Report 5: Meiosis in angiosperm , microsporogenesis

INTRODUCTION :
Meiosis is the second important kind of nuclear division. It resembles mitosis in many ways but the consequences of meiotic divisions are very different from those of mitotic divisions. While mitotic division may occur in almost any living cell of an organism, meiosis occurs only in special cells. In animals, meiosis is restricted to cells that form gametes (eggs and sperm). Each species has a characteristic number of chromosomes per somatic cell. Fruit flies have 8; normal humans have 46. They exist as homologous pairs (partners) that are similar in size and shape and carry the same kinds of genes. Thus humans have 23 homologous pairs. The full complement of 46 chromosomes is referred to as the diploid number (referring to the fact that each kind of chromosome is represented twice). In higher organisms when an egg is fertilized the egg and sperm fuse to form a single cell called a zygote which develops into a new organism. If the egg and sperm were both diploid (46 chromosomes each in the case of humans) then the resulting zygote would be tetraploid. This would be an intolerable situation, so a mechanism has evolved to insure that each gamete (egg or sperm) contains only one representative of each homologous pair (or half the diploid number). This is referred to as the haploid number.
In this experiment, the meiotic process is occurs in angiosperm. Pollen mother located in anthers undergo meiosis and form microspores. The haploid nucleus of each microspore subsequently divides mitotically to give rise to a three nucleate male gametophyte. Pollen grains in the anthers are transferred to the stigma of the pistil by insects or wind. There they germinate and begin to grow a pollen tube, which passes down the style and into the ovary of the pistil. The pollen tube carries the sperm nuclei to the ovule. The pollen tube enter the ovule through the ovule through a pore in the integuments called the micropyle; once inside the ovule, the pollen tube burst and frees the sperms nuclei inside the female gametophytes. The pollen tube nucleus then disintegrate.
Double fertilization occurs in angiosperms. One of the sperm nuclei carried into the gametophyte unites with the egg to form diploid zygote subsequently divide mitotically to give rise embryo. The second sperm nucleus unites with the two polar nuclei of the female gametophytes.

LEARNING OUTCOMES:
1. Prepare an aceto-orcein squash of anthers from a flower buds.
2. Demonstrate the stage of meiosis in microsporogenesis.
3. Identify the basic features of meiosis

MATERIALS
1. 70% ethyl alcohol
2. Absolute ethyl alcohol
3. Acetocarmine stain in dropping bottle
4. Bunsen burner
5. Cover slip
6. Teasing needles
7. Forceps
8. Compound Microscope
9. Scalpel or razor blade
10. Slides
11. Supply of immature flower buds (chives)
12. Watch glass

METHODS
1. An individual flower bud was remove from the storage container and was placed in a watch glass. A few drops of 70% ethyl alcohols were added to keep the anther moist.
2. Individual flower buds moist in alcohol were continually kept to dissect the anthers from the bud.
3. Some anther was transfer to a clean microscope slide and was placed in a drop of aceto-orcein stain. A teasing needle was use to macerates the anthers, freeing the microsporocytes from the anther walls.
4. A cover slip was applied and was heat over the Bunsen burner. Stain was added to prevent dying.
5. Paper toweling was use to cover the slide after heating and was press down firmly with thumb. This will flatten the microsporocytes and make it possible to observe the chromosome in the various meiotic stages.
6. If anther from more than one flower is crushed, different stages are likely to be observed. The slide was observed by using the compound microscope.

RESULT

MEIOTIC STAGES	NAME OF PLANT : CHIVES / KUCHAI
	SLIDE 1	SLIDE 2	SLIDE 3
PROPHASE 1	a
LEPTONEMA
ZYGONEMA
PACHYNEMA
DIPLONEMA
DIAKINESIS
METAPHASE I
ANAPHASE I
TELOPHASE I	a	a
PROPHASE II
METAPHASE II
ANAPHASE II
TELOPHASE II	a	a
MATURE POLLEN GRAIN

