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  Section: Biotechnology Methods » Cell Biology and Genetics
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Breeding Experiments in Drosophila Melanogaster

Cell Biology and Genetics
  Cell Cycles
  Meiosis in Flower Buds of Allium Cepa-Acetocarmine Stain
  Meiosis in Grasshopper Testis (Poecilocerus Pictus)
  Mitosis in Onion Root Tip (Allium Cepa)
  Differential Staining of Blood
  Buccal Epithelial Smear and Barr Body
  Vital Staining of DNA and RNA in Paramecium
  Induction of Polyploidy
  Mounting of Genitalia in Drosophila Melanogaster
  Mounting of Genitalia in the Silk Moth Bombyx Mori
  Mounting of the Sex Comb in Drosophila Melanogaster
  Mounting of the Mouth Parts of the Mosquito
  Normal Human Karyotyping
  Black and White Film Development and Printing for Karyotype Analysis
  Study of Drumsticks in the Neutrophils of Females
  Study of the Malaria Parasite
  Vital Staining of DNA and RNA in Paramecium
  Sex-Linked Inheritance in Drosophila Melanogaster
  Preparation of Somatic Chromosomes from Rat Bone Marrow
  Chromosomal Aberrations
  Study of Phenocopy
  Study of Mendelian Traits
  Estimation of Number of Erythrocytes [RBC] in Human Blood
  Estimation of Number of Leucocytes (WBC) in Human Blood
  Culturing Techniques and Handling of Flies
  Life Cycle of the Mosquito (Culex Pipiens)
  Life Cycle of the Silkworm (Bombyx Mori)
  Vital Staining of Earthworm Ovary
  Culturing and Observation of Paramecium
  Culturing and Staining of E.coli (Gram’s Staining)
  Breeding Experiments in Drosophila Melanogaster
  Preparation of Salivary Gland Chromosomes
  Observation of Mutants in Drosophila Melanogaster
  ABO Blood Grouping and Rh Factor in Humans
  Determination of Blood Group and Rh Factor
  Demonstration of the Law of Independent Assortment
  Demonstration of Law of Segregation

Life cycle of Drosophila melanogaster
Drosophila melanogaster is a common fruit fly used as a test system and has contributed to the establishment of the basic principles of heredity. It is also called the “Cinderella of Genetics”. Drosophila melanogaster is a dipterous, holometabolous insect. It has a characteristic larval stage preceded by the egg and succeeded by the pupalstage.

Egg is about 0.5 mm in length, ovoid in shape, and white. Extending from the anterior dorsal surface, there is a pair of egg filaments. The terminal portion of these filaments are flattened into spoonlike floats. This floats keep the egg from sinking into the semi-liquid medium.

The larva hatches out from the egg. It is white, segmented, and wormlike. The head is narrow and has black mouth parts (jaw hooks). The larva undergoes 2 moults, so that the larval phase consists of 3 instars. After this stage, the larva crawls out of the medium and finally attaches to the inner drier surface of the bottle. This culminates in pupation.

Soon after the formation of the “pupal horn” from the anterior spiracle, the larval body is shortened and the skin becomes hardened and pigmented. The pupa is considered a reorganization stage. During this process, most of the
adult structures are developed from the imaginal disc. A fully transformed adult fly emerges out through the anterior end of the pupal case. At the times of eclosion, the fly is greatly elongated and light in color, with wings yet to be unfolded. Immediately after this, the wings unfold and the body gradually turns dark and brown. After 6 hours of emergence, the adult fly attains the ability to participate in reproduction.

The body is divided into head, thorax, and abdomen. The head has a pair of compound eyes and a pair of antennae. The thorax is divided into 3 segments— prothorax, mesothorax, and metathorax, each with a pair of legs. The mesothorax has a pair of wings and the metathorax has a pair of halters. The abdomen is segmented in 4 or 5 sections in males and 6 or 7 in females. The abdominal tip in males is darkly pigmented.

Morphology of Drosophila Melanogaster
The body of an adult Drosophila melanogaster is divided into 3 parts, namely the head, thorax, and abdomen.
  1. Head. The head is composed of 6 fused segments, a pair of antennae with
    plumose aristae, and a licking proboscis without mandibles. On the dorsal
    side of the head between the compound eyes are 3 simple eyes called
    ocelli. Bristles are found on the head.
  2. Thorax. It is composed of 3 fused segments, namely the prothorax,
    mesothorax, and metathorax. All 3 segments have a pair of wings. The
    metathorax has halters (reduced wings).
  3. Abdomen. The abdomen consists of 7 or 8 visible segments in the female
    and 5 or 6 segments in male.
Differentiation between Male and Female Drosophila

Media Preparation
Drosophila melanogaster, like other animals, requires an optimum temperature for its survival, growth, and breeding. The optimum temperature for the maintenance of Drosophila melanogaster is between 20°C to 25°C. The temperature around and above 31°C makes the flies sterile and reduces the oviposition, and may result in death. At any lower temperature, the life cycle is prolonged and the viability may be impaired.

