THE SURFACE DECONTAMINATION OF SEEDS TO PRODUCE AXENIC SEEDLINGS

A variety of sterilizing agents were tested to develop a standard procedure for surface decontaminating seeds to produce axenic seedlings. The use of calcium hypochlorite (0.5% phosphate buffer, pH 6) for 10 min followed by three sterile water rinses was among the most effective agents, and it did not injure some species as did sodium hypochlorite, formaldehyde, ethylene oxide and mercuric chloride. Some species contained internal microbes requiring severe treatments which killed or injured the seedling, while other species were "decontaminated" with a sterile water rinse. The percentage of seeds with internal microbes varied considerably among plant species, seed lot, and the length of seed storage. Thus, with seeds not easily decontaminated, screening of additional seed lots would be more profitable than testing additional decontamination agents. Release of microbes from the seed's interior is associated with germination, and microbial testing must last at least 11 days. Nutrient agar permitted growth, although the seedlings outgrew petri plates too quickly for adequate certification. These seedlings were transferred to nutrient agar in quart jars in which an internal pool of broth was periodically agitated to permit microbial sampling of the leaves while the plant grew. IT is generally recognized that microorganisms in the environment can influence a plant's nutrition, physiology, and health. Microbes influence plant nutrition by fixing nitrogen, solubilizing nutrients from rocks (Arrieta and Grez, 1971), aiding root absorption of some minerals, and competing with plants for compounds in the substrate (Barber, 1968). Both pathogens and opportunistic microbes can invade and obscure a susceptible plant's normal physiology while non-pathogens may manufacture plant hormones, metabolize plant exudates, and perhaps induce some disease resistance (Black, 1968; Keen, 1975). Microbes may inhibit the growth of plants under some circumstances (Lindsey and Baker, 1967) and stimulate growth under others (Bolton, Bozarth, and Walkinshaw, 1973). For the study of plant processes which can either be altered by microbes, or of a plant's relationship to its environment, the plants should be free from all other life forms (axenic). Decontamination of plants to produce axenic plants has been accomplished by using four approaches: decontamination of seeds (Lindsey, 1967), aseptic removal of seeds from fruits (White, 1943), aseptic removal of embryos from seeds (Miflin, 1969), and direct decontamination 1 Received for publication 6 October 1978; revision accepted 19 February 1979. We thank Nina Ghering and Jim Koevenig for their assistance with various phases of this project. of organs of mature plants (Baker and Phillips, 1962). Although the first two techniques are usually adopted, there is little agreement as to which sterilizing solution or its dosage is most effective with plant tissues. Evaluations of the efficacy of decontaminating agents by using inert strips covered with fungal spores showed that 5% hypochlorite (the agent most frequently used by botanists) required a 25-min contact time to achieve sterility. Decontamination with 70% ethanol (its most effective concentration) also required 25 min. Although not often used with plants, 5% solutions of formalin or phenol were effective decontaminators after 10and 15-min contact times, respectively (Kruse et al., 1964). Application of either ethylene oxide (850 mg/l) (Ernst and Doyle, 1968) or formaldehyde gas (10.6 mg/l) for 4-6 hr (Sweet, 1971) is effective with surfaces which are hard to wet. The killing effectiveness of many disinfectants may be improved by altering the disinfection procedures. Elevating the temperature to 20 C during disinfection (Segall, 1968) and acidifying to pH 6 (Segall, 1968; Sykes, 1965) greatly improved the effectiveness of hypochlorite. A wetting agent reduced the contact time and the concentration of hypochlorite, formaldehyde and phenol (Kruse et al., 1964), while buffering and sonication improved glutaraldehyde's effectiveness (Sierra and Boucher, 1971). Although numerous procedures for seed decontamination exist in the literature, few publi-