research tools and information necessary to perform functional genomics study are being developed worldwide. Saturation mutagenesis is a powerful strategy that enables exploration of gene function on a genome-wide scale. We reviewed conventional mutant resources of tomato, including the ethyl methanesulfonate (EMS)-mutagenized population of the ‘M82’, which was developed in Israel (Menda et al., 2004). Recently, a miniature cultivar, ‘Micro-Tom’, has attracted the attention of tomato researchers due to its small size and rapid life cycle, which make it an ideal experimental organism for handling in the laboratory. We are developing a unique mutant resource, 10,000 EMS-mutagenized and gamma-ray-irradiated lines derived from ‘Micro-Tom’. Distinct mutant phenotypes, including plant size, leaf shape, and fruit morphology, are described. The status of newly developed tools for using this mutant resource, such as an efficient transformation method and the Targeting Induced Local Lesions In Genomes (TILLING) system, are also summarized.

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Productivity and quality of tomato (Solanum lycopersicum) fruit largely depend on the quantity and composition of its metabolites. Recent development of mass spectrometry technology has facilitated the simultaneous detection of a large number of metabolites. This opens the possibility of investigating the mechanisms of metabolic regulation that affect fruit productivity and quality, in conjunction with genetic and omics approaches. In this article, we review recent progress in the analysis of tomato fruit metabolites based on comprehensive metabolic profiling and the identification of genes and quantitative trait loci that alter tomato fruit metabolite profiles.

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Tomato (Solanum lycopersicum) is a model plant of the Solanaceae family. Various biological aspects of tomato, such as the mechanisms of its development and physiology, have been investigated with molecular biological approaches. In addition, the number of genome sequences and expression sequence tags in the International Nucleotide Sequence Databases has been rapidly increasing. Genome and transcript sequence data have facilitated other large-scale omics studies using bioinformatics analyses. Recently, omics data, including experimental materials, sequences, gene expression, gene and protein functions, and metabolic pathways have become available from various web databases. This wealth of comprehensive online resources allows for the extraction of essential new biological information. In this review, we summarize the current status of omics databases for tomato researchers.

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The apple AFL (Apple FLORICAULA LEAFY) 1 and 2 gene promoter linked β-glucuronidase (GUS) clearly displayed staining at the meristems of the shoot apexes, lateral axils, and leaf primordia in apple trees. The GUS staining of AFL1 promoter revealed that the AFL1 gene was also expressed at a vegetative meristem, and the staining patterns of AFL2 promoter were almost the same in the culture shoots and two-year-old trees. Quantitative RT-PCR analysis also showed that both genes were expressed at floral buds, where the expression of each was higher than the vegetative expression individually and increased with floral developments. These results showed the similarity in the expression patterns of the two AFL genes, and this expression was not affected by the vegetative growth condition, but was regulated at floral development. In situ hybridization of AFL genes supported the results of the GUS staining. The specific probe of each AFL gene showed almost the same expression pattern at the meristem of shoot apexes, lateral axils, and leaf primordia from the seedling, culture shoots, and watersprouts. These results indicated that the promoter GUS analysis and the in situ hybridization can be combined for the analysis of AFL gene expression. The cooperative usage of the GUS analysis and in situ hybridization suggested that they are powerful tools for apple promoter analysis.

