LECTURE 5 COURSE: RESEARCH METHODOLOGY

METHODOLOGY OF GREENHOUSE EXPERIMENTS

NEPTUN Code: KEGMNNOM143E Prof. Katalin SÁRDI

Pot experiments: developed from the 18th century (Boussingault, 1802-1882, Home, van Helmont and Boyle etc.). Main objecitve: experiments in controlled environments studying plant nutrition, ion uptake of roots, effects of nutrient sources on productivity, relationships between root uptake, soil characteristics and nutrients etc.

IMPORTANT Results can not be directly transferred for practice (i.e. fertilizer recommendations). They serve as a background for field experiments.

Advantages: - controlled environment (partly or completely) - rapidity, lower costs - accuracy, better reproduction - ability for characterizing soil nutrient budget (exhaustive experiments) Disadvantages: - Natural soil profile cannot be ensured - Optimum watering → constant and therefore better solubility of nutrients compared to field conditions - Effects of nutrients are enhanced - Limited soil volume

Greenhouse Experiments Greenhouse experiments are used for several reasons:
to quantify nutrient availability in soil
to sudy the nutrient supplying capacity of soil (exhaustion experiments)
to study interactions between selected nutrients
to study the efficiency of nutrient/fertilizer additions
to study crop responses to increasing nutrient rates → maximum biomass production, nutrient content etc.
to compare selected soils, crop varieties, fertilizer sources etc.
influences of residual nutrients on feshly applied fertilizers/nutrient elements (e.g. Phosphorus)

Classification of greenhouse experiments By pot size, by growing media etc. Pot size: a.) SMALL POTS CHAMINADE methodology: (Chaminade, R. 1960) referred as standard method: 1 kg of air-dried soil test plant: perennial ryegrass, (Lolium perenne L.) 1,000 seeds per pot growth, development, biomass production, nutrient uptake and accumulation can be studied in several cuts (usually 4-6 cuts in a year)

Pot experiment with Lolium perenne (Chaminade method) before cutting







Pot size: b.) MEDIUM SIZE POTS Commonly used for cereals: 5-6 kg of soil MITSCHERLICH methodology (Mitscherlich, E.A. 1909) developed for cereal test plants "Mitscherlich containers" have a double bottom, into which excess fertilizer flows







C.) BIG SIZE POTS/CONTAINERS FOR TALL PLANTS - up to 15-20 kg soil, various methodologies

MITSCHERLICH TYPE POTS

MITSCHERLICH TYPE POTS

Greenhouse experiment , MITSCHERLICH pots (Spring barley L.)

Greenhouse experiment Corn (Zea mays L.)

MITSCHERLICH POTS WITH FRAMES

TEST PLANTS
Requirements: rapid, growth, intensive

nutrient accumulation, more cuts/harvests
GRASSES: e.g. perennial ryegrass
CEREALS: wheat, barley, oats
OTHER MONOCOTS: corn, sorghum
DICOTS: Sunflower, mustard, lettuce

PREPARATION OF SOIL Steps of soil preparation: drying – usually at room temperature passing on a 2 (or max 5) mm sieve At the bottom of the pot: - a gravel layer +plastic net - draining tube Equal weights must be ensured: drain tube

pot + gravel + net +

Soil surface: fine, 3-4 cm below the upper level of the pot

Preparation of soil

Pot experiment after sowing

Nutrient rates commonly used in pot experiments N mg/kg

P mg/kg

K mg/kg

100-300

15-150

100-150

150-300

10-140

100-200

test plant

Corn

soil weight

4.5 kg /pot

perennial 1.0 kg/pot ryegrass ________________________________________________

NUMBER OF PLANTS TO BE GROWN PER POT E.g. 20 cm Ø pot: - cereals: 8-12 plants - corn, sunflower: 4-6 plants - alfalfa, red clover: 10-12 plants Seed cover: - pure sand - at a 0.5 cm layer - filter paper: should be kept moist until seed emergence Seed number per pot: 25-30 % more than required number of plants. After 4-6 days of emergence, number of plants should be equaled. - Arrangement of pots a.) usually randomized b.) re-arranged frequently

WATERING A.) Soil moisture usually should be kept at 60-70 % WHC (water holding capacity) B.) At setting: amount of water should be added in 2-3 portions C.) usually daily watering is required however, during hot summer periods twice in a day may be necessary C.) Weight control usually once or twice in a week

Observations during plant growth: Daily notes on the general conditions of plantsoccurance of plant diseases and/or pests should be controlled Pest control - manually, mechanical way, without application of chemicals

Plant growth and phenology parameters -Date of sowing/planting -Days after sowing (DAS), days after emergence (DAE) -Growth patterns to be studied - Growth stages – scales e.g Feekes, BBCH scale (Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie) etc. -Most common parameters studied -Average plant height, number of leaves, leaf area, Leaf area index (LAI), number of new stems etc .

Corn plants grown with and without N in a pot experiment

Corn roots receiving nitrate and ammonium nutrition as a single N source

Most common parameters to be determined at the harvest: Average plant height (cm) Fresh weight (g per pot) Average root length, Root fresh weight (cm, g/pot) Shoot:root ratio Dry matter (DM) weight (g/pot) Number of seeds, fruits etc. per plants Yield fresh and DM weight (g/pot)

Parameters to be determined after the harvest Laboratory analyses and calculations from the results


Nutrient concentrations of plant samples 

Macro- and microelements: N,P,K, Ca,Mg,S Fe, Mn, Zn, Cu, B, Mo, Co etc.




Amounts of Nutrients taken up by plants (mg per pot) = calculated from DM weight and nutrient concentration




Soil nutrient contents e.g. Nmin, plant available amounts of macroand microelements etc.




Soil sampling: from the centre of the pot 

Soil samples must be free of root particles!

Sand and Hydroponic cultures Developed and introduced by Hoagland and Arnon in 1950. Hydroponics: Nutrient solutions used as a growth media l. e.g. Hoagland, Knop, Hewitt solutions etc. Complete or deficient solutions depending on the subject of the experiment IMPORTANT pH value should be kept constant, it should be controlled continuously. Sand must be free of any contamination

Hydroponics

Source: Eötvös University

Hydroponics

Hoagland hydroponic solutions












Hoagland solution 1 (per liter of nutrient solution): 5 ml of 1 M KNO3 5 ml of 1 M Ca(NO3)2 1 ml of 1 M KH2PO4 2 ml of 1 M MgSO4 1 ml of micronutrient stock solution 1 to 5 ml of 1000 mg/liter iron from iron chelate (FeEDTA, Fe-DTPA, or Fe-EDDHA) Hoagland Solution 2 Similar to Hoagland Solution 1, plus ammonium therefor pH can be kept lower

Fitotrone (fully automatized, completely controlled environment chambers or rooms)

Evaluation of Experimental Results General requirements: • Treatments are applied in replicates (minimum of 4) for statistical evaluation. • Experimental results should be analyzed by using computer program packages e.g. SPSS • Most commonly used statistical analyses: • ANOVA (ANalysis Of VAriance) Multifactorial Analysis of Variance, High-Dimensional Analysis of Variance, HANOVA), Duncan test etc. • Results are compared to untreated/unfertilized control, significant differences (LSD5 % ) or correlation between determinant factors (coefficient values, r or R2) are expressed. • Regression analyses are also commonly used