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By
B. Pavan Kumar Naik
1st Ph. D Horticulture
Credit Seminar on
SELF INCOMPATIBILITY IN
VEGETABLES
Annamalai University
Faculty Of Agriculture
Department Of Horticulture, Chidambaram
Chairperson
Dr. J. Sam Ruban
Associate Professor
Department of Horticulture
Members
1. Dr. Arumugam Shakila
Professor
Department of Horticulture
2. Dr. Y. Anbuselvan
Professor
Department of Genetics & Plant Breeding
CONTENT
• Introduction
• Key breakthrough in timeline of research
• Main features of self-incompatibility
• Mechanism of SI
• Bateman classification of self-incompatibility
• Lewis classification of self-incompatibility
• Methods to asses self incompatibility
• Molecular model of SI in Brassicaceae
• Molecular model of SI in Solanaceae
• Need to use of self-incompatibility in the hybrid breeding
• Significance of self-incompatibility (Hybrid production)
• Temporary suppression of self incompatibility
• Limitations in exploiting self-incompatibility
INTRODUCTION
• First reported by Koelreuter (1850)
• Growers (1989) has defined self-incompatibility as “the
prevention of fusion of fertile (functional) male and female
gametes after self-pollination.
• Self-incompatibility is a genetic mechanism of avoiding
self-fertilization that leads to natural out breeding where
self-recognition and rejection is the rule.
• It promotes heterozgosity and prevents inbreeding
depression.
Key breakthroughs in the timeline of research on Self-
Incompatibility
MAIN FEATURES OF SI
• SI important outbreeding mechanism
• SI species do not produce seed on self pollination
• SI maintains high degree of heterozygosity & reduces homozygosity
• SI results due to morphological, genetic, physiological and
biochemical causes
Bateman (1952) Classification of self-incompatibility :
1. Complementary systems of self-incompatibility
2. Oppositional system of self-incompatibility
When pollen and pistil belong to same SI they produce certain chemicals which will prevent
pollen germination and growth. In a compatible reactions no chemicals are inhibiting normal growth
and development of pollen will be resulting in a successful fertilization.
Pollen of one SI group Stigma of other SI group
stimulate germination and
growth of pollen tube results
successful fertilization
Classification of Self-incompatibility
Lewis, 1954
Self-
Incompatibility
Heteromorphic system Homomorphic System
Gametophytic
(Solanaceae)
Sporophytic
(Brassicaceae)
HETEROMORPHIC SYSTEM
• In the heteromorphic system, there are morphological differences
among the flowers of the same plant. eg., primula (Primula
species) there are two types of flowers pin (ss) having long style
and short stigma and thrum having a short style and long stigma
(Ss).
• This character is controlled by a single gene S; having two alleles.
• The alleles for short style are dominant over long style.
• The only compatible mating is between pin and thrum flowers and
progeny have pin and thrum flowers in 1:1 ratio.
CROSSES RESULTS
Phenotype Genotype Genotype Phenotype
Pin x Pin ss x ss Incompatible mating
Pin x Thrum ss x Ss 1 Ss : 1 ss 1 Thrum: 1 Pin
Thrum x Pin Ss x ss 1 Ss : 1 ss 1 Thrum : 1 Pin
Thrum x Thrum Ss x Ss Incompatible mating
Conti…
• In some plant another situation called tristyly occurs. In this case style are of three length:
Ex. Brinjal
HOMOMORPHIC SYSTEM
• Self incompatibility results due to physiological causes rather than differences in
flower morphology.
• In this system, the plants do not have differences in the length of style and stamens or
other floral parts. This system is very much important in crop plants.
It can operate in various ways,
 The pollen grains do not germinate on the stigma of same flower.
 If they germinate the pollen tube fails to penetrate the stigma as in Rye, Cabbage and
Radish.
 The pollen grains may germinate but there is retardation of pollen tube growth.
 In some cases, there is slow rate of pollen tube growth and it rarely reaches the ovary
in time to effect self fertilization.
Gametophytic system
• Incompatibility reaction of pollen is determined by its own genotype.
• S-allele in style show co-dominance
Sporophytic system
• In this system the self-incompatibility is governed by a single gene, S, with multiple
alleles.
