Lepidopteran larvae are the most injurious pests of Chinese cabbage production. We attempted the development of transgenic Chinese cabbage expressing the potato proteinase inhibitor II gene (pinII) and bioassayed the pest-repelling ability of these transgenic plants. Cotyledons with petioles from aseptic seedlings were used as explants for Agrobacterium-mediated in vitro transformation. Agrobacterium tumefaciens C58 contained the binary vector pBBBasta-pinII-bar comprising pinII and bar genes. Plants showing vigorous PPT resistance were obtained by a series concentration selection for PPT resistance and subsequent regeneration of leaf explants dissected from the putative chimera. Transgenic plants were confirmed by PCR and genomic Southern blotting, which showed that the bar and pinII genes were integrated into the plant genome. Double haploid homozygous transgenic plants were obtained by microspore culture. The pinII expression was detected using quantitative real time polymerase chain reaction (qRT-PCR) and detection of PINII protein content in the transgenic homozygous lines. Insect-feeding trials using the larvae of cabbage worm (Pieris rapae) and the larvae of the diamondback moth (Plutella xylostella) showed higher larval mortality, stunted larval development, and lower pupal weights, pupation rates, and eclosion rates in most of the transgenic lines in comparison with the corresponding values in the non-transformed wild-type line.

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Inter-sectional hybrids were successfully obtained by the reciprocal crosses between 11 cultivars (including 6 diploids and 5 tetraploids) of Begonia semperflorens (SS & SSSS genomes) and B. ‘Orange Rubra’ (RR genome) with the aid of in vitro culture of mature or immature seeds on MS medium containing 0.1 mg l^-1 α-naphthylacetic acid, 0.1 mg l^-1 6-benzyladenine, 10 mg l^-1 gibberellic acid, 30 g l^-1 sucrose and 2.5 g l^-1 gellan gum. Embryo rescue as ovary culture with immature seeds 12^th–16^th day after pollination (DAP) generally gave higher efficiency of plantlet formation, but in some cross combinations, culture of mature seeds (30 DAP) resulted in higher yield of plantlets. Flow cytometric analysis revealed that they were consisted of the plants with various genomic combinations (RS, RR, RSS, RRS, RRSS and RRRRSS) as estimated by the DNA contents of both parents. Hybridity of these plants with various genomic combinations including RR was confirmed by random amplified polymorphic DNA analysis. These results suggested that unreduced gamete formation and spontaneous chromosome doubling were involved in the hybrid formation of various ploidy levels and genomic combinations. These hybrids showed various levels of intermediate traits between both parents according to the genomic compositions, and some of them had desirable characters of both parents.

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Pollen-mediated transgene flow is a major concern for the production of genetically modified (GM) rice. Cleistogamy is a useful tool for preventing this form of gene flow. We previously identified the cleistogamous rice mutant superwoman1-cleistogamy (spw1-cls) and determined its molecular genetic mechanism. In the present study, we cultivated spw1-cls over five years to examine effects of cleistogamy on agronomic traits. Simultaneously, we cultivated cleistogamous backcross lines created by continuous backcrossing with “Yumeaoba” (a japonica cultivar) as the recurrent parent and by application of a DNA marker. In these experimental cultivations, spw1-cls and its backcross lines showed almost equal or slightly lower, but acceptable, agronomic traits compared with each control line. We also conducted natural crossing tests in paddy fields to assess the gene containment capability of spw1-cls. In a series of field experiments, there was no natural crossing between spw1-cls (pollen donor) and pollen recipient lines, but the wild-type donor and recipient lines were crossed. Thus, the cleistogamy of the spw1-cls mutation is able to inhibit natural crossing effectively, without significant loss of commercial benefits, such as yield. We conclude that spw1-cls cleistogamy is a practical tool for gene containment in GM rice cultivation.

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Grain size is an important yield component in rice, however, genes controlling the trait remain poorly understood. Previously, we have shown that an antagonistic pair of basic helix-loop-helix (bHLH) proteins, POSITIVE REGULATOR OF GRAIN LENGTH 1 (PGL1) and ANTAGONIST OF PGL1 (APG), is involved in controlling rice grain length. Here, we report the involvement of another atypical bHLH protein gene, POSITIVE REGULATOR OF GRAIN LENGTH 2 (PGL2), in the regulation of rice grain length. Overexpression of PGL2 in the lemma/palea increased grain length and weight in correlation with the level of transgene expression. Observation of the inner epidermal cells of lemma of PGL2-overexpressing lines revealed that the long grain size is caused by an increase in cell length. PGL2 interacts with a typical bHLH protein APG, a negative regulator of rice grain length and weight, in vitro and in vivo. It was reported that overexpression of BU1 (BRASSINOSTEROID UPREGULATED 1), the closest homolog of PGL2, caused an increase in grain length. However, we detected no interaction between BU1 and APG. These findings suggest that PGL2 and PGL1 redundantly suppress the function of APG by forming heterodimers to positively regulate the rice grain length, while the pathway through which BU1, the closest homolog of PGL2, controls grain length is independent of APG.

