Daylily (Hemerocallis) as a model system for the study of ethylene-insensitive flower senescence : tissue culture and aspects of the development of proteolytic enzyme activities, with special emphasis on leucine aminopeptidase.

A micropropagation technique has been developed using petal and ovary explants from floral buds of several daylily cultivars. The growth regulators NAA and BA or 2iP in ½ strength MS was used for the initiation of shoot primordia while rooting was carried out in growth regulator-free ½ strength MS. Many plantlets of cv. Stella D'oro were grown successfully in the glasshouse to supply flowers for the experiments on senescence in this study. The role of proteolytic enzymes with special emphasis on leucine aminopeptidase (LAP), in senescence of ethylene-insensitive flowers was studied using daylily flowers. During bud development the LAP activity began to increase and reached a maximum 6 hr after the flower was fully open. Increase in endopeptidase activity began only after the flower was fully open and reached a maximum value 12 hr later. As the flower senesced, both enzymes decreased. In contrast, carboxypeptidase level remained constant throughout flower development. Therefore, LAP appears to be responsible, at least for the initiation of proteolysis during senescence of petals, while endopeptidase may be responsible for the massive proteolysis that occurred during later stages of senescence. Carboxypeptidase on the other hand does not seem to play a special role during senescence. There was no evidence for the presence of endogenous inhibitors or promoters against the 3 proteolytic enzymes studied here nor the occurrence of low-pH extractable forms of the 3 enzymes in daylily petals. When experiments were carried out to determine the effect of numerous chemicals on vase life of daylily flowers, cycloheximide was the only chemical that delayed senescence of daylily flowers. Cycloheximide treatment also retained the high levels of LAP and lap mRNA associated with full bloom flowers. In contrast to flowers, several chemicals including ethrel, cycloheximide, BA, sucrose, fructose were effective in retaining the green colour of daylily leaf discs for a longer time than those kept in water while ABA accelerated yellowing of leaf discs. GA3 kept the green colour of the leaf discs a little longer than water controls. Silver thiosulfate, ascorbic acid, casein hydrolysate, glyphosate, kinetin and IAA caused yellowing of leaf discs around the same time as water controls. 8-hydroxyquinoline caused browning of leaf discs, so did the glucose in 8-hydroxyquinoline. Green leaf discs treated with cycloheximide and ethrel had lower levels of LAP than the yellow ones in water. Yellow leaf discs in ABA also had lower levels of the enzyme than the green ones in water. Yellow leaf discs in silver thiosulfate had similar levels of LAP to those kept in water. In contrast to flower senescence, yellowing of daylily leaf discs was not always accompanied by a reduction in LAP. Therefore, LAP appears to have different roles during senescence of leaves and flowers. IEF gel analysis revealed that daylily extracts had 2 closely run LAP isozyme bands of pI 5. LAP in daylily is a thermolabile, metallo enzyme, whose activity was enhanced by Mn++. EDTA, PMSF, NEM and Zn++ inhibited enzyme activity while Mg++ and leupeptin had no effect. It also did not need SH-protecting agents for activity. The optimum pH for the enzyme activity was 8. Dot blot hybridization of mRNA from daylily petals using lap cDNA probe from Arabidopsis thaliana showed that LAP in daylily petals is under developmental regulation. Petals from buds and full bloom flowers of daylily had equal amounts of lap mRNA while those from senesced flowers had very low amounts of lap mRNA

Investigation of tropane alkaloids in genetically transformed Atropa belladonna L. cultures

The purpose of the present study was to determine the tropane alkaloid content of genetically transformed hairy root cultures of Atropa belladonna L. Determination of alkaloids was performed by HPLC method. Samples were extracted with chloroform - methanol - cc. ammonia 15:5:1 (v/v/v). Crude extracts were purified on Extrelut columns. HPLC separation was performed on Luna C8 reversed phase column. An isocratic mixture of acetonitrile - 30 mM phosphate buffer - methanol 12.2:79.7:8.1 (v/v/v) was used as eluent. Peaks were identified by addition of standards and diode-array detection. Hyoscyamine, scopolamine and apoatropine were determined by external standard method at 210 nm. We measured the alkaloid content of genetically transformed in vitro cultures (hairy roots and reorganised plants) cultivated on Gamborg B5 basic media. The highest hyoscyamine and scopolamine content was found in hairy root clone #K-5 (0,223 m/m%) and in hairy root clone #K-4 (0,018 m/m%) respectively. Alkaloid contents were higher in the hairy roots than in the reorganised plants