Masahiro Mii

Micropropagation of Phalaenopsis and Doritaenopsis by culturing shoot tips of flower stalk buds.

Green Protocorm-like Bodies (PLB) with high multiplication capacity were induced from shoot tips of flower stalk buds having 1 or 2 leaf primordia using New Dogashima Medium (NDM) containing 0.1 mg l−1 α-naphthaleneacetic acid (NAA) and 1 mg 1−1 6-benzylaminopurine (BAP). These PLB were subcultured on the same medium. More than 10,000 PLBs were obtained from a few buds on a single flower stalk within one year. After transfer onto NDM containing no plant growth regulator (PGR), the PLB developed into plantlets. The micropropagation method formulated in this study was applicable to 12 different genotypes. These results suggest that the methodology could be used on a commercial scale for vegetative propagation of Phalaenopsis and Doritaenopsis.

Agrobacterium-mediated genetic transformation of a phalaenopsis orchid.

Abstract 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 β-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.

Production of transgenic plants of the Liliaceous ornamental plant Agapanthus praecox ssp. orientalis (Leighton) Leighton via Agrobacterium-mediated transformation of embryogenic calli

Abstract A system for producing transgenic plants was developed for the Liliaceous ornamental Agapanthus praecox ssp. orientalis (Leighton) Leighton via Agrobacterium -mediated genetic transformation. Leaf-derived embryogenic calli were inoculated with A . tumefaciens strain EHA101/pIG121Hm or LBA4404/pTOK233, both of which harbored the binary vector carrying the neomycin phosphotransferase II (NPTII), hygromycin phosphotransferase (HPT) and intron-containing β-glucuronidase (GUS-intron) genes in the T-DNA region. Following co-cultivation, the calli were transferred to a medium containing 1 mg l −1 picloram (PIC), 50 mg l −1 hygromycin and 500 mg l −1 cefotaxime, on which several hygromycin-resistant (Hyg r ) cell clusters were obtained 5–6 weeks after transfer. Agrobacterium strain, co-cultivation period and acetosyringone (AS) treatment during co-cultivation affected the number of Hyg r callus lines produced: the best result was obtained when embryogenic calli were co-cultivated with LBA4404/pTOK233 for 7 days in the presence of 20 mg l −1 AS. Hyg r calli were transferred to the same medium, but lacking PIC, for inducing somatic embryos. Somatic embryos thus obtained developed into complete plantlets following their transfer to a medium without PIC and antibiotics. All of them were verified to be stable transformants by GUS histochemical assay, PCR and Southern blot analyses.

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.

Induction of embryogenic callus and cell suspension culture from shoot tips excised from flower stalk buds of Phalaenopsis (Orchidaceae)

SummaryEmbryogenic calluses were induced from 73% of Phalaenopsis shoot-tip explants excised from flower stalk buds by culturing for 7 mo. on New Dogashima Medium (NDM) containing 0.5 μM α-naphthaleneacetic acid (NAA), 4.4 μM 6-benzylaminopurine and 29.2 mM sucrose. The sucrose concentration was increased to 58.4 mM 4 mo. after initiation of the callus culture. These calluses were successfully subcultured as cell suspension cultures in liquid NDM supplemented with 5.4μM NAA and 58.4 mM sucrose. By simply reducing the sucrose concentration to 29.2 mM, the cells grew into plantlets through a developmental process similar to that of Phalaenopsis seedlings. The occurrence of somaclonal variants was less than 10% in six out of eight genotypes examined. These results suggest that the embryogenic callus and cell suspension culture could be utilized as the materials for micropropagation and breeding of Phalaenopsis orchids.

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

Abstract 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.

Transformation of sweet potato (Ipomoea batatas (L.) Lam.) plants by Agrobacterium rhizogenes

Abstract Transgenic sweet potato plants were obtained after Agrobacterium rhizogenes -mediated transformation. Leaf disks of in vitro plants were inoculated with different Agrobacterium rhizogenes strains. Numerous hairy roots were induced on leaf disks by both agropine-type and mikimopine-type strains. Whole plants transformed with Ri-T-DNA were regenerated from the hairy roots in five cultivars. These plants had wrinkled leaves, altered shape of flowers, reduced apical dominance, shortened internodes, small storage roots and abundant, frequently branching roots that showed reduced geotropism. Transgenic sweet potato plants possessing both NPT II gene and GUS gene were also obtained from the hairy roots by infection with Agrobacterium rhizogenes containing the binary vector pBI121 in addition to the wild-type Ri-plasmid.

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

Abstract 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.

Somatic embryogenesis and plant regeneration from immature seed-derived calli of rugosa rose (Rosa rugosa Thunb. )

Abstract A procedure for plant regeneration from immature seed-derived calli of rugosa rose (Rosa rugosa Thunb.) via somatic embryogenesis is described. Embryogenic calli were initiated from immature seeds 2–3 weeks after anthesis on Murashige and Skoog (MS) medium without growth regulators. Induced calli had a white, friable and nodular appearance with several proembryos. These calli were subcultured at 20-day intervals on MS medium containing 0.1–0.2 M galactose on which they grew rapidly; but somatic embryogenesis was inhibited. Somatic embryos were again induced from the subcultured calli after transferring to MS medium containing 0.1 M M fructose or sucrose but lacking growth regulators. After transferring these embryos (1–2 mm) to MS medium containing 0.1 M sorbitol, 3% of them germinated and grew into plantlets which showed sustained growth on the MS medium containing only 0.1 M sorbitol as the sole carbon source.

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.