When Vicia hajastana Grossh. cells or protoplasts were cultured at a high population density (ca. 5000 cells or protoplasts/ml), they were able to grow in a mineral-salt solution supplemented with sucrose (or glucose), a few vitamins, and 2,4-dichlorophenoxyacetic acid. They were not able to survive when cultured at a low population density unless the medium was supplemented with zeatin, naphthalene-1-acetic acid, nucleic-acid bases, amino acids, other sugars, sugar alcohols, and organic acids. Vicia cells were able to grow at an initial population density of 25-50 cells/ml in this defined medium. The population density could be lowered to 1-2 cells/ml with good growth when the mineral-salt medium was enriched with organic acids, sugars, sugar alcohols, coconut water, and casamino acids. The protoplasts also grew best in a medium enriched with these supplements. Three individual protoplasts were isolated and each one was cultured in a separate dish containing 4 ml of this medium. Within 30-40 days, each one had grown indefinitely and formed a mass of cells (ca. 10(7)).

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Protoplasts of Vicia hajastana Grossh. obtained from suspension-culture cells and Pisum sativum L. obtained from leaves adhered tightly to each other in concentrated solutions of high-molecular-weight polyethylene glycol (PEG). The adhesion occurred non-specifically between the free protoplasts from the same species as well as from the different species and genus. It was enhanced by enrichment of the PEG solution with calcium. Very few heteroplasmic fusions occurred during the period when the protoplasts were incubated in the PEG solution. However, many heterokaryons (up to 10%) were formed soon after the PEG solution was diluted out. The same phenomena were also observed in protoplasts from suspension-culture cells of Glycine max L. and from leaves of Hordeum vulgare L. Vicia and soybean protoplasts obtained from cultured cells regenerated cell walls and underwent sustained cell division after such treatment. Some Vicia-pea heterokaryons divided once. Over 10% of the soybean-barley hybrids divided in 7 days. Some divided 4-5 times and formed small clusters of cells in 10 days. The hybrids were recognizable because they contained chloroplasts from the leaf protoplast and exhibited morphological characters typical of the chlorophyll-less cells. None of the protoplasts from pea and barley leaves, either with or without PEG treatment, underwent cell division during the period of observation. The mechanism of adhesion and fusion of the protoplasts has been discussed.

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