チン ドンポー

Lilies (species and cultivars of the genus Lilium) are economically important ornamental plants because of their large and attractive flowers. The establishment of an efficient transformation system of lilies is desired for their genetic improvement with respect to ornamental and marketable qualities such as novel flower color and plant form as well as benefits for commercial cultivation such as virus resistance and environmental stress tolerance. In Lilium, genetic transformation has been achieved by electroporation, and biolistic and Agrobacterium-mediated transformation. However, the transformation efficiency varies according to the methods, species or cultivars. In the initial trials, only the biolistic method was used for Lilium genetic transformation, but eventually, Agrobacterium-mediated method has been incorporated as a successful approach for the genetic transformation of lilies in addition to a large number of monocots with better understanding of the process of monocot transformation, and availability of superior constructs, strains and vectors. This article summarizes the reported gene-transfer methods for Lilium, with an emphasis on the recent progress in introducing new traits into lily species in our laboratory.

In Arabidopsis, flowering is delayed under red light and induced under far red light and blue light. Studies suggest that the florigen, FLOWERING LOCUS T, is involved in the control of light quality-associated flowering in Arabidopsis. In petunia, similar to Arabidopsis, flowering is delayed under red light and induced under blue light, however its mechanism still remains unknown. Here we isolated a gene which has 75% amino acid sequence similarity with Arabidopsis FT (AtFT), named PehFT. By overexpressing PehFT in Arbidopsis and petunia, we tested its ability to induce flowering. Also, by conducting expression analyses of PehFT under different light quality treatments, we tested its response to light quality. We concluded that PehFT, like AtFT, is a gene which responds to photoperiod and light quality, but unlike AtFT, is not the main gene controlling the light quality-associated flowering.

Dendrobium orchid is one of the most popular cut flower and potted plants. In this study, a protocol for efficient genetic transformation of D. nobile-type orchids was established by co-cultivating 21 day-old protocorms for 3 days with Agrobacterium tumefaciens strain EHA101 carrying pIG121Hm harboring β-glucuronidase (GUS) gene as reporter gene and hygromycin phosphotransferase (hpt) gene as selectable marker gene. After selection of the infected protocorms on New Dogashima (ND) medium containing 10 gl−1 maltose, 30 mgl−1 hygromycin and 20 mgl−1 meropenem for 3 months followed by the culture on hygromycin-free recovery medium for 1 month, secondary protocorm-like bodies (PLBs) produced on this medium were again transferred onto secondary selection (regeneration) medium. Plantlets were successfully regenerated from these secondary PLBs after the transfer. The highest transformation efficiency of 27.3% was obtained when protocomrs were inoculated with 10 times diluted Agrobacterium solution (OD600=0.1) for 300 min. Transformation of the selected plants was confirmed by GUS assay, PCR and Southern blot analysis. This protocol could be adopted to produce transgenic D. nobile-type orchids with various traits such as novel flower color and resistances to biotic and abiotic stresses.

Agrobacterium-mediated genetic transformation system was established in Dendrobium Formidible 'Ugusu' by inoculating PLBs with A. tumefaciens strain EHA101 (pIG121Hm) harboring hygromycin phosphotranferase (hpt) and neomycin phosphotranferase II (nptII) genes as selectable marker gene and β-glucuronidase (gus) gene as a reporter gene. For obtaining the optimum conditions for the transformation, several factors such as the stage of PLBs after subculture, bacterial concentrations, kind of inoculation medium, inoculation time and inoculation condition (with or without rotary shaking), were examined. After inoculation, PLBs were cocultivated for 3 days and then transferred for selection onto 2.5 g l⁻¹ gellan gum-solidified ND medium containing 10 g l⁻¹ maltose, 20 mg l⁻¹ hygromycin and 20 mg l⁻¹ meropenem. Hygromycin-resistant plantlets were regenerated from secondary PLBs after 4 months of selection. Transformation of these plants was confirmed by GUS histochemical assay, PCR and Southern blot analyses. The highest transformation efficiency of 18.5% was obtained when PLBs 3 weeks after subculture were inoculated with 1 : 10 diluted bacteria (OD₆₀₀≈0.1) with liquid medium containing only 10 g l⁻¹ maltose and 100 M acetosyringone with shaking for 30 min.