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Japanese apricot (Prunus mume Sieb. et Zucc.) exhibits the S-RNase-based gametophytic self-incompatibility (SI) system, which is controlled by a single polymorphic locus called the S locus containing the pistil S (S-RNase) and pollen S genes (SFB/SLF for S haplotype-specific F-box gene/S locus F-box gene). This study was conducted to elucidate the unknown molecular basis of self-compatibility (SC) in a selection line 1K0-26, an offspring of ‘Benisashi’ × ‘Koshinoume’, to explore the possible use of this line for SC breeding in Japanese apricot. Controlled pollination and segregation analyses demonstrated that 1K0-26 has an intact SI S⁷ haplotype and a pollen-part mutant SC S^3′ haplotype. Cloning and DNA sequence analysis of the S^3′ locus revealed a 7.1 kb insertion in the pollen determinant SFB^3′, which makes a premature stop codon to produce transcripts for truncated dysfunctional SFB, just like the mode of mutation in SFB^f, another SC S haplotype in Japanese apricot. The inserted sequence appeared to be a non-autonomous retroposon with long terminal repeats, a part of which is identical to part of the inserted sequence to SFB^f. Taking advantage of the similarity of the inserted sequences to the two SC S haplotypes of distinct origin, S^3′ and S^f, we have developed a universal PCR marker for the marker-assisted selection for SC in Japanese apricot. We surveyed 86 Japanese apricot cultivars and lines using the PCR marker, and it appeared that 38, 1, and 3 appeared to have only S^f, only S^3′, and both S^f and S^3′, respectively. Marker-assisted selection for SC in Japanese apricot and the possible use of these cultivars and lines for future SC Japanese apricot breeding programs are discussed.

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Marker-assisted selection (MAS) using 5 DNA markers linked to the pear scab resistance derived from ‘Kinchaku’ (Vnk) was examined in a pear breeding program. The relationship between DNA markers and scab resistance was investigated. All individuals without both DNA markers of STS-OPW2 and STS-OPO9 derived from ‘Kinchaku’ showed scab symptoms with heavy sporulation on leaves. Individuals possessing these markers exhibited no symptoms, or necrosis with or without weak sporulation on petioles which was localized and did not affect growth, and were classified as resistant. Hence, elimination of individuals that have neither STS-OPW2 nor STS-OPO9 would be a suitable strategy to select for scab resistance in Japanese pear breeding when introgressing Vnk from ‘Kinchaku’.

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Japanese pear exhibits gametophytic self-incompatibility controlled by a single S-locus with multiple alleles. The S-locus encodes an S-RNase as a stylar product, and 10 S-RNase alleles (S₁ to S₉, and S_k) have been cloned from major cultivars. To investigate the diversity of S-alleles in Japanese pear, we analyzed the S-genotypes of three local cultivars (‘Senryo’, ‘Kuroki’, and ‘Hogyoku’). Two S-RNase fragments were amplified from each cultivar by genomic PCR with S-RNase-specific primers. A cleaved amplified polymorphic sequence (CAPS) marker system to distinguish S₁- to S₉-RNases assigned S₃- and S₂-RNase alleles to ‘Senryo’ and ‘Kuroki’, respectively. Cloning and sequencing of the other S-RNase genes identified S_e-RNase of European pear in ‘Senryo’, S₁₂-RNase of Chinese pear in ‘Kuroki’, and S₃₀-RNase of Chinese pear and S_k-RNase in ‘Hogyoku’. Therefore, S-genotypes of ‘Senryo’, ‘Kuroki’, and ‘Hogyoku’ were assigned as S₃S_e, S₂S₁₂, and S_kS₃₀, respectively. These results revealed that Japanese pear has some of the same S-alleles as European and Chinese pears as well as S₁- to S₉- and S_k-alleles.

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The expression of seedlessness derived from ‘Mukaku Kishu’ (Citrus kinokuni hort. ex Tanaka) was investigated histologically. Neither perfect nor imperfect seeds found in seedy cultivars/offspring were formed in ‘Mukaku Kishu’ and its seedless descendants. Among ‘Mukaku Kishu’ and its seedless descendants, the fertilized seed type (termed “type A seeds”) with an immature, soft seed coat was specifically observed. In most seedless cultivars/offspring, all embryos of type A seeds were arrested at the zygote or proembryo stage, and embryo development was arrested 10 weeks after pollination. These results indicate that the expression of Mukaku Kishu-type seedlessness is characteristic of a forming type A seed with an immature seed coat and an embryo arrested at an early stage. Endosperm abortion in type A seeds was not observed. Therefore, we demonstrated that an arrested embryo development in the type A seed is not caused by endosperm abortion. Because the expression of Mukaku Kishu-type seedlessness is not controlled by the embryo genotype, type A seed production would not be caused by gene expression in embryos but by that in maternal tissues, more likely in the immature seed coat tissue.