QUESTION AND ANSWER
1. Does crossing over occur before or after chromosome duplication in cells going through meiosis?
Crossing over occurs after the chromosome duplicate because the chromosome duplicate during the S phase.
2. What visible characteristics of chromosome indicate that they have undergone crossing over during meiosis?
The formation of chiasma where the place occur the crossing over.
3. During meiosis, when does the chromosomes disjunction occur? When does chromatid disjunction occur?
Anaphase I.Anaphase II.
4. In flowering plants such as kuchai, two nuclei from the pollen grain participate in the events of fertilization. With which nuclei from the female gametophyte do these nuclei combine? What tissues are formed from the fertilization events? This event is called double fertilization.
The pollen grain contains 2 nuclei will develop to form two sperm microsporocyte. One of the sperm fuse with ovum and another one fuse with polar nuclei. This will form triploid endosperm tissue that provides food for the seed.

DISCUSSIONS:
From this experiment, I can observed the prophase I, telophase I and telophase two. In prophase I, I determined this stages because of the chromosome are condensed and the color of the center of the cell are darker then each cell. For telophase I, I observed that the cell is dividing into 2 clusters and for telophase II I observed that there are four clusters in one cells.
In prophase I of meiosis I, there are 5 sub-stages. The first one is leptotene where the chromosome starts to condense. Followed by zygotene was the pairing of the homologous chromosome ad become closely associated during synapsis and the formation of the Synaptonemal complex. The third sub-stage is pachytene were the crossing over occur, forming chiasmata, which hold homologous chromosome together. In diplotene, the Synaptonemal complex start to disappear and in the last stage, diakinesis, the Synaptonemal complex is completely disappearing.
In metaphase I, the pair of homologous chromosome aligns along the metaphase plate in double row. The formation of chiasmata before are to help keep the pairs together and position the pairs such that only one side of each homologue’s centromere faces outward toward of the cell’s poles. Thus kinetochore microtubules attach to only one side of each centromere; a kinetochore microtubules from one pole of the cell attaches to one homologue of the chromosome while a kinetochore microtubules from the other cell poles attaches to other homologue.
In anaphase I, kinetochore microtubules shorten and homologous pairs are pulling apart. One duplicated homologue goes to the other poles. Sister chromatids do not separate. This is in contrast to mitosis, where duplicated homologues line up individually on the metaphase plate, kinetochore microtubules from opposite poles of the cell attach to opposite sides of one homologue’s centromere, and sister chromatids are pulled apart in anaphase.
In telophase I, the separated homologues form a cluster at each poles of the cell, and the nuclear membrane re-forms around each daughter cell nucleus. Cytokinesis may occur. The resulting two cells have half the number of chromosome as the original cell. Each chromosome is still in the duplicated state and consists of two sister chromatids, but sister chromatids are not identical because crossing over has occurred.
During prophase II, a new spindle apparatus forms in each cells and the nuclear membrane breaks down and disappears. In metaphase II, a complete spindle apparatus is in place in each cell. Chromosome consisting of sister chromatids joined at the centromere aligns along the metaphase place in each cell. Now kinetochore microtubules from opposite poles attach to opposite side of the same centromere.
When microtubules are shortening in anaphase II, the centromeres split and sister chromatids are pulled to opposite poles of the cells. In telophase II, the nuclear membranes re-form around four different clusters of chromosomes. After cytokinesis, four haploid cells are produce. NO two cells are alike due to the genetic recombination (crossing over) that occurred during prophase I.

CONCLUSION:
As a conclusion, we are able to prepare an aceto-orcein squash of anthers from the flower bud. We also able to demonstrate the stages of meiosis in microsporogenesis. We are also able to identify the basic features of meiosis as it occurs in angiosperm.

REFERENCES: 
Brooker, R. J. (2009). Genetics Analysis & Principle. Third edition: Mc Graw Hill international edition.
Eldra P. Solomon, Linda R. Berg, and Diana W.Martin. (2005). Biology. Third Edition: Thomson Brooks/Cole.
Kenneth R. Miller,and Joseph Levine. (2004). Prentice Hall Biology. Teacher's Edition: Pearson Publishing.
Campbell O'N. (2005). Biology. Seventh Edition: Benjamin Cumming.