Abstract
Investigations were conducted on the strongly infested by microbes embryos isolated from seed stratified in non-sterile conditions or from tree buds. The results showed, that PPM (Plant Preservative Mixture, produced by Plant Cell Technology, Inc.) was very useful for increasing the percentage of sterile cultures disinfected in a 5% solution of NaOCl. The best way of applying PPM turned out to be adding the substance directly to the growing medium. At the concentration of 2 or 4 cm<sup>3</sup> x dm<sup>-3</sup> of PPM there was an 30% increase in percentage of sterile of in vitro cultures developed from embryos and 70-80% of cultures developed from buds, as compared with the control without PPM. At the range of concentrations employed in this study, no negative effects of PPM upon the development and growth of callus were observed.
Highlights
Obtaining sterile cultures is a prerequisite for in vitro plant culturing
In the experiment no I, in which the entire embryos were sterilized, the PPM added to the growing medium in the amount of 2 cm3 × dm-3 or 4 cm3 × dm-3 only delayed the development of microbes; after 30 days of growing, none of these cultures were free of microbes
Similar results were obtained in the experiment no III, where apart from other treatments the fragments of embryos soaked in a culture medium containing 10 cm3 × dm-3 of PPM were encapsulated
Summary
Obtaining sterile cultures is a prerequisite for in vitro plant culturing. There are relatively few methodical papers dealing with sterilization of plant material. Seeds and buds of European beech are difficult to sterilize, because they are usually strongly infested by microbes, and because infiltration ability of the sterilizing agent into folded cotyledons in embryos and folded leaves in buds is limited (Kraj and Dolnicki 1998; Nadel et al 1991). Sterile cultures are usually obtained by applying HgCl2 (Ahuja 1984; Chalupa 1985, 1987, 1990; Kraj and Dolnicki 1998; Nadel et al 1991), which is strongly toxic for humans and believed to be a mutagenic factor. To reduce the infestation of in vitro cultures the antibiotics and fungicides were applied; these substances are specific towards certain species of microbes, can be decomposed in an autoclave, they can be toxic or detrimental in other way for the cultures of vascular plants (Guri and Kishor 1998)
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