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Persimmon (Diospyros kaki Thunb.) cultivars are classified into 4 types depending on the relationship between astringency of the mature fruit and the effect of seeds on the loss of astringency, and only pollination-constant and non-astringent (PCNA)-type persimmons stably lose fruit astringency as a part of fruit development. This is a recessive trait, regarded to be controlled by a single locus, namely, the AST locus, which has a polysomic nature. Thus far, we have identified 2 restriction fragment length polymorphism (RFLP) markers, namely, A1 and A2, each of which is separately linked to a different AST allele, and proved that the RFLP markers were useful for selecting PCNA-type persimmons. This study was conducted to convert the RFLP markers into polymerase chain reaction (PCR)-based markers. We isolated and characterized genomic regions corresponding to each RFLP marker by inverse PCR. Two primer pairs, E4/E9r and E4/A2r, were designed to generate 2 sequence characterized amplified region (SCAR) markers, i.e., PCR-A1 and PCR-A2, respectively. The PCR-A1 and PCR-A2 markers cosegregated with the A1 and A2 markers, respectively, in ‘Nishimura-wase’-derived progenies. Although the primer pair E4/A2r did not produce the PCR-A2 marker in the FU-275, which is a progeny derived from ‘Aizumishirazu’, all non-PCNA-type offspring but no PCNA-type offspring showed the PCR-A1 marker using the primer pair E4/E9r. Thus, it was revealed that the SCAR markers were useful for selecting PCNA-type offspring in these progenies. On the other hand, disruption of the relationship between the markers and the AST locus was observed in the KU-325, derived from ‘Kurokuma’, indicating that the selection of PCNA-type offspring using PCR-based markers is not effective for progeny derived from ‘Kurokuma’. Herein, we discuss the possibility of applying PCR-based markers to genetic studies.

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We have developed a CiFT co-expression system for the rapid evaluation of transgenic citrus flowers and fruit. In order to co-express a target gene and the CiFT gene, a CiFT co-expression vector was constructed by inserting the fragment containing a chimeric target gene into the binary vector harboring P35S::CiFT. This system was applied to metabolically engineer aroma components in trifoliate orange (Poncirus trifoliata L. Raf). In order to reduce the limonene level, a gene encoding limonene synthase (CitMTSE1) from satsuma mandarin (Citrus unshiu Marc.) was introduced into trifoliate orange in an anti-sense orientation with the CiFT co-expression vector. Transgenic plants flowered extremely early, and began to produce normal fruit within 2 years of Agrobacterium infection. Transcripts of transgenes were accumulated in reproductive tissues of transgenic plants, and endogenous transcripts for limonene synthase were reduced. The ratio of limonene to other monoterpenes was significantly decreased in flowers and fruit of transgenic plants. These results clearly indicated that the CiFT co-expression system is a useful tool for the functional analysis of genes in citrus flowers and fruit. Such analyses are important for several applications, including the metabolic engineering of aroma components in citrus.

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To obtain a peel-specific promoter useful for citrus transgenic research, the 5' flanking region of a citrus d-limonene synthase gene (CitMTSE2), which showed a high degree of expression in the peel of mature fruits, was isolated from the satsuma mandarin (Citrus unshiu Marc.) Bacterial Artificial Chromosome (BAC) library. Promoter activity was characterized by particle bombardment, agroinjection and Arabidopsis transformation analyses. Approximately 5 kbps in the 5' upstream region (PCuMTSE2) contained several cis-motifs relating to guard cell-specific and stress-related genes. PCuMTSE2 fused to the uidA gene was directly incorporated into tissues from citrus and its relatives using particle bombardment. The results showed that PCuMTSE2 conferred β-glucronidase (GUS) activity in fruits rather than in seed and leaf tissues. Agroinjection analysis using citrus fruits showed PCuMTSE2 has promoter activity in peel but not in juice sacs. In a transgenic Arabidopsis-incorporated PCuMTSE2::uidA construct, GUS activity was detected in the joint between the stalk and silique. These results suggest that PCuMTSE2 could be utilized as a promoter regulating the quantitatively preferential expression in peel, and is useful for studies of manipulation by genetic engineering in citrus.