• Incompatibility reaction of pollen is governed by the genotype of plant,
• It was 1st reported by Hughes and Babcock in 1950
Mechanism of SI
1. Pollen-stigma interaction
2. Pollen tube – style
interaction
3. Pollen tube – ovule
interaction
Dresselhausa and Franklin-Tong, 2013
https://getrevising.co.uk/revision-cards/plant-reproduction
METHODS TO ASSESS SELF INCOMPATIBILITY
• Pollination method
• Cytological method
• Molecular method
Narayanapur et al. 2018
Pollination Method
• Pollination methods varies depending upon the type of self-
incompatibility and crop.
• For example, in cabbage, after selfing, the self-incompatibility is
assessed by counting the number of seeds after harvest.
• After selfing wait for 60 days till the pod maturity and count the
number of seed set per pod.
• If more number of seeds are formed then it is self-compatible, if
less seeds are formed then it is incompatible.
RESEARCH STUDY
• Ma, et,al. developed B. napus Self-incompatible line i.e, S-1300.
• Their result shown that salt solution can overcome the self-incompatible of S-1300 and this can
be amplified by salt spraying with bee or hand pollination.
Chaozhi Ma et al. 2009
Cytological Method
• In cabbage, the self-incompatibility reaction is assessed by number of pollen
tube penetration in the style.
• Stigma and style are squashed on a microscope after 48 hours of pollination.
Aniline blue is the stain used.
• It will get accumulated in the pollen tube and become fluorescent when
irradiated with UV light.
• If there is no or less penetration of pollen tube in the style, then it is
incompatible.
• If penetration of pollen tube is intermediate, then it is semi-compatible and
penetration by many pollen tube in the style then it is compatible.
Narayanapur et al. 2018
Assessment of self-incompatibility in cabbage by cytological
method
FIG: Floral morphology and pollination behaviour during flower development in A. thaliana and A. halleri. Appearance and inner
morphological structure of flower buds, with floral developmental stages (Smyth et al., 1990). Flower buds of Old-1 and W302
were emasculated at developmental stage 12 and incubated at 22°C on 1% agar medium until appropriate developmental stages.
Each emasculated pistil at developmental stages 12, 13, 14, and 15 was self-pollinated in W302 and cross-pollinated with pollen of
W302 in Old-1 to test the female SI function of Old-1. Scale bar, 1 mm. Self-incompatible and self-compatible floral stages are
highlighted with blue and yellow boarders, respectively, in self-pollinated pistils of A. halleri and A. thaliana pistils pollinated with
A. halleri pollens. Arrows indicate growing pollen tubes. Scale bar, 0.1 mm. (Keita Suwabe et al. 2020)
RESEARCH STUDY
Molecular basis of self-incompatibility
• Self-incompatibility system has been studied extensively in case of
Solanaceae and brassicaceae family of vegetables.
• It involves two proteins located at S locus. One is male determinant
and other is female determinant.
S. locus controlling self-incompatibility
The S-locus complex and a model of self-incompatibility in Brassica. June B. Nasrallah, 1977
List of the identified female and male determinant genes
Family Types of SI Male
determinant
Female
determinant
Brassicaceae SSI SP11/SCR SRK
Solanaceae GSI SLF/SFB S-RNase
Muthuselvi and Praneetha, 2019
S locus/ S related genes and their function/s in
Brassicaceae members
S-locus related gene Gene function Species name
SLG Doubtful B. oleracea
SRK Female determinant B. oleracea
SP11/SCR Male determinant B. rapa
MLPK Positive regulator B. rapa
ARCI Positive regulator B. napus
THLI Negative regulator B. napus
KAPP Putative SRK interactor B. oleracea
Calmodulin Putative SRK interactor B. oleracea
sp locus Putative suprpressor B. napus
Narayanapur and Minimol, 2018
The number of alleles and type of self-incompatibility identified
in different plants
Crop No. of S alleles Type of SI Reference
B. oleraceavar. capitata 50 Sporophytic Bassett (1986)
B. campestris 30 Sporophytic Singh (2012)
Raphanus sativus 32 Sporophytic SI Karron et al. (1989)
Raphanus raphanistrum 9 Sporophytic SI Sampson (1964)
Molecular model of SI in Brassicaceae
• Two genes SP11/ SCR is present, SP11 is male determinant and SRK
is the female determinant. SLG is promoter of incompatibility
reaction.
