A short video I made of my process for making stomatal peels from bluebunch wheatgrass seedlings.
Author Archives: alexandk
Stomatal Peels (procedure)
Nine populations were selected that represent three different degrees of aridity. Climate data for each collection site will be gathered using a program called Climate WNA. These data will be used to group each population into one of three groups according to an aridity index. This aridity index is calculated using the ratio of mean annual precipitation (P) and mean annual potential evapotranspiration (PET) and is currently used by the United Nations Environment Programme (2006) to categorize arid regions.
Twenty-four replicates from each collection site were planted in a randomized block design and grown in a growth chamber set to 61 degrees F with diurnal lighting in twelve hour cycles. Plants will be grown under optimal conditions until leaves develop that are conducive to stomatal measurements (approx. 3 weeks).
The central portion of the longest leaf from each plant will be coated in nail varnish on both the adaxial and abaxial surfaces. Once dry, the varnish will be peeled away from the grass blade and mounted onto microscope slides.
Stomates will be counted manually using a compound microscope at 200X magnification. All stomates within a predefined area will be counted at three different locations along the length of the midrib and averaged.
References:
United Nations Environment Programme. 2006. “The Desert Biome: A Global Perspective.” Global Deserts Outlook chapter 1. http://www.unep.org/geo/gdoutlook/016.asp.
Stomatal Density
A potentially important seedling trait is stomatal density. Woodward (1987) found that stomatal density and distribution may affect gas exchange and associated relationships with environmental factors such as light, CO2, and water status.
Xu and Zhou (2008) found that stomatal density increased, but the number of stomata per-leaf decreased with water stress, and this change in stomatal density was correlated to changes in specific leaf area water use efficiency in another species of perennial grass.
Stomatal density may therefore be an important adaptive trait for bluebunch wheatgrass in the Great Basin. We aim to fill the knowledge gap surrounding seedling stomatal density in bluebunch wheatgrass to determine whether adaptation to climate exists for this trait.
Stomate Study
Hypothesis & objective:
We hypothesize that stomatal density is a driver of drought tolerance in young bluebunch wheatgrass plants and that the variability in this trait is caused by natural selection and local adaptation. Our objective is to determine the range of variability in stomatal density in populations along an aridity gradient and to tie this trait to selection through climate. Using an exploratory approach, we will measure stomatal densities from young plants (grown from seed-sources along an aridity gradient) and determine their relationship to climate in terms of aridity.
Expected results and interpretations:
We predict that there is a negative correlation between stomatal density and aridity. If this prediction is supported, then there is evidence that natural selection has reduced stomatal density in arid environments as compared to less arid environments. If this prediction is not supported, then we must conclude that stomatal density does not change in relation to aridity.
References:
Woodward, F. I. 1987. “Stomatal Numbers Are Sensitive to Increases in CO2 from Pre-Industrial Levels.” Nature 327 (6123): 617–18. doi:10.1038/327617a0.
Xu, Zhenzhu, and Guangsheng Zhou. 2008. “Responses of Leaf Stomatal Density to Water Status and Its Relationship with Photosynthesis in a Grass.” Journal of Experimental Botany 59 (12): 3317–25. doi:10.1093/jxb/ern185.
Root & Shoots (scanning)
After monitoring all of the bluebunch wheatgrass seeds for germination and allowing them to grow for 10 days, it is time to digitally scan the roots and shoots for analysis.
Each seedling was removed from the sand-filled conetainer so the roots could be cleaned and prepared for scanning. The shoot and roots were severed from the seed and scanned separately.
To analyze root morphology, my mentor and collaborator Holly Prendeville will be inputting the root images into specialized root software called WinRhizo.
The shoot portion of the plant will be measured using ImageJ. For this portion of the experiment, I will measure the length and width of the longest leaf on each plant. These data will be used in conjunction with biomass data to calculate leaf shape, and root-to-shoot ratio for each seedling in the experiment.
After the roots and shoots were both scanned all of the plant parts were placed into envelopes for drying and eventual dry-biomass measurements.
Root & Shoots (day 1)
To explore local adaptation in bluebunch wheatgrass, we have designed an experiment to look at early (10-day) root and shoot development of seeds collected across 4 different seed zones in the Great Basin.
Today Partric and I planted the first round of 98 seeds into sand filled tubes. We used sand as a planting medium to make the eventual root-cleaning and imaging easier.
We will monitor each seed for germination and harvest each seedling once it is 10 days old for root and shoot measurements.
Research Overview
Featured
Introduction:
Despite the growing number of scientists, federal and state agencies, private citizens, and non-profit organizations working to restore damaged ecosystems in the Great Basin, intact native plant communities continue to decline.
The shift away from native-perennial to invasive annual-grass dominated systems has reduced biodiversity, increased wildfire severity and frequency, and has expedited desertification.
To combat this ecosystem overhaul, the most up-to-date and relevant science must be used to guide the restoration of Great Basin plant communities. Improving native plant establishment rates in the restoration setting is one of the biggest challenges faced by land managers.
Bluebunch wheatgrass (Pseudoroegneria spicata) is a commonly used native species in restoration but seedling establishment is modest. The goal of our study is to fill knowledge gaps surrounding seedling adaptation to climate and soils and to provide seed producers with zone specific harvest recommendations.
We hypothesize
1) Bluebunch wheatgrass is adapted to differing soil conditions
2)Through the process of selection, early plant traits such as root-to-shoot ratio, stomatal density, and leaf length have evolved in response to local climate.
3) The timing and duration of the seed production in bluebunch wheatgrass varies with climate and population.
We will complete four studies. The soils study will utilize existing phenotypic trait data and soils maps to explore links between soil order, soil series, plant traits, seed zones, and ecoregions. We predict that phenotypic trait divergence will be correlated to soil gradients that exist across seed zones.
The seedling study will relate phenotypic trait variability in twenty-four bluebunch wheatgrass populations to existing seed zones. We predict that seed zones will account for the observed variability in these traits.
The stomate study will compare population stomatal density to aridity. We predict that there is a negative correlation between stomatal density and aridity.
Lastly, we will examine seed production phenology at a common garden near the Crooked River Grasslands. We predict that both the timing and duration of the seed production in bluebunch wheatgrass will vary with climate and seed zone.
Information obtained from this work will either support current seed zones for bluebunch wheatgrass or create better seed zones, and help land managers to achieve higher success rates in restoration.