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. Received: 19 September 1996 / Revision received: 3 January 1997 / Accepted: 24 February 1997     

Invading cancer cells are predominantly in G0/G1 resulting in chemoresistance demonstrated by real-time FUCCI imaging

Invasive cancer cells are a critical target in order to prevent metastasis. In the present report, we demonstrate real-time visualization of cell cycle kinetics of invading cancer cells in 3-dimensional (3D) Gelfoam® histoculture, which is in vivo-like. A fluorescence ubiquitination cell cycle indicator (FUCCI) whereby G₀/G₁ cells express a red fluorescent protein and S/G₂/M cells express a green fluorescent protein was used to determine the cell cycle position of invading and non-invading cells. With FUCCI 3D confocal imaging, we observed that cancer cells in G₀/G₁ phase in Gelfoam® histoculture migrated more rapidly and further than cancer cells in S/G₂/M phases. Cancer cells ceased migrating when they entered S/G₂/M phases and restarted migrating after cell division when the cells re-entered G₀/G₁. Migrating cancer cells also were resistant to cytotoxic chemotherapy, since they were preponderantly in G₀/G₁, where cytotoxic chemotherapy is not effective. The results of the present report suggest that novel therapy targeting G₀/G₁ cancer cells should be developed to prevent metastasis.     

Somatic hybridization between Dianthus chinensis and D. barbatus through protoplast fusion

Summary Protoplasts isolated from leaf mesophyll cells of Dianthus chinensis and D. barbatus were fused by polyethylene glycol (PEG). Calli exhibiting vigorous growth were selected from the PEG-treated protoplasts and shoots were regenerated from one of these calli after 5 months of culture. These shoots readily rooted and continuously produced flowers in the in-vitro condition. The data on flower color, chromosome number, and esterase isozyme patterns indicated that this plantlet was an interspecific somatic hybrid. The hybridity of the plantlet was also confirmed by nuclear rDNA analysis. This report provides the possibility of applying the somatic hybridization technique for the genetic improvement of the genus Dianthus.     

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

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.     

Plant regeneration from cell suspension-derived protoplasts of Saintpaulia ionantha Wendl

Summary Friable calli were induced on leaf segments of Saintpaulia ionantha Wendl. on B5 medium containing 1 mg l^–1 2,4-D and 2 g l^–1 casein hydrolysate. Cell suspension cultures were readily established from these friable calli and protoplasts could be isolated from the cells with yields of 1–3×10⁷/g f. wt.. By culturing in 0.1 % gellan gum-solidified B5 medium supplemented with 1 mg l^–1 2,4-D and 0.1 M each of sucrose and mannitol at a density of 1×10⁵/ml, the protoplasts divided within 6 days and formed macro-colonies after 2 months of culture. Shoot regeneration from protoplast-derived calli was obtained by sequential treatment of the calli with plant growth regulators: initially with 1 mg l^–1 each of NAA and BA for 2 months followed by 0.01 mg l^–1 NAA and 5 mg l^–1 BA for 4 months. Regenerated plants were established after rooting of the shoots on half-strength MS medium, and successfully transferred to the greenhouse. The regenerated plants grew into flowering stage and showed the same phenotype as the parent plant.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - f. wt fresh weight - MES 2-(N-morpholino)-ethanesulfonic acid - MS Murashige and Skoog (1962) - NAA -naphthaleneacetic acid - PE plating efficiency     

Development of a method to analyze single cell activity by using dielectrophoretic levitation

In cell fusion and genetic recombination, although the activity of single cells is extremely important, there is no method to analyze single cell activity. Development of a quick analyzing method for single cell activity is desired in various fields. Dielectrophoresis (DEP) refers to the force exerted on the induced dipole moment of an uncharged dielectric and/or conductive particle by a nonuniform electric field. By applying DEP, we obtained experimentally a relationship between the cell activity and the dielectric property, Re[K(omega)], and examined how to evaluate the single cell activity by measuring Re[K(omega)] of a single cell. A cone and plate electrode geometry was adapted in order to achieve the feedback-controlled DEP levitation. The single cell is exposed to a nonuniform field induced by the cone and plate electrode, and a more polarizable cell is moved to the direction of the cone electrode by the DEP force. The cell settles in the position where the DEP force and gravity are balanced by controlling applied voltage. This settled position, measured on the center axis of the cone electrode, depended on the dielectric constant of the cell. From these results, the relationship between the specific growth rates in cell growth phase and the dielectric properties Re[K(omega)] was obtained. Furthermore, the effect on the cell activity of various stresses, such as concentration of carbon dioxide, temperature, etc., was examined.     

