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Ultrastructure and protein patterns of horseradish (Armoracia lapathifolia Gilib.) in vitro tissue culture

Leaf fragments of in vitro propagated horseradish (Armoracia lapathifolia Gilib.) plants (Fig 1.) were infected with a wild octopine-strain B6S3 of Agrobacterium tumefaciens to induce primary crown gall tumours. The tumour tissue has been subcultured on hormone free nutrient medium. During subcultivation two phenotypically different transformed tissue lines were established: one unorganized (TN) and other organized teratoma line (TM). The TM tissue produced malformed shoots with hyperhydrated leaves (Fig. 2). This shoots have never develop roots and spontaneously formed unorganized tissue, which subsequently produced new shoots. The TN tumour line grew in an undifferentiated way producing abundant yellow greenish unorganized tissue (Fig. 3). The TM shoots were found to be octopine positive, but the octopine signal was weaker than the signal of the TN tissue.1 The aim of the present study was to compare in vitro grown horseradish plantlet leaves, tumour and teratoma tissues with regard to morphology, ultrastructure and protein patterns with the intention to connect the structural and biochemical data. Protein extracts were prepared by grinding the tissue in a cold Tris/HCl buffer (0.1 M, pH 8.0). The homogenates was centrifuged and a protein content of postmitochondrial fraction was determined spectrophotometrically. Proteins were separated by SDS-polyacrylamide gel electrophoresis and silver stained. For ultrastructural analyses, the tissue was fixed with 1% glutaraldehyde in cacodylate buffer (pH 7.2) and postfixed with 1% OsO4 in the same buffer. After dehydration in ethanol, the tissue was embedded in Spurr's resin. Semi-thin sections of fixed material were stained with 2% toluidine blue and examined using light microscope „ Zeiss Axiovert 35“ . Ultrathin sections were stained with uranyl acetate and lead citrate and examined using a FEI Morgagni 268D electron microscope. Ultrastructural studies revealed differences between the leaf and crown-gall tissue. The leaf cells contained typical chloroplasts with well-developed grana-tylakoids and 2-3 large starch grains (Fig. 4). Teratoma plastids were mostly roundish in shape. Their thylakoid system was usually less-developed than in the leaf chloroplasts, with thylakoids often being dilated (Fig. 5). In tumour cells, plastids varied in size and shape, from oval to ameboid-like (Fig. 6). Their thylakoid system was often strongly reduced and usually consisted of few tylakoids which extended through the central stroma. In some plastids, 1-2 starch grains were also noticed. Nuclei of tumour cells were usually irregularly shaped, with deep invaginations of the nuclear envelope (Fig. 7). The SDS-PAGE showed a few tissue-specific bands. The 110, 70 and 23 kDa polypeptides were specific for leaf extracts (arrow) (Fig. 8). Tumour and teratoma tissues coincided in the majority of bands and showed three distinct polypeptides of about 115, 33 and 20 kDa which were not detected in the leaf (asterisk). The polypeptide of 63 kDa was detected in teratoma tissue only (spot). The bends of 64 and 65 kDa were remarkable in tumour tissue (oval). The 54 and 16 kDa polypeptides, most likely subunits of Rubisco, were quantitatively dominant in the leaf (rectangle symbol). The 80, 44 and 42 kDa polypeptides were expressed more intensively in teratoma and tumour tissue than in the leaf (arrowhead). The results obtained show tissue specific ultrastructural characteristics of plastids and nuclei as well as specific electrophoretic protein bands. Fig. 1. Horseradish plantlet. Fig. 2. Teratoma tissue. Fig. 3. Tumour tissue. Fig. 4. Chloroplast from a horseradish leaf cell. Bar = 1 μ m. Fig. 5. Chloroplast from a teratoma cell line. Bar = 1 μ m. Fig. 6. Chloroplast from a tumour cell line. Bar = 1 μ m. Fig. 7. A part of a nucleus from a tumour cell line. Bar = 0.25 μ m. Fig. 8. SDS-electrophoretic protein pattern. Reference: 1. Krsnik-Rasol, M. Ham, A. (1992) Period Biol. 94:105-110.

ultrastructure of leaf and crown-gall tissues; protein patterns;

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