• SRK acts in the plasma membrane of papilla cells of stigma while
SP11 get expressed in the anther tapetum during the maturation of
pollen grain.
• In a SI reaction, when pollen grain land on stigma SP11 will bind
with SRK and leads to autophosphorylation resulting in prevention of
pollen tube growth.
• But in a compatible reaction, SP11 is not get activated and hence,
normal pollen germination and fertilization takes place.
Vijayakumar et al. 2018
Molecular model of SI in Solanaceae
• In Solanaceae family, SLF/ SFB is the male determinant and S-RNase is the
female determinant.
• During SIC reaction, when pollen falls on stigma, S-RNase is produced and
enter into the stigmatic surface.
• They will degrade the RNA encoding the enzyme for pollen tube growth and
result in death of pollen tube.
• In incompatible reaction also RNase is produced and it enter the stigma but it
goes and forms a complex with SLF.
• Hence, RNA encoding enzyme for pollen tube growth is not disturbed and
results in normal pollen tube growth and fertilization.
SELF INCOMPATIBILITY FINAL
Significance of self-incompatibility
• Self-incompatibility effectively prevents self pollination.
• As a result, it has profound effect on breeding approaches and
objectives.
Hybrid production
• Self-incompatibility is primarily exploited for hybrid production.
• If self-incompatible lines are available tedious process of emasculation
can be avoided.
• If both lines are self-incompatible, they can be utilized for forward and
reverse crosses.
• By using self-incompatibility double cross and three way cross hybrids
can be produced.
Single, Double and three way cross hybrid seed production
FIG: Application of self-incompatibility in practical plant breeding. Sporophytic incompatibility is widely
used in breeding of cabbage and other Brassica species. Single-cross hybrids are more uniform and easier
to produce. The top cross is commonly used. A single self-incompatible parent is used as female, and is
open pollinated by a desirable cultivar as the pollen source. (Gulzar et al. 2012)
RESEARCH STUDY
Chaozhi Ma et al. 2009
a b c d
Visualization of seed set of self-incompatible line S-1300 in different conditions a:
selfing in isolation; b:selfing and bud-pollinating; c: propagation by salt solution;
d: hybrid seed production
RESEARCH STUDY
M. NIEUWHOF, 1974
From 30 varieties and strains of
cauliflower the degree of self-
incompatibility was tested. It was
found that the early cauliflower
varieties show a low level of self-
incompatibility and most of the late
cauliflower varieties a higher level of
self-incompatibility.
Conti…
• The different reaction of the self-compatible and
the more self-incompatible varieties is also
reflected after bud-pollination.
• When bud-pollinating the first group of varieties
seed-set was mostly lower than when selfing
freshly opened flowers, on an average 19 %.
• This indicates that when no incompatibility barrier
is present the conditions for seed production are
more favourable after selfing freshly opened
flowers than after selfing the young buds.
• But when such a barrier is present, as in self-
incompatible varieties, bud-pollination is more
effective.
• Thus, the average seed-set of the self-incompatible
varieties was 89% higher after bud-pollination
than after selfing open flowers.
M. NIEUWHOF, 1974
Conti…
• It was clearly demonstrated that early
cauliflower varieties are very self-compatible.
• After selfing the early varieties observed a
somewhat lower seed set than after crossing.
This might indicate the presence of a low
degree of self-incompatibility in this group of
varieties.
• The autumn and winter varieties possess a
distinctly higher degree of self-incompatibility
than the early cauliflower varieties.
M. NIEUWHOF, 1974
RESEARCH STUDY
P. K. SINGH, 2012
Conti…
P. K. SINGH, 2012
Conti…
• Hybrid seed production using self-incompatibility involves the
development and maintenance of parents which are homozygously
selfincompatible but cross-compatible, so the degree of self-
incompatibility and stability in the inbred lines is of utmost
importance. This is largely dependent on environmental factors,
especially temperature.
• Uniform, effective cross-pollination may be of great importance for
obtaining a high quantity of hybrid seed in self-incompatible types.
• The two inbred lines, Desi Red and Khasi Kata, can be used as
parental lines to produce single cross hybrid seed.
