A Day in the Lab: Plant Data Collection

Jered Korfhage and Katie Sagarin

After harvesting biomass and collecting data last week, the next step was to process the samples. This was also the last step we needed to take before we could analyze our data and draw conclusions about our results. Hopefully we'll see that salt has had a significant detrimental effect on our plants.

Dr. Drenovsky struggles to open
a tin can with a hammer.

The soil collected in tins in last week’s lab had been dried completely. Some lids had even cemented closed, to the point where a hammer was required to dislodge them. After struggling to remove some of the lids, we carefully measured 40 grams of soil from each tin, and placed each sample into a labeled plastic bottle. We added 200 ml of distilled water to each bottle to create a 1:5 mixture. All of the samples were put in a shaker for fifteen minutes to mix the soil and dissolve any salts into the water. These then sat for an hour to make sure everything dissolved, and then were shaken again to make the liquid uniform in composition.

Soil in the shaker

This mixture of soil and water was then vacuum-filtered (Watch how to vacuum filter here). This involved hooking a flask up to a vacuum to separate the soil from the liquid, a small portion of which was poured into a small, labelled vial. Electrical conductivity (EC) was measured for each vial by inserting the probe of an electrical conductivity meter into the liquid (Watch how to use an EC meter here). Electrical conductivity can be used to estimate the amount of dissolved salt in the mixture (more information here).

Vacuum filtration

The EC measures we observed matched our expectations. We saw a trend of increasing EC with increasing salt treatment. In other words, the 0 mM-treated soil had a lower EC than the 50 mM which had a lower EC than the 100 mM-treated soil.

The aboveground plant material and roots collected last week had also been dried. Each sample was weighed to the nearest 0.1 gram, unless it was lighter than 0.1 gram in which case it was measured to the nearest 0.001 gram. All samples were gently brushed to remove soil and sand before weighing. The biomass observed also matched our expectations. The 100 mM-treated plants were either dead or small, whereas the 0 mM-treated plants were alive and sometimes thriving.

Measuring electrical conductivity

At the end of the lab, each group entered their EC, aboveground, and below-ground biomass data into the same Excel spreadsheet, for later analysis. Based on our observations, we feel optimistic that our data will support our hypotheses.

 Results! Effects of Salinity on Stressed Common Roadside Plants

By: Natalie Fry and Meghan Kelley

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Eariler this semester of Fall 2013, in the blog entitled "Trade-off of Salting Winter Roads: Human Safety vs. Plant Survival," our Plant Ecology class at John Carroll University set up a laboratory experiment in order to test different concentrations of salt on common roadside plants to determine how tolerant certain species (Purple Coneflowers, Black-Eyed Susans, Shasta Daisies, and Mexican Asters) are to man-made stressors, such as salted winter roads. During this last week of November 4, 2013, we were able to begin assessing the results on our study plants.
 

The first step before harvesting dead and alive plants is to record the observations of how plant individuals of each species look, by examining characteristics such as leaf senescence or dropped leaves, leaf coloration abnormalities like tip yellowing, and morphological drying, wilting, or withered plant leaves and stems, as can be seen in the picture below. For more information about how plants cope with salt, visit the page for the Society of Experimental Biology.

 

Qualitatively, of the four species, it appeared that the Purple Coneflower (ECPU) did not do quite as well with the salt stress as some of the other plant species.  For example, the Shasta Daisy (CHMA) in this photo is clearly green and growing better at the highest salt concentration than the Purple Coneflower grew at the intermediate salt concentration. Observations such as these were recorded in addition to the counting of plants that lived and died for each species, for each salt concentration.  Overall, as stated in the previous blog, five experimental blocks were created with replicates of each species for the salt treatments- however, as an example, only one block will be used to indicate some preliminary observational results.  Of the twelve original plants in this block, only five survived, and three of those five were from controls that were not salt stressed.  Therefore, we can say that these four common roadside plants are not all amenable to winter salinity treatments of roads that can accumulate toxic salt levels over time. However, more tests were also performed in this lab to further strengthen this claim or to add additional information about physiological tolerance in these species.

For more definitive quantitative measurements, our class used a LICOR machine, which includes a clamp for leaves in a 2x3 centimeter frame in order to measure the amount of light captured for photosynthesis as well as other functions linked to photosynthesis. The LICOR machine used in this study is pictured below, showing its easy to read digital monitor for assessing measurements.

