Importance of co-cultivation medium pH for successful Agrobacterium-mediated transformation of Lilium x formolongi.

An efficient system for Agrobacterium-mediated transformation of Lilium x formolongi was established by preventing the drastic drop of pH in the co-cultivation medium with MES. Meristematic nodular calli were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm which harbored intron-containing beta-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransfease II (NPTII) genes. After three days of co-cultivation on 2 g/l gellan gum-solidified MS medium containing 100 microM acetosyringone, 30 g/l sucrose, 1 mg/l picloram and different concentrations of MES, they were cultured on the same medium containing 12.5 mg/l meropenem to eliminate Agrobacterium for 2 weeks and then transferred onto medium containing the same concentration of meropenem and 25 mg/l hygromycin for selecting putative transgenic calli. Transient GUS expression was only observed by adding MES to co-cultivation medium. Hygromycin-resistant transgenic calli were obtained only when MES was added to the co-cultivation medium especially at 10 mM. The hygromycin-resistant calli were successfully regenerated into plantlets after transferring onto picloram-free medium. Transformation of plants was confirmed by histochemical GUS assay, PCR analysis and Southern blot analysis.

Plant regeneration from cell suspension-derived protoplasts of Phalaenopsis.

Protoplasts isolated from cell suspension culture of Phalaenopsis "Wataboushi" were cultured by (a) embedding in gellan gum-solidified hormone-free 1/2 New Dogashima medium (1/2 NDM) containing 0.44 M sorbitol, 0.06 M sucrose and 0.1 g/l L-glutamine (standard method) and (b) beads method using beads of gellan gum or sodium alginate as the gelling agents which were surrounded by liquid NDM. Although, the two beads methods gave less frequency of initial protoplast division than the standard method, the former finally resulted in higher frequency of microcolony formation than the latter. The highest frequency of microcolony formation (23%) was obtained when protoplasts were embedded in 1% Ca-alginate beads and subcultured every two weeks by replacing the surrounding liquid culture medium with a decrease in sorbitol concentration by 0.1 M. Colonies visible to the naked eyes were observed within 2 months of culture and the regenerated calluses were transferred onto hormone-free NDM supplemented with 10 g/l maltose and 0.3% (w/v) gellan gum, on which PLBs were formed and proliferated profusely. The PLBs were regenerated into plantlets after changing the carbon source to 10 g/l sorbitol and successfully acclimatized to greenhouse conditions.

Genome size, karyotype, meiosis and a novel extra chromosome in Torenia fournieri, T. baillonii and their hybrid.

Torenia is a suitable model plant to study plant fertilization because of its protruding embryo sac. However, information on the genomes and chromosomes of this species is limited. We determined the genome sizes of T. fournieri Linden and T. baillonii Godefr as 1.71 pg x 10(8) bp and 1.67 x 10(8) bp, respectively. The small genome size of these species suggests their superiority as the targets for molecular cloning studies. Furthermore, karyotypes of T. fournieri and T. baillonii were determined using FISH probed with 5S rDNA, 45S rDNA and species-specific centromere repetitive sequences. Although the two species have similar genome size, number of chromosomes, centromere repeats and 5S rDNA loci were varied. Observation of meiosis in the F(1) hybrid revealed that all chromosomes except one of T. fournieri paired well with the chromosomes of T. baillonii throughout the entire length of the chromosomes including species-specific centromeric regions. One exceptional chromosome of T. fournieri behaved as a univalent and was not always required for gametogenesis. The present results provide the basis for the molecular genetics in Torenia.

Cryopreservation of immature seeds of Ponerorchis graminifolia var. suzukiana by vitrification.

Ponerorchis graminifolia var. suzukiana is a terrestrial orchid that is an endangered species native to Japan, and it germinates more readily in immature seeds than in mature seeds. To preserve this orchid, an efficient protocol was established for the cryopreservation of immature seeds of P. graminifolia var. suzukiana. When immature seeds of 6 weeks after pollination, which showed higher germination and protocorm formation than mature seeds, were precultured on New Dogashima (ND) medium with 0.3M sucrose for 3 days and cryopreserved by vitrification method (treated with PVS2 for 60 min), the viability after preservation as assessed with 2,3,5-triphenyltetrazolium chloride staining test was about 86%. Immature seeds thus treated showed equal rates of germination and protocorm formation to the untreated control immature seeds, and they developed into normal plantlets on ND medium.

Cryopreservation of immature seeds of Bletilla striata by vitrification.