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Tomato plants were grown using a nutrient film technique to evaluate the effects of salinity on the distribution of photosynthates and sucrose-degrading enzymes in fruit in relation to sink strength. From first flower anthesis until harvest of the 2nd truss, saline treatment of sodium chloride added to the standard nutrient solution to an EC of 8.0 dS/m was applied. The average fruit weight in the salinity treatment was 59% of the control at the mature red stage. Total soluble solids (Brix%) were 6.5% in the control and 9.2% in the salinity treatment. The photosynthetic rate per unit leaf area was decreased by salinity treatment. Salinity decreased ¹³C assimilation in the whole tomato plant, while it increased the transport and distribution of ¹³C assimilates to the fruit significantly. The activities of soluble and cell wall-bound acid invertase were not affected by salinity; however, the activity of sucrose synthase in fruit exposed to the salinity was higher than that in the control during fruit development. We concluded that the increase in total soluble solids in fruit subjected to salinity was not only a result of condensation caused by reduced water uptake, but active transport of photosynthates to the fruit due to increase in sucrose-degrading enzyme activities, especially sucrose synthase.

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Nursery plants of watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) were planted in the field on April 19, 2006. To examine the correlation between temperatures around fruit during the latter half of the fruit developmental period and sugar accumulation in fruit, fruits were covered with polyethylene (PE) film, infrared-ray-blocking (IRB) film, and black nylon mesh (BNM) from 20 to 48 days after anthesis (DAA). Relative to the untreated control, higher fruit temperatures were detected by PE film-covering, but the temperatures remained low by IRB and/or BNM film-covering. Fruit growth was significantly accelerated by PE film-covering, but IRB and BNM film-covering resulted in a reduction of fruit growth. Cell enlargement at the intermediate and outer regions of fruit progressively intensified in the former as compared with the latter. Higher fructose and sucrose contents were detected in the intermediate and outer regions of fruit by IRB film-covering, while PE film-covering decreased glucose and fructose contents in all regions of fruit. These results suggest that the active cell enlargement caused by higher temperature during the latter half of the fruit developmental period is closely correlated with the reduction of sugar content in fruit, and that higher sugar content can be produced by lowering temperature with IRB film-covering in this period.

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Ploidy levels of in vitro shoots produced through anther culture of asparagus were determined by flow cytometry (FCM) analysis instead of counting the chromosome number of root tips that were difficult to regenerate. Of the 110 anther-derived shoots, there were 83 diploids, 21 tetraploids, 3 octaploids, and 3 mixoploids, but no haploids. When flower buds of different length were used as starting material for anther culture, the richest variation in polyploidy originated from short flower buds (1.0–1.5 mm). This result suggested that polyploids might develop by the fusion of nuclei during pollen-mother cell division before the tetrad stage. We used scanning electron microscopy (SEM) to measure the stomatal length of in vitro and field-grown shoots in order to clarify the differences among polyploids determined by FCM analysis. When the developmental stage of tissue was taken into account, we found statistically significant differences in stomatal length among all polyploids (diploid, tetraploid, and octaploid) for both stems and phyllodes (P < 0.001). Stomata on stems were longer than those on phyllodes, and mature tissue had longer stomata than immature tissue. Morphological observations with SEM found that immature tissue had guard cells set below epidermal cells, and were poorly developed. Field-grown diploids had much shorter stomatal length than in vitro diploids. Male plants grown in the field had significantly longer stomata than female plants in both their immature stems and mature phyllodes (P < 0.001). These results indicated that stomatal length can be a reliable ploidy index by removing any other influential factors such as sex, environment and developmental stage.

