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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,
wild senna, ringworm cassia, guajava, ketepeng badak, flor del Secreto, Tarantana, man-slabriki
and gelenggang. (Tropilab, Inc., n.d.)
The persistent shrub can grow up to six feet tall (one to two meters) and consists of
yellow waxy, erect spikes that may bear resemblance to big, golden candles prior to full
blossoming. The bilateral leaves are substantial, alternately arranged and folds upon itself during
nighttime. In addition, around eight to twenty oblong-elliptically shaped leaflets measuring
about two to four inches embrace the leaves. The trunk or branches grow generally upright with
no thorns but is thin and easily damaged. (Gilman et al., 1993) Its flowers are made up of oblong
sepals while the fruits are smooth, winged and tetragonal. (Philipine Herbal Medicine, n.d.)
Moreover, the fruit is a pod measuring up to six to twelve inches and brown while the seeds are
square in form and tiny. (Gilman et al., 1993)
Cassia alata L. is a host plant for caterpillars, particularly the sulphur caterpillars which
includes the orange barred sulphure. However, different parts of the plant, mainly the leaves
have specialized features. The leaves are composed of laxative properties, such as saponin, that
can be used as such. (Gilman et al., 1993) In addition, it also consists of chrysophanic acid which
is a fungicide used to treat infections like ringworms, scabies and eczema. (Philippine Herbal
Medicine, n.d.)
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In general, the most significant part of Cassia alata L. that can be used in multiple
occasions is the leaves. As previously mentioned, the leaves contain certain chemicals such as
saponins, anti-fungal and anti-micrombial properties that aid in curing certain kinds of diseases.
In addition to this, the leaves are made up of a particular chemical called chrysophanic acid,
which is a fungicide that can be used to treat fungal infections such as athlete’s foot (Tidea
pedis), ringworms, scabies and eczema. Moreover, the leaves have also been said to be sudorific,
diuretic, purgative and are being used in a similar way as senna. The leaves can also be utilized
to cure bronchitis and asthma. (Filipino Herbs Healing Wonders, n.d.)
As this plant contains saponin, it can be particularly useful as a laxative as well as a tool
to exorcise parasites in the intestine. In Africa, the leaves are boiled and used to fight against
high-blood pressure. In South America, however, the leaves are also utilized to treat a broad
spectrum of illnesses from stomach problems, fever, asthma to snake bites and venereal ailments
such as syphilis and gonorrhea. (Tan, 2001) Aside from the leaves, both the roots and the flowers
may also be employed for specific medicinal practices. Plant extracts from Cassia alata L. is
also usually used as part of the ingredients in creating lotions, soaps and shampoos. (Philippine
Herbal Medicine, n.d.)
A specific example of a product wherein Cassia alata L. is a main ingredient is the soap
that is manufactured by Natural Resources Malaysia. This company incorporated fresh leaves
from Cassia alata L. in their product through the use of cold process soap making method.
Moreover, in an effort to maximize the plant’s antiseptic ability, the manufacturers also included
scented oils such as tea tree and peppermint. This natural handmade soap claims to be able to
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treat eczema, ringworm and psoriasis. In addition to this, it can also cure itchy skin due to its
antipuritic effect. (TradeKey, 2009)
Other products wherein this particular plant was used were in the rapid weight loss tea
and anti-acne lotion by Bonanzle. According to this company, the effect of placing few pieces of
Cassia alata L. leaves in a cup of hot water could lead to rapid weight loss. It was seen that plant
extracts from Cassia alata L. were found as a key ingredient in most dieter’s teas. This plant has
the dual effect of acting as a stimulant, which decreases an individual’s appetite as well as the
laxative property that accelerates the movement of food in the system thereby preventing
absorption of substantial calories. As the leaves of Cassia alata L. also functions as an anti-
septic, it has been found to be very effective in removing acne, prickly heat rash as well as other
skin irritations. (Bonanzle, n.d.)
Objective
In general, this paper aims to examine and distinguish the morphology of both the male
and female gametophyte. However, greater focus will be lent towards the further scrutiny of the
different developmental stages of the male gametophyte.
