 Methods for Measuring Transpiration Efficiency Conventional transplanted rice production has been estimated to require on average 2,500 liters of water to produce 1 kilogram of grain. This water requirement includes the demands of evapotranspiration as well as seepage and percolation from the soil. Increasing the water use efficiency of rice crops will help address the expected future constraints in water availability. The amount of water transpired by the plant per unit of carbon assimilated is termed Transpiration Efficiency. This is a component of evapotranspiration that is controlled by the plant and contributes to crop water consumption at the field level. Therefore, selecting for high transpiration efficiency may be a way to breed for rice crops with lower water requirements. In this video, we will explore three approaches to measuring Transpiration Efficiency. Gravimetry, gas exchange, and bicarbon isotope discrimination. These approaches can be used to screen for genetic variation in Transpiration Efficiency. In this part, we will describe how to determine the Transpiration Efficiency of plants using the Gravimetric method or by rating. The main advantage of using containers such as pots and cylinders in determining Transpiration Efficiency is that the exact amount of water accessible to the plant can be controlled. Different physiological measurements can also be done in this type of method. Here, we will use PVC cylinders of 16 cm in diameter and 18 cm in height. To set up a greenhouse study, use cylinders sealed at the bottom but with a hold plug with a removable stopper in case draining is desired. All cylinders are cleaned, dried, and checked for leaks. It is important for all cylinders to have a uniform amount of dry soil and degree of compaction, resulting in the same volume of soil in each cylinder. To do this, dry-print house soil is sealed through a 3.5 mesh with about 5.6 mm opening. Before filling the cylinder, the empty weight is recorded. A plastic liner can also be inserted inside the cylinder for root studies. A laptop connected to the weighing balance is used for more efficient recording. After recording the empty pot weighing, the weighing balance is paired to zero before filling the cylinder with soil. The cylinders are then filled with the same amount and compaction of soil to the same height in each cylinder. The cylinders are continuously filled with water until no bubbles are coming out. Let it stand for 2 hours, then loosen the plugs to gradually drain water out overnight. In the morning, put the plugs back and weigh the cylinders to determine its weight at filled capacity. The cylinders are labeled before sewing. QR or barcodes are used to facilitate faster and less erroneous data collection. Cylinders are covered with plastic and sealed with masking tape to reduce evaporation of water, so that the change in weight can be mostly attributed to transpiration. Note that compared to the weight of the cylinder, filled with soil and water, increases in dry weight of the plant are considered to be negligible. Cylinders are weighed at a regular interval, usually three times a week, and re-watered as necessary with defined amounts of water to reach a predetermined target weight. For drought treatments, we don't advise to allow the plants to undergo a progressive drydown because larger plants will take up water faster and therefore become drought stressed faster, affecting their water-uptake rates. Here is an example of a file with corresponding target weights for each weighing date. This Excel sheet shows how targeted weights are calculated and how plant water-uptake can be monitored from covered pots. To schedule a controlled progressive drydown in drought stress treatment, researchers can decide on the rate of soil moisture decline over time. Soil moisture levels are determined as a percent of filled capacity, drying down from 100% of filled capacity. In this example, we plan for soil moisture to decline by 10% every two to three days, which can be seen in the formulas for target weight that show the target percent of filled capacity times the amount of water at filled capacity plus the weight of the dry soil and empty pot. In well-watered treatments, a constant target weight at every weighing date can be used, either at filled capacity or above filled capacity for lowland drys. The actual weight is determined by weighing just before great watering on each date. At the end of the experiment, the shoots are harvested and placed in 70 degrees Celsius oven for three days. Water-uptake on each weighing date is determined as the difference between the target weight and the actual weight upon weighing. Cumulative water-uptake is determined as the sum of water-uptake values on each date. We often exclude the first one or two weighing dates from the measurement, since that is when the plants are very small and water-uptake values are not exact. Transpillation efficiency is calculated as kilograms per liter or the weight of the dry shoot per liter of water transpired over the growth period. What we have here now is the Lycor 6400XT, a handheld instrument that can be used to measure photosynthetic and transpiration activities at plant level. It is done by computing the rate of change of carbon dioxide and water, filled time with a part of the leaf enclosed in the chamber. Air supply entering the system is modified to control condition by the mechanisms installed in the instruments. So temperature humidity, carbon dioxide concentration can be set by