An efficient protocol was established for the cryopreservation of immature seeds of a terrestrial orchid, Bletilla striata. Immature seeds collected 2-4 months after pollination (MAP) were treated using three different cryogenic procedures: (1) direct plunging into liquid nitrogen, (2) vitrification, and (3) vitrification with preculture. When immature seeds collected 3 MAP and 4 MAP were precultured for 3 days on New Dogashima medium supplemented with 0.3 M sucrose and cryopreserved by vitrification, the survival rate after preservation, as assessed by staining with 2,3,5-triphenyltetrazolium chloride, was 92% and 81%, respectively. Immature seeds thus treated showed no decrease in germination rate relative to untreated immature seeds, and they developed into normal plantlets in vitro.

Horticultural characterization of Angelonia salicariifolia plants transformed with wild-type strains of Agrobacterium rhizogenes.

Genetic transformation was carried out with wild-type strains of Agrobacterium rhizogenes for introducing a dwarf trait into the Scrophulariaceous ornamental plant, angelonia (Angelonia salicariifolia). Leaf segments of two angelonia genotypes (Ang.1 and Ang.2) were co-cultivated with mikimopine-type strains of A. rhizogenes. Adventitious roots that showed vigorous growth and increased lateral branching when cultured on half-strength Murashige and Skoog's (MS) basal salts medium lacking plant growth regulators (PGRs) after co-cultivation were selected as putatively transformed lines. All of these selected lines produced mikimopine. Adventitious shoots were efficiently induced from putatively transformed root segments on half-strength MS basal salts medium containing 1 mg l(-1) benzyladenine (BA) under continuous illumination (24-h photoperiod), and the shoots easily rooted following their transfer to half-strength MS basal salts medium lacking PGRs. The transgenic nature of regenerated plants was confirmed by Southern hybridization. Transformed plants frequently died during their acclimatization, and acclimatized plants of eight transformed lines grew very slowly for 1-5 months after transplantation to the greenhouse. Plants of two transformed lines of Ang.2 flowered 4-6 months after transplantation. These transformed plants exhibited phenotypic alterations such as dwarfness and smaller leaves. There were no apparent alterations observed in the number, shape, and size of the flowers. Pollen fertility of the transformed plants was 60-80% based on aceto-carmine staining. These results indicate the possibility of applying A. rhizogenes-mediated transformation for introducing a dwarf trait into angelonia.

Unilateral compatibility and genotypic difference in crossability in interspecific hybridization between Dianthus caryophyllus L. and Dianthus japonicus Thunb.

Reciprocal interspecific crosses were carried out between six lines of Dianthus caryophyllus L. and one line of Dianthus japonicus Thunb. Although no seed was set when D. japonicus was used as the seed parent, six seedlings were successfully obtained from 2,380 immature ovules by applying the embryo-rescue technique. However, they showed seed parent-like morphology and no evidence for the hybridity by flow cytometry and RAPD analyses. When six lines of D. caryophyllus were used as seed parents, a total of 192 seedlings were successfully obtained without using the embryo-rescue technique. Among these seedlings, 12 out of 25 progenies obtained from the carnation line '98sp1651' were confirmed to be the hybrids. The remaining 13 progenies of this line, and the total 167 progenies obtained from the other carnation lines, had carnation-like morphology without any evidence of hybridity by flow cytometry and RAPD analyses. The progenies confirmed as hybrids had intermediate characters of the parents with respect to leaf width and flower size, but they had a uniform flower color, reddish purple, which was different from that of either parent. Since the hybrids obtained in the present study have some profitable characters such as vigorous growth in summer time, upright robust stem, broad leaves and early flowering, they are expected to be used for the breeding of carnation which is suitable for growing under the Japanese climate.

Ti- and cryptic-plasmid-borne virulence of wild-type Agrobacterium tumefaciens strain CNI5 isolated from chrysanthemum (Dendranthema grandiflora Tzvelev).

