中村 郁郎

Climate change, with its attendant negative effects, is expected to hamper agricultural production in the coming years. To counteract these negative effects, breeding of environmentally resilient plants via conventional means and genetic engineering is necessary. Stress defense genes are valuable tools by which this can be achieved. Here we report the successful cloning and functional characterization of a melon Y3SK2-type dehydrin gene, designated as CmLEA-S. We generated CmLEA-S overexpressing transgenic tobacco lines and performed in vitro and in vivo drought and salt stress analyses. Seeds of transgenic tobacco plants grown on 10% polyethylene glycol (PEG) showed significantly higher germination rates relative to wild-type seeds. In the same way, transgenic seeds grown on 150 mM sodium chloride (NaCl) recorded significantly higher germination percentages compared with wild-type plants. The fresh weights and root lengths of young transgenic plants subjected to drought stress were significantly higher than that of wild-type plants. Similarly, the fresh weights and root lengths of transgenic seedlings subjected to salt stress treatments were also significantly higher than wild-type plants. Moreover, transgenic plants subjected to drought and salt stresses in vivo showed fewer signs of wilting and chlorosis, respectively. Biochemical assays revealed that transgenic plants accumulated more proline and less malondialdehyde (MDA) compared with wild-type plants under both drought and salt stress conditions. Finally, the enzymatic activities of ascorbate peroxidase (APX) and catalase (CAT) were enhanced in drought-and salt-stressed transgenic lines. These results suggest that the CmLEA-S gene could be used as a potential candidate gene for crop improvement.

Dioscorea rotundata is an economically important food crop in many tropical countries as many people in this region depend on it for food and livelihood. Viral diseases, especially Yam mosaic virus (YMV), constitute a major constraint in the cultivation of this crop as they perpetuate through generations in the vegetatively propagated planting materials. Getting resistant or at least virus-free planting materials for farmers thus becomes crucial. This study was aimed at eliminating YMV in Dioscorea rotundata by cryotherapy of axillary buds. Enlarged axillary buds of YMV-infected TDr 2269 were frozen in liquid nitrogen for 1 h, re-warmed at 40 °C and cultured to regenerate plantlets. Approximately 76.33% plantlet regeneration and 100% YMV eradication were obtained for cryo-treated buds, against 95% and 0% obtained respectively for non-treated buds. RT-PCR and RT-qPCR analyses did not reveal detectable quantity of YMV in treated plants but did in control plants. Plants from cryo-treated buds showed no mosaic symptoms and produced slightly more tubers, and heavier mini-tubers (20.48±3.11 g) under greenhouse conditions contrary to non-treated plants that showed severe mosaic symptoms with significantly smaller tubers (1.91±0.39 g) (P < 0.05). This is the first report showing the elimination of YMV from infected white yam stock plant by cryotherapy and would be useful for producing clean planting materials.

Controlling root-knot nematodes (Meloidogyne spp.) using RNA interference (RNAi)-based molecular strategy is currently gaining consideration as a safer alternative to the use of chemical nematicides. However, identifying target genes whose knockdown in the parasites can effectively protect host plants is critical to success of this strategy. In this study, we transformed tomato plants to express hairpin RNA of Meloidogyne incognita-specific sequence (MiPA) of PolA1, an essential single-copy nuclear gene encoding the largest subunit of RNA polymerase I enzyme. We then evaluated nematode resistance in T1 transgenic lines for efficacy of host plant-mediated silencing of PolA1 gene in invading nematodes. We observed a significant reduction in nematode egg masses per plant root, eggs per egg mass and overall parasite multiplication in the transgenic plants compared to the wild type. Transgenic plants also had a reduced number of nematode galls per plant root than wild type but not significant. Although agronomic traits evaluated at the early growth stage were comparable in transgenics and wild types of tomato plants, production of MiPA-specific siRNAs in transgenic plants as well as the significant reduction of nematode PolA1 gene expression in feeding nematodes isolated from roots of transgenic plants provided evidence of host-induced gene silencing. Put together, our results are an indication that silencing of PolA1 gene is useful for reducing propagation of root-knot nematodes in tomato hosts. Its application to other crops may be possible and useful in improving agricultural productivity.

Oryza rufipogon Griff. is a perennial species of wild rice widely distributed along the channels and rivers of the Mekong Delta, Vietnam. This study attempted to find centers of diversity among wild rice populations in this area and their inter-relationships. The highest genetic diversity was found in the Dong Thap population and the lowest in the Can Tho population. Maternal diversity evaluated using chloroplast INDELs detected ten plastid types, five of which were novel relative to other Asian countries. The mitochondrial genome suggested two unique deletions. One 699-bp deletion via short tandem repeats was accompanied by another deletion including orf153. All accessions carrying the mitochondrial type were found in a particular plastid type. This unique maternal lineage was confined to specific channels where it showed vigorous vegetative growth in comparison to upstream areas where various maternal lineages and maximum genetic diversity occurred. This area along the Mekong Delta is a center of not only nuclear but also maternal diversity.

Molecular evolution has dominated taxonomic studies for decades, replacing traditional methods for identification and classification. However, there is a need for better markers to resolve the problems that have limited their usefulness. In this report, we introduce the protein tag (Ptag) sequence, a highly polymorphic amino acid sequence within the C-terminal region of the largest subunit of RNA polymerase I, as a new systematic tag sequence for delineating the evolutionary history of medically important fungi. As Ptag sequences are highly polymorphic between species and low within species, 42 fungal species representing the main taxonomic groups in the phyla Ascomycota and Basidiomycota were tested. The phylogenetic tree inferred from the Ptag sequences showed high consistency with the accepted classification of the Assembling the Fungal Tree of Life (AFTOL) project. Moreover, it could resolve the interspecies phylogenetic relationships of the tested taxa. In contrast, the phylogeny inferred from the nucleotide tag (Ntag) sequence, encoding the Ptag peptide, displayed lesser discriminatory power in resolving the phylogenetic relationships among distantly related taxa. In the case of closely related fungal species, the phylogenetic trees for Ptag and Ntag sequences were consistent with the tree for ITS1 sequences of 11 dermatophytic species. Taken together, the Ptag sequences should contribute to inferring phylogeny among species whereas the Ntag sequences should be useful to analyze variations among closely related species to resolve taxonomic issues in fungi.