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.

Protocol of plant regeneration from leaf and stem explants was established in dahlia (Dahlia×pinnata) 'Yamatohime' in in vitro culture. Nodular calli were successfully induced from leaf and stem explants on 30 g l⁻¹ sucrose-containing MS medium supplemented singly with one of the cytokinins, 6-benzyladenine (BA), zeatin or thidiazuron (TDZ) and the highest frequency of nodular callus induction was obtained on medium containing 10 mg l⁻¹ TDZ. The nodular callus induced on this medium subsequently produced a tissue with numerous shoot primordial structures called 'mass of shoot primordia' (MSP), which was maintained at least for more than 2 years on the same medium. Shoot induction occurred after transfer the MSP onto hormone-free MS medium, in which replacement of sucrose by maltose was necessary. The shoots excised from the MSP were rooted on hormone-free MS medium and successfully grown under greenhouse conditions. The regenerated plants showed the same morphological characteristics to those of the control plants and flowered normally.

An efficient method of plant regeneration from seed-derived callus was established in ravenna grass [Erianthus ravennae (L.) Beauv.], which is an ornamental tall grass as well as an important biomass crop. For callus induction, mature seeds were cultured on media containing different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) (0.5, 1, 2 and 4 mg l⁻¹) and 6-benzyladenine (BA) (0, 0.1 and 0.2 mg l⁻¹) on callus induction using MS medium (Murashige and Skoog 1962) supplemeted with 1 g l⁻¹ casamino acids, 30 g l⁻¹ maltose and 8 g l⁻¹ agar as a basal medium. The highest callus induction was obtained on medium supplemented with 2 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ BA, where 96% of explants formed callus. During the subculture of the calli on the same medium for 4 months, 3 types of calli showing different growth rate, color and morphology were differentiated. By using these 3 types of calli, effects of different concentrations of BA or 6-furfurylaminopurine (kinetin) (0, 0.1, 0.5, 1, 2 and 4 mg l⁻¹) and naphthaleneacetic acid (NAA) (0, 0.25, 0.5 and 1 mg l⁻¹) as plant growth regulators on shoot regeneration were evaluated using MS medium with 1 g l⁻¹ casamino acids as a basal medium. The highest frequency of shoot formation was obtained in type A callus, which had white and compact nature, on medium containing 30 g l⁻¹ sucrose, 1 mg l⁻¹ BA and 0.1–0.25 mg l⁻¹ NAA, where 89.3% of the calli formed shoots.

An efficient system for Agrobacterium-mediated transformation was established in dahlia (Dahlia×pinnata) 'Yamatohime'. Mass of shoot primordia (MSP) induced on MS medium supplemented with 10 mg l⁻¹ TDZ were inoculated with Agrobacterium tumefaciens strain EHA101 (pIG121-Hm) harboring both β-glucuronidase (GUS) and hygromycin resistant genes. After 2 days of co-cultivation, the MSP were transferred to a selection medium containing hygromycin with meropenem for bacterial elimination. Shoots were successfully regenerated from survived MSP on hormone-free medium without hygromycin and they rooted on hormone-free medium containing hygromycin. The hygromycin-resistant plants thus obtained showed histochemical blue staining for GUS. Transformation of plants was confirmed by PCR and Southern blot analyses. Transgenic 'Kokucho' were also produced by using the same transformation procedure, suggesting wide applicability of this Agrobacterium-mediated transformation procedure for other dahlia cultivars.

Hairy roots were induced from leaf segments of Crotalaria spectabilis, which is used as a green manure crop antagonistic to nematodes, by infection with a mikimopine type wild strain of Agrobacterium rhizogenes A13 (MAFF02-10266). For initial proliferation of hairy roots after induction, addition of gibberellin A₃ to the medium was indispensable. These roots exhibited vigorous growth and abundant lateral branching on half-strength Murashige and Skoog (1/2MS) medium without plant growth regulators. The adventitious shoots were induced from 24% of root segments 2 months after transfer onto B5 medium containing 5 mg l^－1 6-benzyladenine. The axillary buds excised from these shoots produced roots 1 month after transfer onto 0.4% (wv^－1) gellan gum-solidified 1/2MS medium containing 30 mM fructose without plant growth regulators. Regenerated plants were successfully grown under greenhouse conditions. Infection of hairy roots of C. spectabilis with root-knot nematodes Meloidogyne hapla and M. incognita led to the growth inhibition of these nematodes.

