Shigetaka Ishii

Somatic hybrid plants obtained by protoplast fusion between Citrus sinensis and Poncirus trifoliata

SummarySomatic hybrid plants of Rutaceae were obtained by protoplast fusion between Citrus sinensis Osb. (‘Trovita’ orange) and Poncirus trifoliata. Protoplasts isolated from embryogenic cells of C. sinensis and from leaves of P. trifoliata, and the culture of fusion products in the presence of high concentrations of sucrose were essential requirements for the selection of hybrids. Green globular embryoids derived from protoplasts resulted in the regeneration of trifoliate plants. Other morphological characters of these plants were intermediate between both parents. The chromosome number in one of the hybrid plants was 36, which was the sum of C. sinensis (2n=18) and P. trifoliata (2n=18). EcoRI restriction analysis of rDNA confirmed the presence of parental nuclear DNAs in the hybrid.

A somatic hybrid plant obtained by protoplast fusion between navel orange (Citrus sinensis) and satsuma mandarin (C. unshiu)

A somatic hybrid plant was obtained by protoplast fusion between navel orange and satsuma mandarin. Protoplasts isolated from nucellar calli of navel orange and from leaves of satsuma mandarin were fused by the PEG method. The fusion products were cultured in a Murashige & Tucker medium containing 0.6 M sucrose. In this medium, some colonies developed into whole plants through embryogenesis. One of the regenerated plants was shown to be a hybrid, which was proven by restriction endonuclease analysis of nuclear ribosomal DNA. The chromosome number of the hybrid was 36. Both parents have a chromosome number 2n=18.

Somatic hybridization in Citrus: navel orange (C. sinensis Osb.) and grapefruit (C. paradisi Macf.)

SummaryProtoplasts of navel orange, isolated from embryogenic nucellar cell suspension culture, were fused with protoplasts of grapefruit isolated from leaf tissue. The fusion products were cultured in the hormone-free medium containing 0.6 M sucrose. Under the culture conditions, somatic embryogenesis of navel orange protoplasts was suppressed, while cell division of grapefruit mesophyll protoplasts was not induced. Six embryoids were obtained and three lines regenerated to complete plants through embryogenesis. Two of the regenerated lines exhibited intermediate morphological characteristics of the parents in the leaf shape. Chromosome counts showed that these regenerated plants had expected 36 chromosomes (2n=2x=18 for each parent). The rDNA analysis using biotin-labeled rRNA probes confirmed the presence of genomes from both parents in these plants. This somatic hybridization system would be useful for the practical Citrus breeding.

Generation of active oxygen species during enzymic isolation of protoplasts from oat leaves

SummaryProtoplasts were isolated from oat (Avena sativa L.) leaves by the combination of highly purified preparations of pectin lyase, xylanase, and cellulase C1. During the enzymic isolation, superoxide radical (O−2) was generated from the tissues. Both the protoplasts themselves and the cell walls, exposed to enzyme treatment, produced O−2. Hydrogen peroxide (H2O2) apparently accumulated in the reaction mixture due to the spontaneous dismutation reaction of O−2, while a part of H2O2 may have been produced directly from cell walls by the action of enzymes. Singlet molecular oxygen (1O2) generated in the reaction mixture was detected by cholesterol oxidation in small unilamellar liposomes. It seems likely that1O2 may be generated by the peroxidase-H2O2-halide system during enzymic treatment of the leaves.

Fertile fruit trees obtained by somatic hybridization: navel orange (Citrus sinensis) and Troyer citrange (C. sinensis x Poncirus trifoliata)

SummaryNucellar cell suspension protoplasts of navel orange (Citrus sinsensis Osb.) were chemically fused with mesophyll protoplasts of Troyer citrange (C. sinensis x Poncirus trifoliata) and cultured in hormone-free Murashige and Tucker medium containing 0.6 M sucrose. Two types of plant were regenerated through embryogenesis. One type showed intermediate mono-and difoliate leaves and the other types was identical to Troyer citrange. The regenerated plants with intermediate morphology were demonstrated by chromosome counts and rDNA analysis to be amphidiploid somatic hybrids. Five clones of these somatic hybrids were grafted in the field. After 4 years, they set flowers having a morphology intermediate between those of the two parents. The pollen grains showed high stainability and sufficient germinability, and were larger than those of Troyer citrange. The fruits of the somatic hybrids were large and spherical with thick rinds. Most of them contained seeds with normal germinability. These results indicate that somatic hybridization is a useful tool for Citrus breeding.

Enzymatic extraction and linkage analysis of pectic polysaccharides from onion

Abstract Pectin lyase was superior to polygalacturonase for the extraction of onion cell wall pectic polysaccharides. Exhaustive treatment of onion tissue with pectin lyase solubilized 89% of the total uronides of the tissue. The galacturonides released from the tissue were separated into three fractions (10.7, 5.3 and 84%, in order of MW) by gel filtration on Sephadex G-100. The low MW fraction was a mixture of oligogalacturonides. High and intermediate MW fractions were purified by DEAE-Sephadex column chromatography. The intermediate MW fraction was a rhamnogalacturonan II type component which contained 3- and 3,4-linked rhamnose. Methylation analysis showed that the pectic polysaccharides of onion resembled those of potato tuber.

Isolation and characterization of cell-wall pectic substances from potato tuber

Abstract Exhaustive treatment of potato tuber tissues by purified endo-polygalacturonase from Aspergillus japonicus solubilized 95% of the total uronides of