Specifically, the study aims to:
1. Evaluate and characterize the cross-section of the female gametophyte;
2. Fully understand the totality of the said plant’s developmental stages;
3. Contribute additional information with regards to the various uses of Cassia alata; and
4. Make aware to the community about its effective uses against certain diseases.
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Significance
A study concerning an in-depth research on the plant Cassia alata is quite beneficial due
to the limited information that has been discovered regarding this plant. Due to its anti-microbial
and anti-fungal components, further studies pertaining to its various medical uses can be a
significant contribution to the scientific community. In addition, this study could also be
supplementary basis that may contribute to the taxonomy and characterization of the
development of C. alata. On the other hand, this paper can also aid in popularizing or spreading
the many uses of C. alata products to combat illnesses that range from stomach problems to
ringworms.
Scope and Limitation
This study will focus mainly on the flower morphology of the plant Cassia alata L. The
general bulk of the obtained information will come from the cross-sections of both the ovary and
the anther with the use of an Olympus CX 21 binocular compound microscope as well as
Olympus SZ61 stereomicroscope. Specifically, the life cycles of both reproductive parts will be
distinguished and observed. In addition, the specimens acquired will come from one specific
source acquired from the Manila Seedling Bank. However, the remaining parts such as the leaves
and petals will not be considered as part of the study.
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REVIEW OF RELATED LITERATURE
Related taxonomic studies
Table 1. Taxonomy of Cassia alata L .(binomial name: Senna alata )
Scientific classification
Kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliopsida
Subclass: Rosidae
(unranked): Eurosids I
Order: Fabales
Family: Fabaceae
Subfamily: Caesalpinioideae
Tribe: Cassieae
Subtribe: Cassiinae
Genus: Senna
Species: S. alata
Cassia alata L. (also known as Senna alata or “Candle Bush”), classified under the genus
Senna, which is a large genus of flowering plants in the family Fabaceae, subfamily
Caesalpinioideae. This diverse genus is native throughout the tropics, with a small number of
species reaching into temperate regions, and there is an estimated number of 350 species
belonging to this genus. This genus is characterized to be conspicuous legumes with a
characteristic yellow flower, and consists of shrubs, as in the case of Senna alata, subshrubs,
herbs, or a small tree.
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This species is more generally classified to the subfamily Caesalpinioideae, which are
mainly trees distributed in the moist tropics. Their flowers are zygomorphic, but are very
variable, and nodulation is rare in this subfamily, and where it does occur nodules have a
primitive structure. This subfamily belongs to the third largest family of flowering plants next to
Orchidaceae and Asteraceae, and this is Fabaceae or Leguminosae or otherwise known as the
“legume or pea family”. Those belonging to this family range in habit from giant trees to small
annual herbs, with the majority being herbaceous perennials. Plants have indeterminate
inflorescences, which are sometimes reduced to a single flower. The flowers have a short
hypanthium and a single carpel with a short gynophore, and after fertilization produce fruits that
are legumes (Bibsy et al. 1994).
Morphology
Cassia alata L. or synonymously known as Senna alata, is more commonly known in the
Philippines as the Akapulko plant. It is an erect, perennial, shrubby legume which grows up to 6
feet tall. It has dark green even pinnately compound leaves which have orange rachis on stout
branches. Each leaf has 16-28 leaflets measuring 2-4 inches are alternate bilateral in arrangement
(see Fig. 1). Leaves are obviate in shape and are evergreen. (Quisimbing 1978; Gilman and
Watson 1993)
Its golden yellow flower with about 4cm in diameter, is arranged in a inflorescence
column that resembles yellow candlesticks. Flowers are tepals because of the
indistinguishableness of the petals and sepals (Quisimbing 1978) and pseudo-papilionaceous
(common among all sub-family members of Caesalpinioideae). (Watson and Dalwitz 1992)
There are two sets of sepals once a young bud is maturing and the outermost layer falls off as the
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flower matures and opens up. The flowers are actually hypogynous but are appears to be
perigynous because of the hypanthium; a floral structure consisting of the bases of the sepals,
petals, and stamens fused together (a characteristic evident among members of family Fabaceae).