Key message: Forced expression of rice45S rRNAgene conferred ca. 2-fold increase of above-ground growth in transgenicArabidopsis. This growth increase was probably brought by cell proliferation, not by cell enlargement. Abstract: Recent increase in carbon dioxide emissions is causing global climate change. The use of plant biomass as alternative energy source is one way to reduce these emissions. Therefore, reinforcement of plant biomass production is an urgent key issue to overcome both depletion of fossil energies and emission of carbon dioxide. Here, we created transgenic Arabidopsis with a 2-fold increase in above-ground growth by forced expression of the rice 45S rRNA gene using the maize ubiquitin promoter. Although the size of guard cells and ploidy of leaf-cells were similar between transgenic and control plants, numbers of stomata and pavement cells were much increased in the transgenic leaf. This data suggested that cell number, not cell expansion, was responsible for the growth increase, which might be brought by the forced expression of exogenous and full-length 45S rRNA gene. The expression level of rice 45S rRNA transcripts was very low, possibly triggering unknown machinery to enhance cell proliferation. Although microarray analysis showed enhanced expression of ethylene-responsive transcription factors, these factors might respond to ethylene induced by abiotic/biotic stresses or genomic incompatibility, which might be involved in the expression of species-specific internal transcribed spacer (ITS) sequences within rice 45S rRNA transcripts. Further analysis of the mechanism underlying the growth increase will contribute to understanding the regulation of the cell proliferation and the mechanism of hybrid vigor.

An increase in plant biomass production is desired to reduce emission of carbon dioxide emissions and arrest global climate change because it will provide a more source of energy production than fossil fuels. Recently, we found that forced expression of the rice 45S rRNA gene increased aboveground growth by ca. 2-fold in the transgenic Arabidopsis plants. Here, we created transgenic tobacco plants harboring the rice 45S rRNA driven by the maize ubiquitin promoter (UbiP::Os45SrRNA) or cauliflower mosaic virus 35S promoter (35SP::Os45SrRNA). In 35SP::Os45SrRNA and UbiP::Os45SrRNA transgenic tobacco plants, the leaf length and size were increased compared with control plants, leading to an increase of aboveground growth (dry weight) up to 2-fold at the early stage of seedling development. Conversely, leaf physiological traits, such as photosynthetic capacity, stomatal characteristics, and chlorophylls and RuBisCO protein contents, were similar between the transgenic and control plants. Flow cytometry analysis indicated that the transgenic plants had enhanced cell-proliferation especially in seedling root and leaf primordia. Microarray analysis revealed that genes encoding transcription factors, such as GIGANTEA-like, were more than 2-fold up-regulated in the transgenic plants. Although the mechanism underlying the increased growth has yet to be elucidated, this strategy could be used to increase biomass production in cereals, vegetables, and bio-energy plants.

The Brassica genus comprises various important species, of which three diploid species, B. rapa (A genome), B. nigra (B), and B. oleracea (C), yielded three different pair-wise amphidiploids: B. juncea (AB), B. napus (AC), and B. carinata (BC), showing the “triangle of U”. Although DNA sequences of many genes have been analyzed to reveal the relationships between A, B, and C genomes, the phylogeny of any single-copy nuclear gene has not supported the entire relationships of U’s triangle. Most nuclear genomic sequences of plants have genetically recombined between alleles in inter-specific hybrids, while we recently found that intron 19 and nucleotide tag (Ntag) sequences of the single-copy nuclear PolA1 gene, encoding RNA polymerase I’s largest subunit, had rarely recombined during the introgressive hybridizations in Aegilops speltoides. Because phylogenetic analysis including recombined sequences cannot reveal the phylogeny before the recombination occurred, only analysis of non-recombinational DNA sequences can resolve the true evolutionary route. In this study, the phylogenetic relationships of the PolA1 gene in the six Brassica species were clearly consistent with U’s triangle. In addition, two groups of B. napus were shown divergently to have originated from the amphidiploidization between B. oleracea and two progenitors of B. rapa.

Optimum conditions for obtaining tetraploid were investigated in raphanobrassica, the intergeneric hybrid between radish (Raphanus sativus) and kale (Brassica oleracea var. acephala) by treating in vitro plants with an anti-mitotic agent, amiprophosmethyl (APM). Initially, no tetraploids but hexaploids and octaploids were induced by the treatments. Although the leaves of these polyploids of raphanobrassica showed chlorosis during subcultures in in vitro conditions, the chlorosis could be successfully prevented by the ethylene inhibitors, both AVG and AgNO3Based on this result, AVG was added into medium used for the culture after the chromosome doubling treatment, which subsequently resulted in increased survival rates of the treated plant materials as well as increased production rates of polyploids including tetraploid. These polyploid plants showed obviously different characters from the original diploid plant. The tetraploid plant had bigger sizes in shoot, flower and leaf, and more number of leaves than the diploid. On the other hand, the hexaploid and octaploid plants had smaller sizes in shoots and leaves, and less number of leaves than the diploid. Concentration of glucosinolates, functional substances of Brassicaceae crops, did not significantly differ between diploid and tetraploid of raphanobrassica, but reduced in hexaploid and octaploid.

Brassica rapa L. used to be classified into two species, Brassica campestris and B. rapa.These two species can cross with each other, but no molecular marker exists to discriminate between them. Therefore, these two species are now combined into one species, B. rapa. Because nuclear genomes have been genetically recombined through repeated inter-specific hybridisation, most DNA sequences have lost the characteristics unique to each species. Thus, in order to trace an evolutionary lineage, it is important to analyse a DNA sequence that rarely recombines during meiosis in inter-specific hybrids. Sequences of the nineteenth intron of the PolA1 gene in 33 accessions of B. rapa, encoding the largest subunit of RNA polymerase I, showed either short (S; 183 bp) or long (L; 242 bp) type sequences. Nucleotide tag (Ntag) sequences, the protein-coding sequence (ca. 1.2 kb) from exons 19 – 21 of the PolA1 gene, in 20 accessions, were also classified into the same S- or L-types. Detailed sequence analysis showed that 13 single nucleotide polymorphisms (SNPs) in the 5’-half of the Ntag sequence did not recombine between the S- and L-types. These data suggest that two ancestral lineages were present and were probably involved in the origin of Chinese cabbage because accessions of B. rapa var. pekinensis had PolA1 gene sequences of either the S- or L-type, or both. Two accessions of B. napus var. rapifera (2n = 38) showed both the S- and L-types of B. rapa (2n = 20), indicating that this species might be an allotetraploid between the two ancestral lineages, followed by two chromosome deletions.

