Takeshi Taniguchi

Developmental electron microscopy and histochemistry of somatic embryo differentiation in sugarcane

Abstract Embryogenic callus formation and growth of somatic embryos of sugarcane ( Saccharum officinarum L.) were followed by electron microscopy in order to investigate the correlation between the morphological sequence of events and some ultrastructural aspects of storage reserve accumulation. Observations of the differentiation symptoms showed that the embryos pass through distinct morphological stages, superficially reminiscent of those in grass zygotic embryos; with structural bipolar embryonic orientation showing distinctively clear embryo axes and scutellum. Histochemical and ultrastructural examinations revealed that both tissues accumulated significant amounts of the major storage reserves which were sequestered in membrane-bound units, though some differences in storage protein accumulation were observed between them at the later stages of embryo development. Taken together, the developmental and ultrastructural implications of these results are of interest since they show that sugarcane somatic embryos can recapitulate events in zygotic embryos and may, probably, have significance in their utility in synthetic seed technology.

Regeneration of plantlets from petiole callus of wild viola (Viola patrinii DC.).

Plantlets were regenerated from 5-year subcultured compact callus derived from petiole tissues of wild viola (Viola patrinii DC.) but not from 5-year subcultured friable callus. Regeneration occurred most efficiently on medium that contained two-fold diluted basal salts of Murashige and Skoogs (MS) medium, 5 × 10−6 M 1-naphthaleneacetic acid and 10−6 M kinetin. The effect of dilution of MS basal salts could also be achieved solely by two-fold dilution of the potassium dihydrogen phosphate in the mixture.The present study revealed that dilution of MS basal salts, in particular of potassium dihydrogen phosphate, was important for the regeneration of wild viola. Moreover, although the callus had been subcultured for 5 years, regeneration of plantlets from callus was still possible. In addition, scanning electron microscopy revealed that details of the process of plant regeneration from subcultured callus varied with the age and source of callus and differed from that reported in rice.

Callus formation and scanning electron microscopy of plantlet regeneration in African rice (Oryza glaberrima Steud)

Abstract Callus growth parameters such as induction frequency, fresh weight and plant regeneration potentials of ten genotypes of African rice ( Oryza glaberrima ) on Murashige and Skoog (MS) basal medium supplemented with organic nutrients and different levels of two auxin sources as well as the morphological pathway of development leading to plantlet regeneration were studied by scanning electron microscopy. Each of the two synthetic auxins tested: 2,4-dichlorophenoxyacetic acid (2,4-D) and 3,6-dichloro- o -anisic acid (dicamba) was found to have appreciably different effects on the callusing ability of the genotypes at different concentrations. Generally, dicamba was observed to stimulate the formation of a mainly greenish, hard and nodular compact callus phenotype which yielded a higher plant regeneration frequency than 2,4-D in most of the genotypes. Sequential electron microscopy of the callus during growth and differentiation demonstrated that different developmental features were present on the surfaces before the eventual initiation of shoot and root primordia. On the basis of the structural data, the histogenetic event is best ascribed to the non-synchronized, independent growth of callus leading to organogenesis of shoot and root meristems.

Stepwise degradation of cauliflower mosaic virus by pronase.

Abstract Treatment of 32 P-labeled cauliflower mosaic virus (CaMV) with 0.02% pronase for 2–5 min at 37° degraded the viral protein shell, producing particles of about 100 S and finally nucleic acid free from protein. Incubation of 35 S-labeled CaMV under the above conditions resulted in the degradation of virus particles to about 100 S and finally protein remaining in the top of the gradient. The slower-sedimenting particles show the regular loss of protein moiety in a stepwise fashion with the time of incubation in pronase, suggesting that CaMV particles consist of some layers of protein shells. Tobacco mosaic virus (TMV), however, is not degraded by the same treatment with pronase as CaMV. Pronase digestion is rather specific for some viruses. The structure of CaMV is discussed in comparison with that of polyoma virus and simian virus 40.

Flow dichroic spectra of tobacco mosaic virus and their protein assemblies

Ultraviolet flow dichroisms of tobacco mosaic virus (TMV), TMV-RNA and TMV-protein were measured using three strains of TMV. 1. Large positive dichroisms were observed with three strains of TMV, namely ordinary, bean form of bean, and tomato strains (TMV-OM, TMV-B and TMV-T, respectively) at about 255, 276, 284 and 290 nm. The positive dichroisms were confirmed with reconstituted protein assemblies of TMV-OM and TMV-B at about 276, 284 and 290 nm where tyrosine and tryptophan residues of these proteins contribute. These results show that the electronic transition moments of their base groups and aromatic groups are nearly parallel to the polymer axis. It is suggested that there is a strong interaction between the base groups of RNA and aromatic groups of amino acid residues in TMV. 2. A small negative dichroism was observed near 296-300 nm with intact TMV-OM and TMV-T and with the reconstituted protein polymer of TMV-OM. But negative dichroism was not observed either with the intact virus or the protein assembly of TMV-B. 3. Isolated RNA from TMV-OM, TMV-B and TMV-T showed no dichroism. The configuration of RNA in TMV appears to be imposed on it by its packing with the protein.

