Exercise 8 Aim: To study the blastula stage of embryonic development in mammals, with the help of permanent slide, chart, model or photograph Principle:The zygote undergoes a fewcycles of mitotic divisions to form a solid ball of cells called morula. The cells continue to divide and at a later stage a cavity is formed within it. This stage is blastula. The internal structural details of blastula can be observed in its transverse section. Requirement: Permanent slide, chart/model of T.S. of blastula, compound microscope, lens cleaning fluid and paper Procedure Observe the slide under lower magnification of the microscope. In case of chart/models/photographs, note the feature of blastula in your practical record and draw labelled diagram. Observation In transverse section, the blastula appears as a sphere with a cavity, called blastocoel within it (Fig. 8.1). Notice an outer layer of blastomeres called trophoblasts. A cellular mass, adhered to the trophoblast is present on one end of the blastula. It is called inner cell mass. Fig.8.1 Blastula stage of a mammal EXERCISE 8 Questions 1. What are the differences between blastula and morula? 2. What are the main structures you observe in T.S. blastula? 3. Match the stages in column I with features in column II Column I Column II (a) Trophoblast (i) Dividing cells of the morula (b) Morula (ii) Outer layer of blastula (c) Blastocoel (iii) Solid ball of cells (iv) Cavity 31 Exercise 9 Aim: To verify Mendel's Law of Segregation Principle: When two pure lines with contrasting forms of a particular character (phenotypes) are crossed to produce the next generation (F1 generation), all the members of the progeny are of only one phenotype i.e. of one of the two parents. The phenotype that appears is called dominant, and the one that does not appear is called recessive. When the F1 plants are selfed, the progeny i.e. the F2 generation is in the ratio of 3 dominant: 1 recessive (¾: ¼ or 75%: 25%). This reappearance of the recessive phenotype in F2 generation verifies law of segregation. Requirement: 64 yellow and 64 green plastic beads, all of exactly same shape and size, (when beads are not available, pea seeds may be coloured using paint, these beads represent the gametes of a specific trait), plastic beakers/petri dishes and a napkin/hand towel Students have to work in pairs to perform the experiment. The following steps are to be strictly followed in the sequence mentioned below. (i) Put 64 yellow beads in one beaker/petridish and 64 green beads in the other to represent respectively male and female gametes. Let the yellow bead be indicated by ‘Y’ and green bead by ‘y’. (ii) Take a bead from each container and place them together (it represents fertilisation) on the napkin spread before you on the table. (One student to take out beads and to put in the hands of the other student who will put them on the table). (iii) Just like the previous step, continue to pick beads and arrange them in pairs. Thus 64 pairs of beads are obtained representing the 64 heterozygous F progeny. 1 Note that all the F1 individuals are represented by one yellow and one green bead. (iv) Put 32 F progeny in one petridish and the remaining 32 in another 1 petridish (representing the F1 males and females). (v) Stir the beads of each petridish with a pencil/pen for about 10 times taking care that no bead falls off. EXERCISE 9 (vi) To obtain the F2 generation, one student would withdraw one bead from one beaker labelled male and one from the other beaker labelled female keeping his/her eyes closed (to ensure randomness), and put them together in the stretched palm of the partner, who will put them together on the napkin spread over the table. Continue this process till all the beads are paired. Thus 64 offsprings of F2 are obtained. (vii) Note the genotype (YY or Yy or yy) of each pair, and their possible phenotype. (viii) Have six repeats of the experiment (steps i to vii) with partners changing their roles. Pool all the data from the six repeats together. (ix) Calculate the genotypic and phenotypic ratios of your pooled data. Note that larger the sample size, more accurate is the result. Record the result in the following table: Generation Repeat No. Total no. of individuals Genotype (s) YY Yy yy Phenotype (s) F1 1. 2. 3. 4. 5. 6. Total F2 1. 2. 3. 4. 5. 6. Total Phenotypic Ratio: in F1……. in F2……. Genotypic Ratio: in F1……. in F2……. 33 LABORATORY MANUAL: BIOLOGY The results are so because each diploid individual contains two copies of every gene - one copy on each of the two homologous chromosomes. These two copies of the gene may be of similar type (YY or yy) or are dissimilar Yy. The former (YY or yy) are called homozygous for that particular character, and the Yy are called heterozygous ones. The pure lines in the above cross are homozygous ones, which contributed only one copy of their gene (as a result of meiosis) to their F1 progeny to restore its diploid nature with genotype Yy (heterozygous) where only one form(allele) is expressed (dominant) and the other form (allele) is not expressed (recessive). This is the phenomenon of Dominance. When the F1 individuals are crossed together to raise the F2 generation, each F individual produces two types of gametes: 50% having dominant 1 allele, and the remaining 50% having recessive allele. These gametes undergo random fusion during fertilisation to produce the F2 generation. According to simple probability of mixing of opposite sex gametes (sperms and ova), offsprings of three genotypes are likely to appear as follows: [(half of gametes of Y type + half of remaining gamete y type) X (half gametes of Y type + half of remaining gamete of y type)] = One-fourth of F2 individuals of YY phenotype + half of F individual Yy type + one-fourth of F individul of yy type. Among 22 these proportion of dominant phenotype would be � YY+ � Yy = � yellow and recessive phenotype � yy i.e. green phenotypes in 3:1 or 75%:25% ratio. This ratio of 3:1 in the F suggests that in the F heterozygotes, the 21 recessive allele does not get destroyed and remains only in the recessive (dormant) state to get an opportunity to express itself when it has separated from the influence of the dominant allele (Y). This is called Law of Segregation of the alleles. Questions 1. Do you expect the same results in terms of 3:1 ratio in F if you had started with 2 smaller number of beads (say 10 beads)? 34