The genetic similarity between pTiBo542 and pTiCNI5, which are harbored, respectively by the supervirulent Agrobacterium tumefaciens strain A281 and by the highly tumorigenic wild-type strain CNI5 isolated from chrysanthemum was investigated by Southern hybridization. pTiCNI5 and pTiBo542 exhibited highly similar hybridization patterns in both BamHI- and EcoRI-digested plasmids by using four vir-region-specific probes, whereas similarity in these two plasmids was not observed by probing with five TL-DNA-specific probes. The characteristics related to tumor formation of cryptic plasmids carried by strain CNI5 were investigated by using single-plasmid-cured derivatives. pTiCNI5-cured derivatives predictably failed to utilize agropine and mannopine and failed to induce tumors on chrysanthemum and tobacco leaf explants, while pAtCNI5a-, pAtCNI5c- and pAtCNI5d-cured derivatives could utilize these opines similar to the parent strain CNI5. Interestingly, pAtCNI5c- and pAtCNI5d-cured derivatives showed low tumorigenicity in comparison with strain CNI5 or with the pAtCNI5a-cured derivative. These results suggest that the highly virulent phenotype of strain CNI5 may be due to one or more vir genes, which exhibit cartographic similarity to those of pTiBo542. The results also suggest that the gene(s) related to tumor formation of strain CNI5 may exist not only on pTiCNI5 but also on cryptic plasmids pAtCNI5c and pAtCNI5d.

Plant regeneration from cell suspension-derived protoplasts of Primula malacoides and Primula obconica.

Protoplasts were isolated from cell suspension cultures of Primula malacoides cv. 'Lovely Tokyo' and P. obconica cv. 'Aalsmeer Giant White'. P. obconica protoplasts were embedded in 0.1% (w/v) gellan gum-solidified discs comprising MS medium supplemented with 3 mg/l of 2,4-D or picloram, 0.1 mg/l of zeatin, 0.2 M glucose and 0.2 M mannitol, and surrounded by a liquid medium of the same composition except for the addition of 0.1% (w/v) activated charcoal. The protoplasts formed visible colonies, which were transferred to the regeneration medium containing 30 g/l of sucrose, 0.1 mg/l of picloram and 2 mg/l of zeatin for shoot induction. P. malacoides protoplasts formed visible colonies when cultured in disc culture using 0.1% (w/v) gellan gum-solidified MS medium containing 5 mg/l of 2,4-D, 1 mg/l of NAA, 0.1 mg/l of zeatin and 0.4 M glucose. Small calli were transferred to MS medium supplemented with 5 mg/l of zeatin for shoot regeneration. The shoots of both species readily rooted on plant growth regulator-free 1/2 MS medium and successfully acclimatized to greenhouse conditions. The protoplast-derived plants showed some alterations in morphological characteristics from those of the in-vitro-germinated control plants.

Improved plant regeneration from cultured leaf segments in peanut (Arachis hypogaea L.) by limited exposure to thidiazuron.

Bud primordia were induced from leaf segments, which were harvested from young seedlings of Spanish type peanut (Arachis hypogaea L. cv. Chico), on 0.8% agar-solidified medium containing Murashige and Skoog (MS) basal salts supplemented with B5 vitamins, 1 mg/l NAA and various cytokinins such as benzyladenine (BA), isopentenyladenine (2ip), kinetin (KIN), chloropyridylphenylurea (4PU), thidiazuron (TDZ), zeatin (ZTN) in different concentrations. Among the cytokinins tested, TDZ was found to be the most efficient for inducing bud primordia. However, continuous culture on TDZ-containing media induced abnormal development of these primordia, and they failed to grow into plantlets. Histological observations revealed that the malformation most often obtained was a shoot-like structure which lacked shoot apical meristem (SAM) and had disorganized vascular bundles. For normal shoot regeneration, it was necessary to limit the culture period of the explants on TDZ-containing medium to 7 days at 10 mg/l or 21 days at 1 mg/l and then transfer them onto plant growth regulator-free medium. The percentage of conversion from shoot buds to shoots was 34.7%. When shoots were removed from the explants and transferred onto basal medium containing 1 mg/l NAA, all regenerated shoots readily rooted and successfully acclimatized. All of the acclimatized plants produced viable seeds in the greenhouse condition.

Polysomaty analysis in diploid and tetraploid Portulaca grandiflora.

Polysomaty analysis of the succulent portulaca (Portulaca grandiflora Hook.) plant was carried out using flow cytometry. For both diploid and tetraploid plants, mature leaf tissue was found to have a higher level of polysomaty than young leaf tissue. Mesophyll (MP), bundle sheath (BSP) and water storage protoplasts (WSP) were isolated from leaf tissues of diploid portulaca plants. WSP had a higher degree of endopolyploidization than MP and BSP. The ploidy distribution was also variable in different floral organs. Tetraploid plants artificially induced by colchicine treatment showed a decline in the degree of polysomaty compared to diploid plants. Tetraploid plants had more spherical leaves, a larger number of petals and lower pollen fertility than diploid plants.

Highly tumorigenic Agrobacterium tumefaciens strain from crown gall tumors of chrysanthemum.

