GROWTH OF EDIBLE CHLOROPHYLLOUS PLANT TISSUES IN VITRO

HILDEBRANDT, A. C., J. C. WILMAR, H. JOHNS, and A. J. RIKER. (U. Wisconsin, Madison.) Growth of edible chlorophyllous plant tissues in vitro. Amer. Jour. Bot. 50(3): 248-254. Illus. 1963.-Plant callus cultures were attempted from roots, stems, leaves or excised embryos of 32 species of plants on a basal mineral salts-sucrose agar medium (T-medium), on T-medium + coconut milk + a-naphthaleneacetic acid + calcium pantothenate (C-medium) and on C-medium + 2, 4-dichlorophenoxyacetic acid (D-medium). Embryos on Tor C-medium generally produced normal plants, while on D-medium, they often produced callus only. Fresh isolates of carrot, endive, lettuce, parsley, red kidney bean, and navy bean gave moderate to excellent callus on C-medium. Parsley and navy bean also produced excellent callus on D-medium. Strains of callus from potato, tomato, grape and rose also grew well on Cor D-medium. In the light, red pigmentation developed on rose, parsley, and grape callus. Chlorophyll formation was inhibited on D-medium, but on C-medium more or less chlorophyll was initiated in callus from carrot, endive, lettuce, pea, potato and certain rose varieties. Chlorophyll formation was also strong in endive callus on T-medium supplemented with casein hydrolysate, i-inositol and NAA. The amount and type of sugar used in C-medium influenced the amount of growth and were critical in relation to chlorophyll formation. Carrot tissues in constant light produced abundant chlorophyll and were still growing on media without added sugar after 6 weeks. PLANT tissue culture methods offer many possibilities for studies of growth and differentiation. Cell, tissue and organ cultures of many species are possible (Gautheret, 1959). Synthetic liquid or agar media, or media supplemented with complex extracts provide the necessary substrates. The nutritional and other environmental conditions necessary for growth vary with the origin and type of tissue. Callus cultures growing in liquid shake cultures or on agar media have been used for over 20 years in studies (reviewed by Riker and Hildebrandt, 1958) of growth of normal and diseased tissues. Mass cultures of algae (Krauss, 1962) have been studied as methods of producing food for humans. Large-scale production of callus tissues of several species of higher plants as sources of food have been described (Byrne and Koch, 1961). The possibilities of producing callus tissue for food and gas exchange in difficult locations, including space travel, make additional information necessary. Chlorophyll-containing tissues in culture would be useful also for research on autotrophic growth, metabolism, and disease. 1 Received for publication August 3, 1962. 2 This work was supported in part by the U.S. Army Quartermaster Food and Container Institute, Contract DA19-129-QM-1817 (01 5123), by the American Cancer Society, by the National Institutes of Health, and by the Research Committee of the Graduate School from funds supplied by the Wisconsin Alumni Research Foundation. Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. The authors wish to acknowledge assistance from Drs. P. K. Chen and M. V. Rajagopal in isolations of tissues from carrot, endive, lettuce, cucumber, red kidney bean, navy bean and spinach, and to Mr. Eugene Herrling for preparation of the illustrations. The purpose of this study was to develop cultures from a variety of edible plant tissues in vitro, to compare them with established cultures without chlorophyll, and to clarify some requirements for chlorophyll formation and for growth of these green tissues. MATERIALS AND METHODS Tissue and organ isolations were attempted after surface disinfection with a 15-60 sec dip in 95% ethyl alcohol followed by 1-10 min in a 1:10 dilution with water of commercial 6.4% sodium hypochlorite and 1-2 min in each of 7 changes of sterile distilled water. Isolations of tissues were attempted from roots, stems, and leaves. Cross sections of leaf petioles and stems were used as sources of callus. Embryos also were dissected out and grown in vitro as sources of callus or normally differentiated plants. The latter sterile plants were used as sources of sterile plant parts in order to obtain callus cultures. Callus cultures were attempted from stems and leaves of asparagus, beet, Brussels sprouts, cabbage, carrot, cauliflower, celery, cucumber, horseradish, lettuce, parsley, pea, potato, rhubarb, spinach, Swiss chard and tomato. Callus cultures also were attempted from parts of aseptically grown seedlings or excised embryos of asparagus, barley, beet, carrot, cucumber, endive, Idaho refugee bean, lettuce, mustard, navy bean, oats, pea, red kidney bean, rice, rye, spinach, squash, sunflower, Swiss chard, tomato and wheat. In addition, certain established callus cultures from tomato, grape, 9 varieties of rose (Coyier, 1961), and potato (Kumar, Hildebrandt, and Riker, 1961) were included in the growth tests.