Lilium cv Acapulco was transformed with a defective cucumber mosaic virus (CMV) replicase gene (CMV2-GDD) construct using Agrobacterium tumefaciens. Four lines were analyzed for gene expression and resistance to CMV-O strain. Expression of the CMV2-GDD gene in the transgenic plants was confirmed by reverse transcription PCR (RT-PCR). When these four lines were mechanically inoculated with CMV-O, no signal of coat protein (CP) messages using RT-PCR was detected in newly produced leaves of two transgenic lines. Dot-immunobinding assay (DIBA) of CP was performed to examine the presence of the CMV in the newly produced leaves of challenged plants. Results, similar to those obtained with RT-PCR of the CP messages, were observed in DIBA. Therefore, our results imply that the two lines show increased levels of resistance to CMV, and CMV-GDD replicase gene is an effective construct that has protection against CMV in Lilium. © 2011 Springer Science+Business Media B.V.

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.

The use of antibiotic or herbicide resistant genes as selection markers for production of transgenic plants and their continuous presence in the final transgenics has been a serious problem for their public acceptance and commercialization. MAT (multi-auto-transformation) vector system has been one of the different strategies to excise the selection marker gene and produce marker-free transgenic plants. In the present study, ipt (isopentenyl transferase) gene was used as a selection marker gene. A chitinase gene, ChiC (isolated from Streptomyces griseus strain HUT 6037) was used as a gene of interest. ChiC gene was cloned from the binary vector, pEKH1 to an ipt-type MAT vector, pMAT21 by gateway cloning and transferred to Agrobacteriumtumefaciens strain EHA105. The infected tuber discs of potato were cultured on hormone- and antibiotic-free MS medium. Seven of the 35 explants infected with the pMAT21/ChiC produced shoots. The same antibiotic- and hormones-free MS medium was used in subcultures of the shoots (ipt like and normal shoots). Molecular analyses of genomic DNA from transgenic plants confirmed the integration of gene of interest and excision of the selection marker in 3 of the 7 clones. Expression of ChiC gene was confirmed by Northern blot and western blot analyses. Disease-resistant assay of the marker-free transgenic, in vitro and greenhouse-grown plants exhibited enhanced resistance against Alternaria solani (early blight), Botrytis cinerea (gray mold) and Fusarium oxysporum (Fusarium wilt). From these results it could be concluded that ipt gene can be used as a selection marker to produce marker-free disease-resistant transgenic potato plants on PGR- and antibiotic-free MS medium. © 2010 Springer-Verlag.

Cotyledonary explants of two "Egusi" genotypes, 'Ejagham' and NHC1-130, were co-cultivated with Agrobacterium tumefaciens strain EHA101 carrying either plasmid pIG121-Hm harbouring genes coding for beta-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII) or plasmid pBBRacdS harbouring these same genes along with a gene coding for 1-aminocyclopropane-1-carboxylate (ACC) deaminase. Six weeks after co-cultivation, more than 35% of explants produced shoots in both cultivars. A DNA fragment corresponding to the gus gene or the selection marker nptII was amplified from genomic DNA extracted from leaves of regenerated plant clones rooted on hormone-free MS medium containing 100 mg/l kanamycin, suggesting their transgenic nature. Southern blot analysis confirmed successful integration of one to three copies of the gus gene. Transformation efficiencies of cultivar NHC1-130 with EHA101(pIG121-Hm) and EHA101 (pIG121-Hm, pBBRacdS) were 3.8% and 10%, respectively, which were higher than those obtained for cultivar 'Ejagham' of 2.4% and 5.7%, respectively. Co-cultivation medium containing 5 mg/l BA was effective for obtaining high transformation efficiency for both cultivars as compared with that without it. © 2010 Springer Science+Business Media B.V.