• The lines should be grown in alternate rows or with two to four rows
of each, and adequate insect pollination should be ensured. The F1
populations of such hybrids will be uniform.
P. K. SINGH, 2012
RESEARCH STUDY
• SI has been reported to be preferred over male sterility in crop
species with entomophilous pollination since pollen-collecting bees
rarely visit male-sterile plants.
• Nonetheless, SI also may have disadvantages, Ex. F1 hybrids of
two SI parents are also SI and this may be undesirable for crop
production.
• SI F1 hybrids are not a handicap for ornamental or vegetable crops,
but it may hinder seed production (e.g., oilseed rape).
• Consequently, breeding programs favor not only SI female lines,
but also SC F1 hybrids (Kaothien-Nakayama et al., 2010)
Muñoz-Sanz JV et al. 2020
SI based systems as alternatives to androsterility for producing F1
hybrids. Self-(in)compatible parents and F1 hybrids are indicated.
Hybrids developed using self-incompatibility
CROPS HYBRIDS
Cauliflower Pusa Hybrid 2, Pusa Kartik Sankar, Snow queen, Snow
king
Cabbage Pusa Synthetic, Pusa Cabbage 1, Meenakshi, BRH-5,
H-43, H-44
Chinese cabbage Hamburg-3
Raddish Pusa Chetaki, Pusa Desi, Half Red, Chinese Pink, BDI-
689
Need to use of self-incompatibility in the hybrid breeding of
vegetable crops
• Used in hybrid seed production.
• Self incompatible line may be inter planted with a self compatible line. Seed
from only the self-incompatible line would be hybrid.
• The production of double cross and triple cross hybrids can be produced in
case of Brassicas.
Level of SI in Brassica oleracea L.
Crop Level of SI Reference
Kale and
Round headed cabbage
High Thompson, 1965
Nelson, 1927
Broccoli High to moderate Nelson, 1927
Thompson and Taylor, 1965
Autumn and winter
cauliflower
Moderate Watts, 1963
White sprouting broccoli
and brussels-sprout
Moderate to low Johnson, 1955
Green kohl rabi and purple
sprouting broccoli
Low Nelson, 1927
Early summer cauliflower Very low Watts, 1963,
TEMPORARY SUPPRESSION OF SELF
INCOMPATIBILITY
 Bud Pollination
 Increased CO2 concentration
 Salt (NaCl) sprays
 High temperature treatment
RESEARCH STUDY
BUD POLLINATIONS
RESEARCH STUDY
-Cabin et al. 1995
RESEARCH STUDY
SALT (NaCl) SPRAYS
-Carafa and Carratu, 1997
RESEARCH STUDY
Conti…
Self pollen grain germinate and pollen tubes penetrate the stigmatic papillae after stigma treatment with
0.5 M NaCl.
Conti…
RESEARCH STUDY
INCREASED CO2 CONCENTRATION
-Dhaliwal and Malik, 1982
RESEARCH STUDY
Conti…
RESEARCH STUDY
SHORT-TERM HIGH TEMPERATURE TREATMENT
Okazaki and Hinata, 1987
RESEARCH STUDY
Conti…
Many pollen tubes penetrated the pistil in self
pollination after the 40oc - 15 min treatment (x
100)
Callose formed in papillae (arrows) in which
selfed pollen tubes had penetrated after
treatment (x 200). Abnormal pollen tubes
growing in pistil treated at 50o C for 15 min ( x
200)
Limitations in exploiting self-incompatibility
• Continues selfing will lead to inbreeding depression.
• Continues inbreeding may lead to complete loss of the inbred lines.
• Hybrid seed would be expensive if the self-incompatible lines are
difficult to maintain.
• Environmental factor reduce or totally overcome SI.
• Preferential visit of pollinating insects.
• Transfer of S-allele is tedious and complicated.
THANK YOU
REFERENCES
1. Bateman, A. J. (1955) Heredity 9, 52–68
2. Watanabe M, Suwabe K, Suzuki G. 2012. Molecular genetics, physiology and biology of self-incompatibility in
Brassicaceae. Proc. Jpn. Acad., Ser. 88(10): 519-535.
3. Hee-Jeong Jung , Nasar Uddin Ahmed, Jong-In Park , Mi-Young Chung, Yong-Gu Cho, Sup Nou. 2013. Molecular Genetic
Aspects of Self-incompatibility in Brassicaceae. Plant Breed. Biotech, 1(3):205-217.
