Ashley Huffman, Andrew Dyjak, and Karl Jackson

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The Effects of Proximity to Unrelated and Kin Plants on Determining Wheatgrass (Triticum aestivum) Root Growth

Ashley Huffman, Andrew Dyjak, and Karl Jackson

Department of Biological Science

Saddleback College

MissionViejo, CA 92692

28 April 2014

Plant growth, especially root development, can be effected by the type and proximity of plants neighboring it. A proposed question arose regarding distance to kin plants and whether this would inhibit or promote root development. It was predicted that wheatgrass plants grown near unrelated plants of the same species would produce larger roots than those grown next to kin plants.Liquid Sunshine wheatgrass seeds were planted into three different groups (with kin Liquid Sunshine wheatgrass seeds, with unrelated Common wheatgrass seeds and with unrelated Red Hard wheatgrass seeds) and the roots were then measured after twenty days. The mean length of the Liquid Sunshine wheatgrass plant roots grown in the first tray, or kin group, was 10.83cm (N = 10). The mean length of the Liquid Sunshine wheatgrass plant roots grown in the second tray, with the Common wheatgrass plants, was 13.41cm (N = 10). The mean length of the Liquid Sunshine wheatgrass plant roots grown in the third tray, with the Red Hard wheatgrass plants, was 12.79cm (N = 10). Wheatgrass plants grown within close proximity tounrelated plants of the same species do not produce larger roots when compared to those grown next to kin plants (p = 0.172026, one-tailed ANOVA).

Introduction

There has been a prevalent association between root development and plant relatedness observed by many scientific studies, yet the extent of this relationship and its impact on agriculture is still highly debated. Based on the research by Biedrzycki & Bais, (2011) it was found that plants can, in fact, recognize other plants in their surroundings based on relatedness. Biedrzycki & Bais, (2010) noted that plant–plant interactions have been well documented and range from negative interactions, such as allelopathy, to positive interactions such as the release of volatile compounds as a warning to other plants. Fang et al., (2013) found that belowground tissues, such as roots, can not only detect, but also respond to changes in their environment; exhibiting adaptation in their morphology and physiology in response to environmental stimuli, such as alteration in total root length, root system volume, and root depth.

Further analysis by Milla et al., (2009) found that certain plants can identify their kin in competitive settings through root recognition, and can, in turn, react by decreasing their root growth when competing with relatives. A specific example of this was illustrated by Dudley & File (2007) which reported that an annual plant exhibited kin recognition, as the plant produced more roots when grown in pots with strangers (plants of the same species, but grown from seeds collected from different mother plants) versus being grown with kin plants (plants grown from seeds collected from the same mother plant).

In another related study, Murphy & Dudley (2009), it too produced similar results in which they analyzed data that indicated plants exhibited both increased competition toward stranger plants and reduced interference (cooperation) toward kin plants. The broader impacts of this topic have also been investigated in the study by Milla et al., (2009) which asserted the generalization that kin recognition may act as a widespread, major microevolutionary mechanism in plants to reduce competition among related plants and create competition between unrelated plants.Our current study addresses many of these issues and will focus primarily on the question of whether wheatgrass plants grown within close proximity to unrelated plants of the same species will produce larger roots as opposed to those grown next to kin plants.

Materials and Methods

The experiment was performed at the Biology Greenhouse at Saddleback College in Mission Viejo, California from 2-21April 2014. There were four sets of 10 Liquid Sunshine wheatgrass plants (Triticum aestivum) used in this study (N = 10).Prior to planting, these 40 Liquid Sunshine wheatgrass seeds were harvested from one mature organic Liquid Sunshine wheatgrass plant.All of the wheatgrass seeds were planted on 2 April 2014 following the same planting procedure. Three large plastic planting trays (20.32cm x 20.32cm) were used with twenty wheatgrass seeds per tray, arrangedin the following order; the first tray was the kin group and had twenty organic Liquid Sunshine wheatgrass seeds from the same plant, the second tray had ten organic Liquid Sunshine wheatgrass seeds and ten organic Common wheatgrass seeds with alternating planting, and the third group had ten organic Liquid Sunshine wheatgrass seeds and ten organicHard Red wheatgrass seeds again with alternating planting. The setup and arrangement of the planting is shown in Figure One.

Tray 1 Tray 2 Tray 3

LS x LS LS x CW LS x RHW

Figure 1.Diagram of planting arrangement.LS = Liquid Sunshine Wheatgrass; CW = Common Wheatgrass; RHW = Red Hard Wheatgrass(Note: Diagram not to scale).

The planting method used was obtained from the organic seed packets (Botanical Interests & Handy Pantry Seed Co.) and all trays were filled with Foxfarm Natural & Organic Ocean Forest Potting Soil and respectively labeled. Using Figure One, all seeds were planted with 1/4 inch (0.635cm) spacing between each seed and planted 1/8 inch (0.318cm) deep. The seeds were then carefully covered with soil and watered with de-ionized water. All trays/groups were watered three times a week with de-ionized water and were originally planted towards the middle of the greenhouse, but once sprouting occurred (on day 8) were relocated to a window location in the greenhouse. After the growth period of twenty days passed, the roots were cut, washed, dried, measured and then weighed and compared to determine the outcome of the hypothesis.

On 21 April 2014, all Liquid Sunshine wheatgrass plant roots were cut, washed using de-ionized water to ensure no soil remained in the roots and then blotted dry with a paper towel. Each of the plant’s longest rootswere measured using a metric ruler to the nearest 0.1cm and recorded into the lab notebook. Due to the roots being extremely small, thin, and intertwined with the other roots, it was difficult to measure each root on the plant, so the longest root was measured and recorded. In addition, the roots were very small and a more precise scale was needed to individually weigh each root. Therefore, the total weight for the roots of all 10 plants in each group was found to the nearest 0.01g with an analytic balance (Scout Pro) in a 150ml. All data were transferred to MS Excel (Microsoft Corporation, Redmond, Washington) where all further statistical manipulations were performed.

