Female Gametophyte and Early Seed Development in Peperomia (Piperaceae)

Introduction
The scientists are testing the lineage of Piperales to get the overall structure of female
gametophyte and then to see how that can impact seed development. Female gametophyte
development impacts the function of seed development since what happens in the gametophyte
helps us see how seed develop internally as well as how the offspring is produced.
Scientists already know that all female gametophytes in Peperomia contain one egg cell.
What the scientists dont know is whether the overall structure of the gametophyte is 9-celled or
10-celled. To find the results, three species of Peperomia will be tested. Those three species
include P. dolabriformis, P. jamesoniana, and P. hispidula.
In the investigation, standard light microscopy will be combined with high resolution 3-D
computer reconstruction techniques to describe female gametophyte and early seed development
in the three species I mentioned above. P. hispidula reportedly contains three cells at maturity,
with one egg cell, one synergid, and a 14-nucleate central cell, but nothing has been described in
P. dolabriformis and P. jamesoniana.
Materials and Methods
The methods used to obtain information were plant collection, histology, 3-D computer
reconstruction, and microspectrofluorometry.
In plant collection, inflorescences of P. dolabriformis and P. jamesoniana were collected
from greenhouses at the University of Colorado. Inflorescences of P. hispidula were collected
from Costa Rica and sent to the University of Colorado. In the lab, inflorescences were
immersed in a modified PIPES buffer. This caused dissection of smaller pieces of about twelve
flowers that could then be used for chemical fixation.

In histology, there were two different fixed solutions that the dissected flowers could
have been put into for twelve hours. Those solutions are either 3:1 95% ethanol: acetic acid or
4% paraformaldehyde, 2.5% glutaraldehyde, and 4% acrolein. Specimens were sectioned into
5m thick sections using a rotary microtome and glass knives. Flowers fixed into the 3:1
solution were stained with a solution of 0.25 gm-1 of 4, 6-diamidino-2 phenylindole (DAPI)
and 0.1 mgml-1 phylenediamine. Flowers fixed with the other solution were stained with 0.1%
aqueous toluidine blue. Then slides of these flowers were viewed with a microscope.
In 3-D computer reconstruction, the 5m thick sections in the fixed solution of
paraformaldehyde, glutaraldehyde, and acrolein were photographed. By exciting the toluidine
blue stain with a helium/neon laser, images were created. The 5 m thick sections were
photographed along the Z-axis so that eight-ten 0.5m thick optical sections could be viewed.
In microspectrofluorometry, flowers fixed into the 3:1 solution were stained with DAPI
for one hour at room temperature. Within two hours, DNA level measurements of DAPI stained
nuclei could be performed. This method was basically used to get measurements of DNA levels.
Results
In the 3 species of Peperomia, results were collected during megaspongenesis, female
gametophyte development, fertilization, microspectrofluorometry, and endosperm/perisperm
development.
The P. dolabriformis megasporocyte has a dense cytoplasm and normally has a single
vacuole. Meiosis I occurs in the center of the cell when the megasporocyte is about 50m long
and 30m wide. During the division, the vacuole and phragmoplast are perpendicular to each
other. The phragmoplast results in a cell plate that expands outward from between the two nuclei

toward the cell wall but the plate aborts before coming in contact with the syncytium. The cell
wall formed during meiosis I is no longer visible but the phragmoplast is still present between
each megaspore pair. This results in cells plates expanding toward periphery of female
gametophyte. As cell wall aborts, all four polar nuclei become situated into a single megaspore
cell.
During female gametophyte development, the female gametophyte has a tetrahedral
shape. By this time, the majority of the cells volume is the central vacuole. When the diameter
of the female gametophyte is about 75m, each nucleus goes through mitosis again. Sixteen
nuclei are produced and cell walls start to form. Synergid are cells not going through
fertilization. In the mature female gametophyte of P. dolabriformis and P. jamesoniana, 10 cells
were observed which included an egg cell, two synergid cells, six accessory cells, and a central
cell. These two species also contained seven polar nuclei in the central cell. The mature female
gametophyte in P. hispidula contains 3 cells which include an egg cell, a synergid cell, and a
central cell. This species doesnt have accessory cells but its secondary nucleus has a product of
fourteen polar nuclei.
Fertilization was observed in P. jamesoniana and P. hispidula, but not P. dolabriformis.
In female gametophytes of P. hispidula, the polar nuclei fuse before fertilization while in P.
jamesoniana, they fuse after. Female gametophytes of P. jamesoniana contain two synergid
before fertilization and one after. In P. hispidula, the female gametophyte contains one synergid
before and after fertilization.
Microspectrofluorometry was again used for the DNA contents of the fertilized female
gametophytes in Peperomia. Results for P. hispidula was a 30C DNA content and a 15N central

cell. P. jamesoniana contains a 16C DNA content and a 8N central cell. All of these results were
observed in the primary endosperm nuclei during prophase.
In the endosperm/perisperm development, starch starts to form right after fertilization. It
forms in the cytoplasm, close to the female gametophyte in the nucellus cell. Once the seed starts
to develop, the starch grains get larger as well as more oval-shaped so they get farther from the
gametophyte. In P. jamesoniana, endosperm was observed up to 8-celled stage while in P.
hispidula, it was observed up to 20-celled stage. The majority of the seed volume is made up by
the perisperm in both of the species. The most mature embryos were observed in P. hispidula,
containing four cells.
Discussion
Scientists data confirms that in Peperomia, female gametophytes contain a three-celled
egg apparatus at maturity. In P. dolabriformis and P. jamesoniana, female gametophytes are 10celled, with an egg cell, two synergid, six accessory cells, and seven polar nuclei. P. jamesoniana
has a three celled egg apparatus before fertilization and a two celled egg apparatus after
fertilization.
We are the first to reinvestigate female gametophyte development in P. hispidula since
the original claim that egg cells are fertilized through direct penetration of the pollen tube in this
species (Johnson 1914). A three celled configuration has been confirmed in P. hispidula.
Conclusions
Micrographic evidence supports the existence of a 10-celled configuration in several
female gametophytes in Peperomia. Scientists do feel like more investigation has to be done
since previous data has been proven incorrect in this investigation.

Bibliography
Eric N. Madrid, William E. Freidman. "Female gametophyte and early seed development in
Peperomia (Piperaceae)." American Journal of Botany (2009): 14.