4. Gulzar S. Sanghera, Waseem Hussian, Gurpreet Singh and G .A. Parray. 2012. Molecular Perspective to Understand the
Phenomenon of Self-Incompatibility and Its Utilisation in Crop Plants. INROADS, Vol. 1, No. 2.
5. Anubhav Thakur1 and K.C. Dhiman. 2021. Self-Incompatibility in Vegetable Crops – A Review. Just Agriculture, Vol.1
Issue-10: 2582-8223.
6. Suwabe K, Nagasaka K, Windari EA, Hoshiai C, Ota T, Takada M, Kitazumi A, Masuko-Suzuki H, Kagaya Y, Yano K,
Tsuchimatsu T, Shimizu KK, Takayama S, Suzuki G and Watanabe M (2020) DoubleLocking Mechanism of Self-
Compatibility in Arabidopsis thaliana: The Synergistic Effect of Transcriptional Depression and Disruption of Coding
Region in the Male Specificity Gene. Front. Plant Sci. 11:576140. doi: 10.3389/fpls.2020.576140.
7. June B Nasrallah, 2017. Plant mating systems: self-incompatibility and evolutionary transitions to self-fertility in the mustard
family. Current Opinion in Genetics & Development, 47:54–60.
8. June Nasrallah B. Recognizing self in the self incompatibility response. Plant Phy. 1987; 125:105-108.
9. June Nasrallah B. Cell Cell signaling in the self incompatibility response. Current Opinion in Plant Biology. 2000; 3:368-
373.
10. Nasrallah, J. B. 1997. Evolution of the Brassica self-incompatibility locus: A look into S-locus gene polymorphisms.
Proceedings of the National Academy of Sciences, 94: 9516-9519.
Conti…
11. Verma, S. K., Anokhe, A., Singh, A., Kumar, R., Kumari, S. and Debbarma, R. (2018). Self-incompatibility a Mechanism for
Controlled Pollination in Vegetable Crops. International Journal of Current Microbiology and Applied Sciences, 7: 4602 – 4607.
12. Muthuselvi, R. and Praneetha, S (2019). Molecular basis of self-incompatibility in vegetable crops. International Journal of
Chemical Studies, 7(4): 506-510.
13. Dresselhaus, T. and Franklin-Tong, N. (2013). Male–Female Crosstalk during PollenGermination, Tube Growth and
Guidance, andDouble Fertilization. Molecular Plant, 6(4):1018-1036.
14. Robert J. Cabin, Ann S. Evans, Diane L. Jennings, Diane L. Marshall, Randall J. Mitchell, and Anna A. Sher. (1996). Using
bud pollinations to avoid self-incompatibility: implications from studies of three mustards.. Can. J. Bot. 74: 285-289.
15. Yong Yang ID , Zhiquan Liu, Tong Zhang, Guilong Zhou, Zhiqiang Duan, Bing Li, Shengwei Dou, Xiaomei Liang, Jinxing
Tu, Jinxiong Shen, Bin Yi, Tingdong Fu, Cheng Dai and Chaozhi Ma. (2018). Mechanism of Salt-Induced Self-Compatibility
Dissected by Comparative Proteomic Analysis in Brassica napus L. International Journal of Molecular Sciences, 19, 1652;
doi:10.3390/ijms19061652.
16. Carafa, A. M. and Carratu, G. (1997). Stigma treatment with saline solutions: Anew method to overcome self-incompatibility
in Brassica oleracea L. Journal of Horticultural Science, 72(4): 531-535.
17. Xintian Lao,, Keita Suwabe, Satoshi Niikura, Mitsuru Kakita, Megumi Iwano, and Seiji Takayama. (2014). Physiological and
genetic analysis of CO2-induced breakdown of self-incompatibility in Brassica rapa. Journal of Experimental Botany, Vol. 65,
No. 4, pp. 939–951.
18. Dhaliwal, A. S. and Malik, C. P. (1982). Effect of CO2 in ovecroming self-incompatibility bar¡ in Brassica campestris L. var.
toria . Proe. Indian Ac, ad. Sci. (Plant Sci.), Voi. 91: 227-234.