Lilium × formolongi was genetically engineered by Agrobacterium-mediated transformation with the plasmid pCrtZW-N8idi-crtEBIY, which contains seven enzyme genes under the regulation of the CaMV 35S promoter. In the transformants, ketocarotenoids were detected in both calli and leaves, which showed a strong orange color. In transgenic calli, the total amount of carotenoids [133.3 μg/g fresh weight (FW)] was 26.1-fold higher than in wild-type calli. The chlorophyll content and photosynthetic efficiency in transgenic orange plantlets were significantly lowered; however, after several months of subculture, they had turned into plantlets with green leaves that showed significant increases in chlorophyll and photosynthetic efficiency. The total carotenoid contents in leaves of transgenic orange and green plantlets were quantified at 102.9 and 135.2 μg/g FW, respectively, corresponding to 5.6- and 7.4-fold increases over the levels in the wild-type. Ketocarotenoids such as echinenone, canthaxanthin, 3′-hydroxyechinenone, 3-hydroxyechinenone, and astaxanthin were detected in both transgenic calli and orange leaves. A significant change in the type and composition of ketocarotenoids was observed during the transition from orange transgenic plantlets to green plantlets. Although 3′-hydroxyechinenone, 3-hydroxyechinenone, astaxanthin, and adonirubin were absent, and echinenone and canthaxanthin were present at lower levels, interestingly, the upregulation of carotenoid biosynthesis led to an increase in the total carotenoid concentration (+31.4%) in leaves of the transgenic green plantlets. © 2010 Korean Society for Plant Biotechnology and Springer.

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≈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 μM 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.

The technologies allowing the production of transgenic plants without selectable marker genes, is of great interest in public and environmental safety. For generating such marker-free transgenic plants, possibility has been offered by Multi-Auto-Transformation [MAT] vector system, which combines positive selection, using the isopentenyl transferase (ipt) gene, with a site-specific recombination that generates marker-free plants. In this study Agrobacterium tumefaciens strain EHA105 harboring an ipt-type MAT vector, pMAT21, containing lacZ, gus genes and the removable cassette in the T-DNA region was used to produce marker-free transgenic Kalanchoe blossfeldiana Poelln., employing ipt gene as the selectable marker gene. Co-cultivated explants were cultured on hormone- and selective agent-free MS medium, and 85% of the regenerated shoots showed ipt-shooty phenotype with GUS expression. Forty-one morphologically normal shoots were produced during the subculture. More than ninety percent of the normal shoots were ipt -, gus - but lacZ + as determined by PCR analyses. These results indicate that the ipt phenotype was clearly distinguishable from non-transgenic as well as transgenic marker-free shoots. This study opens interesting perspective for the generation of marker-free transgenic K. blossfeldiana with objective useful transgene. © 2009 Springer Science+Business Media B.V.

Gametosomatic hybridization has so far been achieved by the fusion between male-gametic (microspore tetrad or young-stage pollen) protoplasts and somatic protoplasts but no successful results have been reported on the use of female gamete so far. In this paper, we demonstrate for the first time the successful gametosomatic hybridization by using female gametophyte (egg cell) as the gametic-haploid partner instead of male gametophyte based on the method established for isolation of female germ units from ovules in Petunia in our previous report. Using P. hybrida strains, each single protoplast from egg cell and mesophyll cell were manually collected by a micropump-connected microcapillary and were then fused together as an individual pair by electrofusion. Each heterokaryon thus produced was transferred into Millicell culture plate placed in a Petri dish containing nurse cells, where cell division and microcolony formation took place. Among the fusion combinations tested, only fusants between mesophyll protoplast and egg cell protoplast from the same Petunia strain could regenerate and develop into the complete plants, whereas inter-varietal combinations failed to grow after developing into microcolonies or to regenerate plants. The flowers of a triploid hybrid line (2n=3x=21), confirmed by flow cytometric analysis and chromosome observation, were smaller and shorter than those of the parent with original color, and showed male sterility. These results indicate the intactness of isolated female gametophyte protoplasts, which might enable not only to produce triploid plants with various genomic combinations through gametosomatic hybridization but also to conduct fundamental studies on in vitro fertilization with isolated sperm cell protoplasts.