 

Next, a Scholander Pressure Bomb was used to measure the water potential of different leaves of the species that lived, which were only the Shasta Daisy, the Black-Eyed Susan, and the Mexican Aster for one block, not the Purple Coneflower.  The leaf is placed in the pressure bomb upside down and sealed, in order to allow the pressurized nitrogen gas to force water to the center of the leaf petiole. This water movement has to be monitored by someone to determine the correct point of pressure of the plant's water potential, as seen in lower right photo.

Finally, the aboveground (stems and leaves) and the belowground (roots) biomass were harvested along with a soil sample for the purpose of drying the organic materials to weigh for our final results.  Aboveground biomass was clipped with shears at the soil surface, and roots were collected by filtering soil through a metal sieve into a bucket. These will be weighed at a later date. Following the analysis of all data collected in this lab, statistics will be run to make conclusions about this study.  Some discussionary points to help examine the results could include the use of fluorescent lights in the greenhouse in place of sunlight, which was not available during the measurements of this study. Seen in the picture below, plants were harvested under these conditions indoors. Also, possible sources of error along with relevant plant biology could be discussed for drawing further conclusions as to which of the four plant species is the most salt tolerant and would therefore be more likely to survive along midwest and northern roads.

 

 

 

Additional Links:

 

University of California: "Salt Injury in Strawberries" (2012)

 

North Carolina State University: "Got Salt? Winter Weather May Be Toxic to Plants" (2013)

 

 

 

 

 

 

 



Physiological Ecology: An Introduction to Methods

Dr. Drenovsky explaining
components of the LICOR-6400

On the 31^st of October 2013, the BL 435/535 class became acquainted with the LICOR-6400 and the pressure bomb so that for next week’s lab we will be able to harvest and record data of our greenhouse experiment.  The purpose of the LICOR-6400 is to measure gas exchange in plants while the Scholander-type pressure bomb measures leaf water potential. 

Students taking measurements
with the LICOR-6400

In order to collect data using these two machines, learning how to set them up was important.  Our professor gave us a thorough explanation for each machine.  The class was split in two groups to use the two LICOR-6400 machines, affectionately known as LUCY and OSCAR.  We were able to gain hands-on experience as our professor explained how to connect the parts of the instrument.  Although the LICOR-6400 can be easily set up, there is a pre-checklist that must be done before using the machine.  Noting the chamber, the cables, the console, and the chemical tubes were instructions given to us so that we become more aware of the machine before beginning our measurements. We were then able to begin measurements.  Extra samples of the study species for our greenhouse experiment were used.  Due to overcast weather conditions, additional lighting was used to increase photosynthetic rates.  The leaf of a specimen was placed in the chamber, which was held there until the conditions inside the chamber equilibrated.  Upon equilibration, the data was logged in the machine.  We had to repeatedly check during measurements that the Infrared Gas Analyzers (IRGAs) were matching to ensure accuracy.  This simply means to ensure that the carbon dioxide reference and sample are read as equivalent as when a balance is zeroed before weighing something.

Preparing specimens for
 pressure bomb measurements

Before the lab groups were able to use the pressure bombs, samples of our study species in the greenhouse experiment were extracted and placed in a plastic bag inside a cooler until needed.  The cut end of the specimen was placed inside the narrow chamber; this required patience to guarantee that the cut end was exposed on the other side of the chamber.   While one person took charge of the pressure dial for recording the measurements, the other person ensured that the rate valve was never closed completely and controlled the arrow dial while observing the pressurized specimen.  In addition, safety was stressed during this experiment.  When using the pressure bomb, all students had to wear goggles for protection.

The final part of collecting sample data on both machines was practice for our next lab, where we will be using the plants that we have been growing throughout the semester.  Prior knowledge of using these machines in this lab session increases the accuracy of data collection for our greenhouse experiment next week.

For more information on the LICOR-6400:  http://hydrology1.nmsu.edu/teaching/soil698/student_material/photosynthesis/6400.html

For more information on the Scholander pressure bomb:  http://www.pmsinstrument.com/resources/making-the-measurement-of-the-plant-sample

Written by:  Danny Dlugos

                    Jaylan Craig