A wild-type Agrobacterium tumefaciens strain CNI5 isolated from crown gall of chrysanthemum (Dendranthema grandiflora Tzvelev) was characterized. Strain CNI5 was classified into biovar 1, based on physiological and biochemical characteristics, and was resistant to ampicillin. Strain CNI5 induced tumors at a higher frequency and on a larger area of explants in most tested plant species, especially in chrysanthemum cultivars, than the octopine-type strain C58C1cmr (pTiB6S3). Agropine and mannopine were detected in tumors induced by strain CNI5 and were specifically catabolized by this strain. Strain CNI5 harbored five plasmids including one plasmid that shared sequence similarity to TL-DNA of the octopine-type Ti plasmid and four cryptic plasmids.

Plant regeneration from hairy roots induced by infection with Agrobacterium rhizogenes in Crotalaria juncea L.

Hairy roots were induced from leaf segments of Crotalaria juncea, 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). These roots exhibited vigorous growth and abundant lateral branching on half-strength Murashige and Skoog (1/2MS) medium without phytohormones. The adventitious shoots were induced from 30% of root segments 3 months after transfer onto medium containing 3 mg/l benzyl adenine. These shoots produced roots 1 month after transfer onto 1.2% agar-solidified 1/2MS medium without phytohormones. Regenerated plants were successfully grown under greenhouse conditions. The transgenic nature of the regenerated plants was confirmed by Southern-blot analysis.

Cryopreservation of Doritaenopsis suspension culture by vitrification.

Cells of a suspension culture of Doritaenopsis cv. New Toyohashi were placed in a mixture of 2 M glycerol and 0.4 M sucrose for 15 min at room temperature and then dehydrated with a vitrification solution (PVS2) for 1-3 h on ice and plunged into liquid nitrogen. The highest viability (64% by 2,3,5-triphenyltetrazolium chloride stainability) was obtained when the cells were precultured in liquid New Dogashima medium with 0.1 M sucrose and 1.0 mg/l abscisic acid for 1 week at 25  °C in the light. Dehydration by PVS2 was important for the cryopreservation of Doritaenopsis cells. Protocorm-like bodies were induced from cryopreserved cells without morphological variations.

Agrobacterium-mediated genetic transformation of a phalaenopsis orchid.

Genetically transformed plants of a phalaenopsis orchid [Doritaenopsis Coral Fantasy×Phalaenopsis (Baby Hat×Ann Jessica)] were regenerated after cocultivation of cell clumps with Agrobacterium tumefaciens strains LBA4404 (pTOK233) and EHA101 (pIG121Hm) that harbored genes for ß-glucuronidase (GUS) and hygromycin resistance. The efficiency of transformation was markedly increased by 10 h cocultivation of cell clumps with A. tumefaciens that had been induced with 200 µM acetosyringone, and by inclusion of 500 µM acetosyringone in the cocultivation medium. Hygromycin-resistant cell clusters (0.5-3 mm in diameter) were selected from the infected cell clumps after 4-6 weeks of culture on agar (8 g/l)-solidified new Dogashima medium (NDM) containing 20 g/l sucrose, 0.1 mg/l naphthaleneacetic acid, 1.0 mg/l benzyladenine (BA), 50 mg/l hygromycin and 300 mg/l cefotaxime. The cell clusters proliferated 4 weeks after transfer onto the same medium. To induce callus greening, the carbon source was changed from sucrose to maltose. The green calli obtained produced protocorm-like bodies (PLBs) after 4 weeks of culture on phytohormone-free NDM medium. Regeneration of transgenic plantlets was enhanced by incubating PLBs on NDM medium supplemented with 0.1 mg/l abscisic acid, followed by partial desiccation for 10-30 min. Successful transformation was confirmed by histochemical GUS assay, PCR analysis and Southern hybridization of transformants. With this transformation system, more than 100 hygromycin-resistant phalaenopsis plantlets were produced about 7 months following infection of the cell aggregates.

Isolation of embryo sacs from Dianthus ovules by enzymatic treatments and microdissection.

