Hiromichi Morikawa

A novel method for transformation of intact yeast cells by electroinjection of plasmid DNA

SummaryIt was found that plasmid DNA (YEp13) can be introduced into intact yeast cells by electric field pulses. The frequency of transformation by this electroinjection method depend upon the initial electric field intensity, the capacitance of the electric discharge capacitor, and the number of pulses applied. A maximum number of transformants (90±20/μgDNA) was obtained by three successive pulses with an initial intensity of 5 KV/cm and with a capacitance of 1 μF. The present electroinjection method is simple, and transformants can be obtained within 2 to 3 days after transformation treatment.

Gene delivery into cultured plant cells by DNA-coated gold particles accelerated by a pneumatic particle gun.

SummaryTransient expression of the β-glucuronidase (GUS) gene in suspension-cultured cells of tobacco was obtained after gene delivery with a pneumatic particle gun device driven by compressed air. Both the pneumatic pressure used to accelerate the particles as well as the distance between the point of release of the particles from the device and the location of the target cells could be varied. Efficiency of gene delivery was tested by accelerating pressure from 63 to 200 kg/cm2 and distances from 5 to 15 cm. At 150 kg/cm2 and 10cm, 618±49 blue spots of GUS-expressing cells were evenly distributed over the surface of the cell layer (3.5 cm diameter). The frequency of transient gene expression was estimated to be more than 1.2×10-3, which is comparable to, or even higher than, that obtained by the gunpowder-driven device.

Transient expression of foreign genes in plant cells and tissues obtained by a simple biolistic device (particle-gun)

SummarySuccessful transient expression of genes (luciferase and β-glucuronidase) in cultured tobacco cells and adventitious shoots of egg-plant hypocotyls can be obtained by use of a simple biolistic device. This device is driven by controlled gas pressure from a cylinder of nitrogen gas. It has the advantages of being free of explosive heat and of avoiding cell damage caused by expanding gas, due to the “self-sealing” effect of the projectile.

Entrapment of Lavandula vera cells with synthetic resin prepolymers and its application to pigment production

SummaryCultured cells of Lavandula vera were entrapped with photosensitive synthetic resin prepolymers (PVA-SbQ). PVA-SbQ-entrapped cells grew well inside gel matrices and synthesized de novo blue pigments in the presence of l-cysteine as an inducer. The entrapped cells were superior to calcium alginate-entrapped cells judging from cell growth and total pigment productivity. Release of the pigments, which were almost insoluble in water, from the gels was markedly enhanced by the increase in hydrophilicity of the cell-entrapping gels. The entrapped cells could be used repeatedly for the pigment production.

Stable transformation of cultured tobacco cells by DNA-coated gold particles accelerated by gas-pressure-driven particle gun.

SummaryStable transformation of suspension-cultured cells of tobacco with plasmid DNA (pCaMV-NEO) harbouring the neomycin phosphotransferase II (npt-II) gene was achieved using a previously described gas-pressure-driven particle acceleration device. The cells were bombarded with DNA-coated gold particles accelerated by the device, and callus containing the introduced gene was selected in the presence of geneticin disulphate. The geneticin-resistant callus exhibited npt-II enzyme activity, and Southern analysis confirmed the stable integration of the foreign gene into the tobacco genome. Transformants were obtained at a rate of more than 1.9 × 10−4.

Interspecific hybrid plant formation by electrofusion in Nicotiana

SummaryWe obtained complete hybrid plants by electrofusion of mesophyll protoplasts from Nicotiana glauca and N. langsdorffii. Electrofocusing analysis of Fraction I proteins isolated from the leaves of these plants confirmed their hybridity. Cytological analysis indicated that the chromosome number (2n) of these plants is between 60 to 66, suggesting that they are the products of triple fusion. All plants were fertile and set viable seeds after self pollination. As we did not use an AC field for electrofusion, the present results indicate that an AC field is not essential for obtaining hybrid plants with electrofusion.

An Intergeneric Hybrid Cell Line of Duboisia hopwoodii and Nicotiana tabacum by Protoplast Fusion

Summary Fusion of protoplasts from Nicotiana tabacum mesophyll cells and protoplasts from Duboisia hopwoodii suspension culures was induced by polyethylene glycol treatment. Heterokaryons were isolated wih a micropipette and were cloned individually in small nylon mesh chambers, around which mesophyll protoplasts of tobacco were culured as nurse cells. One putative intergeneric hybrid cell line was obtained, and the hybrid nature of this line was confirmed by isozyme analyses, analysis of fluorescent compounds, and cytological investigations. The chromosome numbers in the hybrid varied from ca. 70 to ca. 240. Shoots with heterogeneous morphology were induced from the hybrid callus.

Effect of Temperature on Membrane Fluidity and Protoplast Fusion

SummaryThe fusion of plant protoplasts is greatly enhanced by a rise in temperature in the presence of the fusion-inducing agents polyethylene glycol or polyvinyl alcohol. Kinetic analysis of this reaction reveals that membrane fluidity is important in regulating the fusion process.