Woon Teck Yap
Section: M1-3, E53-220
Meeting 5
Out-of-class Exercise 2
1
Introduction
Artificial fertilization has long been employed as a method of producing new colour
variations in ornamental plants. It is observed that whenever members of the same
species are cross-fertilized, particular hybrid forms appear with astounding consistency
and regularity. In an attempt to elucidate the mechanism behind this phenomenon,
experiments involving the hybrids and their progeny were undertaken and are described
herein.
Many earlier researchers such as Kölreuter, Gärtner, Herbert, Lecoq and Wichura have
tried to determine the mechanism of inheritance behind hybrid reproduction.
In
particular, Gärtner has made some very interesting and valuable observations in Die
Bastarderzeugung im Pflanzenreiche regarding the remarkably fertile hybrids Aquilegia
atropurpurea canadensis, Lavatera pseudolbia thuringiaca, Geum urbanorivale and
Dianthus caryophyllus, chinensis and japonicus. The observations that Gärtner gathered
from his transformation experiments involving these plant species have led him to believe
that plant species are confined within certain evolutionary boundaries beyond which they
cannot change, even with hybridization via artificial cross-fertilization. This is in direct
opposition to the current opinions of naturalists who dispute the stability of present plant
species and firmly believe in a continuous evolution of vegetation i.e. plants evolve and
change constantly over time, by hybridization, to yield new plant species.
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Woon Teck Yap
Section: M1-3, E53-220
Meeting 5
Out-of-class Exercise 2
In addition, Wichura has recently published some results regarding his research on the
hybrids of the Willow. Research on hybrids is time-consuming, laborious and difficult,
with observation requiring several years or more. For this reason, no one has yet come
up with a suitable working model or “universal law” to describe the formation and
development of botanical hybrids. Such a model can be formulated if and only if detailed
experiments are made on a variety of plant species over long time periods and if the data
so collected are analyzed quantitatively.
At the moment, there are no published records of any experiments that have been
adequately and satisfactorily carried out to determine the number of different forms of
hybrid progeny and the separate generations in which these progeny occur. In addition,
statistical analyses of the relationships between the various forms of hybrid progeny have
not yet been performed. Such detailed theoretical and experimental data are required to
answer questions regarding the history and formation of any living species on earth.
This paper describes detailed experiments on a small plant group, which were carried out
carefully and painstakingly over a period of 8 years. It is left to the reader’s judgement as
to whether the experiments were suitably planned, conducted and executed, with regard
to obtaining valid results and sufficiently detailed analyses.
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Woon Teck Yap
Section: M1-3, E53-220
Meeting 5
Out-of-class Exercise 2
2
Materials and Methods
2.1
Growth of Pea Plants
All pea plants were grown in either garden beds or pots. Pea plants were maintained in
their natural upright position by means of sticks, branches of trees or string stretched
between them. For each particular experiment, i.e. set of cross-fertilization experiments,
a specific number of potted pea plants were placed in a greenhouse during the blooming
period to serve as controls for unwanted, random cross fertilization by insects.
2.2
Selection of Suitable Pea Plants
All experimental plants described in this paper were of the genus Pisum. They possessed
constant differentiating characteristics and produced hybrids and offspring that showed
no marked decrease in fertility in successive generations. All experimental plants were
derived from 34 more or less distinct varieties of peas obtained from several seedsmen.
These peas were grown and the resulting pea plants were then subjected to a 2-year trial,
in which the characteristics of the latter were carefully observed. Of these pea plants, 22
that exhibited highly similar characteristics were then selected and cultivated for the
entire 2-year period. All pea plants used in subsequent experiments were then derived
from these 22 plants.
2.3
Characteristics of Pea Plants Examined
Characteristics that were easy to follow and classify were chosen for careful observation
in the pea plants that were cultivated. The 7 characteristics thus chosen were as follows:
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Woon Teck Yap
Section: M1-3, E53-220
Meeting 5
Out-of-class Exercise 2
1. Surface morphology of ripe seeds: Seeds were either classified as round and smooth
or irregularly angular and deeply wrinkled. Seeds that were classified as round and
smooth were either round or roundish and any depressions that occurred on the
surface were always shallow.