(Watson and Dalwitz 1992) C. alata L. is a perfect and complete flower. Each flower has two
long banana-shaped anthers (which are tetrasporangiate), 3 projections for its stigma, 4 stomiums
that serve as the exit point of pollen, a nectary, and a long pointed stalk that will eventually
become the fruit pod of the future seeds known as the Gynophore (see Fig. 2). (Bracegirdle and
Miles 1973; Krommenhoek et. al. 1979; Bowes 1996) (Watson and Dalwitz 1992)
Pollen grains of C. alata are 27um polar length and 26um in equatorial length in 3000x
magnification through Scanning Electron Microscope. It is tricolpate with a prolate spheroidal
shape. Membrane of pollen grains are smooth with the 2um thick exine and a finely articulate
sexine with granulate muri and lumina. (Jugadilla-Bulalacao 1997) Anthers dehisce and pollen
are viable during early morning or mid-day as an evolutionary technique to increase plant–
pollinator interaction success. (Sarala et. al. 1998)
Receptacle houses unicarpellate ovary but forms a fruit separate from the receptacle.
Other organs found in the receptacle are vascular bundles, style extension towards the ovary and
other various parts of the ovary itself (e.g. placenta, locules, etc.) (see Fig. 3). Placentation of the
flower is lateral or marginal. The ovule type is amphitropous. (Watson and Dalwitz 1992) The
egg cell’s development is a monosporic polygonum 7-8n type. (Esau 1898; Esau and Everts
2006) An axis of produces 4-winged pods (i.e. legume) which grows at about 6-12 inches
containing 50-60 flattened, triangular seeds. At a young age, the pods are green, but eventually
harden and turn brown as they mature. (Quisimbing 1978; Gilman and Watson 1993)
Male Gametophyte Development Overview: Angiosperms in General
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Microsporogenesis and Microgametogenesis are two processes that lead to the formation
of the male gametophyte: the pollen grain. These pollen grains are produced in the anther locule
(Masukoet al., 2006). Male gametophyte development begins with the sporophytic cells
(diploid), which give rise to the sporogenous initials, also known as the pollen mother cells
(McCormick, 1993). These pollen mother cells undergo meiosis, which then results in tetrad
haploid cells; dyad cells may also be found, and these cells will eventually divide again to form
the tetrad of haploids (McCormick, 1993). These cells will eventually part, thus becoming
uninucleate microspores, which then undergo asymmetrical (also determinative) mitosis, which
results in a larger vegetative cell and a smaller generative cell; the vegetative cell completely
houses the generative cell (McCormick, 1993). The vegetative cell will not undergo another
round of mitosis, while the generative cell will give rise to two sperm cells. The vegetative cell
will be the “power source” for the further development of the pollen grain, while the sperm cells
will fertilize the female polar nuclei and egg (double fertilization) (McCormick, 1993). This
vegetative cell plus the two sperm are now collectively called as a pollen grain, the male
gametophyte.
Male Gametophyte Development in Caesalpinioideae
Cassia alata L. belongs to the genus Cassia, sub-tribe Cassiinae, tribeCassieae, sub-
family Caesalpinioideae and family Fabaceae (Tucker, 1993). The development of the male
gametophyte of other model legumes will be discussed; these are similar to the male
gametophyte development of C. alataL. The development of pollen of plants belonging to
Caesalpinioideae follows that of the general flow of angiosperm
microsporogenesis/gametogenesis. In the ontogeny of the pollen of Lotus japonicus, a model
legume, the pollen develops in the anther locule (Masukoet al., 2006). Like the flow of pollen
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development in general, the pollen mother cells undergo meiosis, which gives rise to microspore
tetrads, which then part to become uninucleates (McCormick, 1993; Masukoet al., 2006). The
uninucleates will then undergo mitosis, which will give rise to the vegetative and generative cells
(McCormick, 1993; Masukoet al., 2006). The vegetative will stay as it is, while the generative
cell will undergo a second round of mitosis to become sperm (McCormick, 1993; Masukoet al.,
2006). A study conducted by Banks et al (2006), the legume Duparquetia (which belongs to the
same sub family Caesalpinioideae) exhibits not only tetrads, butdyads and “triads”. These cells
are arranged in a tetrahedral conformation; this is why some tetrads are wrongly interpreted as
“triads”, as three of the four cells at a time may be facing up (thus concealing the fourth), rather
than clearly showing the four nuclei present in the tetrad (Banks et al., 2006). Again, these
members of the family Caesalpinioideae exhibit the same general ontogeny of the male
gametophyte, as with angiosperms in genera
Female Gametophyte Development Overview: Angiosperms in General
There are two processes involved in the formation of the female gametophyte:
megasporogenesis and megagametogenesis. Female gametophyte development occurs in the
ovary, specifically in the ovule (Esau, 1977). The nucellar cells, located at the heart of the ovule,
will mostly degenerate; only one cell will enlarge and differentiate into what we call as the
megaspore mother cell (Kennell and Horner, 1985, Esau, 1977). This megaspore mother cell will
undergo meiosis, first forming a megaspore dyad, then finally a linear megaspore tetrad (Kennell
and Horner, 1985; Esau, 1977; Rembert, 1969). Three of the four cells will degenerate, leaving
only one functional megaspore, the one nearest the chalazal end (Kennell and Horner, 1985;
Esau, 1977). This functional megaspore will undergo three rounds of mitosis (first will divide
into two, then into four, finally into eight). The eight resulting nuclei will migrate into their
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respective sites (three in the chalazal end, three in the micropylar end, and two in the center).
