Biomedical subjects
Publications and source records attributed to F Constabel.
Biotechnology is described as methodology to enhance the formation and accumulation of desirable natural products and possible product modification in medicinal plants. Micropropagation, cell and hairy root culture as well as gene technology are being highlighted.
By inserting nitrocellulose strips into agarose gels alongside the electrophoresed lanes and passing an electric current perpendicularly in the direction of the strips, highly efficient transfer of DNA bands onto the membrane in the form of concentrated dots is achieved. DNA detection limits by this technique are enhanced, at least three times as visualized by ethidium bromide fluorescence and at least twice more by radiolabeling.
Treatment of one cell line (No. 615) of Catharanthus roseus c.v. Little Delicata with an elicitor preparation of autoclaved and homogenized Pythium aphanidermatum culture resulted in rapid accumulation of indole alkaloids. Alkaloid formation was preceded by rapid transient increases in the extractable activities of the enzymes tryptophan decarboxylase and strictosidine synthase. The induction of these two enzyme activities occurred when cells were transferred to alkaloid production medium or treatment with fungal elicitors. Treatment of this cell line with translational or transcriptional inhibitors prevented the Pythium-induced increases of enzyme activity as well as alkaloid accumulation. When cells were transferred to alkaloid production medium the induction of strictosidine synthase activity preceded that of tryptophan decarboxylase by many hours even when cells were also treated with Pythium elicitor. Results suggested that tryptophan decarboxylase induction proceeds only when endogenous tryptamine levels were decreased by two-third. The internal cellular level of tryptamine, therefore, could regulate expression of tryptophan decarboxylase, whereas induction of strictosidine synthase or of another enzyme in the biosynthetic pathway could control channeling of tryptamine into alkaloids. The results demonstrate that fungal elicitors can be used to facilitate studies of the factors which regulate expression of indole alkaloid pathway enzymes and their ultimate pathway products.
A procedure for the quantitation of the delivery of liposome contents into Catharanthus roseus protoplasts has been developed. The method is based on the uptake of liposome encapsulated methylumbelliferyl beta-D-glucoside and its enzymatic hydrolysis to yield fluorescent methylumbelliferone. Since the free glucoside is not taken up by the protoplasts to a significant extent, the delivery of material in the nanomole range can be measured with ease.
The freezing behavior of dimethylsulfoxide (DMSO) and sorbitol solutions and periwinkle (Catharanthus roseus) cells treated with DMSO and sorbitol alone and in combination was examined by nuclear magnetic resonance and differential thermal analysis. Incorporation of DMSO or sorbitol into the liquid growth medium had a significant effect in the temperature range for initiation to completion of ice crystallization. Compared to the control, less water crystallized at temperatures below -30 degrees C in DMSO-treated cells. Similar results were obtained with sorbitol-treated cells, except sorbitol had less effect on the amount of water crystallized at temperatures below -25 degrees C. There was a close association between the per cent unfrozen water at -40 degrees C and per cent cell survival after freezing for 1 hour in liquid nitrogen. It appears that, in periwinkle suspension cultures, the amount of liquid water at -40 degrees C is critical for a successful cryopreservation. The combination of DMSO and sorbitol was the most effective in preventing water from freezing. The results obtained may explain the cryoprotective properties of DMSO and sorbitol and why DMSO and sorbitol in combination are more effective as cryoprotectants than when used alone.
A procedure for cryogenic storage of alkaloid producing cell lines of periwinkle, Catharanthus roseus (L.) G. Don., has been developed. The procedure differs from established cryopreservation protocols in several aspects. Specifically, 4-day-old suspension subcultures of three cell lines were precultured in nutrient media supplemented with 1 molar sorbitol for 6 to 20 hours. The cells were then incubated in nutrient media with 1 molar sorbitol plus 5% DMSO in an ice bath for 1 hour and, thereafter, were frozen in this solution at a cooling rate of 0.5 degrees C per minute to -40 degrees C prior to immersion in liquid nitrogen (LN). After rapid thawing in a 40 degrees C water bath, the regrowth of LN stored cells was achieved by transferring them without washing onto filter paper discs over nutrient media solidified with agar for a period of 4 to 5 hours. The filter paper discs with the cells were then transferred to fresh media of the same composition for regrowth. The viability immediately after thawing as evaluated by the 2,3,5-triphenyl tetrazolium chloride method was about 60% of controls. Suspension cultures established from LN stored cells retained the capability for alkaloid synthesis and accumulation.
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Somatic hybridization in higher plants has come into focus since methods have been established for protoplast fusion and uptake of foreign DNA and organelles by protoplasts. Polyethylene glycol (PEG) was an effective agent for inducing fusion. Treatment of protoplasts with PEG resulted in 5 to 30% heterospecific fusion products. Protoplasts of different species, genera and even families were compatible when fused. A number of protoplast combinations (soybean + corn, soybean + pea, soybean + tobacco, carrot + barley, etc.) provided fusion products which underwent cell division and callus formation. Fusion products initially were heterokaryocytes. In dividing heterokaryocytes, random distribution of mitotic nuclei was observed to be accompanied by multiple wall formation and to result in chimeral callus. Juxtaposition of mitotic nuclei suggested nuclear fusion and hybrid formation. Fusion of heterospecific interphase nuclei was demonstrated in soybean + pea and carrot + barley heterokaryons. Provided parental protoplasts carry suitable markers, the fusion products can be recognized. For the isolation and cloning of hybrid cells, fusion experiments must be supplemented with a selective system. Complementation of two non-allelic genes that prevent or inhibit growth under special culture conditions appears as the principle on which to base the selection of somatic hybrids. As protoplasts of some species have been induced to regenerate entire plants, the development of hybrid plants from protoplast fusion products is feasible and has already been demonstrated for tobacco.
Multinucleate soybean protoplasts produced by spontaneous fusion during enzyme digestion of the cell wall initiated cell division after approximately 40 h in culture. The structure of these protoplasts during mitosis and cytokinesis was studied with both light and electron microscopes. Most nuclei did not fuse but divided synchronously. Interphase nuclei was commonly connected by short narrow nuclear bridges. At prophase and metaphase the nuclei appeared typical of those in most higher plants; technical difficulties prevented an adequate examination of protoplasts at anaphase. Telophase was characterized by cytokinesis involving phragmoplast and cell plate formation; however, complete partitioning of the cytoplasm by cell plants was not observed. Numerous coated vesicles were present near to or continuous with the cell plate and plasmalemma. The presence of a few dividing protoplasts with at least double the normal chromosome number suggests that some nuclear fusion occurred prior to mitosis. Very little cell wall material was detected at the margin of the dividing protoplasts.
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