2. Colour of the seed albumen (endosperm): The colour of the endosperm of the ripe
seeds fell into either one of two categories – green or yellow. The endosperm was
classified as green so long as it possessed a more or less green tint while it was
classified as yellow if it was either pale yellow, bright yellow or orange-coloured.
3. Colour of the seed-coat: The colour of the seed-coat fell into either one of two
categories – white or grey, grey-brown, leather-brown (with or without violet
spotting). White seeds coats were constantly correlated with white flowers being
observed in the pea plants while grey, grey-brown and leather-brown seed coats
constantly correlated with flowers in which the standards were violet and the wings
were purple. In addition, the axils of the leaves of these latter pea plants possessed a
reddish tint. All grey seed-coats, upon boiling in water, turned dark brown.
4. Morphology of ripe seed pods: Seed pods were either inflated and not contracted in
any places or more or less wrinkled and deeply constricted in the areas between the
seeds.
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Woon Teck Yap
Section: M1-3, E53-220
Meeting 5
Out-of-class Exercise 2
5. Colour of unripe seed pods: Unripe seed pods were either light to dark green or
vividly and intensely yellow. For yellow unripe seed pods, the stalks, leaf-veins and
calyx of the corresponding pea plants were also coloured yellow.
6. Position of flowers: Flowers were either classified as axial or terminal. Axial refers
to flowers being distributed along the main stem while terminal means that the
flowers were bunched at the top of the stem and arranged in a false umbel, in which
the upper part of the stem was more or less widened in cross-section.
7. Length of stem: The length of the stem was either categorized as long or short. Long
stems refer to those in which the long axis was around 6 to 7 feet while short stems
refer to those in which the long axis was around 0.75 to 1.5 feet.
2.4
Artificial Cross-Fertilization
Artificial cross-fertilization between two distinct pea plants was achieved by the careful
opening of the bud before it fully developed into a flower. The keel of the bud was then
removed and each stamen was carefully extracted by means of forceps so as to prevent its
pollen from falling onto the stigma present in the same bud.
The stigma was
subsequently dusted over with foreign pollen obtained from a pea plant exhibiting the
other constant differentiating character under investigation.
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Woon Teck Yap
Section: M1-3, E53-220
Meeting 5
Out-of-class Exercise 2
2.5
Monohybrid Inheritance
2.5.1 Production and Propagation of Monohybrids
Each pair of differentiating characters described above (Refer to Section 2.3) were united
into one pea plant by artificial cross-fertilization (Refer to Section 2.4) of two pea plants
which exhibited constant differentiation in one of the two distinct forms of a particular
character. The seeds thus obtained from such cross-fertilizations were then sown to
produce the monohybrids from which the “first generation from monohybrids” pea plants
were derived.
In total, 7 experiments were carried out in order to unite the 7 different characters under
investigation.
The details of these 7 experiments are listed in Table 1.
Fertilization/cross-fertilization was carried out on only the most vigorous members of a
large number of pea plants of the same variety. In experiments involving reciprocal
crossings, if a particular pea plant served as the seed-bearer in one set of fertilizations,
then it was used to provide pollen in the other set of fertilizations.
Table 1: Details of the 7 experiments in which the 2 different forms of a particular
character were united in the same plant by cross-fertilization.
Expt
Character Observed
1
2
3
4
5
6
7
Surface morphology of ripe seeds
Colour of seed albumen (endosperm)
Colour of seed coat
Morphology of ripe seed pods
Colour of unripe seed pods
Position of flowers
Length of stem
Number of
fertilizations
60
58
35
40
23
34
37
Number of plants
fertilized
15
10
10
10
5
10
10
6
Woon Teck Yap
Section: M1-3, E53-220
Meeting 5
Out-of-class Exercise 2
2.5.2 Production of First Generation Monohybrids from Cross-Fertilization of
Monohybrids
The monohybrids produced by the artificial cross-fertilization of parental pea plants with
constant differentiating characters (Refer to Section 2.5.1) were then artificially crossfertilized with other monohybrids within the same experimental set, as described in
Section 2.4. The seeds thus obtained were then grown to yield the “first generation from
monohybrids” pea plants.