These will then differentiate and specialize into three antipodal cells, two polar nuclei (cells may
or may not be fused) and three egg apparatus, which is composed of two synergid cells and one
egg (Esau, 1977). This final 7 or 8-celled structure is the female gametophyte, the embryo sac;
the type of development explained above is of the Polygonum type (Esau, 1977). According to a
study done by Rembert (1969), members of the Cassia genus also exhibit this monosporic type
of female gametophyte development.
Miscellaneous Processes: Double Fertilization, Embryogeny, Germination of Sporophyte of
Angiosperms in General
After pollination, the newly formed pollen tube will passes through the style, and will try
to reach the ovarian cavity: this is where the embryo sac is located (Esau, 1977). Once the pollen
tube reaches the ovule, it passes through the micropyle; the two synergids degenerate soon after.
One sperm will fertilize the egg, while the second one will fertilize the polar nuclei: this
phenomenon is referred to as double fertilization (Esau, 1997; Solomon et al., 2008). The zygote
(product of the egg and sperm) will develop into the embryo, through a process called
embryogeny; the primary endosperm nucleus will develop into the endosperm through mitosis
(Esau, 1977; Solomon et al., 2008). After a certain time period, the young sporophyte will
germinate and will exhibit morphogenesis and organogenesis, which will result in the highly
organized adult sporophyte (Esau, 1977).
METHODOLOGY
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Acquisition of Cassia alata L. Flowers
The flowers of Cassia alata L. were bought and transported from the Fruit Bearing Plant
Center in the Manila Seedling Bank, Quezon City. The flowers were cut from the stalk, including
both mature and young flowers and some young fruits. After transportation, the flowers were
stored in a moderately lighted, dry place. The stalks were cut diagonally to maximize water
absorption (Hatter n.d.) and were dipped in a 100mL beaker filled with tap water. The flowers
were left there until further use for the experiment.
Macroscopic Analysis of Flower Parts
The collection of flowers (both mature and young) on the stalks was photomicrographed.
An individual flower was used from the collection of flowers and was placed in the Olympus
SZ61 stereomicroscope (10x), were then exposed by removing the sepals and petals respectively
and photographed accordingly. Parts of the flower such as the stigma, anther and other unique
floral parts particular to Cassia alata L. were noted. The naked flower was once again subjected
to the same stereomicroscope (10x) and photomicrographs were taken.
Microsporangium Analysis
Anther Free-hand Sectioning and Microscopy
Anthers, both mature (from open flowers) and young (from buds), were collected and cut
into half. One halved anther was then sectioned as thinly as possible using a sharp razor. Distinct
differences between the young and mature anther were then distinguished. Pollen grains and
sporogenous cells were identified. Both were subjected to the Olympus SZ61 stereomicroscope
(10x) for viewing and photomicrographs are taken.
Pollen Microscopy
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Pollen grains were collected from halved anthers and were placed in a slide. 0.5%
Acetocarmine was added to the pollen on the slide as a stain. The slide was covered with a cover
slip and was passed over the flame of an alcohol lamp a couple of times. The slide was viewed
under the oil immersion objective (1000x) of an Olympus CX 21 binocular compound
microscope. The characteristics (i.e. shape, size, texture, furrows, etc.) of the pollen were noted.
Photomicrographs of the pollen grains were taken.
Squash and Smear Technique
As adapted from the methodology of Tsuchiya 1971, modified by Tolentino, V.S. 1992,
the Squash and Smear Technique for smears of microsporocytes using whole mounts and
without sectioning was done to mature and young anthers. The isolated anther was placed in a
slide and was cut and squashed in order to release the sporogenous cells within them. The sample
was stained with 0.5% acetocarmine. A cover slip was placed slowly by lowering one end with
the other set on the slide. A few more drops of acetocarmine were added in the sides to clear the
bubbles and the slide was heated with few passes over an alcohol lamp. Finally, the cover slip
was tapped gently with a moderately soft and blunt object (e.g. finger, eraser, etc.). The prepared
slide was placed in an Olympus CX 21 binocular compound microscope and was viewed in oil
immersion (1000x) objective. Pollen mother cells, dyads, tetrads, pollen and other cells were
noted. Photomicrographs were taken.
Ovarian Free-Hand Sectioning and Microscopy
The flower’s receptacle was removed. Using a sharp razor, excesses of the stigma and
filaments were removed from the receptacle. The cleaned off receptacle of the flower was put
under a stereomicroscope and was cross-sectioned as thin as possible, disposing of the thicker
portion and placing the thin section of the ovary in a clean slide. The section was viewed with
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the Olympus SZ61 stereomicroscope (10x), distinct parts were noted and photomicrographs were
taken.
Also, in attempt to obtain longitudinal sections for the analysis of megagametogenesis in
ovary, a modified Paraffin method adapted from Chamberlain, 1915. Disregarding the killing
and fixing, clearing and dehydration processes usually done in laborious paraffin methods done
for microtome sectioning, the procedure was skipped to the immersion to paraffin wax, wherein
whole parts of the receptacle was immersed in melted wax. The hardened paraffin blocks were
then sectioned (i.e. in much more ease than regular free-hand sectioning without the wax) with a
sharp razor only. The wax allows for less injury and easier and thinner cutting of the sample.
(Chamberlain, 1915) The sections were cleared off with remaining parts of wax, were viewed
under a stereomicroscope and photomicrographed.
In Vitro Pollen Tube Germination and Microscopy
As adapted from the methodology of Nurhan, H. 2003, pollen were collected from mature
anthers then subjected to 40% sucrose solution with 10mL distilled water and 4ml cane sugar.
The sucrose solution with pollen was left in a petri dish for 24 hours. After the 24 hours, the petri
dish was tilted so that pollen will suspend on the bottom. Thick whitish liquid which signifies the
presence of pollen was collected using a dropper and was eventually placed in a slide. 0.5 %
Acetocarmine was added to the drop of the mixture, was covered with a cover slip and heated.
The slide was placed in an Olympus CX 21 binocular compound microscope and was viewed
under the oil immersion objective (1000x). Pollen tube was observed, noted and
photomicrographed.
RESULTS
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Flower Morphology
Petal
Sepal
Pedicel
Figure 1. Mature open flower (left) and flower bud (right)
*perigynous; presence of tepals
Flower Morphology: Bud Cross-Section
Sepals (calyx) Nectary
Anther
Gynophore
Stomium Stigma Petals (corolla)
Figure 2. C.alata flower bud cross-section (10x)
Flower Morphology: Reproductive Parts
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GynophoreNectaryAnthers (2)
Stomium (4)
Stigma (3)
Receptacle
Figure 4. C. alata anthers
Figure 3. Front view of C.alata without calyx and corolla (10x)
Gynophore
Nectary
Figure 5. Side (Left) and Back (Right) View of C.alata without calyx and corolla (10x)
Reproductive Parts: Male Cross-Section
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Epidermis
Endothecium
Locule
Figure 6. Mature anther cross-section (10x)
Site of PMCs
Endothecium
Figure 7. Young anther cross-section (10x)
Reproductive Parts: Female Cross-Section
-Unicarpellate-Marginal Placentation Style
Locule
Placenta Ovule
Vascular Bundles
Figure 8. Ovary corss-section (10x)Microsporogenesis
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PMC Microspore Dyad
Meiosis I
Meiosis II
Microspore Tetrad Functional Microspore
Figure 9. Process of Microsporogenesis: PMC (top left), Microspore Dyad (top right), Microspore Tetrad (bottom left), Functional Microspore (bottom right)
Microgametogenesis 2-celled MG 3-celled MGFunctional Microspore
Generative Cell
Mitosis I
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
23
Books
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Journals
Banks H, Feist-Burkhart S, Klitgaard B. 2006. The Unique Pollen Morphology of Duparquetia (Leguminosae: Caesalpinioideae):Developmental Evidence of Aperture Orientation Using Confocal Microscopy. Annals of Botany. 98:107-115.
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Kennell J, Horner H. 1985. Megasporogenesis and Megagametogenesis in Soybean, Glycinemax. American Journal of Botany.72, 1553-15634.
Masuko H, Endo M, Saito H, Hakozaki H, Park J, Kawagishi-Kobayashi m, Takada Y, Okabe T, Kamada M, Takahashi H, Higashitani A, Watanabe M. 2006. Anther-specific genes, which expressed through microsporogenesis, are temporally and spatially regulated in model legume, Lotus japonicus. Genes Genet. Syst. 81, 57-62.
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Nurhan, H. 2003. In Vitro Pollen Germination and Pollen Tube Characteristics in Tetraploid Red Clover (Trifolium pratense L.). Turk J Bot. 27 (2003): 57-61.
Rembert D. 1969.Comparative Megasporogenesis in Caesalpiniaceae.University of Chicago Press.130, 47-52.
Ressayre, A; Godelle, B; Mignot, A; Gouyon, Ph (Jul 1998), "A Morphogenetic Model Accounting for Pollen Aperture Pattern in Flowering Plants", Journal of theoretical biology 193 (2): 321–334.
Tucker S. 1996. Trends in Evolution of Floral Ontogeny in Cassia SensuStricto, Senna and Chamaecrista (Leguminosae: Caesalpinioideae: Cassieae: Cassiinae).Botanical Society of America Stable.83, 687-711.
Websites
Bonanzle. N.d. Cassia alata leaves makes rapid weight loss tea. Internet. [cited July 24, 2010] [http://www.bonanzle.com/booths/wallplant/items/CASSIA__ALATA_leaves_Make_RAPID_WEIGHT_LOSS_TEA].
Filipino Herbs Healing Wonders. N.d. Akapulco – Scientific name: Cassia alata L. Internet. [cited on July 24, 2010] [http://www.filipinoherbshealingwonders.filipinovegetarianrecipe.com/akapulko.htm].
Hatter, Kathryn. n.d. How to Cut Flower Stems With a Knife. Garden Guides. Internet. [cited July 24, 2010] [http://www.gardenguides.com/95932-cut-flower-stems-knife.html].
Philippine Herbal Medicine. N.d. Akapulco/Acapulco (Cassia alata). Internet. [cited July 24, 2010] [http://www.philippineherbalmedicine.org/akapulko.htm].
Tan, Ria. 2001. Seven Golden Candlesticks. Internet. [cited July 24, 2010] [http://www.naturia.per.sg/buloh/plants/candlesticks.htm].
TradeKey. 2009. Natural resources: Cassia alata soap. Internet. [cited July 24, 2010] [http://www.tradekey.com/product_view/id/1128828.htm].
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Tropilab, Inc. N.d. Cassia alata L.-candlebush. Internet. [cited July 24, 2010] [http://www.tropilab.com/cassia-ala.html].
Watson L and Dallwitz MJ. 1992 onwards. The families of flowering plants: descriptions, illustrations, identification, and information retrieval. Version: 20th May 2010. http://delta-intkey.com.
Pictures from websites
http://www.greengrowerindia.com/files/products/images/134.jpg
http://database.prota.org/dbtwwpd/protabase/Photfile%20Images%5CLinedrawing%20Senna%20alata.gif
http://farm3.static.flickr.com/2094/2282628653_aa53b8b073.jpg?v=0
http://1.bp.blogspot.com/_olhzL-oEec4/SWdR45ZUkjI/AAAAAAAAAFk/r7Ibl0-9GZw/s400/akapulko.jpg
http://content9.eol.org/content/2009/04/21/07/26145_large.jpg
http://www.tradenote.net/images/users/000/370/326/products_images/439002.jpg
http://www.quinl.com/viewProductDetail/UploadImages/rGP6krY-pro41SennaAlataSlimTea.jpg
http://www.eurasiatrade.ch/images/OTK-Thanyaporn%20Herbs/Senna%20capsules.JPG
http://www.rainiersresearch.com/v2/images/skin_akapulco.png
http://www.orangkampung.com.my/assets/icons/12689412948300772.jpg
http://img.alibaba.com/photo/104643895/JAMU_DIABETES_Akar_Zaitun.jpg
APPENDIX