This document summarizes a case study on sex determination mechanisms in the dioecious plant species Salix viminalis (basket willow). The study involved crossing four female and four male plants in different combinations to observe sex ratios in the offspring. Several crosses showed significant deviations from a 1:1 sex ratio, with some being female-biased and others male-biased. Germination rates were generally high and did not explain the skewed sex ratios. The document discusses possible mechanisms for the biased sex ratios observed, such as meiotic drive, gametic selection, or cytoplasmic sex ratio disorders, but concludes that the exact mechanism in S. viminalis remains unclear based on the results of this study.

1. Sex determination mechanisms in
plants
Pawan Kumar Khati
CAU/CPGS/GPB/M15/02
MSc 2nd year
1

2. Introduction
Mechanisms of sex
determination
Sex determination in plants
Case study
Conclusion
CONTENTS
2

3. Introduction
 Sex-determination system is a biological
system that determines the development of
sexual characteristics in an organism.
 In many cases, sex determination is genetic:
males and females have different alleles or
even different genes that specify their sexual
morphology
 Camerarius (1694) gave first description of
reproductive organs in plants (maize)
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4. Why does sex exist?
 What evolutionary benefit do organisms gain by
developing diploidy and sexual processes?
-Adjusting to a changing environment.
-sexually reproduction allow much more variation.
 What is better about the combining of gametes to
produce a new generation of offspring?
-Combining beneficial mutations.
-Removing deleterious mutations.
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5. Mode of sexuality in flowers
5

6. Mechanisms
 Environmental
 Chromosomal
1. homomorphic chromosomes
Heterogametic male (XX, XY)
Heterogametic female(XY,XX)
2. heteromorphic chromosomes
XX, XY (active Y)
XX, XY1Y2 (X/autosome balance)
X1X1X2X2, X1X2Y1Y2
 Genic
Single locus
Multiple loci 6

7. Environmental sex determination
 Sex determination is either due to the environment or it is
greatly affected by the environment.
 In Equisetum plants,
Optimum condition – female adverse condition – male
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8.  In cucumber, melons, cannabis etc. the sex of flowers is
affected by many environmental factors
 Temperature, day length, ethylene, gibberellic acid and some
ions etc. usually, a treatment with ethylene or gibberellic acid
promotes production of female flower
 In Cannabis- GA3 induces the development of only female
flowers..
8
Contd…

9. Chromosomal sex determination
 It was first noted that the X chromosome
was special in 1890 by Hermann Henking
in Leipzig of firebug testicles.
 It was first suggested that the X
chromosome was involved in sex
determination by Clarence Erwin
McClung in 1901 after comparing his
work on locusts with Henking's and
others.
 Y chromosomes was discovered and
named by Stevens (1908) in drosophila.
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10. XX female, XY male
X and Y chromosomes are identical in morphology and segregate
randomly
 Spinach and Asparagus
 In addition to the major gene affecting sex, the Y chromosome
thought to contain a gene that suppresses the carpel development
and another gene that promotes stamen development.
 Mutations in these genes leads to hermaphrodite individuals and
asexual flowers. Both of this types are known in Asparagus.
 In Asparagus, Y chromosome appears functionally similar to the X
chromosome since YY males have been produced.
10
Homomorphic sex chromosomes

11. XY female, XX male
 Fragaria elateria (wild strawberry)-unisexual and are either
tetraploid, hexaploid or octaploid.
 Females produces 2 types of gametes with respect to sex
chromosomes – heterogametic sex.
 Male produces only one type of gamete with respect to sex
chromosomes – homogametic sex.
Male X diploid
hermaphrodite sp.
males or female
Female X diploid
hermaphrodite sp.
Female and hermaphrodite
or female and male
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Homomorphic sex chromosomes

12. Heteromorphic sex chromosomes
 X and Y chromosomes are distinct in their morphology.
 Generally, they are unable to pair over a significant portion of
their length.
 Silene, Rumex hastatulus, Rumex acetosa and Humulus
japonicus
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13. XX female, XY male
 Found in Cannabis, Silene (white campion), Rumex hastatulus.
 Egg cells have one X chromosome. Half of the pollen grains have
one X and remaining half have a Y chromosome.
 Random union of these gametes produces 50% of XX(female ) and
50% of XY(male) progenies.
 Operates in same manner as that found in mammals.
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Heteromorphic sex chromosomes

