A description of aseptic plant cell culture, including a glossary of terms, has been given and the biosynthesis of phytosterols and steroidal sapogenins reviewed. The method and conditions employed in the induction of callus cultures from the cotyledons of Fenugreek seed are described. The growth of callus cultures has been studied and expressed as the increase in fresh weight, when using partly and fully defined media with different growth regulator regimes. Phytosterols and steroidal sapogenins were detected in cultures maintained for one year on the induction medium. These compounds were isolated and identified using gas liquid chromatography, infared analysis and mass spectrometry. Unsuccessful attempts were made to determine the small quantities of diosgenin and yamogenin present in the callus cultures by a colourimetric assay originally designed for seed samples. A successful assay method was devised which involved separation of the phytosterol and sapogenin fractions by adsorption column chromatography and subsequent assay of Doth fractions by gas liquid chromatography. The conditions affecting the yield of the aglycone sapogenin from the callus tissue have been examined. Considerable differences were observed in the composition of the free sterol and sapogenin components from callus cultures grown either with NAA or 2,4-D. No significant changes in the steroid yield of cultures was caused by altering the vitamin formulation of the medium, introducing a rare amino acid isolated only from Fenugreek seed, or varying the light conditions under which the cultures were grown. Experiments were performed to select a defined medium for the initiation and maintenance of Fenugreek suspension cultures. The basal medium used for callus culture proved satisfactory, but a reduction in the concentrations of the growth regulators was necessary to induce a cell suspension. The sapogenin yield of the suspension cultures was lower than that of the callus culture from which the original explants were taken.
|Date of Award||1974|