An Axenic Plant Culture System for Optimal Growth in Long-Term Studies Amelia Henry, William Doucette, Jeanette Norton, Scott Jones, Julie Chard, and Bruce Bugbee* ABSTRACT The symbiotic co-evolution of plants and microbes leads to difficulties in understanding which of the two components is responsible for a given environmental response. Plant–microbe studies greatly benefit from the ability to grow plants in axenic (sterile) culture. Several studies have used axenic plant culture systems, but experimental procedures are often poorly documented, the plant growth environment is not optimal, and axenic conditions are not rigorously verified. We developed a unique axenic system using inert components that promotes plant health and can be kept sterile for at least 70 d. Crested wheatgrass (Agropyron cristatum cv. CDII) plants were grown in sand within flow-through glass columns that were positively pressured with filtered air. Plant health was optimized by regulating temperature, light level, CO 2 concentration, humidity, and nutrients. The design incorporates several novel aspects, such as pretreatment of the sand with Fe, graduated sand layers to optimize the air–water balance of the root zone, and modification of a laminar flow hood to serve as a plant growth chamber. Adaptations of several sterile techniques were necessary for maintenance of axenic conditions. Axenic conditions were verified by plating and staining leachates as well as a rhizoplane stain. This system was designed to study nutrient and water stress effects on root exudates, but is useful for assessing a broad range of plant–microbe–environment interactions. Based on total organic C analysis, 74% of exudates was recovered in the leachate, 6% was recovered in the bulk sand, and 17% was recovered in the rhizosphere sand. Carbon in the leachate after 70 d reached 255 mgd 21 . Fumaric, malic, malonic, oxalic, and succinic acids were measured as components of the root exudates. A RTIFACTS from axenic culture must be minimized to obtain reliable measurements of plant responses in the absence of microbial organisms. The term “axenic” refers to a system in which all biological populations are defined. Axenic plant culture is the growth of plants in the absence of microbes. Other terms used to describe these types of systems include sterile, aseptic, and gnoto- biotic. Few axenic plant culture systems have optimized the environment for plant growth and most systems have been designed only for short-term studies. The main challenge associated with long-term axenic plant culture is to maintain optimal plant growth conditions in a microbe-free environment. This is imperative to study the effects of various treatments and assure that re- sponses are due to those treatments alone, not artifacts from suboptimal growth conditions. An ideal axenic plant culture system should provide: (i) continuous control of CO 2 , temperature, humidity, and light in the shoot zone; (ii) adequate nutrients, water, and O 2 and absence of light in the root zone; (iii) appropriate mechanical impedance to root elongation; (iv) inert materials; (v) maintenance of sterility; (vi) rigorous tests to verify sterility; (vii) capability to apply treatments such as nutrient stress or inoculation; and (viii) access to collect root-zone leachate. Although it is necessary to avoid unintentional stresses in axenic studies, not all in vitro plant culture systems promote plant health. Agar plates (Heist et al., 2002) or agar with Millipore (Billerica, MA) membranes (Meharg and Killham, 1991) have been used as simple axenic plant culture setups, but do not supply adequate airflow or allow uniform nutrient, water, or O 2 delivery to the root sur- faces. Agar media allow continuous monitoring of steril- ity but do not facilitate long-term studies. It is also difficult to extrapolate results from agar plates to the field. Axenic liquid hydroponic cultures have been widely used (e.g., Mench and Martin, 1991; Groleau-Renaud et al., 1998), particularly for short-term (,1 wk) studies. However, root morphology and growth are significantly altered in hydroponics, compared with the field, due to reduced mechanical impedance and absence of root hairs. The viability of microbes that might be inoculated into a hydroponic system is limited by the lack of sur- faces to colonize. Soil provides a growth medium more like field con- ditions and has been used for axenic plant culture (Whipps and Lynch, 1983). However, soil is difficult to sterilize and structural and geochemical changes occur from autoclaving or g irradiation. Porous solid substrates also provide growth condi- tions similar to the field but are more easily sterilized. Biondini et al. (1988) enclosed plant roots in pots of sterilized fritted clay with ports for solution input and output. Sand has also been used as a growth medium for axenic plant culture (Ayers and Thornton, 1968; Lipton et al., 1987; Hodge et al., 1996). Sand has fewer reactive surfaces than soil or fritted clay. Certain types of com- mercially available sand are more inert than others due to increased purity of silica in their composition, which decreases reactivity. Glass beads are considerably more expensive than sand and may affect nutrient solution composition (Sandnes and Eldhuset, 2003). Sterilization methods most commonly used include autoclaving of the system components, g irradiation or autoclaving of the growth medium, and surface sterili- zation of seeds by soaking in dilute solutions of H 2 O 2 , NaOCl, or HgCl 2 . A. Henry, J. Norton, S. Jones, J. Chard, and B. Bugbee, Department of Plants, Soils, and Biometeorology, Utah State University, Logan, UT 84322; and W. Doucette, Department of Civil and Environmental En- gineering, Utah State University, Logan, UT 84322. Work supported by the Idaho National Environmental and Engineering Laboratory, and by the Utah Agricultural Experiment Station, Utah State Univ. Approved as Journal Paper no. 7552. Received 15 Apr. 2005. *Cor- responding author (bruce.bugbee@usu.edu). Published in J. Environ. Qual. 35:590–598 (2006). Technical Reports: Plant and Environment Interactions doi:10.2134/jeq2005.0127 ª ASA, CSSA, SSSA 677 S. Segoe Rd., Madison, WI 53711 USA Abbreviations: GC-MS, gas chromatography–mass spectrometry; LC- MS, liquid chromatography–mass spectrometry; TOC, total organic carbon. Reproduced from Journal of Environmental Quality. Published by ASA, CSSA, and SSSA. All copyrights reserved. 590 Published online March 1, 2006 Published online March, 2006