Factors Influencing Protoplast Viability of Suspension-Cultured Rice Cells during Isolation Process

Abstract

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...

Lateral and Rotational Mobilities of Lipids in Specific Cellular Membranes of Eucalyptus gunnii Cultivars Exhibiting Different Freezing Tolerance

“…Nevertheless, it should be remembered that for technical reasons (depth of focus of the objective used [6,9,15] or probe internalization [13]), it is extremely difficult to assess that the fluorescence signal which is observed originates only from the plasma membrane. From another point of view, it has been shown that protoplast preparation through enzymic degradation of the cell wall, as usually done, can be damaging for the protoplast itself, either in terms of cell viability (8) or with respect to the structure of the plasma membrane (5) or its lipid composition (2). In addition, production of activated oxygen in the form of superoxide radical 2-has been reported in the course of preparation of protoplasts from rice cells when using macerating enzymes (8).…”

“…From another point of view, it has been shown that protoplast preparation through enzymic degradation of the cell wall, as usually done, can be damaging for the protoplast itself, either in terms of cell viability (8) or with respect to the structure of the plasma membrane (5) or its lipid composition (2). In addition, production of activated oxygen in the form of superoxide radical 2-has been reported in the course of preparation of protoplasts from rice cells when using macerating enzymes (8). The possibility of degradation of phospholipid-like probes (such as NBD-PC) by phospholipases (12) also should not be ignored.…”

Ethylene Biosynthesis-Inducing Xylanase

“…Subsequent work has shown that the enzyme from T. viride also elicits other defense responses in tobacco, including synthesis of pathogenesisrelated proteins (18), tissue necrosis, and electrolyte leakage (3). Other fungal endoxylanases have been shown to elicit defense responses, such as phytoalexin production (10), lipid peroxidation (13), vascular gelling (29), and cell death (5), in various plant tissues. However, with the exception of the enzyme from Phytophthora parasitica f. sp.…”

An Ethylene Biosynthesis-Inducing Endoxylanase Elicits Electrolyte Leakage and Necrosis in Nicotiana tabacum cv Xanthi Leaves

“…Recently, Ishi (13) demonstrated that a macerating xylanase isolated from a commercial preparation of T. viride origin was toxic to rice cell cultures. Lotan and Fluhr ( 16) observed the induction of pathogeniesis-related proteins (i.e.…”