19. A. Palloix, y. Herve, R. B. Knox and C. Dumas. (1985). Effect of carbon dioxide and relative humidity on self incompatibility
in cauliflower, Brassica oleracea. Theor Appl Genet, 70:628-633 .
20. Okazaki, K. and Hinata, K. (1987). Repressing the expression of self-incompatibility in crucifers by short-term high
temperature treatment. Theor Appl Genet, 73: 496- 500.
Conti…
21. Dresselhaus, T.and Franklin-Tong, N. (2013). Male–Female Crosstalk during
Pollen Germination, Tube Growth and Guidance, and Double Fertilization.
Molecular Plant, 6(4): 1018-1036.
22. Narayanapur, V. B., Suma, B. and Minimol, J. S. (2018). Self-incompatibility:
a pollination control mechanism in plants. INTERNATIONAL Journal Of Plant
Sciences, 13(1): 201-212.
23. Johnson, A. 0. 1955. Ann. Rep. Nat. Veg. Res. Sta. for 1955, p. 14.
24. ThompsoN, K. F. 1957. Self-incompatibility in marrow-stem kale. 1.
Demonstration of a sporophytic system. 3'. Genet., 55, 45-60.
25. ThompsoN, K. F., AND TAyLOR, j. s'. 1965. Identical S alleles in different
botanical varieties of Brassica aleracea. Nature, 208, 306-307.
26. Watts, L. N. 1963. Investigations into the breeding System of cauliflower
Brassica aleracea var. batrytis (L.). I. Studies of self-incompatibility. Euphytica,
12, 323-340.
27. Nelson, A. 1927. Fertility in the Genus Brassica. 3'. Genet., 18, 109-136.

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SELF INCOMPATIBILITY FINAL

  • 1. By B. Pavan Kumar Naik 1st Ph. D Horticulture Credit Seminar on SELF INCOMPATIBILITY IN VEGETABLES Annamalai University Faculty Of Agriculture Department Of Horticulture, Chidambaram Chairperson Dr. J. Sam Ruban Associate Professor Department of Horticulture Members 1. Dr. Arumugam Shakila Professor Department of Horticulture 2. Dr. Y. Anbuselvan Professor Department of Genetics & Plant Breeding
  • 2. CONTENT • Introduction • Key breakthrough in timeline of research • Main features of self-incompatibility • Mechanism of SI • Bateman classification of self-incompatibility • Lewis classification of self-incompatibility • Methods to asses self incompatibility • Molecular model of SI in Brassicaceae • Molecular model of SI in Solanaceae • Need to use of self-incompatibility in the hybrid breeding • Significance of self-incompatibility (Hybrid production) • Temporary suppression of self incompatibility • Limitations in exploiting self-incompatibility
  • 3. INTRODUCTION • First reported by Koelreuter (1850) • Growers (1989) has defined self-incompatibility as “the prevention of fusion of fertile (functional) male and female gametes after self-pollination. • Self-incompatibility is a genetic mechanism of avoiding self-fertilization that leads to natural out breeding where self-recognition and rejection is the rule. • It promotes heterozgosity and prevents inbreeding depression.
