Two types of experiments were used for producing data. The first type was based on a study carried out in a greenhouse where plants were grown using hydroponic solutions. Two plant densities, 20 and 40 plants m–2, were considered. The second type of experiment was based on a field study with a plant density of about 300–500 plants m–2.
Greenhouse experiment (low-density culture)
These experiments were extensively described in Kim et al. (1993)
. Therefore only the most important aspects will be included in this document for data analysis purposes.
Plant material and culture
Lucerne seeds (Medicago sativa L. var. Europe) were germinated on sand. After 15 d, when the primary trifoliate leaves appeared, seedlings were transplanted to plastic pots filled with sand and irrigated three times per week with 300 cm3 of a full nutrient solution. The basic nutrient solution contained 0.4 KH2PO4, 1.0 K2SO4, 3.0 CaCl2, 0.5 MgSO4, 0.15 K2HPO4, and 0.2 Fe-Na EDTA in mol m–3, and 14 H3BO3, 5 MnSO4, 3 ZnSO4, 0.7 CuSO4, 0.7 (NH4)6Mo7O24, and 0.1 CoCl2 in mmol m–3 (Kim et al., 1991). Nitrogen was supplied at 1 mol m–3 of NH4NO3 to repress nodule formation. Plants were grown under greenhouse conditions with temperatures of 20 °C (day) and 18 °C (night) and a photoperiod of 16 h (day) and 8 h (night). After three months, plants were defoliated 6 cm above crown level and transferred to a continuously aerated nutrient solution in a plastic container of 8000 cm3 (three plants per plastic container). After 30 d of regrowth, plants were again defoliated 6 cm above crown level and the regrowth after defoliation was studied by harvesting at days 3, 6, 9, 13, and 26 of growth. The density of the culture was either 20 or 40 plants m–2. Throughout the entire experiment, the basic nutrient solution containing 1 mM NH4NO3 was renewed every 3 d and light was supplemented with high pressure sodium lamps (phytoclaude 400 W) supplying approximately 400 µmol photons m–2 s–1 15 cm above crown level.
Plant sampling and analysis
Plant were harvested on the day of defoliation (day 0) and after 3, 6, 9, 13, and 26 d of regrowth, and were separated into leaves and stems. At the same time, and for the experiment with 40 plants m–2 only, three independent samples per date of harvest were taken to determine the leaf area (LA, expressed as cm2 of leaves per plant measured with Li-3100 area metre, Li-Cor, Inc., Lincoln, NE, USA). Leaf and stem tissues were dried at 80 °C for 72 h. The dry weight of each tissue was determined. The leaves and stems were ground to a fine powder and stored in a vacuum with CaCl2 until N analysis. The N concentration of stems and leaves was determined with an N analyser (Roboprep CN, PDZ Europa Scientific Ltd, Crewe, UK).
Field experiment (high-density culture)
This experiment is extensively described in Avice et al. (1997a, b). In this document, only the most important aspects concerning the plant material are described and, the determination of three height categories of plants that present different hierarchical positions in the overall plant population.
Plant material and culture
Lucerne stands (cv. Europe, or cv. Lodi, 5 blocks of 40 x5 m each) were sown in field plots (16.5 cm between rows) in April 1993 in Lusignan, France (46.26° N, 0.07° E). Plant shoots were harvested in July, August, and November 1993. In 1994, plots received 80 kg P ha–1 and 90 kg K ha–1 at the end of February and they were first cut in May. On 6 July, the plants were cut a second time 6 cm above the soil level and the regrowth of the plants was observed at 7, 14, 21, 27, and 35 d after defoliation. The density was 325±13 plants m–2. Between 24 June and 5 August 2004, plants were irrigated (177 mm H2O) to prevent water deficit.
Plant sampling and analysis
Throughout the experiment, sample plants were harvested along 2 m of a row. Plants were separated into leaves and stems (above the level of cutting). After the fresh weight was determined, tissue samples were dried at 70 °C for 72 h, ground to a fine powder, and stored in a vacuum with CaCl2 until N analysis. Moreover, two independent samples were taken on an adjacent 0.5 m row to determine the leaf area index (LAI, expressed as m2 of leaves m–2 of soil), using a leaf area meter.
The changes of N accumulation in shoot dry matter were also studied in field experiments using the hierarchical position of plants within the canopy for light interception (Avice et al., 1997b). The shorter plants, corresponding to the suppressed plant category, were shaded by the taller ones, the dominant plant category that intercepted most of the incident light. Therefore, at any given time during the regrowth period of the lucerne stand, it was possible to identify three categories of plants, according to the height of each plant: dominant (D), suppressed (S), and intermediary (I), representing the level of irradiance intercepted by their leaves. For the purpose of sampling plants from suppressed to dominant position categories during shoot regrowth after defoliation, all plants along 2 m of each row were harvested and then sorted according to their shoot length into one of the three categories: dominant (D), corresponding to the 20% tallest plants, suppressed (S) corresponding to the 20% shortest plants, and intermediate (I), corresponding to the 20% medium-height plants. Individual plant shoots from each category were separated into stem, leaves, and crown. The tissues were dried at 80 °C for 72 h, weighed for dry matter determination, ground to a fine powder, and stored in a vacuum with CaCl2 dessicant until N analysis could be performed. The N concentration of stems and leaves was determined as described above.
Data analysis
The greenhouse experiment was performed with three replicates (each replicate containing three plants) and the results were given as the mean of n=3. The field experiment was designed as randomized complete blocks with five replicates per plot and dominant, intermediate, and suppressed plants were harvested from two independent plots (Avice et al., 1997a, b). The slope value of linear relationships for dominant, intermediate, and suppressed plants were statistically compared using Student's t-test (Statview Student software, Abacus Concepts, Berkeley, CA., USA).
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