Cassava ranks fifth among the starch producing crops of the world, its annual bioethanol yield is higher than for any other crop. Cassava cultivar KU50, the most widely grown cultivar for non-food purposes is susceptible to Sri Lankan cassava mosaic virus (SLCMV). The objective of this work was to engineer resistance to SLCMV by RNA interference (RNAi) in order to increase biomass yield, an important aspect for bioethanol production. Here, we produced transgenic KU50 lines expressing dsRNA homologous to the region between the AV2 and AV1 of DNA A of SLCMV. High level expression of dsRNA of SLCMV did not induce any growth abnormality in the transgenic plants. Transgenic lines displayed high levels of resistance to SLCMV compared to the wild-type plants and no virus load could be detected in uninoculated new leaves of the infected resistant lines after PCR amplification and RT-PCR analysis. The agronomic performance of the transgenic lines was unimpaired after inoculation with the virus as the plants presented similar growth when compared to the mock inoculated control plants and revealed no apparent reduction in the amount and weight of tubers produced. We show that the resistance is correlated with post-transcriptional gene silencing because of the production of transgene specific siRNA. The results demonstrate that transgenic lines exhibited high levels of resistance to SLCMV. This resistance coupled with the desirable yield components in the transgenic lines makes them better candidates for exploitation in the production of biomass as well as bioethanol.

Prunus × yedoensis ‘Someiyoshino’ is the most popular cultivar of flowering cherry in Japan. Although the origin of this cultivar has been considered hybrid between P. pendula f. ascendens and P. lannesiana var. speciosa, the paternity of P. lannesiana has not been clearly proven by molecular analysis. To reveal the origin of ‘Somei-yoshino,’ we analyzed sequences of intron 19 and exon 20 of PolA1, a single-copy nuclear gene encoding the largest subunit of RNA polymerase I. One of two exon 20 sequences found in ‘Somei-yoshino’ was the same as that of P. pendula, whereas the other sequence was shared with several taxa in seven wild species, including P. jamasakura and P. lannesiana. ‘Somei- yoshino’ contained two different haplotypes of the intron 19 sequences; one was the same as that of P. lannesiana, which is endemic to the Izu and Boso Peninsula in Japan. While another haplotype of ‘Somei-yoshino’ was different from that of P. pendula by two SNPs but identical to one of two haplotypes of P. pendula ‘Komatsuotome,’ which is a cultivar found in the Ueno Park, Tokyo. These results indicated that ‘Someiyoshino’ probably originated by the hybridization of cultivars derived from P. pendula and P. lannesiana.

In May 2011, numerous poppy plants closely resembling Papaver bracteatum Lindl., a type of narcotic plant that is illegal in Japan, were distributed directly from several large flower shops or through online shopping throughout Japan, including the Tokyo Metropolitan area. In order to better identify the narcotic plants, the relative nuclear DNA content at the vegetative stage was measured by flow cytometric (FCM) analysis in 3 closely-related species of the genus Papaver section Oxytona, namely P. orientale, P. pseudo-orientale, and P. bracteatum, based on the difference between the chromosome numbers of these species. The results showed that the nuclear DNA content differed between these 3 species, and that most of the commercially distributed plants examined in this study could be identified as P. bracteatum. The remaining plants were P. pseudo-orientale, a non-narcotic plant. In addition, the FCM results for the identification of P. bracteatum completely agreed with the results obtained by the morphological analysis, the inter-genic spacer sequence of rpl16-rpl14 (PS-ID sequence) of chloroplast DNA, and the presence of thebaine. These results clearly indicate the usefulness of FCM analysis for the identification of P. bracteatum plants, including when they are in their vegetative stage.

Multi-auto-transformation vector system has been one of the strategies to produce marker-free transgenic plants without using selective chemicals and plant growth regulators and thus facilitating transgene stacking. In the study reported here, retransformation was carried out in marker-free transgenic potato CV. May Queen containing ChiC gene (isolated from Streptomyces griseus strain HUT 6037) with wasabi defensin (WD) gene (isolated from Wasabia japonica) to pyramid the two disease resistant genes. Molecular analyses of the developed shoots confirmed the existence of both the genes of interest (ChiC and WD) in transgenic plants. Co-expression of the genes was confirmed by RT-PCR, northern blot, and western blot analyses. Disease resistance assay of in vitro plants showed that the transgenic lines co-expressing both the ChiC and WD genes had higher resistance against the fungal pathogens, Fusarium oxysporum (Fusarium wilt) and Alternaria solani (early blight) compared to the non-transformed control and the transgenic lines expressing either of the ChiC or WD genes. The disease resistance potential of the transgenic plants could be increased by transgene stacking or multiple transformations. © 2014 Springer Science+Business Media.

In early spring, the flowering of cherry trees is taken as a good sign for farmers to initiate rice cultivation in Japan. In the era before calendars, the timing of cultivation was very important for growing rice plants in temperate regions. Nine native species of flowering cherry (Prunus subgenus Cerasus) are present in Japan and they are classified into three groups: Yamazakura, Miyamazakura, and Edohigan. More than 250 cultivars of Japanese flowering cherry have been selected or bred from these wild species. Two species, Oshimazakura and Edohigan, have specially contributed to the breeding of flowering cherry cultivars. While Edohigan is distributed in most areas of Japan, Oshimazakura (of the Yamazakura group) is an endemic species found around the Izu and Boso Peninsulas. ‘Somei-yoshino,’ Prunus × yedoensis, is the most popular cultivar and now comprises 80-90% of all flowering cherry trees planted in Japan. ‘Someiyoshino’ was probably created through hybridization between Edohigan and Oshimazakura in the Edo era. In this paper, the diversity and breeding of Japanese flowering cherry, including the origin of ‘Somei-yoshino,’ are described along with the political and horticultural backgrounds.

Tetraploid plants were successfully induced for the first time in Mitracarpus hirtus L., by overnight immersion of shoot meristems in 0.1 % colchicine solution, followed by in vitro culture leading to plant regeneration. Examination of ploidy level by flow cytometric analysis and counting chromosome number at metaphase confirmed that original diploid plant (WT1) contained chromosome number as 2n = 2x = 28, whereas 2n = 4x = 56 was observed in the tetraploids induced with colchicine treatment (CC102 and CC110). Thicker root formation, larger stomata (1.3-2 times), and lower density of stomata (1.7-4 times) were observed in these tetraploid plants. After transplantation to the pot, tetraploid plant (CC110) showed higher fresh weights of aerial part and leaves (1.5 and 1.4 times respectively) than diploid. However, the methanolic extracts from leaves of tetraploid line CC102 showed inhibition against human pathogenic bacterium, S. aureus while WT1 and CC110 showed no activity. GC-MS revealed 40 unique compounds present in CC102, but absent in WT1 and CC110. Through hierarchical clustering analysis the 40 unique compounds in CC102 formed a cluster group found to correlate with anti-S. aurens activity. These results suggested that tetraploid M. hirtus CC102 created in this study provides a novel source of compounds useful in fighting infectious disease. © 2014 Springer Science+Business Media Dordrecht.

