MINERAL OIL OVERLAY FOR CONSERVATION OF PLANT TISSUE CULTURES

CAPLIN, SAMUEL M. (Calif. Inst. Technol., Pasadena.) Mineral oil overlay for conservation of plant tissue cultures. Amer. Jour. Bot. 46(5): 324-329. Illus. 1959.-Used successfully by other workers for conservation of stock cultures of bacteria and fungi, mineral oil (m.o.) may also be used to conserve plant tissue cultures. Prolonged growth under m.o. does not appear to affect the ability of carrot tissue cultures to grow subsequently on subculture in air. In contrast with growth under liquid nutrient, which permitted little or no growth at depths greater than 15 mm., growth under m.o. was maintained at approximately 1'4 of that of the controls up to 45 mm., the greatest depth tested. Better growth under m.o. is attributed to the 4 to 5 X greater solubility of oxygen in m.o. than in water. Insensitivity to depth under m.o. is ascribed to maintenance by diffusion of the oxygen concentration at the surface of the tissue. Under m.o. in the light, cultures became green and grew better, probably due to increased oxygen availability as the result of photosynthesis. Heavy m.o. appears to be equally as good as light oil for use as an overlay. Because of the slower growth of cultures under m.o., subculture periods may be lengthened from the order of 4-6 weeks to as many months, with attendant savings in effort, time, and materials. LIQUID PETROLATUM, commonly known as mineral oil, is now utilized widely for the conservation of cultures of various microorganisms. By covering cultures on agar with oil, 2 results are obtained: (1) the rate of growth is reduced, and (2) loss of water by evaporation from the agar medium may be virtuallv eliminated. The net result is that subculturing may be done less frequently without apparent loss of viability. Attendant savings in expenditure of time, effort, and materials, particularly where large stocks of cultures are maintained, may be substantial. Further, losses through contamination may be considerably reduced. The first use of mineral oil was made in 1914 as an overlay to conserve bacteria (Lumiere and Chevrotier), and only much later first used for the maintenance of fungi (Sherf, 1943), including Actinomycetes, Basidiomycetes (Buell and Weston, 1947), and Phycomycetes (Reischer, 1949). Recently, mineral oil has also been utilized in the continuous culture of animal cells. Clones from single cells were grown by isolation in small droplets of nutrient solution under a layer of mineral oil in Petri dishes (Wildy and Stoker, 1958; Vogt and Dulbecco, 1958). The purpose of the present paper is to report on efforts to determine whether the mineral oil technique could be applied to maintaining tissue cultures of higher plants. Received for publication August 20, 1959. Supported by research grants, Nos. C-755 (C-7) (University of Rochester) and C-3217 (California Institute of Technology), from the National Cancer Institute, Public Healtb Service. This material was presented in part at the meeting of the A.I.B.S., August 1957, Stanford University, Palo Alto, California. The author wishes to acknowledge the benefit of discussions with various members of the Division of Biology at the California Institute of Technology, especially Dr. Henry Klostergaard. MATERIALS AND METHODS. Explants weighing 2.5 mg. from the secondary phloem of carrot root (Caplin, 1956) were placed on 0.8% agar nutrient medium in 22-mm.-diameter test tubes. Nutrient consisted of a basal mixture (White, 1943), modified by replacing iron as sulfate with tartrate, containing 10% coconut milk; pH was adjusted to 6.4. Tubes were capped with aluminum foil. after the method used by Gautheret (1942). MVineral oil (U.S.P., heavy, unless otherwise mentioned) was added aseptically by pipette from a flask of autoclaved mineral oil to test tubes containing sterilized agar medium, all previously cooled to room temperature (in contrast with test tubes, mineral oil overlay on agar medium may be autoclaved together safely in flasks without loss due to "bumping") . All cultures were kept at 260 + 1?0C. Carrots were California-grown 'Long Chantenay' (kindly supplied by the Pieters-Wheeler Seed Company). RESULTS.-Growth of cultures under mineral oil. -The first test of the effect of mineral oil was made in an experiment of 5 mo. duration in weak light using explants from a single carrot. To the cultures, replicated 8-fold, were added 0.5, 1 and 2 ml., respectively, of mineral oil as an overlay, resulting in a maximum depth of 6.2 mm. Eight cultures not covered with mineral oil served as controls. During growth, those not covered with mineral oil grew rapidly, then became discolored and brownish with progressive loss of water from the culture medium. Those under mineral oil grew more slowly but were bright-green at the end of the experiment. The results, shown in fig. 2, in which each point is the final weight of a single culture, indicate that even a shallow overlay of mineral oil considerably reduced the ability of the explants to grow. Further, while the substratum of the cultures growing in air was markedly desiccated, the culture