The routinely used food media for the maintenance of Drosophila melanogaster is cream of white agar medium. The ingredients of this media are:
1000 mL of distilled water
100 gm of wheat flour (sooji)
100 gm of jaggery
10 gm of agar agar
7.5 mL of propionic acid, and
Yeast granule.

Heat-sterilized bottles should be used for preparing culture. Similarly, sterilized cotton has to be used to plug the bottles. As the condition of the medium deteriorates with time, the flies have to be transferred from old to new bottles, with fresh culture medium at least once every 3 weeks.

Take a clean vessel and boil 1000 mL of water. Then add 10 gm of jaggery and stir well. To this add 10 gm of agar agar, which acts as a solidifying agent. Once it boils, add 100 gm of sooji. Then add 7.5 mL of propionic acid, which
acts as an antimicrobial agent. By constant stirring, the medium becomes a viscous fluid. The hot mixture is transferred into the culture bottle. The bottles are left for cooling, yeast is added, and they are plugged with cotton. Bottles are ready to use only after adding the yeast solution.

When flies have to be analyzed, either for routine observation or for experiments, they are anaesthetized to make them inactive. The procedure is to transfer flies from the media bottle to another empty wide-mouthed bottle, referred to as an etherizer. The mouth of this bottle is to be covered with a stopper sprayed with ether. It takes a minute or so to anaesthetize flies. After this, if etherized flies revive before completion of observation, they have to be re-etherized by using re-etherizer. The re-etherizer is an ether-soaked filter paper fitted in a petri plate, which has to be placed over the flies on the glass plate.

Adult flies are 2–3 mm long, while females are slightly larger than males. The males carry a sex comb on the first tarsal segment of the first leg. Males can also be identified by the presence of black pigmentation at the tip of the rounded abdomen. The tip of the female’s abdomen is pointed and nonpigmented. After the separation of the 2 sexes, the unwanted flies should be discarded immediately into the morgue (a bowl of mineral oil or detergent in water).

Isolation of Virgins
In many experiments with Drosophila, it is essential that the sperm of a particular genotype (male) is used for fertilization of a particular female. To ensure this, it is often essential to isolate virgin females. The females can store and utilize the sperms from 1 insemination for a large part of the reproductive life. Females that have any chance of being nonvirgins should not be used for crosses. For ensuring virginity, females are removed before 6 hrs of their emergence and males are removed from the culture bottle before 12 hrs of emergence.
Making and Conducting Crosses To make crosses between different strains 1–10 virgins from the first strain are mated in a culture bottle with the corresponding number of males from other strains. The reciprocal mating of the 2 strains is also done. As soon as the cross is made, the bottle is marked with the nature of the cross and the date of crossing. If the larva does not appear after 5–7 days, then the culture is discarded. If the culture is successful, then the parents are discarded and the already-laid eggs are allowed to develop into adults.

Analysis of the Progeny
The aim is to understand the pattern of inheritance of a character from parents to offspring and to subsequent generations. Therefore, each progeny (F1, F2, and test cross) has to be carefully analyzed and classified according to the phenotype and sex of each individual. Utmost care must be taken to record the number of flies in each category. From each experiment bottle, the counting must be restricted to the first 7 days from the third day of eclosion. A minimum of 200 flies must be analyzed from each of the F1, F2, and test cross progenies.
Statistical Test and Confirmation of Results The data obtained from the analysis of the progeny have to be tested with an established hypothesis. This has to be done to ascertain whether the observed data work with the hypothesis or not. The routinely used statistical test for such an experiment is the Chi square test:

x2 = ε (0 – E)2

The chi square test obtained from this test and the degree of freedom (df ) df = n – 1 are checked for the level of significance in the chi square probability table.

If the calculated value of the chi square is less than the table value at a particular level of significance, the difference between the observed and expected frequency is not significant. Then we have to accept the hypothesis.

On the other hand, when the calculated value is more than the table value, the difference between the observed and expected value is significant, then we have to reject the hypothesis.


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