Establishment of the efficient method for isolating the female germ unit (FGU; egg, synergid and central cell) is useful for the studies on the characterization of each FGU as well as in vitro fertilization and gametosomatic hybridization. In this study, an easy one-step enzymatic procedure was successfully developed to isolate FGU from ovules of Petunia hybrida without releasing the somatic protoplasts from ovular tissues, which could not be achieved in the previous studies. Each FGU was separately liberated after treating the ovules, which were collected from ovaries of flowers one day after anthesis, with an appropriate enzyme solution comprised of 10 g l⁻¹ Cellulase Onozuka R-10, 10 g l⁻¹ Macerozyme R-10, 0.6 M mannitol, 5 mM 2-morpholinoethane-sulfonic acid and 5 g l⁻¹ potassium dextran sulfate, pH 5.8 with 50-rpm shaking for 2 h at room temperature. Isolated FGUs were distinguished by their specific size and characteristics. Fluorescent staining with 4′, 6-diamidino-2-phenylindole could identify the polar nuclei of central cell and the nuclear polarity of egg apparatus cells. After transfer into washing solution supplemented with 0.6 M mannitol using a micropump-connected microcapillary, about 80% of the isolated FGUs were viable for up to 8 h after the isolation, as determining by fluorescein diacetate staining.

Agrobacterium-mediated genetic transformation was applied to produce transgenic plants of spinach (Spinacia oleracea) resistant to 2 pest species, Trichoplusia ni and Autographa nigrisigna. Leaf segments from in vitro spinach plants of cultivar 'Glory' were co-cultivated with A. tumefaciens strain EHA105, which harbored the plasmid pBE2111FMB containing a synthetic cry1Ac gene encoding an insecticidal crystal protein of Bacillus thuringiensis, and neomycin phosphotransferase II and bialaphos resistance genes as selectable marker genes. Kanamycin (Km)-resistant calluses were obtained from co-cultivated leaf segments 1 month after selection on 2.5 g l⁻¹ gellan gum-solidified MS medium containing 30 g l⁻¹ sucrose, 0.5 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg l⁻¹ kinetin, 100 mg l⁻¹ Km and 20 mg l⁻¹ meropenem trihydrate. Regeneration of adventitious shoots from Km-resistant calluses was performed on 2.5 g l⁻¹ gellan gum-solidified MS medium containing 30 g l⁻¹ sucrose, 0.01 mg l⁻¹ 2,4-D, 1 mg l⁻¹ kinetin, 1 mg l⁻¹ gibberellic acid, 100 mg l⁻¹ Km and 10 mg l⁻¹ meropenem trihydrate at 14°C. PCR and Southern blot analyses of genomic DNA isolated from T1 plants confirmed the successful integration of T-DNA into the plant genome. Expression of Cry1Ac protein was confirmed in leaves of transgenic plants by Western blot analysis. Insect bioassays against T. ni and A. nigrisigna performed with T1 plants showed more than 93.3% insect mortality within 1 week. These results suggest that the cry1Ac gene was effectively expressed in spinach.

Agrobacterium strain EHA101 harboring plasmid pIG121-Hm containing the genes for β-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase (nptII) was co-cultivated with leaf explants from in vitro grown spinach plants. Hygromycin-resistant calli were obtained 1 month after selection of transformed cells on 2.5 g l⁻¹ gellan gum-solidified MS medium containing 30 g l⁻¹ sucrose, 0.5 mg l⁻¹ 2,4-D, 2 mg l⁻¹ kinetin, 20 mg l⁻¹ hygromycin and 20 mg l⁻¹ meropenem trihydrate. Regeneration of adventitious shoots was affected by culture temperature, and the highest frequency of shoot formation was 40% when the calli were cultured at 14°C during regeneration process on 2.5 g l⁻¹ gellan gum-solidified MS medium containing 30 g l⁻¹ sucrose, 0.01 mg l⁻¹ 2,4-D, 1 mg l⁻¹ kinetin, 1 mg l⁻¹ gibberellic acid (GA3), 20 mg l⁻¹ hygromycin and 10 mg l⁻¹ meropenem trihydrate. Stable expression of gus gene was indicated by histochemical GUS assay in the leaves and roots of putative transgenic plants. Southern blot analysis of genomic DNA isolated from T0 plants confirmed the successful integration of T-DNA into the plant genome. Segregation of the gus gene in transgenic T1 progeny was confirmed by PCR analysis. These results show that Agrobacterium-mediated transformation combined with plant regeneration at a low temperature, 14°C, can be efficiently used for producing transgenic spinach plants with useful genes.

