engleski

Protein and glycoprotein patterns related to morphogenesis in Mammillaria gracillis Pfeiff. tissue culture

As plants with Crassulacean Acid Metabolism (CAM), cacti are highly affected by artificial environmental conditions in the tissue culture. In vitro propagated plants of Mammillaria gracillis Pfeiff. (Cactaceae) spontaneously produced callus. This habituated callus regenerated normal and hyperhydric shoots without the addition of growth regulators. In order to compare habituated callus with the tumorous one, cactus cells were transformed with two strains of Agrobacterium tumefaciens: wild strain B6S3 (tumour line TW) and a rooty mutant GV3101 (tumour line TR). Both tumour lines grew vigorously and never expressed any morphogenic potential. The aim of this work was to obtain information about causes and conditions leading to growth of habituated callus and to the spontaneous regeneration in the callus culture in order to contribute to better understanding of cell differentiation and dedifferentiation mechanisms. Gene expression in cactus plants, habituated callus, regenerated shoots and both tumour lines was analysed at the level of cellular and extracellular protein and glycoprotein profiles. Proteins were separated by SDS-polyacrylamide gel electrophoresis and silver stained. Glycoproteins with D-manose in their glycan component were detected on protein blots by concavalin A-peroxidase staining. SDS-PAGE of cellular proteins showed a few tissue-specific bands. The cellular glycoprotein of 42 kDa detected by Con A was characteristic only for undifferentiated tissues (callus, TW and TR tumours) and hyperhydric regenerant, while the same protein was present in all cactus tissues. Patterns of proteins secreted in the nutrient medium of suspension-cultured cells showed differences between untransformed tissues and tumours. In extracts of plant and normal regenerant no extracellular proteins were detected. Among glycosilated extracellular proteins, only two bands were common for all tissues, whereas the seven bands were distinct for both tumour lines. From these results it can be concluded that N-glycosylation of cellular and extracellular proteins was influenced by the developmental stage of cactus tissue.

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