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Lantana (Verbenaceae) has adapted to the natural environment of Taiwan, and is thought to be a naturalized weed. A total of 43 Lantana plants, comprising 1 species of 3 varieties (38 plants collected from 38 sites in Taiwan) and 5 horticultural plants, were collected in Taiwan. In order to study the relationship between wild species and horticultural plants, we used co-HPLC to examine each anthocyanin peak and the content ratio in mature flowers as biochemical characters, along with two morphological characters. As a result of HPLC analysis, five out of six anthocyanin peaks were detected in 42 Lantana plants. Pigments were identified as: 3-glucoside, 3,5-diglucoside, and 3-malonylglucoside of cyanidin, and 3,5-diglucoside and 3-malonylglucoside of peonidin. The anthocyanin ratios in each plant were different; they presented several significant ratios. Based on these results, we can distinguish wild from horticultural plants. The flower color of Lantana will become more diverse through crosses between wild and horticultural plants.

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Thirteen SSR (simple sequence repeat) markers were successfully developed in carnation using an enriched genomic library method. All 13 primer pairs designed could successfully amplify target fragments of the expected size for the original variety ‘Francesco’ or ‘Barbara’. Thirty-nine of 41 carnation varieties, except for mutant varieties, were successfully distinguishable using 32 putative alleles and 15 polymorphic bands produced from 13 SSR markers. ‘U Conn Sim’ and ‘White Sim’, which are bud mutants originated from ‘William Sim’, showed an identical genotype to ‘William Sim’. Three (CB026a, CB057a, and CB060a) of 13 SSR markers were thought to be derived from multi-locus because the 3 SSR markers produced more than 3 discrete amplified fragments. The number of alleles per locus ranged from 1 (CB020a and CB041a) to 9 (CB003a), with an average value of 3.2. The values of the observed heterozygosity (H_O) and the expected heterozygosity (H_E) ranged from 0 to 0.59 (mean 0.30) and 0 to 0.68 (mean 0.34), respectively. The power of discrimination (PD) ranged from 0 (CB020a and CB041a) to 0.93 (CB026a) (mean 0.49). The parentage of 2 carnation varieties from intraspecific crosses was analyzed using 10 SSR markers, and the hybridity of the progenic varieties was confirmed. SSR markers were very effective for variety discrimination and determination of carnation parentage.

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To investigate the mechanism of browning occurring during the processing of semi-dried ‘Atago’ persimmon (Diospyros kaki Thunb.) fruit, the changes in several components related to enzymatic and non-enzymatic browning were determined. The browning of semi-dried persimmons developed when the water content of fruit decreased to about 50% or less, and hand massage (HM) treatment promoted both the drying process and development of browning. The polyphenol content as a substrate of polyphenol oxidase (PPO) markedly decreased during the first 7 days of drying before the occurrence of browning. PPO activity gradually decreased and was lower in dry + HM than dry treatment alone during drying. The ascorbic acid (AsA) content decreased and HM accelerated the reduction by 7 days of drying. Dehydroascorbic acid (DHA) also decreased with stagnation in the mid-stage during 14 days of drying and was lower in dry + HM than dry treatment alone during the latter period of drying. Furfural, a non-oxidative degradation product of AsA, started to increase from 4 or 10 days of drying in dry + HM or dry treatment, respectively. Reducing sugars, fructose and glucose, markedly increased for the first 4 days with a concomitant decrease in the sucrose content, and were higher in dry + HM than dry treatment. The content of amino acids gradually decreased, and no difference was observed between treatments. Hydroxymethylfurfural (HMF), as an intermediate product from fructose to brown pigments, decreased during drying and was lower in dry + HM than dry treatment alone. These results suggest that the oxidative and non-oxidative degradation of AsA would contribute markedly to the browning of semi-dried persimmon fruit, and enzymatic browning by PPO and the Maillard reaction between amino acids and reducing sugars might not play an important role.

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