Difference in Nucellar Length and Width Ratios in Two Diverging Ecotypes of Arabidopsis thaliana (L.) Heynh

Introduction Arabidopsis thaliana is a member of the Brassicaceae family. Traits

such as excessive seed production and rapid flowering characterize it as a colonizing species that grows in recently disturbed habitats. It inhabits over 800 different ecotypes, making it highly adaptable to different environments, as well as an ecologically important model organism for study (ABRC, 2009).

Both stages of female gametophyte development, megasporogenesis and megagametogenesis (Figure 1), have been investigated using Herr’s Clearing Fluid, which clears the cells in the ovule (Smith, 1973). This method, combined with newer technologies allows for quantitative data to be collected for nucellar growth within the ovule (Amato, 2010).

The difference in the mean lengths and widths of the nucellus can be used to generate a Fibonacci sequence, which is related to exponential growth found in nature (Cervantes et al. 2010) Using two key Phi values that have been found to be consistent in angiosperms, different ecotypes are able to be compared (Herr, 2010).

The nucellar length and width measurements can be used to calculate a sequence of divisions that reach the Phi value, or the Golden Ratio of 1.618 at the 11th division and 1.6180339 at the 18th division (Herr, 2010).

Materials and Methods Seeds were obtained from two ecotypes in Libya and Durham,

England and vernalized for 7 days in 4° C. Plants were grown in quads under artificial light; upon flowering the pistils were placed into FPA50 for 24 hours.

The pistils were dehydrated in 70-100% EtOH for 10 minutes each and then cleared using the Herr’s Clearing Fluid; where they remained for storage.

The cleared whole ovulary was dissected using a Nikon SMZ 1000 microscope and ovules were placed under a Raj slide for viewing and measurement using a Nikon Eclipse80i phase contrast microscope. These measurements were taken for each of the 7 stages of megasporogenesis and megagametogenesis(Figure 2.a-e).

Beth Schenning, York College of Pennsylvania

Results The nucellar length and width measurements were used to calculate a

sequence of divisions that reach the Golden Ratio (Table 1). The ratios in the four stages of megagametogenesis did reach the

expected Phi values for both ecotypes, except for the Libya functional spore and 4-nucleated stages where Phi was not reached at the 11th or 18th division, respectively.

The three stages of megasporogenesis never reached the expected Phi values at the appropriate divisions for either ecotype.

Hypothesis• The expected Phi value will be reached by the 11th and 18th

divisions in the nucellus during the haploid generation of the megagametophyte in both diverging ecotypes.

• The expected Phi values will not be reached by the 11th or 18th divisions due to variations in the diploid generation during megasporogenesis for either ecotype.

Conclusion• Overall, the hypothesis was supported by the results of the

Fibonacci sequence. A majority of the stages during megagametogenesis for both ecotypes were found to follow the Fibonacci sequence, with the exception of the functional spore and 4-nucleated stages of the Libya ecotype.

• Variation in the diploid generation of megasporogenesis inhibited the length and width ratio to follow the sequence for both ecotypes.

• It is concluded that the haploid gamete is so conserved in its structure and composition that it is uniform among the studied A. thaliana ecotypes using the Fibonacci sequence.

Literature Cited• ABRC. 2009. The Ohio State University. http://abrc.osu.edu/•Amato, Dena. 2010. Does the environment affect the size of the female

megagametophyte stages in Arabidopsis thaliana (L) Heynh ecotypes. York College Senior Thesis.

•Cervantes, E., Martin, J., Ardanuy, R.,and Angel Tocino. 2010. Modeling the Arabidopsis seed shape by a cardioid: Efficacy of the adjustment with a scale change with factor equal to the Golden Ratio and analysis of seed shape in ethylene mutants. Journal of Plant Physiology. 167(5): 408-410.

• Herr, John. 2008, January 22. Email Interview. • Smith, B. B. 1973. The use of a new clearing technique for the study of early ovule development, megasporogenesis, and megagametogenesis in five species of Cornus L. American Journal of Botany 60(4): 322-338.• Smith, B, B. 1975. A quantitative analysis of the megagametophyte of five

species of Cornus L. American Journal of Botany 62(4): 387-394.

AcknowledgementsI would like to thank Dr. Bruce Smith for his irreplaceable knowledge and support through the senior thesis project.

Figure 1. Process of megagametogenesisand megasporogenesis.

a b

c d

e

Figure 2. Durham ecotypes, 1000x. a) Megaspore mother cell stage. b) Tetrad stage.

Figure 2. Libya ecotypes, 1000x. c) 2-nucleated stage d) 8-nucleated stage.

Figure 2e. Durham ecotype, functional spore stage, 1000x.

Table 1. Mean length to width measurements at 19 divisions of megagametogenesis for the Durham ecotype in a Fibonacci sequence.

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