2.5.3 Production of Second Generation Monohybrids from First Generation
Monohybrids
Seeds resulting from the artificial cross-fertilization of first generation monohybrid pea
plants (Refer to Section 2.5.2) were grown and their offspring were meticulously
observed, with respect to the 7 differentiating characters under investigation. These
offspring were termed the “second generation from monohybrids” pea plants.
2.6
Dihybrid Inheritance
2.6.1
Production and Propagation of Dihybrids
Dihybrid pea plants were obtained by artificial cross-fertilization (Refer to Section 2.4) of
two parental pea plants which exhibited constant differentiation in one of the two distinct
forms for each of the two characters observed – the surface morphology of the ripe seeds
and the colour of the seed albumen (endosperm). The seed-bearing parental pea plant,
henceforth known as the seed plant, gave seeds that were round and smooth with yellow
7
Woon Teck Yap
Section: M1-3, E53-220
Meeting 5
Out-of-class Exercise 2
albumens. The pollen-providing parental pea plant, henceforth known as pollen plant,
gave seeds that were irregularly shaped and wrinkled with green albumens. The smooth,
round seeds with yellow albumens thus obtained from the cross-fertilization of the truebreeding parental pea plants were then sown to produce the dihybrids from which the
“first generation from dihybrids” pea plants were derived.
2.6.2 Production of First Generation Dihybrids from Cross-Fertilization of
Hybrids
The dihybrids produced by the artificial cross-fertilization of parental pea plants, each
with 2 constant differentiating characters (Refer to Section 2.6.1) were then artificially
cross-fertilized with other dihybrids within the same experimental set, as described in
Section 2.4. The seeds thus obtained were then grown to yield the “first generation from
dihybrids” pea plants.
2.6.3 Production of Seeds from First Generation Dihybrid Pea Plants
The first generation dihybrid pea plants were allowed to self-fertilize so as to produce
ripe seeds.
The seeds thus obtained were then observed in terms of the surface
morphology of the ripe seed and the colour of the seed albumen (endosperm).
8
Woon Teck Yap
Section: M1-3, E53-220
Meeting 5
Out-of-class Exercise 2
3
Results
3.1
Monohybrid Inheritance
3.1.1
Characteristics of Monohybrids – Dominant Forms of Differentiating
Characters
For all 7 of the crosses that had been performed (Refer to Sections 2.3 and 2.4), it has
been observed that with respect to a specific form of a particular differentiating character,
the hybrids closely resembled one of the parental pea plants to such an extent that both
the hybrid and the parental pea plant were virtually indistinguishable. In fact, the other
form of the character exhibited by the other parental pea plant was never observed or
rarely observed with certainty. As such, the observed form of a particular differentiating
character that was exhibited by the hybrid and one of its parents was termed dominant
while the form exhibited by the other parent was termed recessive. A list of the 7
differentiating characters investigated and their respective dominant forms is presented in
Table 2.
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Woon Teck Yap
Section: M1-3, E53-220
Meeting 5
Out-of-class Exercise 2
Table 2: List of 7 differentiating characters of the pea plants investigated and their
corresponding dominant forms.
Expt
1
2
3
4
5
6
7
3.1.2
Differentiating Character
Dominant Form
Surface morphology of ripe seeds: Round and Round and smooth, with and
smooth or angular and wrinkled
without shallow depressions
Colour of seed albumen (endosperm): Green or
Yellow
yellow
Grey/grey-brown/leatherColour of seed coat: White with white flowers or
brown with violet-red
grey/grey-brown/leather-brown
with
purple
blossoms and reddish spots
flowers
in leaf axils
Morphology of ripe seed pods: Inflated or
Simple inflated
wrinkled
Colour of unripe seed pods: Green or yellow with
Green
similarly-coloured stems, leaf-veins and calyx
Position of flowers: Axial or terminal
Axial
Length of stem: Long (6 – 7 feet) or short (0.75 –
Long
1.5 feet)
Characteristics of the First Generation Monohybrids from CrossFertilization of Monohybrids
The first generation of pea plants obtained from the cross-fertilization of hybrids as
described in Section 2.5.2 was subjected to careful observation, with respect to the 7
differentiating characters under investigation. It was noted that in this generation, there
appeared both kinds of pea plants i.e., those with the dominant form of the differentiating
character under investigation and those with the corresponding recessive form. It was
also fairly obvious that the number of pea plants exhibiting the dominant form of a
particular character to that exhibiting the corresponding recessive form was in a 3:1 ratio.