While Arabidopsis bears only one MET1 gene encoding the DNA methyltransferase that is mainly responsible for maintaining CG methylation after DNA replication, rice carries two MET1 genes, MET1a and MET1b, expressed in actively replicating and dividing cells, and MET1b is more abundantly expressed than is MET1a. A met1a null mutant displayed no overt phenotypes, implying that MET1b must play a major role in the maintenance DNA methylation. Here, we employed two met1b null mutants, generated by homologous recombination-mediated knock-in targeting and insertion of endogenous retrotransposon Tos17. These MET1a/MET1a met1b/met1b homozygotes exhibited abnormal seed phenotypes, which is associated with either viviparous germination or early embryonic lethality. They also displayed decreased levels of DNA methylation at repetitive CentO sequences and at the FIE1 gene locus in the embryos. In addition, independently isolated knock-in-targeted plants, in which the promoterless GUS reporter gene was fused with the endogenous MET1b promoter, showed the reproducible, dosage-dependent, and spatiotemporal expression patterns of GUS. The genotyping analysis of selfed progeny of heterozygous met1a met1b null mutants indicated that weakly active MET1a seems to serve as a genetic backup mechanism in rice met1b gametophytes, although the stochastic and uncoordinated activation of epigenetic backup mechanisms occurred less efficiently in the met1b homozygotes of rice than in the met1 homozygotes of Arabidopsis. Moreover, passive depletion of CG methylation during the postmeiotic DNA replication in the haploid nuclei of the met1a met1b gametophytes in rice results in early embryonic lethality. This situation somewhat resembles that of the met1 gametophytes in Arabidopsis. © 2014 Springer Science+Business Media Dordrecht.

Fusarium wilt is a soil-borne disease causing substantial yield losses in various crops and vegetables. We have previously reported the synthetic chitinase (NIC) gene (1.2 kb), in which codon usage of fungus, replaced with that of plant, conferred resistance against Botrytis cinerea. In this study, the NIC or GUS gene was linked to two root-specific promoters, LjNRT2 or AtNRT2.1 (nitrate transporter 2), derived from Lotus japonica and Arabidopsis thaliana, respectively. Transgenic tobacco lines expressing LjNRT2-GUS and LjNRT2-NIC, and tomato lines expressing AtNRT2.1-NIC, were produced by Agrobacterium-mediated transformation. GUS histochemical staining was observed in vascular regions of the roots but was conspicuously absent in the leaves of transgenic plants. Western blot analysis showed the production of NIC proteins in the roots but not in the leaves of transgenic tobacco and tomato lines. These results indicate that LjNRT2 and AtNRT2.1 promoters expressed transgenes in a root-specific manner. When in vitro whole plant resistance assay against Fusarium oxysporum was conducted, transgenic plants showed increased levels of resistance compared to non-transgenic plants. Antifungal activities of the root extract against spore germination of F. oxysporum showed lower CFU (colony-forming unit) than those of the leaf extract. Root colonization assay against F. oxysporum showed much lower CFU values in the roots of transgenic plants than in those of non-transgenic plants. These results suggest that NIC gene triggered by the root-specific promoters successfully expressed only in the roots and conferred increased levels of resistance against the root pathogen, F. oxysporum.

Key message: Marker-free transgenic eggplants, exhibiting enhanced resistance to Alternaria solani, can be generated on plant growth regulators (PGRs)- and antibiotic-free MS medium employing the multi-auto-transformation (MAT) vector, pMAT21 - wasabi defensin, wherein isopentenyl transferase (ipt) gene is used as a positive selection marker. Abstract: Use of the selection marker genes conferring antibiotic or herbicide resistance in transgenic plants has been considered a serious problem for environment and the public. Multi-auto-transformation (MAT) vector system has been one of the tools to excise the selection marker gene and produce marker-free transgenic plants. Ipt gene was used as a selection marker gene. Wasabi defensin gene, isolated from Wasabia japonica (a Japanese horseradish which has been a potential source of antimicrobial proteins), was used as a gene of interest. Wasabi defensin gene was cloned from the binary vector, pEKH-WD, to an ipt-type MAT vector, pMAT21, by gateway cloning technology and transferred to Agrobacterium tumefaciens strain EHA105. Infected cotyledon explants of eggplant were cultured on PGRs- and antibiotic-free MS medium. Extreme shooty phenotype/ipt shoots were produced by the explants infected with the pMAT21-wasabi defensin (WD). The same PGRs- and antibiotic-free MS medium was used in subcultures of the ipt shoots. Subsequently, morphologically normal shoots emerged from the Ipt shoots. Molecular analyses of genomic DNA from transgenic plants confirmed the integration of the WD gene and excision of the selection marker (ipt gene). Expression of the WD gene was confirmed by RT-PCR and Northern blot analyses. In vitro whole plant and detached leaf assay of the marker-free transgenic plants exhibited enhanced resistance against Alternaria solani. © 2013 Springer-Verlag Berlin Heidelberg.

Cucumber mosaic virus (CMV) is a tripartite, positive sense RNA virus causing infections and yield losses to many plant species. Here, we generated a construct containing inverted repeat of 1,793 bp fragment of defective CMV replicase gene derived from RNA2 of cucumber mosaic virus strain O (CMV-O). The replicase gene was modified by deleting a 9 bp region between nucleotides 1909-1918. This caused a deletion in the active centre motif of polymerases, producing defective translated product 9 nucleotides shorter than the full length protein. The RNAi construct containing inverted repeat of the defective gene was used to produce transgenic tobacco lines expressing CMV-derived double-stranded RNA via Agrobacterium-mediated transformation. Of the four transgenic lines inoculated with CMV-O or CMV-Y in vitro and ex vivo, three lines (T1, T4 and T5) showed immunity to both strains of CMV as no symptoms were detected, whereas one line (T7) exhibited high resistance with mild symptoms limited to inoculation portions. No virus could be detected in uninoculated new leaves of the transgenic lines after RTPCR and Dot-immunobinding assay analyses. Small interfering RNAs present in transgenic lines before and after virus challenge indicates that the resistance was acquired through RNA silencing. © Springer Science+Business Media 2013.