Microscopic Observations of Fusion Process of Rice and Lettuce Protoplasts

Protoplasts were isolated from rice callus and lettuce cotyledons. Their fusion process was studied by light and electron microscopy. Lettuce protoplast fusion could be easily produced by either of polyethylene glycol and electric methods. The transformation of fusion bodies during the fusion process was more easily observed by using the electric method than chemical one. After electric pulse application, the fusion of lettuce protoplasts was finished within about one hr, judging from their externals. On the other hand, the fusion of rice protoplasts occurred in a very short time after electric pulse application. Fusion was performed between lettuce protoplasts and rice protoplasts. Rice protoplasts contain no chloroplasts and lettuce protoplasts contain them. Therefore, both protoplasts could be easily distinguished under a light microscope. When the fusion between rice and lettuce protoplasts was induced by electric pulse application, chloroplasts in lettuce protoplasts moved into rice protoplasts within about one hr. Electron micrographs showed that the components from both protoplasts was located separately in the fusion bodies. Big rice fusion bodies produced by fusion of rice protoplasts contained some round-shaped particles. Their size was almost the same as that of the original protoplasts. These results indicate that it take a long time until the cytoplasm of rice protoplasts mix with the cytoplasm of lettuce protoplasts in the fusion body.

Thermally induced conformational changes of tobacco mosaic virus and their protein assemblies

The measurement of the fluorescence intensity of tryptophan residues showed that a reversible transition in the local structure took place between 20 degrees C and 30c in intact virus particles and reconstituted protein assemblies of the ordinary strain and the tomato strain of tobacco mosaic virus. During this transition the overall polymer structure was maintained. In the case of the bean strain of tobacco mosaic virus, however, the fluorescence intensity did not show any transition in the same temperature range. Such a difference between different strains gave some information on the location of tryptophan residues possibly involved in the local structure change. The fluorescence polarization of intact virus particles showed no change in the whole temperature range, but the polarization of the reconstituted protein assembly of the ordinary strain showed a transition at the same temperature as the fluorescence intensity. This suggested a difference in the freedom of the local structure between intact virus particles and reconstituted protein assemblies. Oligomers of these virus proteins were stable up to 45c and above this temperature, began to make an irreversible transition where the secondary structure of the monomer was partially broken but the oligomer structure was retained.

The accumulation in plants of chromatographically separated components of tobacco mosaic virus

Abstract Chemically purified tobacco mosaic virus preparations were separated into several components by stepwise elution of a cation-exchange resin (Amberlite IR-120) and ECTEOLA cellulose columns. The amounts of the components in preparations from source plants infected for different periods was determined. Two of the components separated by ECTEOLA cellulose chromatography started to accumulate a day later than the others.

Effect of Field Drainage on Root Lodging Tolerance in Direct-Sown Rice in Flooded Paddy Field

Abstract To elucidate the effect of drainage of paddy fields on root lodging tolerance in direct-sown rice, we measured the pushing resistance (R), diameter of hill at the base (Dm), shoot dry weight (Ws) and root dry weight (Wr), in rice varieties grown using several irrigation management schemes that differed in the frequency and length of field drainage during the growing season. Soil hardness was also monitored to investigate the relationship between the variance of soil physical properties caused by different irrigation treatments and root lodging tolerance. Pushing resistance moment (Rh), i.e., product of pushing resistance (R) and height of pushed part of hill (h), showed higher values in rice grown in fields drained more frequently or for longer periods. A similar pattern was found in rice grown in field plots where root penetration to the subsoil layers was prevented by laying an unwoven cloth between the topsoil and subsoil layers. Higher values for pushing resistance efficiency based on root dry weight (Kr : Rh/Wr/Dm) were also found in plots subjected to more frequent or prolonged drainage, irrespective of rice variety. Soil hardness was progressively increased by each field drainage during the growing season, and showed a highly significant relationship with Kr. The above results suggest that field drainage increases the root lodging tolerance in direct-sown rice through improvement of anchoring ability caused by increased soil hardness.

Effect of salts on the adsorption of tobacco mosaic virus to tobacco leaf cell debris

Abstract The amount of tobacco mosaic virus (TMV) adsorbed to tobacco leaf cell debris was estimated at different concentrations of salts and of TMV. When the medium contained sodium phosphate buffer (0.0084–0.033 M , pH 7.0), ammonium phosphate (0.025 M ), sodium chloride (0.025–0.1 M ), or calcium chloride (0.025 M ), the amount of bound TMV was enhanced as compared with a medium containing no salts. The percentage increase of bound TMV caused by phosphate decreased with increasing concentration of TMV. The effect of phosphate in this in vitro system is similar to its effect on numbers of local lesions produced on Nicotiana glutinosa leaves by TMV inoculation.