14. Active -Y chromosome
 Historically Y chromosome was considered to contain
degenerate genes or no genes
 This idea was based on some of the discoveries
In drosophila,
Flies without Y chromosome (XO)-viable
but flies without X chromosome (YO-YY)-inviable
 1959- Y in man is strongly male determining, that drastically
changed the earlier conclusions
 XXY, XXXY, XXXXY are males phenotypically in human.
 Also in mice, cats and other mammals
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15.  In plants, Y chromosome tends to be large
 The presence of a single Y chromosome can suppress female
development when three X chromosomes are present.
 X to Autosome ratios have no profound effects on the sex
determining factors present on the Y chromosome
15
Contd…

16. Female suppression region
Male promoter region
Male fertility region
Pairing region
Differential
region of X
X Y
16

17. X1X1X2X2 female , X1X2Y1Y2 male
 Found in some strains of Humulus lupulus
 Eggs have X1X2 chromosome constitution
 Males produces X1X2 and Y1Y2 pollen grains
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Heteromorphic sex chromosomes

18. Genic sex determination
 Found both in monoecious and dioecious
 Sex of an individual is governed by genes
Single locus - a single gene plays major role in sex
determination
Multiple loci – two or more genes
 Operating in maize, papaya, mercury, etc.
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19. Bisexual flower
sepals
petals
carpel
Sterile
perianth
Schematic diagram of the four floral whorls (1 to 4) and
three regions (A to C) of homeotic gene action
Stephen L. Dellaporta' and Alejandro Calderon-Urrea,1993

20.  It is possible that sex determination genes might selectively
affect the action of homeotic genes in one whorl.
 ex: stamen development is altered, without secondary effects
on carpel formation.
 In Arabidopsis homeotic mutation, flo70 replaces stamens
with carpels,
 Unisexual flowers often pass through a “bisexual stage” in
which all floral organs are initiated.
20
Contd…

21. Developmental steps affected by sex
determination process in maize
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Sexual development of male and female florets in the
inflorescences of maize
Stephen L. Dellaporta' and Alejandro Calderon-Urrea,1993

22. Developmental steps in maize.
• lnitiation of branch meristems or spikelet intials
on the inflorescence meristem.
• Spikelet initials bifurcate to form two spikelets
• Each floral primordium initiates an outer lemma
and an inner palea, three stamen initials and a
central gynoecium composed of three fused
carpels . Up to this point, floral development in
both ear and tassel inflorescences is nearly
identical. The action of sex determination genes
causes selective abortion of preformed floral
organs.
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23. Objective :To understand sex biasness in the absence of ecological disturbance
in dioecious species ‘Salix viminalis’ .
Experimental Material - Salix viminalis [Basket willow]
These are multi stemmed shrub growing to between 3 and 6 m.
Male and female catkins are borne on separate plants.
Commonly used in basketry. Other uses: effluent treatment in wastewater
gardens, and for water purification.
Case Study-1

24. Materials and methods: The four females and four males used as
parents in the breeding population were selected based on earlier crosses
between them which had led to varying sex ratios
•Sex determination experiment:
Crosses first grown in pots , and seedlings were transferred to the field.
Next year during flowering, sex ratio was observed.
4 female
×4 male
Sex
determination
experiment
13
crosses
6 crosses were
female biased.
2 crosses were
male biased.
5 crosses were
intermediate
Result

25. Sex determination experiments:
Of the 13 crosses 8 showed significant deviations from a 1 : 1 sex ratio. Six
crosses were female-biased, and 2 crosses were male-biased.

26. •Germination experiment: A total of 200 seeds per cross were used in
the germination experiment. Seeds were placed in petridishes at room
temperature in a greenhouse. After 24 hours, the number of seeds per
cross that had developed a cotyledon were observed
Ten of the crosses showed high germination frequencies, varying between 86%
and 100%, while seeds from 2 crosses (2 x6 and 3x6) germinated poorly.

27. In species with sex chromosomes, the following reasons have been suggested as possible
explanations of biased sex ratios
•Fitness differences between males and females,
•Sexual Lability,
•Gametic Viability Selection,
•Meiotic Drive and,
• Cytoplasmic sex ratio disorders
Discussion and Conclusions from the study:
In the current study, overall germination and survival frequencies were high, and no
trend in favor of one sex was observed. therefore Fitness variation is not the likely
major cause of the observed deviations from balanced sex ratios.
In our experiment none of the plants appeared to be hermaphrodites and/or have
labile sex expression. Moreover, no sex change, from male to female or the converse,
was observed.
Because distortion does not take place during germination or flowering, the skewed sex
ratios obtained in this study were not distorted during the diploid phase.