Antifungal peptides are a potential group of defense molecules that have been utilized to develop resistance to various plant pathogens. Wasabi defensin (WD) gene (0.5 kb) consists of cysteine-rich peptides that show potent growth inhibition of pathogenic filamentous fungi, such as Botrytis cinerea. Under regulation by the root-specific LjNRT2 or AtNRT2.1 promoter, WD gene was expressed in the roots of transgenic tobacco and tomato plants by Agrobacterium-mediated transformation. The regenerated plants showed stable integration of the transgene, with different insertion sites, and the transgene was expressed in the root tissues but not in the leaf tissues. This result confirmed that WD protein accumulated only in the roots of transgenic plants. In a bioassay for resistance to Fusarium oxysporum, all transgenic plants showed increased resistance to the fungus as compared to non-transformed plants. Protein extracts from root and leaf tissues were assayed for antifungal activity and the activity was express as the number of colonies formed per cm(2) (CFU cm(-2)). The CFU values of the root and leaf extracts of control plants did not show significant differences. In contrast, the CFU values of the root extracts of the transgenic plants were significantly lower than those of the leaf extracts and much lower than those of control. These results suggest that LjNRT2 and AtNRT2.1 promoters triggered the antifungal gene expression in the roots and conferred increased resistance to the root pathogen F. oxysporum. In the view of bio-safety, the root-specific expression of the transgene is desirable because the roots of tomato are not edible.

Cucumber mosaic virus is an important plant pathogen with a broad host range encompassing many plant species. This study demonstrates the production of transgenic potato lines exhibiting complete resistance to cucumber mosaic virus strain O and Y by post transcriptional gene silencing. Two constructs were used, one, pEKH2IN2CMVai, contains inverted repeat of 1,138 bp fragment of a defective CMV replicase gene derived from RNA2 of cucumber mosaic virus strain O (CMV-O), while the other, TRV-based VIGS vector (pTRV2CMVai), contains the same fragment of the replicase gene, but without inverted repeat. These constructs were used to produce transgenic potato lines of cultivar 'Danshaku', a susceptible genotype to CMV. Transgenic lines derived from pEKH2IN2CMVai accumulated small interfering RNA (siRNA) before and after virus challenge, whereas those derived from pTRV2CMVai showed siRNA expression after virus challenge. When transgenic lines were challenged with CMV-O or CMV-Y, four lines exhibited complete (100 %) resistance to both strains, whereas the other lines had high levels of resistance. Infectivity of CMV-O was lower than that of CMV-Y in the highly resistant plants. There were no significant differences with regard to resistance between plants derived from pEKH2IN2CMVai and those obtained from pTRV2CMVai. The presence of CMV-specific siRNA in the resistant phenotypes indicates that the resistance was acquired through RNA silencing. © 2013 Springer Science+Business Media Dordrecht.

Background: The perennial, Oryza rufipogon distributed from Asia to Australia and the annual O. meridionalis indigenous to Australia are AA genome species in the Oryza. However, recent research has demonstrated that the Australian AA genome perennial populations have maternal genomes more closely related to those of O. meridionalis than to those found in Asian populations of O. rufipogon suggesting that the Australian perennials may represent a new distinct gene pool for rice. Results: Analysis of an Oryza core collection covering AA genome species from Asia to Oceania revealed that some Oceania perennials had organellar genomes closely related to that of O meridionalis (meridionalis-type). O. rufipogon accessions from New Guinea carried either the meridionalis-type or rufirpogon-type (like O. rufipogon) organellar genomes. Australian perennials carried only the meridionalis-type organellar genomes when accompanied by the rufipogon-type nuclear genome. New accessions were collected to better characterize the Australian perennials, and their life histories (annual or perennial) were confirmed by field observations. All of the material collected carried only meridionalis-type organellar genomes. However, there were two distinct perennial groups. One of them carried an rufipogon-type nuclear genome similar to the Australian O. rufipogon in the core collection and the other carried an meridionalis-type nuclear genome not represented in the existing collection. Morphologically the rufipogon-type shared similarity with Asian O. rufipogon. The meridionalis-type showed some similarities to O. meridionalis such as the short anthers usually characteristic of annual populations. However, the meridionalis-type perennial was readily distinguished from O. meridionalis by the presence of a larger lemma and higher number of spikelets. Conclusion: Analysis of current accessions clearly indicated that there are two distinct types of Australian perennials. Both of them differed genetically from Asian O. rufipogon. One lineage is closely related to O. meridionalis and another to Asian O. rufipogon. The first was presumed to have evolved by divergence from O. meridionalis becoming differentiated as a perennial species in Australia indicating that it represents a new gene pool. The second, apparently derived from Asian O. rufipogon, possibly arrived in Australia later.

PolA1, a single-copy nuclear gene encoding the largest subunit of RNA polymerase I, comprises highly polymorphic intron 19 and nucleotide tag (Ntag) sequences. We analyzed these sequences in 42 accessions, which differed in ploidy, of Triticum-Aegilops and Hordeum species. The lengths of the intron 19 sequences were ca. 110 bp long in Triticum-Aegilops species, except in four Sitopsis species, Ae. longissima, Ae. searsii, Ae. sharonensis, Ae. speltoides, which had introns similar in length to those of Hordeum species, i. e., ca. 240 bp long. Phylogenetic analyses of the Ntag sequences showed that the four Sitopsis and remaining Triticum-Aegilops species were classified into two discrete Hordeum and Triticum clades, respectively. The A and D genome-specific Ntag sequences of polyploid wheats were highly homologous with those of T. urartu and Ae. tauschii, respectively. In Ae. bicornis, another Sitopsis species, two accessions had the short intron 19 and Triticum-type Ntag sequence, which were highly homologous with those of the B genome in polyploid wheats, whereas one accession contained the long intron 19 and Hordeum-type Ntag sequences. In contrast, partial sequence analyses revealed that the three accessions of Ae. bicornis shared highly homology to single-copy DMC1 and EF-G genes. The discrepancy between these results indicates that the Sitopsis species were probably established by hybrid speciation including ancient introgressive hybridization between progenitors of Triticum-Aegilops and Hordeum. Although many researchers have proposed Ae. speltoides as a candidate for the B genome donor, our data suggest the existence of diploid B genome species in the past that were responsible for the origin of both polyploid wheats and Sitopsis species, including Ae. speltoides. © 2012 Springer Science+Business Media B.V.