  • 4. Key breakthroughs in the timeline of research on Self- Incompatibility
  • 5. MAIN FEATURES OF SI • SI important outbreeding mechanism • SI species do not produce seed on self pollination • SI maintains high degree of heterozygosity & reduces homozygosity • SI results due to morphological, genetic, physiological and biochemical causes
  • 6. Bateman (1952) Classification of self-incompatibility : 1. Complementary systems of self-incompatibility 2. Oppositional system of self-incompatibility When pollen and pistil belong to same SI they produce certain chemicals which will prevent pollen germination and growth. In a compatible reactions no chemicals are inhibiting normal growth and development of pollen will be resulting in a successful fertilization. Pollen of one SI group Stigma of other SI group stimulate germination and growth of pollen tube results successful fertilization
  • 7. Classification of Self-incompatibility Lewis, 1954 Self- Incompatibility Heteromorphic system Homomorphic System Gametophytic (Solanaceae) Sporophytic (Brassicaceae)
  • 8. HETEROMORPHIC SYSTEM • In the heteromorphic system, there are morphological differences among the flowers of the same plant. eg., primula (Primula species) there are two types of flowers pin (ss) having long style and short stigma and thrum having a short style and long stigma (Ss). • This character is controlled by a single gene S; having two alleles. • The alleles for short style are dominant over long style. • The only compatible mating is between pin and thrum flowers and progeny have pin and thrum flowers in 1:1 ratio. CROSSES RESULTS Phenotype Genotype Genotype Phenotype Pin x Pin ss x ss Incompatible mating Pin x Thrum ss x Ss 1 Ss : 1 ss 1 Thrum: 1 Pin Thrum x Pin Ss x ss 1 Ss : 1 ss 1 Thrum : 1 Pin Thrum x Thrum Ss x Ss Incompatible mating
  • 9. Conti… • In some plant another situation called tristyly occurs. In this case style are of three length: Ex. Brinjal
  • 10. HOMOMORPHIC SYSTEM • Self incompatibility results due to physiological causes rather than differences in flower morphology. • In this system, the plants do not have differences in the length of style and stamens or other floral parts. This system is very much important in crop plants. It can operate in various ways,  The pollen grains do not germinate on the stigma of same flower.  If they germinate the pollen tube fails to penetrate the stigma as in Rye, Cabbage and Radish.  The pollen grains may germinate but there is retardation of pollen tube growth.  In some cases, there is slow rate of pollen tube growth and it rarely reaches the ovary in time to effect self fertilization.
  • 11. Gametophytic system • Incompatibility reaction of pollen is determined by its own genotype. • S-allele in style show co-dominance
  • 12. Sporophytic system • In this system the self-incompatibility is governed by a single gene, S, with multiple alleles. • Incompatibility reaction of pollen is governed by the genotype of plant, • It was 1st reported by Hughes and Babcock in 1950
  • 13. Mechanism of SI 1. Pollen-stigma interaction 2. Pollen tube – style interaction 3. Pollen tube – ovule interaction Dresselhausa and Franklin-Tong, 2013
  • 15. METHODS TO ASSESS SELF INCOMPATIBILITY • Pollination method • Cytological method • Molecular method Narayanapur et al. 2018
  • 16. Pollination Method • Pollination methods varies depending upon the type of self- incompatibility and crop. • For example, in cabbage, after selfing, the self-incompatibility is assessed by counting the number of seeds after harvest. • After selfing wait for 60 days till the pod maturity and count the number of seed set per pod. • If more number of seeds are formed then it is self-compatible, if less seeds are formed then it is incompatible.
  • 17. RESEARCH STUDY • Ma, et,al. developed B. napus Self-incompatible line i.e, S-1300. • Their result shown that salt solution can overcome the self-incompatible of S-1300 and this can be amplified by salt spraying with bee or hand pollination. Chaozhi Ma et al. 2009
  • 18. Cytological Method • In cabbage, the self-incompatibility reaction is assessed by number of pollen tube penetration in the style. • Stigma and style are squashed on a microscope after 48 hours of pollination. Aniline blue is the stain used. • It will get accumulated in the pollen tube and become fluorescent when irradiated with UV light. • If there is no or less penetration of pollen tube in the style, then it is incompatible. • If penetration of pollen tube is intermediate, then it is semi-compatible and penetration by many pollen tube in the style then it is compatible. Narayanapur et al. 2018
  • 19. Assessment of self-incompatibility in cabbage by cytological method
  • 20. FIG: Floral morphology and pollination behaviour during flower development in A. thaliana and A. halleri. Appearance and inner morphological structure of flower buds, with floral developmental stages (Smyth et al., 1990). Flower buds of Old-1 and W302 were emasculated at developmental stage 12 and incubated at 22°C on 1% agar medium until appropriate developmental stages. Each emasculated pistil at developmental stages 12, 13, 14, and 15 was self-pollinated in W302 and cross-pollinated with pollen of W302 in Old-1 to test the female SI function of Old-1. Scale bar, 1 mm. Self-incompatible and self-compatible floral stages are highlighted with blue and yellow boarders, respectively, in self-pollinated pistils of A. halleri and A. thaliana pistils pollinated with A. halleri pollens. Arrows indicate growing pollen tubes. Scale bar, 0.1 mm. (Keita Suwabe et al. 2020) RESEARCH STUDY
  • 21. Molecular basis of self-incompatibility • Self-incompatibility system has been studied extensively in case of Solanaceae and brassicaceae family of vegetables. • It involves two proteins located at S locus. One is male determinant and other is female determinant.