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One of major objectives of crop breeding is conferring resistance to diseases and pests. However, large-scale phenotypic evaluation for many diseases and pests is difficult because strict controls are required to prevent their spread. Detection of disease resistance genes by using DNA markers may be an alternative approach to select potentially resistant accessions. Potato (Solanum tuberosum L.) breeders in Japan extensively use resistance gene H1, which confers nearly absolute resistance to potato cyst nematode (Globodera rostochiensis) pathotype Ro1, the only pathotype found in Japan. However, considering the possibility of accidental introduction of the other pathotypes, breeding of resistant varieties is an important strategy to prevent infestation by non-invading pathotypes in Japan. In this study, to evaluate the prevalence of resistance genes in Japanese genetic resources, we developed a multiplex PCR method that simultaneously detects 3 resistance genes, H1, Gpa2 and Gro1-4. We revealed that many Japanese varieties possess not only H1 but Gpa2, which are potentially resistant to other pathotypes of potato cyst nematode. On the other hand, no genotype was found to have the Gro1-4, indicating importance of introduction of varieties having Gro1-4. Our results demonstrate the applicability of DNA-marker assisted evaluation of resistant potato genotypes without phenotypic evaluation.

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Due to its critical importance in crop yield, the photoperiodic regulation of flowering time is considered an important trait in sorghum breeding programs. In this study, quantitative trait loci for flowering time were detected using an F₂ population derived from a cross between Kikuchi Zairai, a late-flowering cultivar originating from Japan and SC112, an early-flowering cultivar originating from Ethiopia. F₂ plants were grown with their parents under a natural day length and a 12 h day length. Two linkage maps were constructed using 213 simple sequence repeats markers. Nine quantitative trait loci controlling flowering time were identified in F₂ plants grown under a natural day length, whereas 7 QTLs were identified under a 12 h day length. Five QTLs controlling flowering time were shared under both of the day length conditions.

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To investigate the mode of inheritance of apomixis in Chinese chive, the degrees of diplospory and parthenogenesis were evaluated in F₁ and BC₁ progenies derived from crosses between amphimictic and apomictic diploids (2n = 16, 2x). The F₁ population was generated by crossing three amphimictic diploids 94Mo13, 94Mo49 and 94Mo50 with an apomictic diploid KaD2 and comprised 110 diploids and 773 triploids. All the diploid F₁ plants examined were completely or highly eusporous and completely syngamic. All the triploid F₁ plants examined were highly diplosporous and highly parthenogenetic. KaD2 could not transmit its high level of apomixis via monoploid pollen grains. The BC₁ population, generated by crossing 94Mo49 with apomictic triploids found in the F₁ offspring, exhibited heteroploidy; it comprised haploid, diploid, triploid, tetraploid and various aneuploid individuals. In this generation, clear segregation was observed between diplospory and parthenogenesis. Analysis of the BC₁ population suggests that diplospory and parthenogenesis are each controlled by single dominant genes, D and P, respectively. However, all the BC₁ plants characterized as parthenogenetic were diplosporous. The absence of phenotypically eusporous parthenogenetic plants can be explained by assuming that the presence of diplospory gene is a prerequisite for the parthenogenesis gene expression in Chinese chive.

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Genome evolution is a continuous process and genomic rearrangement occurs both within and between species. With the sequencing of the Arabidopsis thaliana genome, comparative genetics and genomics offer new insights into plant biology. The genus Brassica offers excellent opportunities with which to compare genomic synteny so as to reveal genome evolution. During a previous genetic analysis of clubroot resistance in Brassica rapa, we identified a genetic region that is highly collinear with Arabidopsis chromosome 4. This region corresponds to a disease resistance gene cluster in the A. thaliana genome. Relying on synteny with Arabidopsis, we fine-mapped the region and found that the location and order of the markers showed good correspondence with those in Arabidopsis. Microsynteny on a physical map indicated an almost parallel correspondence, with a few rearrangements such as inversions and insertions. The results show that this genomic region of Brassica is conserved extensively with that of Arabidopsis and has potential as a disease resistance gene cluster, although the genera diverged 20 million years ago.