Genes encoding pathogenesis-related proteins, such as degrading enzymes of fungal cell wall polysaccharides, have been used to confer enhanced resistance to fungal pathogens of various plants. A new type of endo-β-mannanase gene, amn5A, was isolated from alkaliphilic Bacillus strain (JAMB-602) found in deep-sea sediment. The AMN5A mannanase is active over a wide pH range (pH 7-10) and stable at high temperature. In this study, transgenic tobacco plants expressing the amn5A gene were generated using Agrobacterium-mediated transformation. Polymerase chain reaction (PCR) analysis revealed that the amn5A gene was integrated into the genome of transgenic tobacco plants. Southern blot analysis showed that transgenic plants contained 1-6 copies of amn5A transgenes in their genome. Expression of the amn5A transgene was confirmed by reverse transcription-PCR analysis. Leaf extracts from the transgenic plants showed degradation activity of Konjak mannan. Antifungal assay of detached leaves and in vitro whole plantlets indicated that transgenic plants expressing amn5A gene acquired enhanced resistance to the soil-borne pathogenic fungus, Fusarium oxysporum, compared to untransformed control plants. © 2012 Korean Society for Plant Biotechnology and Springer.

An in-situ conservation site in Laos for a mixture of annual and perennial wild rice, LV27, which is a single swamp with an observation pier has been developed. In order to develop a strategy for evaluation of natural resources, systematic leaf sampling has been conducted and their genetic characteristics measured with 16 SSR loci. In order to determine population structure, a small number of individuals localized together were regarded as sub-populations belonging to a single mother population. Annual individuals were clustered at particular peripheral areas of the pond. Perennial individuals were close by and growing within deeper pond water. Scores of observed heterozygosity (Ho) were not significantly different between annual and perennial sub-populations, but relatively lower in annual ones. Genetic distance among annual and perennial sub-populations in close juxtaposition at peripheral sites showed that annuals were clustered against perennials. In addition, comparison of perennial sub-populations peripheral areas and inside the swamp, found they clustered together and were some distance from annual ones. When the genetic components were compared in detail, private alleles were frequently found in annual plants, suggesting there might be restriction of gene flow between annual and perennial types. Partitions of deep water perennial sub-populations identified private alleles in particular areas, suggesting there were some areas with unique polymorphisms. Combining peripheral perennial sub-populations led to the disappearance of most private alleles which implied there is frequent gene flow among perennial sub-populations. This in-situ conservation site allowed us to observe the succession of populations and also to research detailed population structure of a typical wild population and this found wild rice genetic structure in this single swamp is complex. The data obtained will provide valuable insight about how to evaluate wild populations genetically and how to deal with such populations as field collections.

Five novel thionin genes have been isolated from three Brassicaceae species, Brassica oleracea var. acephala, Nasturtium officinale, and Barbarea vulgaris. Comparison of the deduced amino acid sequences shows that these thionin proteins share seven highly conserved cysteine residues. These five thionin genes were, respectively, inserted between cauliflower mosaic virus 35S promoter and nopaline synthase terminator in a binary vector pEKH2. Transgenic potato plants were generated through Agrobacterium-mediated transformation methods. Southern blot analysis of transgenic potato plants indicated successful integration with varying copy number of the thionin genes into the plant genomes. Expressions of thionin transgenes in the transgenic plants were confirmed by RT-PCR and their protein products were immunologically detected by western blot analyses. Antifungal assay using detached leaves from transgenic lines expressing either of the thionin genes showed similar levels of enhanced resistance to Botrytis cinerea as compared to those of non-transformed control plants. Novel thionin genes isolated in this study are probably useful tools to produce transgenic plants resistant to various phytopathogens. © 2012 The Japanese Society for Plant Cell and Molecular Biology.

Disease resistance potential of a synthetic fungal chitinase (NIC) gene was evaluated in transgenic Petunia hybrid employing Agrobacterium tumefaciens-mediated genetic transformation. The NIC gene (1271-bp in length) was synthesized to encode the same amino acid sequence (except (25)Ser to (25)Arg) as that of chitinase 1 gene (chi1) from Rhizopus oligosporus. As 18% of codons in the NIC gene were changed from fungal type (AT-rich) to plant type (GC-rich), the nucleotide sequence of NIC had 82% homology with the chi1 gene. Petunia hybrida 'Danty Lady' was transformed with A. tumefaciens EHA 101 harboring a binary vector plasmid containing NIC (gene of interest) and nptII (selection marker) genes. Putative transgenic plants were produced on MS medium containing kanamycin monosulphate as a selective chemical. PCR analysis revealed that the NIC and nptII genes are integrated into the genome of transgenic plants. Integrations of the transgenes were indicated by Southern blot analysis of genomic DNA from the transgenic plants. Expression of NIC gene at the mRNA level was confirmed by reverse-transcription-polymerase chain reaction (RT-PCR). Western blot analysis detected the accumulation of NIC protein in the leaves of transgenic plants. The transgenic plants exhibited enhanced resistance against Botrytis cinerea (grey mold) as indicated by inhibition of the fungal growth in detached leaves and by the total protein extract. From these results it could be concluded that the NIC gene was successfully integrated into the genome of transgenic petunia plants and produced the NIC protein. Expression of the synthetic chitinase, NIC gene, probably, conferred enhance resistance to B. cinerea in transgenic petunia.

The presence of marker genes conferring antibiotic or herbicide resistance in transgenic plants has been a controversial issue and a serious problem for their public acceptance and commercialization. The MAT (multi-auto-transformation) vector system has been one of the strategies developed to excise the selection marker gene and produce marker-free transgenic plants. In an attempt to produce transgenic marker-free Petunia hybrida plants resistant to Botrytis cinerea (gray mold), we used the ipt gene as a selectable marker gene and the wasabi defensin (WD) gene, isolated from Wasabia japonica (a Japanese horseradish which has been a potential source of antimicrobial proteins), as a gene of interest. The WD gene was cloned from the binary vector, pEKH-WD, to an ipt-type MAT vector, pMAT21, by gateway cloning technology and transferred to Agrobacteriumtumefaciens strain EHA105. Infected leaf explants of P. hybrida were cultured on hormone- and antibiotic-free MS medium. Extreme shooty phenotype (ESP)/ipt shoots were produced by the explants infected with the pMAT21-WD. The same antibiotic- and hormone-free MS medium was used in subcultures of the ipt shoots. Ipt shoots subsequently produced morphologically normal shoots. Molecular analyses of genomic DNA from the transgenic plants confirmed the integration of the gene of interest and excision of the selection marker. Expression of the WD gene was confirmed by northern blot and western blot analyses. A disease resistance assay of the marker-free transgenic plants exhibited enhanced resistance against B. cinerea strain 40 isolated from P. hybrida. © 2010 Springer Science+Business Media B.V.