28. Meiotic drive, which is the differential production of X- and Y-chromosome-bearing
gametes by the heterogametic sex, has been shown to distort the sex ratio.
The sex ratio can also be distorted as a result of gametic selection, the differential
success of X- and Y- chromosome-bearing gametes in accomplishing fertilisation.
If it is assumed that females are heterogametic with respect to sex chromosomes,
meiotic drive and gametic selection
would both lead to variation in the sex ratio of the offspring among crosses sharing the
same father,
while no such variation would be expected among crosses sharing the same mother.
If males are heterogametic, the opposite would be expected.
In this study, that variation in the sex ratio of the offspring exists both among crosses
sharing the same father and among crosses sharing the same mother , making gametic
selection unlikely in S. viminalis.

29. If cytoplasmic sex ratio disorder are assumed to be responsible for sex biasness
This would involve several cytoplasmic factors and nuclear restorer loci.
If sex ratio modifers are nuclear a minimum of two loci independent of
the sex chromosomes are necessary to explain the sex ratios of crosses 1X6 and
4X6.
In conclusion, the skewed sex ratios in this study may not be explained solely by sex
chromosomes.
Environmental, nuclear-cytoplasmic or multi-locus sex determination may provide
explanations to the results.

30. Objective: Sex determination of F2 papaya plants by using RAPD based SCAR primers.
Materials:
 500 RAPD primers (10bp)
 Papaya cultivars; a) Kohopo
b) Sunrise
 SCAR primers
Case study 2

31. Methods
F2 plants from
cross between
Sunrise’ and UH
Line 365
25 plants
(hermaphrodite)
25 female
plants
Bulked
DNA
DNA
isolation
Bulked
DNA
Random amplification
with 500 RAPD
primers
Identification of
Primers giving
reproducible and
sex linked bands
Purification,
Cloning and
sequencing of
polymorphic
bands
Designing
SCAR
primers
Sex
determination

32.  Of the 500 RAPD primers: 3 primers viz T1, T12 and W11 gave
reproducible and sex linked band in hermaphrodite and female plants
 Those three RAPD products was cloned and a portion of their DNA
was sequenced and SCAR primers were design
 SCAR primers were used for PCR in genomic DNA of hermaphrodite
and female plants
Results

33.  SCAR T12 and SCAR W11
produced products in
hermaphrodite and SCAR T1
produce product in all the plants
regardless of plants sex
 SCAR T1 was used as positive
control in sex determination by
SCAR T12 and SCAR W11

34. Objective: Determination of sex in Simarouba glauca by RAPD markers
Materials:
 Simarouba glauca plants
a. Male
b. Female
c. Hermaphrodite
 85 RAPD 10 bp primers
3

35. Methods
Genomic DNA
isolationMale
Female and
Hermaphrodite
plants
PCR with 85 RAPD
10 bp primers
Identification of
reproducible
primers
Identification of
reproducible primers
showing
polymorphism
Use of
selected
primers for
sex
determination
in Simarouba
glauca

36. Results
 Out of 85 RAPD primers, 16 primers gave reproducible bands.
 From 16 reproducible RAPD primers, Five primers: OPU-10 (5’-
ACCTCGGCAC-3’), OPD-19 (5’-CTGGGGACTT-3’), OPU-19 (5’-
GTCAGTGCGG-3’), OPS-05 (5’-TTTGGGGCCT-3’) and OPW-03
(5’-GTCCGGAGTG-3’) produced unique amplicon for sex
differentiation

37. OPU-10 OPD-19 OPU-19
OPS-05 OPW-03

38. Conclusion
• Sex determination in plants is a complex mechanism
involving various factors.
• For crop improvement, a crop to be used in a breeding
program, the determination of sex in seedling stage will
reduced the time as well as input cost.
• Morphological markers like flower type etc. for
confirming sex of the plants requires tedious work and
large area to select sufficient no. of plants.
• Therefore, molecular marker are playing important role in
determining sex in early stages of the plants.

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