Itaru Takebe

Plating of Isolated Tobacco Mesophyll Protoplasts on Agar Medium

SummaryA technique was developed to derive cell and plant clones from isolated mesophyll protoplasts of tobacco. The protoplasts, plated on a fully defined agar medium, divided and grew actively forming visible colonies after one month of culture. Efficiency of colony formation depended on cell density and light condition during incubation. Under standard conditions, 60% of plated protoplasts formed colonies. Upon transfer onto suitable media, these colonies differentiated shoots and roots, and eventually regenerated whole plants. Advantages of mesophyll protoplasts as the source of clones as well as implication of the plating technique for genetical studies are discussed.

Cell Wall Regeneration and Cell Division in Isolated Tobacco Mesophyll Protoplasts

SummaryProtoplasts were isolated from palisade tissue of tobacco leaves by treatment with pectinase and cellulase under aseptic conditions, and were cultured in a synthetic liquid medium. Calcofluor, a fluorescent brightener, was found to be an excellent stain for plant cell walls and was used to demonstrate regeneration of cell walls in these protoplasts. The cultured protoplasts regenerated cell walls by the 3rd day of culture, giving rise to spherical cells. The majority of the protoplasts regenerating cell walls underwent mitosis and cell division. The cycle of mitosis and cell division was repeated 2–3 times during 2 weeks of culture. Some of the nutritional conditions affecting division in the cultured protoplasts were studied.

Delivery of Tobacco Mosaic Virus RNA into Plant Protoplasts Mediated by Reverse-Phase Evaporation Vesicles (Liposomes)

SummaryProtoplasts were isolated from tobacco suspension cultures using a new cellulase preparation. Tobacco mosaic virus (TMV) RNA was encapsulated in reverse-phase evaporation vesicle (REV) liposomes of phosphatidylserine and cholesterol, and was successfully introduced into tobacco protoplasts by treatment of the REV/protoplast mixture with polyvinyl alcohol or polyethylene glycol followed by washing with high pH-high Ca buffer. Delivery of TMV-RNA was monitored by determining the number of infected protoplasts using the immunofluorescence technique. Production of TMV particles in the infected protoplasts was also confirmed by electron microscopy. Because of the high encapsulation efficiency of REV liposomes the amount of TMV-RNA necessary to cause infection in the majority of protoplasts could be reduced to 1/10 to 1/5 that required in the previous study (Fukunaga et al. 1981). The usefulness of the REV-mediated delivery of nucleic acids for genetic manipulation of plant protoplasts is discussed.

Infection of tobacco mesophyll protoplasts by tobacco mosaic virus ribonucleic acid

Abstract Isolated protoplasts from healthy tobacco mesophyll tissue were infected by brief contact with infectious RNA from tobacco mosaic virus (TMV). During subsequent incubation of the protoplasts in a liquid medium, a synchronous multiplication of TMV occurred, and the amount of intracellular virus reached a maximum after 22 hours. Fluorescent antibody staining showed that 3–7% of the protoplasts were infected. Average virus yield per infected protoplast was estimated to be 0.9–5.5 × 10 5 particles.

Mitotic protoplasts and their infection with tobacco mosaic virus RNA encapsulated in liposomes

M-phase and S-phase protoplasts were prepared from tobacco cells in suspension culture after a high degree of synchronization using aphidicolin, a specific inhibitor for eukaryotic DNA polymerase. When TMV-RNA was introduced into these protoplasts mediated by REV liposomes, 37% of M-phase and 26% of S-phase protoplasts were infected as determined by the fluorescent antibody technique. After the 24 hr interval between the introduction of TMV-RNA into protoplasts and the determination of infection, half of the infected mitotic protoplasts formed dumbell-shaped daughter cells. The significance of synchronized protoplasts in genetic engineering of plant cells is discussed in reference to the delivery of DNA into the nucleus.

Ultrastructure of infection of tobacco mesophyll protoplasts by tobacco mosaic virus

Protoplasts isolated from tobacco leaves were inoculated in vitro with tobacco mosaic virus (TMV) and were examined by thin sectioning during 45 hr of incubation. One end of TMV rods adsorbed to the plasmalemma and the virus entered protoplasts by a pinocytotic process. During the next several hours, which coincided with the “eclipse period” in the virus growth curve, virus particles were not detected in the protoplasts. Progeny virus particles were first seen in the ground cytoplasm 6 hr postinfection. Extensive virus synthesis during the subsequent hours resulted in the formation of large virus aggregates in nearly all the protoplasts. No sign of degenerative changes was evident in the ultrastructure of protoplasts even after the virus had multiplied to a maximum level.