The selection marker genes, imparting antibiotic or herbicide resistance, in the final transgenics have been criticized by the public and considered a hindrance in their commercialization. Multi-auto-transformation (MAT) vector system has been one of the strategies to produce marker-free transgenic plants without using selective chemicals and plant growth regulators (PGRs). In the study reported here, isopentenyltransferase (ipt) gene was used as a selection marker and wasabi defensin (WD) gene, isolated from Wasabia japonica as a target gene. WD was cloned from the binary vector, pEKH-WD to an ipt-type MAT vector, pMAT21 by gateway cloning and transferred to Agrobacteriumtumefaciens strain EHA105. Infected cotyledons of tomato cv. Reiyo were cultured on PGR- and antibiotic-free MS medium. Adventitious shoots were developed by the explants infected with the pMAT21/wasabi defensin. The same PGR- and antibiotic-free MS medium was used in subcultures of the adventitious shoot lines (ASLs) to produce ipt and normal shoots. Approximately, 6months after infection morphologically normal shoots were produced. Molecular analyses of the developed shoots confirmed the integration of gene of interest (WD) and excision of the selection marker (ipt). Expression of WD was confirmed by Northern blot and Western blot analyses. The marker-free transgenic plants exhibited enhanced resistance against Botrytis cinerea (gray mold), Alternaria solani (early blight),Fusarium oxysporum (Fusarium wilt) and Erysiphe lycopersici (powdery mildew). © 2011 Springer-Verlag.

Marker-free transgenic tobacco (Nicotiana tabacum) lines containing a chitinase (ChiC) gene isolated from Streptomyces griseus strain HUT 6037 were produced by Agrobacterium-mediated transformation. One marker-free transgenic line, TC-1, was retransformed with the wasabi defensin (WD) gene, isolated from Wasabia japonica. Of the retransformed shoots, 37% co-expressed the ChiC/WD genes, as confirmed by western and northern analyses. Southern blot analysis showed that no chromosomal rearrangement was introduced between the first and the second transformation. Transgenic lines either expressing ChiC or WD, or co-expressing both genes were challenged with Fusarium oxysporum f.sp. nicotianae (Fon). Assessment of in vitro plant survival in the presence of Fon showed that transgenic lines co-expressing both genes had significantly enhanced protection against the fungus (infection indices 0·0-1.·2) compared with corresponding isogenic lines expressing either of the genes (infection indices 2·5-9·8). Whole-plant infection indices in transgenic lines were significantly related (r=0·93, P<0·01) to the extent of root colonization of the host, which ranged from 2·1% to 11·3% in lines co-expressing both genes, and from 16·8% to 37·7% in lines expressing just one of the genes (compared with 86·4% in non-transformed controls). Leaf extracts of transgenic lines also inhibited mycelial growth of Fon in vitro and caused hyphal abnormalities. © 2010 The Authors. Plant Pathology © 2010 BSPP.

Betaine aldehyde dehydrogenase (BADH) is a major oxidative enzyme that converts betaine aldehyde to glycine betaine (GB), an osmoprotectant compound in plants. Japonica rice (salt-sensitive) was genetically engineered to enhance salt tolerance by introducing the OsBADH1 gene from Indica rice (salt-tolerant), which is a GB accumulator. We produced transgenic rice plants overexpressing the modified OsBADH1 gene under the control of the maize ubiquitin promoter. The transgenic rice showed increased OsBADH1 gene expression and OsBADH1 enzyme production, resulting in the accumulation of GB. It also exhibited enhanced salt tolerance in immature and mature transgenic rice seedlings. The adverse effect of salt stress on seed germination, the growth of immature and mature seedlings, water status, and photosynthetic pigments was alleviated in transgenic seedlings. © 2010 Springer Science+Business Media B.V.

Multi-Auto-Transformation (MAT) vector system consists of positive selection, using the ipt or rol gene, with a site-specific recombination and DNA removal system, that generates morphologically normal marker-free transgenic plants without antibiotic selective-agent. This study describes rol-type MAT vector (pMAT101) containing lacZ gene as a model gene and the removable cassette with gus gene in the T-DNA region which was used to produce morphologically normal transgenic Kalanchoe blossfeldiana Poelln. employing rol gene as the selectable marker gene and gus gene as a reporter gene. Leaf explants inoculated with pMAT101 produced hairy roots with GUS expression on agar-solidified, half-strength MS medium without both plant growth regulators and selective agent under dark condition. These hairy roots produced shoots with Ri syndrome such as dwarfism, wrinkled leaves, and an over abundance of roots as a consequence of the morphogenic action of rol gene. They eventually produced morphologically normal shoots without GUS expression on the same fresh MS medium under 16h photoperiod. Molecular analysis of DNA from the hairy roots, shoots with Ri syndrome and morphologically normal shoots revealed that the normal shoots had only lacZ gene, and the removable cassette consisting of rol, R (recombinase) and gus genes was excised. This study indicates that the rol-type MAT vector could be used for the production of morphologically normal marker-free transgenic K. blossfeldiana plants without using selective chemical agents.

Glycine betaine has been reported as an osmoprotectant compound conferring tolerance to salinity and osmotic stresses in plants. We previously found that the expression of betaine aldehyde dehydrogenase 1 gene (OsBADH1), encoding a key enzyme for glycine betaine biosynthesis pathway, showed close correlation with salt tolerance of rice. In this study, the expression of the OsBADH1 gene in transgenic tobacco was investigated in response to salt stress using a transgenic approach. Transgenic tobacco plants expressing the OsBADH1 gene were generated under the control of a promoter from the maize ubiquitin gene. Three homozygous lines of T2 progenies with single transgene insert were chosen for gene expression analysis. RT-PCR and western blot analysis results indicated that the OsBADH1 gene was effectively expressed in transgenic tobacco leading to the accumulation of glycine betaine. Transgenic lines demonstrated normal seed germination and morphology, and normal growth rates of seedlings under salt stress conditions. These results suggest that the OsBADH1 gene could be an excellent candidate for producing plants with osmotic stress tolerance. © Korean Society for Plant Biotechnology and Springer 2009.

Triticum timopheevii (genome symbol AAGG) comprises two subspecies, cultivated ssp. timopheevii, and wild ssp. armeniacum. These two subspecies are considered as allotetraploids of AA genome from Triticum diploid species and SS genome from Aegilops species. The difference in genome symbol (G vs. S) is due to wide genetic variations among four SS genome species, Ae. bicornis, Ae. longissima, Ae. searsii, and Ae. speltoides. In order to study the origin of T. timopheevii, we compared 19th intron (PI19) sequence of the PolA1 gene, encoding the largest subunit of RNA polymerase I. Two different sized DNA fragments containing PI19 sequences (PI19A and PI19G) were amplified both in ssp. timopheevii and ssp. armeniacum. Shorter PI19A (112 bp) sequences of both subspecies were identical to PI19 sequences of two AA species, T. monococcum and T. urartu. Interestingly, the longer PI19G (241-243 bp) sequences of ssp. armeniacum showed more similarity to PI19 sequences of Ae. speltoides whereas ssp. timopheevii showed more similarity to PI19 sequences of other three SS genome species. The results indicated that two subspecies of T. timopheevii, ssp. armeniacum or ssp. timopheevii, might have arisen independently by allotetraploidization of AA genome with Ae. speltoides or one of the remaining three Aegilops species, respectively. © 2009 Springer Science+Business Media B.V.

