Is there an ideal pistil length in which each stage of Megagametogenesis can be observed in Arabidopsis thaliana?

Introduction:• Comparing the haploid stages of seed development provides yet another morphological feature that can be used to determine relationships between and among species by phylogenists. These stages, although difficult to isolate and quantify, can now be studied using the Herr Clearing techniques (Smith 1973).• Ecotypes are members of the same species that appear differently when viewed in varying environmental conditions, i.e., Arabidopsis thaliana (Rédei, 1969).• Arabidopsis thaliana with over 800 ecotypes identified is a good research model because its developmental stages are easily discernible, it’s inexpensive, easy to order, and it goes from seed to seed in less than a month.• I chose to compare three ecotypes looking carefully at the sizes of their pistils and the megagametophytic stages; functional megaspore, 2-nucleate, 4-nucleate, and 8-nucleate stages to see if a single pistil length might contain each of these megagametophytic stages.

Objectives:• Compare pistil lengths of 3 ecotypes against the megagametophytic stages they contain (Webb & Bowman, 1994).

Joy HaringDepartment of Biological Sciences, York College of Pennsylvania

Definitions:Megagametogenesis: the mitotic divisions of a megaspore mother cell that later develops into an embryo sac, all of which is the megagametophyte.Ecotype: genetic variety within a species that arises as a result of adaptions to the local environment. For Arabidopsis thaliana, 800+ ecotypes are known. Pistil: the female reproductive parts of the flower. The pistil is divided into the stigma, style and ovary. The ovary is composed of individual ovules.Haploid: the number of chromosomes that represents ½ the diploid number. Functional megaspore: after meiotic division four cells are formed that are haploid and three of them die. The remaining one is the functional megaspore. Ovules: the structure attached to the placenta of the ovary and eventually becomes a seed.

Table 2. Comparison of Ovary lengths to number of ovules in each developmental stage.

Length of the Pistil in

mm# ovules

FMa

# ovules 2-nucleate

# ovules 4-nucleate

# ovules 8-nucleate

Ovary 1 (Le)b 1.5

Ovary 2 (Le) 2.1 3

Ovary 3 (Le) 2 1

Ovary 4 (Le) 2 1 10

Ovary 5 (Le) 1 7

Ovary 6 (Col)c 1.4

Ovary 7 (Le) 1.1 3

Ovary 8 (Le) 1.8 1 1 1 1

Ovary 9 (Col) 1.3

Ovary 10 (Le) 1.2 11

Ovary 11 (D)d 1.1 6

Ovary 12 (D) 1.1 12

Ovary 13 (D) 1 8

Ovary 14 (D) 1.9 1 3 3

a Functional Megasporeb Le: Landsberg erectac Col: Columbiad D: Dijon

Future Studies:• To work with other ecotypes to determine if there is a universal pistil length or if there is variation within the ecotype.• To compose a table that compares floral stages, appearance of the flower, anther length and its morphology.• To determine ideal pistil lengths in other species and create a table for them modeled after Webb and Bowman (1994).

Literature Cited:

Rédei, G.P. 1969. Arabidopsis thaliana (L.) Heynh. A Review of the Genetics and Biology.

Bibliographia Genetica. 21: 1-151.

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.

Webb, M.C. and Bowman, J.L. 1994. Arabidopsis: An Atlas ofMorphology and Development. Springer-Verlag, New York.

Acknowledgements:

I would like to thank Dr. Smith for his continued expertise, guidance and understanding through the entire Senior Thesis process, and throughout the Biology curriculum.

Grow multiple wells of each

ecotype

At first sign of flowering remove

inflorescence and place in fixative (FPA50

Minimum 24 hrs.)

Continue collections and

fixing until some flowers

go to fruit

Place flowers in dehydration series, 70% to

80% to 85% to 90% to 95% to 100%

EtOH Minimum 10 mins. in each concentration

Herr Fluid

Measure pistil length

View dissected ovule with phase

contrast microscope

Conclusion:• One ovary from the Landsberg erecta ecotype was found to have ovules in each stage of development.

•Another ovary from Landsberg erecta has ovules in two of the stages, indicating more ovaries with pistil lengths in between these two could have more stages.

•From this small sample size, an ideal pistil length cannot be definitely determined.

Methods:

4-nucleate stage:

8-nucleate stage:

Whole PistilFunctional Megaspore

2-nucleate stage

Taken from Webb and Bowman, 1994

Table 1. Stages of embryo sac and ovule development

Approximate floral stage

Stage of embryo sac

development

Ovule morphology

Pistil length (mm)

Appearance of flower

9 Ovule primordia

arise

0.15-0.4

10 Ovule primordia elongate

0.4-0.5

11 Megasporocyte

4 megasporesFunctional megaspore

Inner and outer

integuments initiated

0.5-1.01.0-1.5

Early 12 2-nucleate embryo sac

Funiculus and nucellus

curve; other integument

exhibits asymmetric

growth

1.5-2.0

Mid 12 4-nucleate embryo sac

Integuments grow upwards

around nucellus

Late 12 8-nucleate embryo sac

Outer integument begins to

cover both inner

integument and nucellus

Results: