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
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
 4

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
 8

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
 10

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
 11

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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Gynophore
Nectary
Anthers (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 MG
Functional Microspore
Vegetative Cell
Generative Cell Sperm Cells
Mitosis I Mitosis II

Figure 10. Microgametogenesis: 2-celled male gametophyte (left), 3-celled male gametophyte
*MG= Male Gametophyte
Pollen Grains and Pollen Tube Formation
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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 Tube
Sperm 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
 20

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
 21

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
 22

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

Bibsy FA, Buckingham J, Harborne JB, Zarucchi JL. 1994. Phytochemical dictionary of the
Leguminosae. Boca Raton: CRC Press. p 573.

Bowes B. 1996. Sexual Reproduction: A Colour Atlas of Plant Structure. London, UK: Manson
Publishing Ltd. p 145-147, 150-159.

Bracegirdle B and Miles P. 1973. Reproductive Structures: An Atlas of Plant Structure. London,
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Chamberlain CJ. 1915. Methods in Plant Histology: The Paraffin Method. Chicago, US: Read
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Esau K. 1977. Anatomy of Seed Plants. John Wiley and Sons, N.Y. p. 403-425.

Esau K and Evert RF. 2006. Esau’s Plant anatomy: meristems , cells, and tissues of the plant
body: their structure, function and development / updated by Ray F. Evert; with the assistance of
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Publishing Co., Inc. p377.

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Tolentino VS. 1995. Squash and Smear Technique: Handbook on Botanical Microtechnique.
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(Leguminosae: Caesalpinioideae):Developmental Evidence of Aperture Orientation Using
Confocal Microscopy. Annals of Botany. 98:107-115.
 24

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,
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Reproduction. 1265-1275.

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

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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.

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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].
 25

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