In Vanda orchids, it is important to produce cultivars with economically important traits such as disease and pest resistances and novel flower colors, which are difficult to achieve by conventional cross breeding methods. To realize these breeding objectives, it is now expected to apply genetic transformation technology to introduce useful foreign genes into Vanda orchids. However, there has been almost no information on the genetic transformation of Vanda. Transgenic plants were successfully regenerated after co-cultivating protocorm-like bodies (PLBs) with Agrobacterium tumefaciens strain EHA101 (pIG121Hm) that harbored genes for β-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII). PLBs of 'Tokyo Blue' maintained in liquid New Dogashima medium (NDM) under dark condition, were subjected to transformation experiments. The PLBs inoculated with Agrobacterium produced secondary PLBs 4 weeks after transfer onto 3 g l⁻¹ gellan gum-solidified NDM containing 30 g l⁻¹ maltose, 10 mg l⁻¹ meropenem and 10 mg l⁻¹ hygromycin. Transformation efficiency was increased by prolonged period of infection (240 min) and wounding treatment of PLBs by sonication (5 min) during inoculation. Transformation of the hygromycin-resistant plantlets regenerated from different PLBs was confirmed by histochemical GUS assay, PCR analysis and Southern hybridization. With this transformation system, approximately 17 independent transgenic plants were obtained from 1 g PLBs.

Agrobacterium tumefaciens strain EHA105 harboring an ipt-type MAT vector, pNPI132, was used to produce morphologically normal transgenic Nierembergia caerulea cv. Mont Blanc employing ipt gene as the selectable marker gene. β-glucuronidase (GUS) gene was used as model gene of interest. The MAT vector system is a positive selection system that gives the advantage of regeneration to the transgenic cells without killing the non-transgenic cells. Infected explants were cultured on hormone- and antibiotic-free MS medium, and 65% of the regenerated shoots developed ipt shooty phenotype-morphologically abnormal shoots, within approximately 3 months after co-cultivation. Twenty morphologically normal shoots were produced from 12 transgenic ipt shoots 7 months after co-cultivation. The normal shoots rooted well on hormone-free MS medium. Ninety percent of the normal shoots were ipt -, GUS + and excision+ as determined by PCR and Southern blot analyses. These results indicate that ipt-type MAT vector system can be used successfully in Nierembergia to produce marker-free transgenic plants without using phytohormones and selective chemical agents. © Springer-Verlag 2006.

Transgenic plants over-expressing wasabi defensin gene were successfully produced in Phalaenopsis orchid by Agrobacterium-mediated transformation method. Embryogenic cell suspension culture of Phalaenopsis Wataboushi '#6.13' was infected with A. tumefaciens strain EHA101 carrying a plasmid containing wasabi defensin gene and selectable marker nptII, hpt genes. Plantlets were regenerated through somatic embryogenesis from the calli selected on hygromycin-containing medium. Transformation of plantlets with wasabi defensin gene was confirmed by PCR analysis. Southern blot analysis confirmed successful integration of 1–4 copies of the gene. Production of the 5 kDa wasabi defensin protein with varying levels was confirmed in the leaf extracts of different transgenic clones using Western blot analysis. Most of the transgenic plants showed strong resistance to Erwinia carotovora, which causes soft rot disease in the control plant. These results suggest the usefulness of this gene for conferring the resistance to various diseases of Phalaenopsis and possibly other orchids.