THE EFFECT OF SUCROSE AND OTHER FACTORS ON THE SHOOT‐ROOT RATIO OF ORCHID SEEDLINGS

THE SEEDS of orchids are distinctive in that they contain practically no reserve food material. Knudson (1922) showed by pure culture technique that the function of the symbiotic fungus, which is associated with orchid seedlings under natural conditions, is the provision of a suitable food source for the germinating seeds. The germination of many species has subsequently been obtained asymbiotically on an artificial medium of suitable acidity which contains sucrose, glucose, or certain other sugars as the carbon source (Knudson, 1922, 1925; La Garde, 1929; and Quednow, 1930). The embryo of the mature orchid seed consists of a group of cells which are differentiated into neither growing axis nor cotyledon; nor is there an endosperm. When conditions are favorable the embryo develops into a ball-shaped protocorm. Although this may become green, it remains at least partiallv dependent upon an external food source until a leaf has been formed. Later a shoot and additional leaves differentiate, but no primary root is formed; ultimately adventitious roots appear at the nodes a short distance back from the growing tip. It was noted in this laboratory that increased sugar content in the medium resulted in a marked effect on the growth pattern of orchid seedlings. Seeds of Epidendrum and Cattleya species were germinated and grown on media containing from 0.06 to 0.24 molar sucrose. As the plaints became older, i.e., about a year in age, the increased root growth was highly conspicuous. Shoot growth was notably decreased at those higher concentrations which gave maximum root growth. The following experiments were performed to confirm this phenomenon and to ascertain whether a similar response could be obtained by other means. Orchids are particularly suitable for such investigations because of their normal dependence on an outside source of sugar, and because large numbers of plants can be grown with relative ease. METHODS AND MATERIALS.-The seedlings were cultured uniformly in 4 oz. square screw-cap bottles, the caps of which were lined with paper-cushioned aluminum foil to assure an air-tight seal. Each bottle contained 50 ml. of solution, leaving about 80 ml. as the volume of gas. The various orchid species tested will be noted in the data for each experiment. For much of the work Epidendrum nocturnum was the test plant; preliminary studies showed it to be tolerant of a wide range of sucrose levels. The seeds were disinfected with calcium hypochlorite and then transferred to a small quantity of sterile water, where they were kept in suspension 1 Received for publication October 15, 1948. by gentle agitation; one-half milliliter of this suspension was transferred to each culture bottle. The amount of seeds was so adjusted that several hundred seedlings might be obtained per culture bottle. All of the planting was done in a special chamber observing the usual aseptic procedures. Two basic mineral nutrient solutions were employed, the selection depending on the type of orchid. The composition of solution #5 in millimols per liter was as follows: KH2PO4, 0.882; MgSO4 7H20, 1.058; Ca(NO3)2 4H20, 1.482; NH4NO:3, 2.748 and FePO44H20, 0.146; that of solution #15 was: KH2PO4, 0.804; MgSO477H20, 0.486; MgNH4PO4 6H20, 0.285; CaCl2 2H20, 0.118; NH4NO3, 2.000; (NH4)2SO4, 0.984 and FePO4' 4H20, 0.146. All media contained 1.5 per cent Difco Bacto-Agar and 100 p.p.m. Difco yeast extract. The pH was adjusted to 4.8 with 0.1N HCl by the spot plate method at the time of pouring the media. The cultures were kept at room temperature under constant illumination from daylight fluorescent lamps, at somewhat less than 150 foot candles. Records.-Records for young cultures were made of the percentage germination, the rate of development, and the color. Development was measured by the growth index method (Curtis, 1947) in which five morphological stages, from the swelling of the embryo in the seed to the formation of the first few distinct leaves, were arbitrarily selected and given numerical values of from one to five. The percentage of a population in each of these stages was multiplied by the value given to that respective stage, and the products were added. Thus numerical values of from 100 to 500 indicating the relative development of all viable seeds, were obtained for each orchid species under each treatment. In older cultures such distinct stages in growth were not apparent, so records were kept on the size of the plants and the rate of appearance of roots. At intervals -a representative series of bottles from each experiment was sampled to obtain exact data on size of plants, number of roots, and weights. Sugar analysis.-In certain experiments sug ir analyses were also made. For this the method of Hassid (1937) was followed. Determinations of reducing sugars were made on alcoholic extracts from dried plant samples. The extracts were then hydrolyzed by the method of Lehmann (1931) and analyzed for total sugars. The non-reducing sugar value was represented by the difference between total and reducing sugars. Gas analysis.-Carbon dioxide and oxygen determinations were made using the standard Burel gas analysis apparatus and procedure. These analyses were made on the gas contained in a series of bottles