In this article we will discuss about the Mendel’s experiment with garden pea plant.
In 1856 Mendel began his experiments on plant hybridisation with garden peas in the monastery garden. Although similar work had already been done by contemporary botanists, the significant features of all these experiments had been overlooked because the investigators made overall observations of all inherited characters instead of collecting and analysing data in a systematic, mathematical way.
This is how Mendel achieved what his predecessors could not. First of all he concentrated his attention on a single character in his experiments on inheritance. Secondly, he kept accurate pedigree records for each plant. And third, he counted the different kinds of plants resulting from each cross. Fourthly, he analysed his data mathematically.
Mendel’s success is in part also attributed to his choice of material. The garden pea (Pisum sativum) used in his experiments (Fig. 1.1) offers certain advantages: it is an easily growing, naturally self fertilising plant; it is well suited for artificial cross pollination therefore hybridisation (crossing of two different varieties) is easily accomplished; it shows pairs of contrasting characters which do not blend to produce intermediate types and can be traced through successive generations without confusion.
For example tall and dwarf are a pair of contrasting conditions for the character height; similarly round and wrinkled seeds are contrasting forms for the character seed texture. On self pollination each character breeds true. Mendel worked with seven pairs of characters so that he had 14 pure breeding varieties.
Mendel crossed varieties of edible peas which showed clear-cut differences in morphological characters (Fig. 1.2) such as colour of flowers (red vs. white), shape of pod (inflated vs. constricted), colour of pod (green vs. yellow), texture of seed (round vs. wrinkled), colour of cotyledons (yellow vs. green), flower position (axial vs. terminal) and height of plant (tall vs. dwarf).
He was dusting the pollen of one variety on the pistil of the other. To prevent self-pollination of the female parent, he removed its stamens before the flowers had opened and shed the pollen. After making the cross he would enclose the flowers in bags to protect them from insects and foreign pollen.
Mendel’s first experiments explain how a single gene segregates in inheritance. When Mendel crossed a true breeding tall plant (female parent) with a true breeding plant of the dwarf variety (male parent), he got tall plants like one parent in the first filial generation designated F1.
He used the term “dominant” for the tall character which dominated in the F1 generation, and “recessive” for the character of dwarfness which remained hidden (latent) in the F1 generation.
Self fertilisation of the F1 hybrids produced the second filial generation F2 consisting of a total of 1064 plants of which 787 were tall and 277 were dwarf. That is tall and dwarf plants appeared in F2 in the proportion of 2.84:1 which is roughly equal to 3:1.
When he performed the reciprocal cross by reversing the sexes of the parents, the same results were obtained showing thereby that it did not matter which plant was used as male or as female parent.
Similarly, Mendel crossed pea plants differing in other characters such as colour of flowers (red flowered versus white flowered), texture of seed (round versus wrinkled), colour of cotyledons (yellow versus green). Such a cross which involves only one character from each parent is called a monohybrid cross.
In each case Mendel found one parental character dominating in the F1 hybrid, and after self fertilisation in F2 generation both parental characters appeared in the proportion of three-fourths to one-fourth. He performed each experiment on several thousand plants and counted all the plants in F2 progeny which gave an average ratio of 3:1.
Segregation of Genes:
From his experiments Mendel concluded that each parent contributes one factor for a character to the F1 hybrid. In this way the F1 hybrid has two factors for each character.
When the F1 hybrid forms gametes the two factors separate from each other. There is no mixing up of factors thus emphasizing the purity of gametes. The phenomenon of separation became Mendel’s First Principle and was later termed as the Law of Segregation.
This is explained diagrammatically as follows:
Terms Used in Mendel’s Crosses:
Dominant versus Recessive:
When two pure breeding varieties are crossed, the parental character that expresses itself unchanged in the F1 generation hybrids is dominant: the one that does not appear in F1 but appears in F2 is called recessive. In the above cross three-fourths of the F2 progeny show the dominant character and one-fourth the recessive character.
Factors which control contrasting expressions of a character are said to be alleles or allelomorphs of each other. In the above cross the character in consideration is height, and factors T and t which control tallness and dwarfness are alleles of each other.
Homozygous and Heterozygous:
These terms were coined by Bateson and Saunders in 1902. Mendel had concluded that each character is controlled by a pair of factors. When both factors are identical such as TT and tt, the individual is said to be homozygous for that character. When the factors are different (for example Tt), the term heterozygous is used. Mendel used capital letter of the alphabet to denote dominant factors, and small letters for recessive alleles.