Effect of sugar type on the efficiency of plant regeneration from protoplasts isolated from shoot tip-derived meristematic nodular cell clumps of Lilium x formolongi hort.

Summary Suspension cultures composed of meristematic nodular cell clumps of Lilium x formolongi hort were established from shoot tips placed on MS medium supplemented with 1 mg/l picloram and 30 g/l sucrose, glucose, fructose or sorbitol. Protoplasts isolated from these cultures were embedded in 1 g/l gellan gumsolidified 1/2MS medium with 1 mg/l picloram and the different kinds of sugars at 0.5 M, and cultured at 25 °C in the dark. The highest plating efficiency (13.7%) was obtained when the protoplasts were isolated from the cell clumps which had been subcultured in MS medium containing glucose and were likewise cultured in MS medium supplemented with 0.5 M glucose. Plants were regenerated from the protoplast-derived calli on 1/2MS medium containing 2.5–10 g/l sucrose or 5–10 g/l glucose. These results suggest that the kinds of sugar and concentration are important parameters affecting protoplast isolation, proliferation and plant regeneration in L. x formolomgi hort.Abbreviations FW fresh weight - MS Murashige and Skoog (1962) - 1/2MS medium MS medium containing half strength mineral salts - 1/2MS-0 1/2MS medium containing no growth regulators - NAA 1-naphthaleneacetic acid - p-calli protoplast-derived calli - PE plating efficiency - picloram 4-amino-3,5,6-trichloro-picolinic acid     

Ripply3 is required for the maintenance of epithelial sheets in the morphogenesis of pharyngeal pouches

During tissue development, the morphogenesis of epithelial sheets is regulated by many factors, including mechanical force, although the underlying mechanisms remain largely unknown. In the pharyngeal region of the vertebrate embryo, endodermal epithelium is reiteratively folded outward to form pharyngeal pouches, making partitions between the pharyngeal arches. Ripply3, encoding a member of the Ripply family of adaptor proteins, is required for the pouch formation posterior to the 2nd pharyngeal pouch. In this study, we found that the expression of mouse Ripply3 was specifically activated in accordance with the bending of the endodermal epithelium during the pouch formation. In Ripply3‐deficient embryos, a continuous monolayer of the endodermal epithelium was not maintained posterior to the 2nd pharyngeal pouch. Corresponding to the endodermal region of the deformed epithelium, the activated form of Integrin β1, which was localized at the basal side of the epithelial cells in the wild‐type embryos, was not persistently observed in the mutants. On the other hand, cell proliferation and apoptotic cell death in the endoderm were not obviously affected by the Ripply3 deficiency. Significantly, Ripply3 expressed in cultured cells was found to be preferentially accumulated in the focal adhesions, which are Integrin‐mediated adhesive contact sites transmitting mechanical force between the extracellular matrix and attached cells. Furthermore, Ripply3 promoted the maturation of focal adhesions in these cells. Thus, Ripply3 appears to have been activated to enhance the connection between the extracellular matrix and endodermal epithelial cells, as a mechanism to resist the mechanical stress generated during the bending of the epithelial sheets.     

Transplantation of isolated nuclei into plant protoplasts

The uptake of isolated nuclei from Vicia hajastana Grossh. cells into protoplasts of an auxotrophic cell line of Datura innoxia P. Mill. was induced under the influence of polyethylene glycol and Ca²⁺ at pH 6.8. The frequency of nuclear uptake varied from 0.8 to 2.3% and that of the recovery of prototrophic clones from 10^-5 to 6·10^-4. The prototrophic nuclear fusion products following nuclear uptake could be rescued by initial culture of the protoplasts in non-selective conditions and by the subsequent use of feeder cell layers to support the growth of surviving colonies on a selective medium. The presence of Vicia genomic DNA in some prototrophic clones was confirmed by dot-blot hybridization using Datura and Vicia DNA probes. In certain transformed clones, the recovery of prototrophy was accompanied by the restoration of morphogenetic potential. Welldeveloped shoots typical of wild-type Datura could be regenerated employing an appropriate regeneration medium.Abbreviations MS Murashige and Skoog (1962) - PEG polyethylene glycol