Results

The mean length of the Liquid Sunshine wheatgrass plant roots grown in the first tray, or kin group, was 10.83cm (N = 10). The mean length of the Liquid Sunshine wheatgrass plant roots grown in the second tray, with the Common wheatgrass plants, was 13.41cm (N = 10). The mean length of the Liquid Sunshine wheatgrass plant roots grown in the third tray, with the Red Hard wheatgrass, was 12.79cm (N = 10). After 20 days of germination, it was found that there was no statistical difference between the mean root lengthsof all three groups (p = 0.172026, one-tailed ANOVA). These data are shown in Figure Two.

Figure 2.Comparison of mean root length for Liquid Sunshine wheatgrass plants grown next to kin plants and next to unrelated plants of the same species (N = 10). The mean root length of wheatgrass plants grown next to unrelated plants of the same species is not largerthan the mean root length of wheatgrass plants grown next to kin plants (p =0.172026, single factor ANOVA).

The total weight for all of the Liquid Sunshine wheatgrass plant roots in each tray was also collected and found to be 0.14g for tray 1, 0.27g for tray 2, and 0.32g for tray 3. Since only total weight was recorded, statistical analysis could not be run for these data but a graph has been included to show weight difference among the roots in Figure Three.

Figure 3.Comparison of total weight for Liquid Sunshine wheatgrass plant roots grown next to kin plants and next to unrelated plants of the same species (N = 10).

Discussion

In this study, the results did not support the proposed hypothesis. The Liquid Sunshine wheatgrass plants grown within close proximity to unrelated plants of the same species did not produce larger roots when compared to Liquid Sunshine wheatgrass plants grown next to kin plants.Although the data collected yielded larger numbers in groups 2 and 3 for both mean root length and overall root weight when compared to group 1, statistically this was not a large enough difference to show the hypothesis to be correct.The overall trend of the kin group having the shortest mean root length and lowest total root weight was clearly evident, however, this difference in size was not great enough to definitively conclude that proximity to kin plants effects root size. There are several factors that may have contributed to why these results were obtained.

Despite efforts to ensure a consistent environment by growing all the wheatgrass plants in the same greenhouse and placing them in the same location within the greenhouse, certain conditions observed contradict the belief that this environment was uniform for all trays. For example, although all of the groups were watered the same amount and at the same times, the consistency of how ‘wet’ the soil was would fluctuate. The air circulation within the greenhouse may have caused the soil in certain trays to become dryer and inhibited root growth. In addition, the trays seems to be growing at different rates, with tray 2 seeming to grow the slowest. An uneven distribution of direct sunlight, as well as proximity to air ventilation, may have contributed to why tray 2 seemed to be growing at a slower rate than the other trays.

Although we can conclude that the environmental conditions within the greenhouse may not have been optimal for ideal plant growth, there are other factors which may haveeffected the results as well. Despite pH of the soil not having been measured, it could have been a variable which affected the outcome of this study. More significantly, however, the type of plant used in this experiment is not one that any of the previous research has been based upon. In addition, the research gathered for this studyused different methods of growing and measuring the plants and their roots. As in Dudley & File (2007) which reported that an annual plant exhibited kin recognition, as the plant produced more roots when grown in pots with strangers versus being grown with kin plants. The use of large plastic trays instead of individual pots, as well as the need for a different species of plant, could have negatively impacted the wheatgrass plant’s root growth. This is especially true when taking into account how intertwined and thin the roots of the wheatgrass plants were and furthermore supports the necessity of a different species of plant, perhaps an annual or bean plant, whose roots are much more defined.

Further studies are needed to distinctlydefine the effects of proximity to unrelated and kin plants on determiningroot growth.Suggestions for such future endeavors include using different plants to study whose roots are much more well-defined in addition to using different methods of evaluating them, such as measuring distance of roots from other plants and/or individually weighing and measuring each root.The findings produced from these future experimentscould yield results that would impact agricultural practices currently in place in order to accommodate the need for plants in geographical areas that will not outcompete each other for nutrients.

Literature Cited

Biedrzycki, ML, and Bais, HP. 2011. Kin Recognition: Another Biological Function for Root Secretion. Landes Bioscience Plant Signaling & Behavior vol 5(4): p 401–402.

Biedrzycki ML, and Bais HP. 2010. Kin Recognition in Plants: a Mysterious Behaviour Unsolved. Journal of Experimental Botany vol 61: p 4123–4128.

Dudley, SA, and File,AL. 2007. Kin Recognition in an Annual Plant. Biology Letters vol 3: p 435-438.

Fang, S, Clark, RT, Zheng, Y, Iyer-Pascuzzi, AS, Weitz, JS, Kochian, LV, Edelsbrunner, H, Liao, H, and Benfey, PN. 2013. Genotypic Recognition and Spatial Responses by Rice Roots. Proceedings of the National Academy of Sciences of the United States of America vol 110: p 2670–2675.

Milla, R, Forero, DM, Escudero, A, and Iriondo, JM. 2009. Growing with Siblings: a Common Ground for Cooperation or for Fiercer Competition Among Plants?Proceedings of the Royal Society Lond B Biological Sciences vol 276(1667): p 2531-2540.

Murphy GP, and Dudley SA. 2009. Kin Recognition: Competition and Cooperation in Impatiens (Balsaminaceae). American Journal of Botany vol 96: p 1990–1996.