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Deepwater rice possesses internode elongation ability to avoid drowning under deepwater conditions. Previous studies identified three QTLs regulating internode elongation ability on chromosomes 1, 3 and 12 using different populations. However, these QTLs only induce internode elongation in response to deepwater conditions from the 7-leaf stage and not during the early leaf stage. In this study, we detected two novel QTLs, qTIL2 and qTIL4 regulating deepwater response at the early leaf stage using an F₂ population derived from the cross between NIL1-3-12 carrying the three QTLs regulating deepwater response in T65 (O. sativa ssp. japonica) genetic background and C9285 (O. sativa ssp. indica, deepwater rice). Plants of the BC₂F₂ population derived from NIL1-3-12/C9285 and the RILs of T65/Bhadua (O. sativa ssp. indica, deepwater rice) possessing these QTLs as well as the three QTLs previously identified also showed internode elongation during the early leaf stage. These results indicate that qTIL2 and qTIL4 regulate early internode elongation and function in coordination with the three major QTLs under deepwater conditions. The results presented here would not only help define the mechanism of deepwater response in rice but also contribute in the breeding of deepwater tolerant rice that is adapted to various water depths.

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Tea is one of the most popular beverages in the world and the tea plant, Camellia sinensis (L.) O. Kuntze, is an important crop in many countries. To increase the amount of genomic information available for C. sinensis, we constructed seven cDNA libraries from various organs and used these to generate expressed sequence tags (ESTs). A total of 17,458 ESTs were generated and assembled into 5,262 unigenes. About 50% of the unigenes were assigned annotations by Gene Ontology. Some were homologous to genes involved in important biological processes, such as nitrogen assimilation, aluminum response, and biosynthesis of caffeine and catechins. Digital northern analysis showed that 67 unigenes were expressed differentially among the seven organs. Simple sequence repeat (SSR) motif searches among the unigenes identified 1,835 unigenes (34.9%) harboring SSR motifs of more than six repeat units. A subset of 100 EST-SSR primer sets was tested for amplification and polymorphism in 16 tea accessions. Seventy-one primer sets successfully amplified EST-SSRs and 70 EST-SSR loci were polymorphic. Furthermore, these 70 EST-SSR markers were transferable to 14 other Camellia species. The ESTs and EST-SSR markers will enhance the study of important traits and the molecular genetics of tea plants and other Camellia species.

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To exploit the genetic mechanism of cold tolerance in rice, cold tolerant near-isogenic lines (NILs) were developed by backcrossing Kunmingxiaobaigu (KMXBG), reported to be the most cold-tolerant variety at the booting stage, as donor, with the cold sensitive Japanese commercial japonica variety, Towada. Comparisons of cold tolerance-related traits between five BC₆F₅ NILs and recurrent parent Towada under cold treatment and normal temperatures at the booting stage showed that the differences between the NILs and Towada were significant only for spikelet fertility-related traits. Analyses of cold tolerance in the NILs at the budding (germination), seedling and booting stages indicated both correlated effects and differences. Lines 1913-4 and 1916-1 showed strong and stable tolerance at all three stages. Whole genome marker screening showed that the proportion of genetic background recovery was more than 98%. Seventeen markers from KMXBG were introgressed in two or more NILs, and cold tolerance genes were possibly present in these marker regions. The NILs should be excellent materials for both rice improvement and map-based cloning of cold tolerance QTLs.

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Novel mutant alleles of an ethylene receptor Solanum lycopersicum ETHYLENE RESPONSE1 (SlETR1) gene, Sletr1-1 and Sletr1-2, were isolated from the Micro-Tom mutant library by TILLING in our previous study. They displayed different levels of impaired fruit ripening phenotype, suggesting that these alleles could be a valuable breeding material for improving shelf life of tomato fruit. To conduct practical use of the Sletr1 alleles in tomato breeding, genetic complementation analysis by transformation of genes carrying each allele is required. In this study, we generated and characterized transgenic lines over-expressing Sletr1-1 and Sletr1-2. All transgenic lines displayed ethylene insensitive phenotype and ripening inhibition, indicating that Sletr1-1 and Sletr1-2 associate with the ethylene insensitive phenotype. The level of ethylene sensitivity in the seedling was different between Sletr1-1 and Sletr1-2 transgenic lines, whereas no apparent difference was observed in fruit ripening phenotype. These results suggested that it is difficult to fine-tune the extent of ripening by transgenic approach even if the weaker allele (Sletr1-2) was used. Our present and previous studies indicate that the Micro-Tom mutant library combined with TILLING could be an efficient tool for exploring genetic variations of important agronomic traits in tomato breeding.

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