regulating the airflow in the chamber from the console. So with the chemical tubes that installed at the site. This includes the plasticine and CO2 scrambles. It is powered by two rechargeable batteries placed in the allotted compartments at the right side. Scrubbing mechanism is located on the other side with the CO2 mixer and concentrated CO2 cartridge. So this is the mixer system and the CO2 concentrated cartridge. So this job contains a liquefied pure carbon dioxide as a source of carbon dioxide in the system. Users should read the Lycor 6400 manual to understand how the instrument is operated. The warm-up checklist on the first page of chapter 4 should be run through before each set of measurements. Which takes 5 to 10 minutes and includes checking that temperature, light and pressure sensors. Good blood change for chamber closure and gasket condition play vital role to prevent leaks from and into the sample chamber. At this stage, taking measurements with the leaf samples are now ready. Set and check the desired chamber conditions, light, airflow, reference carbon dioxide, temperature and constant humidity. Clump the leaf into the leaf chamber and monitor the values and graphs in the console LCD. Once the values of photosynthesis and transpiration stabilize, reading should be logged before moving into the next plant sample. The data from the Lycor can be exported as a spreadsheet. For transpiration efficiency, the key measurements are A or the photosynthesis rate and the cond or the stomatal conductance. We calculate the instantaneous transpiration efficiency as A over GS. Carbon isotope discrimination analysis is another method for screening diverse genotypes for transpiration efficiency. The principle behind carbon isotope discrimination is that the different isotopes of carbon in the air, specifically the most abundant, C12 and the higher molecular weight, C13, have different sizes. The size of the carbon atoms affect their ability to diffuse into the stomatal cavity and be incorporated by Rubisco. Environmental factors including soil moisture and light levels can affect stomatal aperture and can be reflected in carbon isotope discrimination levels. In this part, we will show you how to do the leaf collection in the field and prepare the samples for 13C analysis. First, prepare ahead of time QR or barcode labels are placed on paper envelopes to be used during the sampling and kept in order of the plants arranged in the field. In the field, we collect 10 pieces of young, fully expanded leaves randomly in a plot. The leaves are rolled up and tied before placing inside the paper envelopes. For easy handling of dried samples during grinding, especially for drought studies, the youngest leaves tend to show the largest genotypic difference in transpiration efficiency. It is better to avoid including older leaves in your sample collection. The leaf samples are placed in an oven set to 70 degrees Celsius and allowed to dry for 3 days. After 3 days, sample will be ground using a ball mill with less than 18 mesh which is required size of 13C analysis. After every sample, the grinder is cleaned with a paint brush and vacuum to remove sample particles left in the grinder. Then, the sample holder and ball mill are wiped with paper towel with ethanol. Brown rice plant tissue is twid in a thin capsule and dropped by the auto sampler into the furnace at 950 degrees Celsius. In at atmosphere of oxygen, the sample is rapidly combusted and transformed into gaseous carbon dioxide and nitrogen products. The water byproducts from the gas mixture is removed by a trap containing anhydrous magnesium perchlorate. The gas mixture of carbon dioxide and nitrogen gas resulting from combustion are then passed into a gas chromatograph column where the carbon dioxide and nitrogen gas are separated and then bled into a mass spectrometer. In the mass spectrometer, the sample gas molecules are ionized, formed into a beam, accelerated by electric field, deflected in a magnetic field and finally detected according to ion mass ratio. The carbon-13 results from the GC-IRMS analysis are typically provided as small delta carbon-13 values in units of per mill which represent the carbon isotope composition of the plant sample as compared to the reference material which is the BNAP-D-Belenite. The small delta carbon-13 values from plant tissue are negative values and can be converted to big delta carbon-13 values using the equation as shown which factors in the isotope composition of air. Lower big delta carbon-13 values indicate a greater degree of transpiration efficiency. After learning the different ways to measure transpiration efficiency, it is also important to know about measuring water use at field scale. Water use efficiency is the percentage of water supplied to the field that is effectively taken up by the plant. We have the flowmeter here and the valve is measure how much water it would be had in the per plat. Since we are at the farmers' park practice, we open the valve and let the water go without any computation. Since we are computing the volume of water per plat, so we use the flowmeter. We will just simple taking the initial reading and using the gauge inside the plat, we have the stick gauge to measure how much water are already in the field. By the use gauge, we maintain the pipe centimeter and after the pipe centimeter depth in the plat, we will take the final reading. From there, we will take the initial minus the final reading. You can get the volume of water by cubic centimeter.