Mendel’s factors were later replaced by the term ‘gene’ by a Danish botanist Johannsen in 1909. The word genotype refers to the genetic constitution of an individual, whereas phenotype refers to the external appearance or manifestation of a character.
Mendel selfed members of the F2 progeny and found that out of the dominant types, one-third bred true for the dominant character, whereas two-thirds segregated into dominants and recessives in the ratio of 3: 1. All the recessive plants of F2 generation when selfed bred true for the recessive character.
Mendel found similar results in monohybrid crosses with all the seven pairs of contrasting characters in Pisum sativum. After eight years of detailed investigations on thousands of pea plants, Mendel published his results in a paper entitled “Experiments in Plant Hybridisation” in the Proceedings of the Brunn Natural History Society in 1866.
However, his work received no attention for 34 years until three scientists, De-Vries in Holland, Correns in Germany and Tschermak in Austria working independently published their findings in 1900 and confirmed Mendel’s results.
The Test Cross:
Not satisfied with his work, Mendel himself subjected his results to a test. In the cross between tall and dwarf pea plants, the F1 hybrids were all phenotypically tall but their genotypes were not only TT but also Tt. Consider a heterozygous hybrid plant Tt. When it forms gametes, the factors T and t segregate in the gametes in a 1: 1 ratio.
This means that 50% of the gametes of an F1 heterozygous hybrid carry the factor T and 50% the factor t. Mendel crossed such a hybrid plant (Tt) with a plant of the true breeding, dwarf variety (tt). All the gametes of the homozygous dwarf plant carried the recessive factor t.
Every gamete of the recessive parent has 50% chance of combining with a gamete carrying T and 50% chance to combine with a t gamete from the heterozygous parent.
This should result in 50% of progeny showing the tall phenotype and genetic constitution Tt, whereas 50% of the progeny should be phenotypically dwarf with genotype tt as explained diagrammatically below:
Indeed Mendel’s results of this cross agreed with the theoretical expectations thus providing additional experimental proof of the correctness of his interpretations. Such a cross where an individual is crossed to a double recessive parent to test and verify the individual’s genotype is called a testcross or backcross.
In order to determine genotypes of the F2 progeny, Mendel allowed the F2 plants to self- fertilize and produce a third filial or F3 generation. He found that the homozygous F2 tall plants could produce only tall plants on self-fertilisation. This indicated their genotype to be TT.
Similarly the F2 dwarf homozygotes yielded only dwarf plants on selling; their genotype was tt. The F2 heterozygotes on self fertilizing behaved identical to the F1 hybrids and gave rise to tall and dwarf phenotypes in the ratio 3:1. This proved that their genotype was identical to that of F1 hybrids i.e. Tt.
It is noteworthy that the genotypes of the parents are written as TT and tt instead of single T and t. This is in accordance with Mendel’s hypothesis that each parent has two factors for a character. There is also a cytological explanation. The somatic chromosomes of all plants and animals exist in homologous pairs, one member of each pair coming from the paternal parent, other from maternal parent.
A gene is a section of the chromosomal DNA which has information necessary for determination of a specific genetic trait. Suppose a hypothetical gene A occupies a particular site or locus on a given chromosome. The homologous chromosome contains at the identical locus an alternative gene a which controls the same trait as gene A, but in such a way as to produce a different phenotype for the same trait.
The alternative genes at the same locus A and a are also called alleles. It is an astonishing fact that though Mendel knew nothing about genes, he could predict the existence of factors, which later turned out to be genes. During the reduction division of meiosis (Metaphase I), chromosomes of a pair separate and go to the opposite poles. Consequently genes or alleles segregate from each other and pass into different gametes.
Demonstration of Genetic Segregation:
Mendel’s F1 hybrids (Tt) were all tall plants indistinguishable phenotypically. Sometimes homozygous and heterozygous plants show phenotypic differences. There is a seedling character for green pigment in soybeans. The homozygous (GG) soybean plant is dark green, the heterozygous (Gg) plant light green.
The homozygous recessive (gg) produces a golden lethal seedling which dies in early stages due to lack of green pigment. If the heterozygous plants are grown to maturity and self-pollinated, their progeny will again segregate as dark green, light green and lethal golden in the ratio of 1: 2: 1.
Differences between homozygous and heterozygous genotypes can sometimes be observed in the gametes. In rice, sorghum and maize, effect of the gene for waxy endosperm is visible in the pollen grains. Maize kernels which have waxy endosperm produce starch and stain blue with iodine; non-waxy endosperm does not produce starch and stains red with iodine. In maize gene for waxy endosperm is located on chromosome 9.