In addition, transitional forms were not observed in any experiments, confirming the
hypothesis that one form was clearly dominant to the other.
The results of these
observations are summarized in Table 3.
10
Woon Teck Yap
Section: M1-3, E53-220
Meeting 5
Out-of-class Exercise 2
Table 3: Summary of results obtained from the cross-fertilization of hybrids within the
same experimental set, with respect to the differentiating character under investigation,
the number of hybrids observed, the specific plant part observed and the total number of
such observations, the dominant and recessive forms of the specific character observed
and their respective numbers and ratios.
Expt
1
2
3
4
5
Differentiating
Character
Seed surface
morphology
Colour of
albumen
Colour of seedcoats
Pod
morphology
Colour of
unripe pods
Number
Plant
of
Part /
Hybrids Number
Seed /
253
7324
Seed /
258
8023
929
Seed / -
1181
Pod / -
580
Pod / -
6
Flower position
858
Flower /
-
7
Length of stem
1064
Stem / -
Dominat
Form /
Number
Round /
5474
Yellow /
6022
Greybrown /
705
Inflated /
882
Green /
428
Axial /
651
Long /
787
Recessive
Form /
Number
Angular /
1850
Green /
2001
Ratio
Dominant:
Recessive
White / 224
3.15:1
Constricted
/ 299
Yellow /
152
Terminal /
207
Short / 277
2.96:1
3.01:1
2.95:1
2.82:1
3.14:1
2.84:1
The average ratio of dominant forms to recessive forms was found to be 2:98:1, closely
approximating a 3:1 ratio.
3.1.3
Characteristics of the Second Generation Monohybrids Grown from the
Seeds of First Generation Monohybrid Pea Plants
It was observed that those pea plants from the first generation that exhibited the recessive
forms of the differentiating characters produced offspring that consistently exhibited the
recessive forms. In addition, among the pea plants that exhibited the dominant forms in
the first generation, ⅓ of their offspring showed constant transmission of the dominant
11
Woon Teck Yap
Section: M1-3, E53-220
Meeting 5
Out-of-class Exercise 2
forms. The remaining ⅔ of their offspring gave rise to progeny that displayed the
dominant and recessive forms of the differentiating characters in the familiar 3:1 ratio.
Table 4 shows the results obtained following the procedure described in Section 2.5.3 for
the pea plants exhibiting the dominant forms of the 7 characters.
Table 4: Summary of characteristics of second generation pea plants grown from the
seeds of the first generation hybrid pea plants. Pea plants were classified according to the
type of seed from which they were grown, the number of such seeds used, whether they
exhibited only the dominant form or both the dominant and recessive forms of the
differentiating character under investigation and the respective ratios of both types of pea
plants.
Expt
1
2
3
4
5
6
7
Type of
Seed/Plant Used
Round seed
Yellow albumen
seed
Seed with greybrown seed coat
Plant with
inflated pods
Plant with green
pods
Plant with axial
flowers
Tall plant
Number of
Plants/Seeds
Used
565
Number of
Plants with
Both Forms
372
Number of
Plants with only
Dominant Form
193
1.93:1
519
353
166
2:13:1
100
64
36
1.78:1
100
71
29
2.45:1
100
60
40
1.5:1
100
67
33
2.03:1
100
72
28
2.57:1
Ratio
12
Woon Teck Yap
Section: M1-3, E53-220
Meeting 5
Out-of-class Exercise 2
3.2
Dihybrid Inheritance
3.2.1 Characteristics of Dihybrids Obtained from Cross-Fertilization of Parental
Pea Plants Differing in 2 Constantly Differentiating Characters
Let A denote the observed character for a round and smooth seed and a denote the
character for an irregularly-shaped and wrinkled seed. In addition, if B denotes the
observed character for a yellow albumen and b denotes that for a green albumen, then the
character of the parental seed plant would be AB and that of the parental pollen plant
would be ab.
