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Interspecific and intergeneric fusions of plant protoplasts were induced by polyethylene glycol (PEG) 1540 or 4000. The frequency of heterokaryocyte formation (or rate of fusion) was much higher when PEG was eluted with a high pH-high Ca(2+) solution or a salt solution than when it was eluted with a protoplast culture medium. The frequency of heterokaryocyte formation was also affected by the types of enzymes used for wall degradation, duration of enzyme incubation and molality of the PEG solutions.The maximum frequency of heterokaryocyte formation was 23% for V. hajastana Grossh.-soybean (Glycine max L.) and barley (Hordeum vulgare L.)-soybean, 35% for pea (Pisum sativum L.)-soybean, 20% for pea-V. hajastana, 14% for corn (Zea mays L.)-soybean and 10% for V. villosa Roth-V. hajastana.40% of the barley-soybean, corn-soybean and pea-soybean heterokaryocytes divided at least once. Some divided many times and formed clusters of up to 100 cells in 2 weeks. The heterokaryocytes of soybean-V. hajastana, V. villosa-V. hajastana also divided. Of the PEG-treated protoplasts of N. langsdorffii and N. glauca 13.5% developed into tumor-like calli. The morphology of these calli was very much like that of the tumors produced on amphidiploid plants of N. langsdorffii x glauca.Nuclear staining indicated that heterokaryocytes of V. hajastana-soybean, pea-soybean, corn-soybean and barley-soybean could undergo mitosis. Nuclear divisions in a heterokaryocyte were usually synchronized or almost synchronized. Nuclear fusion and true hybrid formation usually occurred during the first mitotic division after protoplast fusion. A hybrid of barley-soybean in third cell division was observed. The frequency of heterokaryocytes which underwent nuclear fusion has not been determined. Multipole formation and chimeral cell colonies were also observed.

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 0.5C per minute to -40°C prior to immersion in liquid nitrogen (LN). After rapid thawing in a 40°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.Plant cells cultured in vitro are potential sources of useful chemicals such as alkaloids (1,5,16,23,31). The production of alkaloids by in vitro cultures can vary among cell lines and in individual lines be fairly stable (2, 4); in other cell lines, alkaloid content and spectra have been found subject to gradual change over years of subculture (6). Instead of repeated selection to maintain a high alkaloid level, cryopreservation would appear as an alternative to overcome stability problems (14, 31).Storage in LN2 followed by recovery of viable plant cells have been reported for many species (7,10,12,14,21,22,28,30) and cells of certain species retained the biosynthetic capacity for biotin (25), anthocyanin (8), and cardenolides (7). However, whether such a cryogenic technique could be applied to the preservation of cell cultures with capability for alkaloid synthesis and accumulation has not been determined and thus served as the primary objective of the present study. A procedure for prolonged cryogenic storage of periwinkle cell line no. 916, which did not produce alkaloids in detectable amounts, has been reported (14). We employed the procedure established for no. 916 (14) as well as other published protocols for the freeze-preservation of cultured plant cells (10,14,21,22,25,26,27,28,29); but none of them was found suitable for any of the alkaloid-producing periwinkle cell lines.In the present paper, we report a method for the successful cryopreservation of alkaloid-producing cell lines of periwinkle. It differs in several respects from previously established protocols.MATERIALS AND METHODS Plant Material. Alkaloid-producing periwinkle (Catharanthus roseus L., G. Don.) cell lines designated PRL nos. 200, 615, and 91601 were used in this study. These cell lines were initiated from callus of anther walls and filaments of p...

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