Mature ovules of Dianthus (Caryophyllaceae) were histologically observed by clearing and serial sectioning to characterize the cells of the embryo sac. The results show that the mature embryo sac was located deep inside the hemitropous ovule due to thick nucellar tissue at the micropylar region. For the isolation of the embryo sacs, ovules were collected from ovaries of flowers 1 day after anthesis, and treated with an enzyme solution for digesting cell walls on a gyratory shaker. After 12 h of enzyme treatment, these ovules were dissected using a glass needle under an inverted microscope to release the embryo sacs. The embryo sacs, characterized by their specific size, were successfully released by these successive treatments. The viability of the embryo sacs was more than 80% as assessed with fluorescein diacetate staining. Fluorescent staining with 4,6-diamidino-2-phenylindole revealed the nuclei of the egg apparatus in the isolated embryo sacs. The procedure for isolating embryo sacs established in this study will offer a new approach to further in vitro studies on fertilization in Dianthus.

Bialaphos stimulates shoot regeneration from hairy roots of snapdragon (Antirrhinum majus L.) transformed by Agrobacterium rhizogenes.

Hairy roots of snapdragon (Antirrhinum ma-jus L.: Scrophulariaceae) induced by a wild-type strain of Agrobacterium rhizogenes were cultured on media containing various concentrations of a phosphinothricin-based herbicide, bialaphos, or plant growth regulators (PGRs). Adventitious shoot regeneration from hairy roots was observed with a low frequency (10%) on half-strength Murashige and Skoog medium. Addition of α-naphthalene-acetic acid in combination with 6-benzylaminopurine, thidiazuron, or zeatin to the medium had no effect on shoot regeneration from hairy roots. Although bialaphos at 0.9 mg l-1 or more was toxic to hairy roots, it significantly increased the shoot regeneration frequency up to 56% at 0.5 mg l-1. In contrast, non-transformed roots and leaves regenerated no shoots on media with or without bialaphos. Regenerated shoots detached from host roots readily developed roots on gellan-gum-solidified medium. Regenerated plants were successfully transferred to the greenhouse, but did not produce seed.

A simple and efficient plant regeneration system for leaf protoplasts ofNierembergia repens by inducing single shoots on the microcolonies.

A simple and efficient protocol for plant regeneration from mesophyll protoplasts ofNierembergia repens is described. The protoplasts divided in modified half-strength MS (/12 MS) medium containing benzylaminopurine (BA) and a-naphthaleneacetic acid (NAA) and formed visible colonies after 2 weeks which produced single adventitious shoots 4 weeks later. Plating efficiency (11.2%), percent colony formation (0.84%), and the number of shoot-forming colonies (368/dish) were highest in /12 MS containing 0.1 mg/l BA and 0.05 mg/l NAA. However, the percentage of colonies with shoot formation was highest (31.8%) in /12 containing 0.05 mg/l BA and 0.01 mg/l NAA. Almost all of the remaining colonies (97.5%) also regenerated shoots upon transfer onto MS medium containing 0.05 mg/l BA. The shoots with 2-3 leaves readily rooted 3-5 days after insertion in /12MS lacking plant growth regulators. Regenerated plantlets were easily established in soil 50 days after protoplast isolation. All the regenerants were normal and possessed diploid chromosome numbers.

Cryopreservation of zygotic embryos of a Japanese terrestrial orchid (Bletilla striata) by vitrification.

The seeds of a Japanese terrestrial orchid (Bletilla striata Rchb.f.) were germinated and cultured on solidified new Dogashima (ND) medium for 10 days. These embryos were then precultured on ND medium supplemented with 0.3 M sucrose for 3 days at 25°C in continuous dark. The embryos were then overlaid with a mixture of 2 M glycerol and 0.4 M sucrose for 15 min at 25°C and finally dehydrated with highly concentrated vitrification solution (PVS2) for 3 h at 0°C prior to immersion into liquid nitrogen for 30 min. After rapid warming, the embryos were washed with liquid ND medium supplemented with 1.2 M sucrose for 20 min and then plated on ND medium. Successfully vitrified and warmed embryos developed into normal plantlets. The rate of plant regeneration amounted to about 60%. This vitrification method appears to be a promising technique for cryopreservation of orchids.

Intergeneric somatic hybrid plantlets between Dianthus barbatus and Gypsophila paniculata obtained by electrofusion.

Hypocotyl-derived protoplasts of Dianthus barbatus that had been pretreated with iodoacetamide were fused electrically with cell suspension culture-derived protoplasts of Gypsophila paniculata that could divide to form callus but could not regenerate shoots under the culture conditions used in this study. Electrofusion-derived calli which produced shoots were selected as putative somatic hybrids, and plantlets were subsequently regenerated from 2 of these selected calli. These plantlets, which in vitro produced flowers precociously, were identified as intergeneric somatic hybrids by nuclear ribosomal DNA analysis. Normal plants have not been established up to the present.