The seeds obtained from the cross-fertilization of the parental pea plants were all smooth
and round, with yellow albumens i.e. AB. Cross-fertilization of 15 dihybrid pea plants,
grown from the smooth, yellow and round seeds, yielded 556 seeds with 4 different
combinations of morphologies and colour. Seeds of different combinations of characters
were frequently found in the same seed pod. Of these 556 seeds, 315 were round and
yellow, 101 were wrinkled and yellow, 108 were round and green and 32 were wrinkled
and green. These seeds were thus of characters AB, aB, Ab and ab, respectively.
3.2.2 Characteristics
of
the
First
Generation
Dihybrids
from
Cross-
Fertilization of Dihybrids
All 556 seeds obtained from the cross-fertilization of the dihybrid pea plants were sown
in order to produce the first generation dihybrid pea plants.
These first generation
dihybrid pea plants were then allowed to self-fertilize and produce ripe seeds. These
13
Woon Teck Yap
Section: M1-3, E53-220
Meeting 5
Out-of-class Exercise 2
seeds were then categorized according to their surface morphologies and the colour of the
seed albumen (endosperm). The characteristics of the seeds and their respective numbers
are summarized in Table 5.
Table 5: Summary of types of seed obtained from the cross-fertilization of parental
dihybrids, the respective numbers of the former and the resultant numbers of plants and
seed-bearing plants obtained from these seeds. The types of seeds obtained from the selffertilization of the first generation dihybrids, the numbers of plants yielding each type of
seed and the characters of the seeds are also listed.
Type of
Seed
Sown
Round &
Yellow
Wrinkled
& Yellow
#
Seeds
Sown
315
101
Round &
Green
108
Wrinkled
& Green
32
#
Resultant
Plants
304
96
108
30
# Seedbearing
Plants
Type of Seed
301
Round & Yellow
Round &
Yellow/Green
Round/Wrinkled &
Yellow
Round/Wrinkled &
Yellow/Green
96
102
30
#
Plants
Character
of Seed
38
AB
65
ABb
60
AaB
138
AaBb
Wrinkled & Yellow
28
aB
Wrinkled &
Yellow/Green
68
aBb
Round & Green
35
Ab
Round/Wrinkled &
Green
67
Aab
Wrinkled & Green
30
Ab
14
Woon Teck Yap
Section: M1-3, E53-220
Meeting 5
Out-of-class Exercise 2
4
Discussion
4.1
Law of Segregation
Referring to sections 3.1.1 and 3.1.2, it is obvious that if a pea plant exhibiting the
recessive form of a particular constantly differentiating character is cross-fertilized to one
exhibiting the dominant form of that character, the resulting monohybrids would only
exhibit the dominant form.
In addition, if these monohybrids are cross-fertilized to one another, their resulting
offspring, F1, exhibit a 3:1 ratio of dominant:recessive forms. If F1 pea plants exhibiting
the dominant form of a particular character are self-fertilized, it is observed that ⅓ of
them give rise only to offspring exhibiting the dominant form while the remaining ⅔ give
rise to offspring exhibiting both the dominant and recessive forms in the familiar 3:1 ratio.
This phenomenon is easily explained by adopting what shall be termed as the “law of
segregation”. Assume that in a normal (i.e. non-pollen/egg) cell of a pea plant, there
exist two distinct and separate units of inheritance for each character exhibited by the pea
plant. Each unit of inheritance can represent either the dominant or recessive form of a
particular character. In addition, assume that when egg cells (in the ovaries of the
flowers) or pollen cells (in the stamen) are formed, each egg/pollen cell only contains one
of the two units of inheritance found in a normal cell. The union of any particular egg
cell with any pollen cell to result in a new pea plant is taken to be totally random.
15
Woon Teck Yap
Section: M1-3, E53-220
Meeting 5
Out-of-class Exercise 2
Pea plants that are constantly differentiating in a particular form of a particular character
are assumed to contain two identical units of inheritance, both representing the same
dominant/recessive form. When a pea plant constantly exhibiting a recessive form (aa) is
crossed with that constantly exhibiting the corresponding dominant form (AA), an egg
cell with A is united with a pollen cell with a and vice versa, where A and a denote the
dominant and recessive units of inheritance, respectively. Each normal cell of the new
hybrid pea plant will contain 2 different units of inheritance, A and a. A cell would
exhibit the dominant form if it contained AA or Aa and the recessive form if it contained
aa.