A homozygous plant with genetic constitution WxWx produces starch in endosperm and stains blue with iodine. In the heterozygous plant (Wx wx) the dominant gene causes starch production and the kernels stain blue with iodine. But kernels on homozygous recessive plants (wx wx) have no starch and stain red with iodine. If anthers of these plants are treated with iodine, the pollen grains stain in a similar way.
In homozygous plants all the pollen grains stain blue. In heterozygous plants 50% of pollen grains stain blue (i.e. those containing Wx), whereas 50% stain red (i.e. pollen grains having wx).
In the homozygous recessive plant, all the pollen grains stain red. If breeding tests are done by self-pollinating the heterozygous F1 plants, the progeny consists of blue staining kernels (WxWx and Wxwx plants) and red staining kernels (wxwx plants) in the ratio 3:1.
The Dihybrid Cross:
Mendel made crosses between pea plants differing in two characters such as texture of seed and colour of cotyledons. Such a cross in which inheritance of two characters is considered is called a dihybrid cross.
First of all Mendel crossed a pea plant that was breeding true for round seeds with a plant that bred true for wrinkled seeds. The F1 indicated that roundness was dominant over wrinkled texture of seed coat. Similarly, by another cross he could determine that yellow colour of cotyledons was dominant over green.
He now used as male parent a plant which bred true for both round and yellow characters and crossed it with a female parent that bred true for wrinkled green. As expected from the results of his single crosses, the F1 was round yellow. When he selfed the F1 hybrids, the F2 progeny showed all the parental characters in different combinations with each other.
Thus plants with round yellow seeds, round green seeds, wrinkled yellow seeds and wrinkled green seeds all appeared in the ratio 9:3:3:1. Reciprocal cross in which the female parent was round yellow and male parent wrinkled green gave the same results.
Mendel applied the principle of a monohybrid cross and argued that in the dihybrid cross the true breeding round yellow parent must be homozygous RRYY, and the wrinkled green parent rryy. Since each character is determined be two factors, in a dihybrid cross there must be four factors present in each parent.
Likewise the F1 hybrid must be RrYy. But the question remained as to how did the four different combinations of parental phenotypes appear in the progeny? Mendel argued that the pair of factors for roundness must be behaving independently of the pair of factors for yellow colour of seeds. In other words, one factor for a character must be passing independently of a factor for another character.
Thus in the F1 hybrids, R and r pass into different gametes.
Now the probability of an R gamete formed is one-half, and of r gamete also one-half. Similar probabilities exist for Y and y gametes. It follows that the probability that R and Y should go to the same gamete is one-fourth, as also of R and y, r and Y, and r and y. Therefore, gametes containing factors RY, Ry, rY and ry should form in equal proportions.
The F1 hybrid producing the four types of gametes mentioned above was selfed. The results expected in the F2 progeny can be predicted by making a checkerboard or a Punnett Square. Gametes produced by one parent are plotted on top of the checkerboard, and gametes of the other parent on the side.
The sixteen squares of the checkerboard are filled up by making various possible combinations of male and female gametes during fertilisation. The phenotypes read out from the checkerboard indicate a 9: 3: 3: 1 ratio exactly as observed by Mendel.
As in the case of the monohybrid cross, Mendel verified his results by performing the test cross. He crossed the F1 hybrid heterozygous for both characters with a double recessive parent (rryy) which should produce only one type of gamete ry. The uniformity in the gametes of the recessive parent determines the differences in the types of gametes produced by the heterozygous parent.
Now the hybrid RrYy produces gametes carrying RY, Ry, rY and ry with equal frequency. It follows that during fertilisation if all these four types of gametes unite with ry gamete of the recessive parent, the resulting progeny should show all the four combinations of characters also in equal proportions. Indeed, Mendel observed the testcross progeny to consist of Round Yellow, Round Green, Wrinkled Yellow and Wrinkled Green plants in the ratio 1:1:1:1.
From the results of his dihybrid crosses, Mendel realised the following facts. At the time of gamete formation the segregation of alleles R and r into separate gametes occurs independently of the segregation of alleles Y and y.
That is why the resulting gametes contain all possible combinations of these alleles, i.e. RY, Ry, rY, ry. In this way Mendel proved that when two characters are considered in a cross, there is independent assortment of genes for each character, and this became the Law of Independent Assortment.