Studies with Detached Lupin Root Nodules in Culture

Abstract

A method has been developed for culturing detached nitrogen-fixing root nodules of lupin (Lupinus angustifolius L.) on a simple nutrient medium. Under the best conditions devised, the acetylene reduction activity of mature detached nodules was maintained at 10 to 25 nmoles of ethylene hr-1 mg-' fresh weight for 3 days. Under A recent report showed that at least one plant tissue, the immature pea cotyledon, can maintain a near normal developmental sequence for several days when excised and cultured in a nutrient medium (17). Since detached soybean root nodules can maintain a linear rate of acetylene reduction for several hr (26), it occurred to us that detached nodules supplied with a nutrient medium might be able to continue normal development and provide a useful third system for studying critical stages in nodule differentiation. MATERIALS AND METHODSThe slow growing, nonacid-producing Rhizobium strain NZP 2257, from the collection of Mr. R. M. Greenwood, Applied Biochemistry Division, D.S.I.R., was grown at 25 C on a medium containing 0.5 g of Difco yeast extract, 0.5 g of KH2PO4, 0.2 g of MgSO4 .7 H20, 0.1 g of NaCl, and 10 g of agar/l, adjusted to pH 6.8 with KOH.Seeds of L. angustifolius L. cv. Bitter Blue were sterilized with a 1:1 (v/v) mixture of 95% ethyl alcohol and 30% (w/w) H202, were germinated at 20 C in 0.5% agar, and were planted in stainless steel troughs of sterile pumice. Each seed was inoculated with an aliquot from a fresh slope culture of Rhizobium suspended in distilled H20. The troughs were placed in a controlled-environment cabinet set at a temperature regime of 25 C day, 20 C night, with a day length of 12 hr, and a light intensity over the 400 to 700 mm range of 130 w m-2. The troughs were watered with a nitrogen-free plant nutrient solution. No attempt was made to maintain sterility in the troughs after inoculation.Plants were prized out for harvesting after the pumice had been loosened with a spatula. Pumice granules adhering to the roots were removed under running water with a brush. The roots were then rinsed three times with distilled H20, drained, and the nodules were excised with forceps.For culture experiments, freshly excised nodules were placed with their broken surfaces down on a medium containing 0.7% (w/v) agar, 6% (w/v) sucrose, 20 ug/ml of D-threo-chloramphenicol, 5 mm potassium acetate, and 5 mm MES, adjusted to pH 5.5 with KOH. The cultures were maintained at 20 C.Acetylene reduction assays for nitrogenase activity (6) were performed at 25 C using 30 to 150 mg (fresh weight) samples of nodule tissue in 16-ml glass vials fitted with rubber serum caps. Each vial contained 90% air and 10% acetylene (v/v). Gas samples (0.3 ml) were withdrawn at intervals up to 1 hr and assayed for ethylene and acetylene using a Perkin-Elmer gas chromatograph, fitted with a 20 X 0.15 cm column of Poropak T maintained at 75 C. The sensitivity of these assays was approximately 0.01 nmole of ethylene hr-' mg-' fresh weight. Lupin nodules incubated in air alone produced less than 0.0...

Carbon Dioxide Fixation by Lupin Root Nodules

“…The previously reported pattern of induction of acetylene reduction activity (14) was confirmed (Fig. IA).…”

“…Bitter Blue, inoculated with Rhizobium strain NZP 2257, was grown and harvested as previously described (14).…”

“…Acetylene reduction activity was assayed as before (14). In vivo C02 fixation was measured by incubating detached nodules at 25 C in a 25-ml Erlenmeyer flask containing a small piece of moist filter paper.…”

Simultaneous Measurement of Nitrogen Fixation Estimated by Acetylene-Ethylene Assay and Nitrate Absorption by Soybeans

“…These data suggest that techniques widely used to measure C2H2 reduction preclude estimation of maximum potential N2 fixation capacity. Too high a nodule-to-volume ratio in the C2H2 incubation vessel can reduce N2[C2H2] fixation rates, even during short assays (6,21), probably through a depletion of available 02. Sprent (19) demonstrated the sensitivity of C2H2 reduction by detached nodules to either too much or too little water on their surfaces.…”