The “law of segregation” proposes that during the formation of egg and pollen cells,
these units of inheritance segregate independently and unchanged into the former,
resulting in pollen and egg cells with A or a. When the monohybrids described above are
cross-fertilized with one another, random unions of egg and pollen cells are obtained,
resulting in normal cells that contain one of the following pairs of units of inheritance,
AA, Aa and aa. By simple probabilistic analyses, the chance of getting an AA or aa cell
is ¼ each while that of obtaining an Aa cell is ½. Since the observed form of an Aa cell
is the same as that of an AA cell, the probability of observing the dominant form is ¾
while that for the corresponding recessive form is ¼. This results in the familiar 3:1
dominant:recessive ratio observed in 3.1.2.
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Woon Teck Yap
Section: M1-3, E53-220
Meeting 5
Out-of-class Exercise 2
In addition, since ⅓ of the pea plants exhibiting the dominant form have AA cells, only
the remaining ⅔ would result in offspring that show both recessive and dominant forms.
Hence, the 2:1 ratio observed.
Thus, all observed results, with regard to monohybrid inheritance, support the proposed
“law of segregation”.
4.2
Law of Independent Assortment
Dihybrid inheritance in pea plants was observed by crossing parental pea plants whose
seeds differed in both surface morphology and albumen colour.
Following the
procedures described in Sections 2.6.2. and 2.6.3, the results in Section 3.2.2 are thus
obtained.
It is noticed that the 9 different characters observed can be classified into 3 essential
groups – those that are constant in both characters and thus do not vary in subsequent
generations, those that are constant in only one character and hybrid in another and thus
vary in subsequent generations with respect to the hybrid character and finally those that
are hybrid is both characters and vary in subsequent generations with respect to both
characters. The first class includes seeds whose characters are AB, ab, Ab and aB while
the second includes seeds whose characters are ABb, aBb, AaB, Aab. The last class
consists of only one member, AaBb.
17
Woon Teck Yap
Section: M1-3, E53-220
Meeting 5
Out-of-class Exercise 2
On the average, each of the forms in the first class appears 33 times while those in the
second and dihybrid classes appears 65 and 138 times, respectively. This 33:65:138 ratio
closely approximates 33:66:132 which reduces to 1:2:4. Using this ratio, a combination
series involving the 9 different forms is derived, being:
AB + Ab + aB + ab + 2ABb + 2aBb + 2AaB + 2Aab + 4AaBb
It is fairly obvious that this combination series separates out into the 2 different
expressions describing monohybrid inheritance with regard to seed surface morphology
and seed albumen colour, respectively:
A + 2Aa + a
B + 2Bb + b
Thus, it seems that the offspring of the hybrids in which several essentially different
characters are combined exhibit the terms of a series of combinations, in which the
developmental series for each pair of differentiating characters are united. In addition,
the relation of each pair of different characters in hybrid unions is independent of the
other differences in the two original parental stocks1. This latter observation shall be
termed as the “law of independent assortment” since it seems that the units of inheritance
representing the different forms of different characters are transmitted independently and
separately to subsequent generations.
18
Woon Teck Yap
Section: M1-3, E53-220
Meeting 5
Out-of-class Exercise 2
5
Conclusion
Using cross-fertilization to monitor 7 differentiating characters in pea plants of the genus
Pisum, it was determined that the two different observed forms of a particular character
may be either dominant or recessive to each other.
In addition, observation of
monohybrid inheritance in these pea plants through 2 generations after the parental
monohybrids, allowed for the formulation of the “law of segregation.”
Dihybrid
inheritance involving seed surface morphology and seed albumen colour resulted in the
production of 9 combinations of the 4 different forms of both differentiating characters.
Analyses of these results led to the establishment of the “law of independent assortment.”
19
Woon Teck Yap
Section: M1-3, E53-220
Meeting 5
Out-of-class Exercise 2
[1]
I shall provide references in the next revision of this paper.
20