Experession and integration of genes introduced into highly synchronized plant protoplasts

SummaryChimaeric genes containing the chloramphenicol acetyltransferase (CAT) coding sequence were introduced into protoplasts of suspension-cultured tobacco cells using improved conditions of electroporation (Okada et al. 1986). CAT activity became detectable in the protoplasts within 3 h, was maximal during a period of 18–36 h after electroporation, and then declined gradually. Alpha-amanitin added to the medium abolished the transient expression of the CAT gene. The closed circular form of input DNA was as effective as the linear form for the transient expression. The suspension culture was treated with aphidicolin, and S, G2, M and G1 phases were identified in the highly synchronized cell cycle obtained by releasing the cells from the inhibition of DNA synthesis. When a chimacric CAT gene was introduced into M phase protoplasts prepared from the synchronized culture, the transient expression of the CAT gene was 3–4 times higher than when it was introduced into protoplasts of other cell cycle phases. The frequency of stable transformation with a chimaeric neomycin phosphotransferase II gene was studied using the same system. G-418-resistant transformants were obtained from M phase protoplasts at frequencies 2–8 times those obtained from protoplasts at other cell cycle phases. The results indicate that the absence of the nuclear membrane in mitotic cells favours delivery to the nucleus of exogenous DNA introduced into the cytoplasm.

Protein synthesis in tobacco mesophyll protoplasts induced by tobacco mosaic virus infection

Abstract Tobacco mesophyll protoplasts inoculated in vitro with tobacco mosaic virus (TMV) were used to study protein synthesis induced or stimulated by virus infection. Ultraviolet irradiation of inoculated protoplasts sufficiently reduced host protein synthesis without markedly affecting TMV replication. In addition to viral coat protein, incorporation of amino acids into at least two high-molecular-weight proteins were demonstrated in infected protoplasts. One of these was unrelated to coat protein, consisted of a single polypeptide of about MW 140,000 and was present in association with some cellular structure less dense than nuclei or chloroplasts. The other protein with molecular weight of about 180,000 also appeared to be unrelated to coat protein, since it was labeled with amino acids not contained in the coat protein. Synthesis of the 140,000-MW protein followed a time course very similar to that of viral RNA replication and attained a maximum incorporation rate 4 hr earlier than coat protein synthesis. Production of progeny virus particles closely followed the course of coat protein synthesis.

Localization and orientation of the VirD4 protein of Agrobacterium tumefaciens in the cell membrane

SummaryThe virD4 gene of Agrobacterium tumefaciens is essential for the formation of crown galls. Analysis of the nucleotide sequence of virD4 has suggested that the N-terminal region of the encoded protein acts as a signal peptide for the transport of the VirD4 protein to the cell membrane of Agrobacterium. We have examined the localization and orientation of this protein in the cell membrane. When the nucleotides encoding the first 30 to 41 amino acids from the N-terminus of the VirD4 protein were fused to the gene for alkaline phosphatase from which the signal sequence had been removed, alkaline phosphatase activity was detectable under appropriate conditions. Immunoblotting with VirD4-specific antiserum indicated that the VirD4 protein could be recovered exclusively from the membrane fraction of Agrobacterium cells. Moreover, when the membrane fraction was separated into inner and outer membrane fractions by sucrose density-gradient centrifugation, VirD4 protein was detected in the inner-membrane fraction and in fractions that sedimented between the inner and outer membrane fractions. By contrast, the VirD4′/′alkaline phosphatase fusion protein with the N-terminal sequence from VirD4 was detected only in the inner membrane fraction. Treatment of spheroplasts of Agrobacterium cells with proteinase K resulted in digestion of the VirD4 protein. These results indicate that the VirD4 protein is transported to the bacterial membrane and anchored on the inner membrane by its N-terminal region. In addition, the C-terminal portion of the VirD4 protein probably protrudes into the periplasmic space, perhaps in association with some unidentified cellular factor(s).

Infection of tobacco mesophyll protoplasts by cucumber mosaic virus

Abstract Protoplasts isolated from tobacco leaves were inoculated with cucumber mosaic virus (CMV) using a method slightly modified from that developed for inoculating tobacco mosaic virus. Multiplication of CMV was demonstrated by staining the protoplasts with fluorescent CMV-antibody, by the electron microscopy of ultrathin sections, and by assaying the infectivity in protoplasts. Seventy to 90% of protoplasts were infected under optimal conditions.