1 Major Project in Partial Fulfillment of the Requirements for the Developmental Biology Course Department of Biology Ateneo de Manila University Documentation of Flower Morphology and Male Gametophyte Development of Cassia alata L. through the usage of Microtechnique Submitted by: Group 8 Leandro Victor L. Arcena (4) Rafael Carlo E. De Guzman (12) Robert Leonard C. Goco (20) Christina Andrea Samantha Nadela (28) Gian Van Paolo V. Tenorio (36) Maria Monica I. Yupangco (44) Katipunan Avenue, Loyola Heights, Ateneo de Manila University, Quezon City 1108 Submitted to: Dr. Vivian S. Tolentino Developmental Biology Professor Date of Submission: August 4, 2010
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Documentation of Flower Morphology and Male Gametophyte Development of Cassia Alata L. Through the Usage of Micro Technique
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Major Project in Partial Fulfillment
of the Requirements for the Developmental Biology Course
Department of Biology
Ateneo de Manila University
Documentation of Flower Morphology and Male Gametophyte Development of Cassia alata L. through the usage of Microtechnique
Submitted by: Group 8
Leandro Victor L. Arcena (4)
Rafael Carlo E. De Guzman (12)
Robert Leonard C. Goco (20)
Christina Andrea Samantha Nadela (28)
Gian Van Paolo V. Tenorio (36)
Maria Monica I. Yupangco (44)
Katipunan Avenue, Loyola Heights, Ateneo de Manila University, Quezon City 1108
Submitted to:
Dr. Vivian S. Tolentino
Developmental Biology Professor
Date of Submission:
August 4, 2010
Venue, Date, and Time of Thesis Proposal Presentation
Ateneo de Manila University Campus, Sec B 107
August 3, 2010
1:30-5:30 AM
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INTRODUCTION
Background
Cassia alata L. belongs to the family Leguminosae and can be found throughout various
locations in the Philippines. This erect, tropical once-a-year herb with pinnately compound,
leathery leaves consist of many names. Locally, it is commonly known as akapulco or acapulco.
However, it can also be recognized as candle bush, fleur palmiste, fleur dartre, candlestick senna,
Figure 10. Microgametogenesis: 2-celled male gametophyte (left), 3-celled male gametophyte*MG= Male GametophytePollen Grains and Pollen Tube Formation
Mitosis II
Vegetative Cell
Sperm Cells
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Mature Pollen Grain Forming Pollen Tube
Appertures
Forming pollen tube
After 4 hours under 40%
sucrose solution
Pollen with Pollen Tube
Pollen TubeSperm Cells
After 24 hours under 40% sucrose solution
Figure 11. Pollen Grains and Formation of Pollen Tube
DISCUSSION
Macroscopic Analysis of Cassia alata L. Flowers
C. alata, which is also known for another scientific name, Senna alata, is under the genus
Senna and like its relatives from genus Cassia; they are all under the bigger sub-family
Caesalpinioideae. Morphological properties of C. alata that are also evident to most of the
members of its genus are to be discussed. The flowers dentified to be tepals (rather than
perianth) because of the similarities in appearance (i.e. color, size and shape) between the calyx
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and corolla. The corolla type is pseudo-pappilionaceous: a petal arrangement that almost consists
of parts of a pappilionaceous flower (i.e. wing, keel, etc.) but still has distinct differences that
requires a different classification. The reproductive parts are composed of three separate short
stigmas with each having their respective styles, two long banana-shaped tetraporangiate anthers,
four modified stomiums (exit points of pollen) in the middle, a long stalk that will eventually
house the seeds after fertilization called the gynophore and a nectary which aids in attracting
insects for pollination. The positioning of the ovary which is actually hypogynous, though
appearing like epigynous, is caused by the fusion of the sepals, petals, and stamens fused
together called a hypanthium. The ovary is unicarpellate with a marginal placentation and an
amphitopous ovule. The female gamete development is a monosporic polygonum 7-8 cell type.
Microsporogenesis
The different stages of microsporogenesis were observed in different developing stages
of the flower, namely developing buds. PMCs (pollen mother cells) were found in the
congregation of the youngest buds located at the topmost inflorescence. One nucleus was evident
and it was fairly large in size implying that it was about to undergo meiosis 1. Microspore dyads
and tetrads were also observed in buds; however, these were located in different parts of the stem
and not in the top congregation of young buds. Dyads showed two nuclei while tetrads appeared
to have either three or four. Some microspore tetrads were observed to have only three nuclei
giving the impression that it was tri-nucleated. This was due to the tetrahedral confirmation of
the nuclei which would sometimes mask the presence of one nucleus since it was at the back.
The separations between nuclei formed by the callose were indicators on how far in either
meiosis 1 or 2 the cell was. Calloses which were not strongly defined would imply that the cell
was still undergoing meiosis 1 or 2 (dyad and tetrad respectively) while well-defined calloses
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would mean that the process of meiosis 1 or 2 had finished. Each nuclei of the tetrad would
eventually become functional microspores and undergo microgametogenesis.
Microgametogenesis
Microgametogenesis was also observed in some flower specimens but these were limited
in large buds and flowering buds. The two-celled male gametophyte had its generative cell
sticking near the inner wall of the cell (which would eventually be the exine) while the
vegetative cell would comprise most of the cell material. The three-celled gametophyte also had
a vegetative cell but now the generative cell had split and had become two sperm cells freely
floating around the cytoplasm; one would be used to fertilize the egg cell while the other would
combine with the polar nuclei. These cells were more apparent when the three-celled
gametophyte was about to become a mature pollen grain. Three partitions could be observed as
the gametophyte was taking the shape of the mature pollen grain.
Pollen Grains and Pollen Tube
Mature pollen grains were found in numerous amounts (almost the whole field of vision
under the microscope) in anthers from fully blossomed flowers of Cassia alata L. and less when
in the budding stages of the flower. Three apertures could be seen in three different regions of
the pollen grain in such a way that they could serve as points forming a “Y” shape. These
apertures would serve as a good indicator on what type of angiosperm the plant is. Dicotyledons
would usually have three apertures (tricolpate) and this would mean that C. alata would be a
dicot (Esau, 1977). Through these structures, the pollen tube was most-likely to emerge once
pollination commenced. The great number of pollen grains in the anther of mature flowers
would connote that the plant was ready to release pollen as most were already mature. The
texture of the pollen grains in the mature anthers was fine-grained/powdery; however, in younger
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anthers (the ones in younger flowers and young buds), the contents (forming pollen) of the anther
were seen in a liquefied state. The contents of the young buds constituted of dyads, tetrads, and
pollen mother cells.
The fully realized pollen tube formed when the grains were submerged in 40% sucrose
solution for 24 hours; when viewed after only four hours, the pollen tubes were still emerging,
giving it a stub-like appearance. Pollen tubes that were submerged for 24 hours created notably
long clear pollen tubes. It was observed that the apertures vanished on the onset the pollen tubes
started to form. The tube was also stained which would connote that the contents of the pollen
(i.e. generative cell) has generated the pollen tube and its contents (i.e. 2 sperm nuclei) may have
passed through the tube. In the process of the pollen tube growth, the apertures vanished and they
could be said to be ephemeral. They were merely thin walled regions of the exine (outer covering
of the pollen grain overlaying the cell wall) where the pollen tube could emerge (Esau, 1977).
The pollen tube of pollen grains of C. alata could have formed in any of the three apertures and
those three would just vanish as the tube began to develop because they are not needed anymore
since apertures are merely the places where the pollen tube is able to break through the very
tough wall of the mature pollen grain. (Ressayre et. al. 1998)
LITERATURE CITED
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