The presence of marker genes conferring antibiotic or herbicide resistance in transgenic plants has been a serious problem for their public acceptance and commercialization. MAT (multi-auto-transformation) vector system has been one of the strategies to excise the selection marker gene from transgenic plants. Agrobacteriumtumefaciens strain EHA105 harboring a rol-type MAT vector, pMAT101, was used to produce morphologically normal transgenic Petunia hybrida 'Dainty Lady' employing rol gene as the selection marker gene. LacZ gene was used as a model gene of interest. Infected explants were cultured on plant growth regulator (PGR)- and antibiotic-free half-strength MS medium. Sixty-five percent of the infected explants produced hairy roots. The hairy roots were separated and proliferated on 1/2 MS hormone-free medium. Shoots produced from the hairy roots on 1/2 MS medium supplemented with benzylaminopurine (BA) and naphthalene acetic acid (NAA) exhibited hairy root syndrome (Ri syndrome) such as dwarfed, reduced apical dominance, short internodes and increased rooting, but subsequently produced normal-looking marker-free shoots. Molecular analysis of DNA from the hairy roots, shoots with Ri syndrome and morphologically normal shoots revealed that the normal shoots had only LacZ gene, and the removable cassette consisting of rol, R (recombinase) and GUS genes was excised. From this study it can be concluded that the chimeric rol genes can be used as a selection marker for Agrobacterinum-mediated transformation of Petunia hybrida and that the production of marker-free normal transgenic plants is possible without using selective chemical agents employing rol-type MAT vector. © 2010 Springer Science+Business Media B.V.

Production of "Egusi" melon (Colocynthis citrullus L.) in West Africa is limited by fungal diseases, such as Alternaria leaf spot and Fusarium wilt. In order to engineer "Egusi" resistant to these diseases, cotyledonary explants of two "Egusi" genotypes, 'Ejagham' and NHC1-130, were transformed with Agrobacterium tumefaciens strain EHA101 harbouring wasabi defensin gene (isolated from Wasabia japonica L.) in a binary vector pEKH1. After co-cultivation for 3 days, infected explants were transferred to MS medium containing 100 mgl-l kanamycin to select transformed tissues. After 3 weeks of culture, adventitious shoots appeared directly along the edges of the explants. As much as 19 out of 52 (36.5%) and 25 out of 71 (35.2%) of the explants in genotype NHC1-130 and 'Ejagham', respectively, formed shoots after 6 weeks of culture. As much as 74% (14 out of 19) of the shoots regenerated in genotype NHC1-130 and 72% (18 out of 25) of those produced in genotype 'Ejagham' were transgenic. A DNA fragment corresponding to the wasabi defensin gene or the selection marker nptII was amplified by PCR from the genomic DNA of all regenerated plant clones rooted on hormone-free MS medium under the same selection pressure, suggesting their transgenic nature. Southern blot analysis confirmed successful integration of 1-5 copies of the transgene. RT-PCR, northern and western blot analyses revealed that wasabi defensin gene was expressed in transgenic lines. Transgenic lines showed increased levels of resistance to Alternaria solani, which causes Alternaria leaf spot and Fusarium oxysporum, which causes Fusarium wilt, as compared to that of untransformed plants. © 2010 Springer-Verlag.

MAT (multi-auto-transformation) vector system has been one of the strategies to excise the selection marker gene from transgenic plants. Agrobacterium tumefaciens strain EHA105 harboring an ipt-type MAT vector, pNPI132, was used to produce morphologically normal transgenic Petunia hybrida 'Dainty Lady' employing isopentenyl transferase (ipt) gene as the selection marker gene. β-glucuronidase (GUS) gene was used as model gene of interest. Infected explants were cultured on Murashige and Skoog (MS) medium without plant growth regulators (PGR) and antibiotics. Shoots showing extreme shooty phenotype (ESP) were produced from the adventitious shoots separated from the explants. Visual selection was carried out until production of morphologically normal shoots (approximately 4 months after infection). Histochemical GUS assay detected GUS gene in both ESP and normal shoots. PCR analysis confirmed the presence of model gene (GUS gene) and excision of the selection marker (ipt) gene in the normal transgenic plants. The insertion sites (1 - 3 for ipt gene and 1 - 2 for GUS gene) were detected by Southern blot analysis using DIG-labeled probes of both genes. These results show that ipt-type MAT vector can be used successfully to produce marker-free transgenic Petunia hybrida plants on PGR- and antibiotic-free MS medium.

Protocorm-like bodies (PLBs) of Cattleya orchid CM2450 cultured either under constant illumination with cool-white-fluorescent lamps or in the dark were cocultivated with Agrobacterium tumefaciens strain EHA105 carrying plasmid pSMAHdN627-ORSV harboring genes coding for Odontoglossum ringspot virus (ORSV) replicase and hygromycin phosphotransferase. PLBs were maintained in liquid New Dogashima (ND) medium and then added to a bacterial suspension culture (OD600 approximate to 0.6) yielding medium dilution ratio of 1 : 10 and incubated for either 30 minutes or 3 h. Hygromycin-resistant secondary PLBs were induced after 4 weeks of culture on 2.5 g l(-1) gellan gum-solidified ND medium containing 1 mg l(-1) naphthaleneacetic acid (NAA), 0.1 mg l(-1) benzyladenine (BA), 10 mg l(-1) hygromycin, 20 mg l(-1) meropenem, 10 g l(-1) sucrose in both light-and dark-cultured PLBs. The number of resistant PLBs generated using dark-cultured PLBs was higher than those cultured under constant illumination. Presence of acetosyringone (AS) in the pre-culture medium was also effective for the transformation. The highest frequency of transformation was obtained when dark-cultured PLBs were pre-cultured with 100 mu M AS for 3 days and inoculated with Agrobacterium liquid culture for 3 h. Transformation of hygromycin-resistant plantlets regenerated from different sites of inoculated PLBs was confirmed by Southern blot hybridization. Transcription of ORSV replicase gene in transgenic lines was successfully confirmed by Northern blot hybridization.