Somatic 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.

Callus cells of rice (Oryza sativa L.) that were actively dividing in suspension culture had lost the ability to divide during the isolation process of protoplasts. Factors influencing the protoplast viability were examined using highly purified preparations of cellulase C,, xylanase, and pectin lyase, which were essential enzymes for the isolation of protoplasts from the rice cells. The treatment of the cells with xylanase and pectin lyase, both of which are macerating enzymes, caused cellular damage. Xylanase treatment was more detrimental to the cells. Osmotic stress, cell wall fragments solubilized by xylanase, and disassembly of cortical microtubules were not the primary factors which damaged the rice cells and protoplasts. The addition ofAgNO3, an inhibitor ofethylene action, to the protoplast isolation medium increased the number of colonies formed from the cultured protoplasts, although the yield of protoplasts was reduced by the addition. Superoxide radical (02-) was generated from the cells treated with xylanase or pectin lyase. The addition of superoxide dismutase and catalase to the protoplast isolation medium resulted in a marked improvement in protoplast viability especially when the non-additive control protoplasts formed colonies with a low frequency. The addition of glutathione peroxidase and phospholipase A2, which have been known to reduce and detoxify lipid hydroperoxides in membranes, to the protoplast culture medium significantly increased the frequency of colony formation. These results suggested that some of the damage to rice protoplasts may be caused by oxygen toxicity. cellular death (4, 29). The toxic effect of pectinases on plant cells was found to be reduced by plasmolysis (25). Thus, the use of osmotica is essential for the isolation of viable protoplasts to reduce the toxic effect of pectinases as well as to prevent protoplasts from bursting. However, it is known that plasmolysis per se gives rise to some cellular damage by osmotic shock (18,20).If plant tissues such as leaves are used as protoplast sources, it should be kept in mind that peeling or cutting ofthe tissues may trigger wound-inducing cellular damage (28).Rice cells, derived from roots that were actively dividing in suspension culture, yielded protoplasts that had lost the ability to divide when isolated using commercially available enzymes. They showed a delay of first division and a low frequency of cell division (2). Only 1 to 2% ofthe cultured protoplasts formed cell colonies. Impurities and other enzyme activities that contaminated the commercial cell wall-degrading enzymes were suggested as some ofthe possible agents that damage protoplasts (8). Ishii and Mogi (16) developed a protoplast isolation procedure using leaf tissues of monocots and dicots and treatment with a combination of highly purified enzymes. Protoplasts were easily isolated from the suspension-cultured rice cells by highly purified preparations of cellulase Cl, xylanase, and pectin lyase. But the use of purified enzymes alone did not gre...

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Pectinesterase was purified from the culture medium of Aspergilhs juponicus completely free from pectin depolymerizing enzymes. The purified enzyme was able to convert high-methoxyl pectin into lowmethoxyl pectin capable of forming strong gels with calcium ion. The maximum gel strength of enzyme-demethylated pectin was obtained at methoxyl content of 6.3%, calcium concentration of 41.8 mg/g of pectin and pH 3.5. The low-methoxyl pectin, however, formed weaker gels at low concentrations of calcium ion and low pH. DEAE-cellulose column chromatography indicated that the enzyme-demethylated pectin was homogeneous with respect to methoxyl groups of the molecules. It was suggested that this homogeneity may be probably responsible for high gel strength of the lowmethoxyl pectin.

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