  • 22. S. locus controlling self-incompatibility The S-locus complex and a model of self-incompatibility in Brassica. June B. Nasrallah, 1977
  • 23. List of the identified female and male determinant genes Family Types of SI Male determinant Female determinant Brassicaceae SSI SP11/SCR SRK Solanaceae GSI SLF/SFB S-RNase Muthuselvi and Praneetha, 2019
  • 24. S locus/ S related genes and their function/s in Brassicaceae members S-locus related gene Gene function Species name SLG Doubtful B. oleracea SRK Female determinant B. oleracea SP11/SCR Male determinant B. rapa MLPK Positive regulator B. rapa ARCI Positive regulator B. napus THLI Negative regulator B. napus KAPP Putative SRK interactor B. oleracea Calmodulin Putative SRK interactor B. oleracea sp locus Putative suprpressor B. napus Narayanapur and Minimol, 2018
  • 25. The number of alleles and type of self-incompatibility identified in different plants Crop No. of S alleles Type of SI Reference B. oleraceavar. capitata 50 Sporophytic Bassett (1986) B. campestris 30 Sporophytic Singh (2012) Raphanus sativus 32 Sporophytic SI Karron et al. (1989) Raphanus raphanistrum 9 Sporophytic SI Sampson (1964)
  • 26. Molecular model of SI in Brassicaceae • Two genes SP11/ SCR is present, SP11 is male determinant and SRK is the female determinant. SLG is promoter of incompatibility reaction. • SRK acts in the plasma membrane of papilla cells of stigma while SP11 get expressed in the anther tapetum during the maturation of pollen grain. • In a SI reaction, when pollen grain land on stigma SP11 will bind with SRK and leads to autophosphorylation resulting in prevention of pollen tube growth. • But in a compatible reaction, SP11 is not get activated and hence, normal pollen germination and fertilization takes place.
  • 28. Molecular model of SI in Solanaceae • In Solanaceae family, SLF/ SFB is the male determinant and S-RNase is the female determinant. • During SIC reaction, when pollen falls on stigma, S-RNase is produced and enter into the stigmatic surface. • They will degrade the RNA encoding the enzyme for pollen tube growth and result in death of pollen tube. • In incompatible reaction also RNase is produced and it enter the stigma but it goes and forms a complex with SLF. • Hence, RNA encoding enzyme for pollen tube growth is not disturbed and results in normal pollen tube growth and fertilization.
  • 30. Significance of self-incompatibility • Self-incompatibility effectively prevents self pollination. • As a result, it has profound effect on breeding approaches and objectives.
  • 31. Hybrid production • Self-incompatibility is primarily exploited for hybrid production. • If self-incompatible lines are available tedious process of emasculation can be avoided. • If both lines are self-incompatible, they can be utilized for forward and reverse crosses. • By using self-incompatibility double cross and three way cross hybrids can be produced.
  • 32. Single, Double and three way cross hybrid seed production FIG: Application of self-incompatibility in practical plant breeding. Sporophytic incompatibility is widely used in breeding of cabbage and other Brassica species. Single-cross hybrids are more uniform and easier to produce. The top cross is commonly used. A single self-incompatible parent is used as female, and is open pollinated by a desirable cultivar as the pollen source. (Gulzar et al. 2012)
  • 33. RESEARCH STUDY Chaozhi Ma et al. 2009 a b c d Visualization of seed set of self-incompatible line S-1300 in different conditions a: selfing in isolation; b:selfing and bud-pollinating; c: propagation by salt solution; d: hybrid seed production
  • 34. RESEARCH STUDY M. NIEUWHOF, 1974 From 30 varieties and strains of cauliflower the degree of self- incompatibility was tested. It was found that the early cauliflower varieties show a low level of self- incompatibility and most of the late cauliflower varieties a higher level of self-incompatibility.
  • 35. Conti… • The different reaction of the self-compatible and the more self-incompatible varieties is also reflected after bud-pollination. • When bud-pollinating the first group of varieties seed-set was mostly lower than when selfing freshly opened flowers, on an average 19 %. • This indicates that when no incompatibility barrier is present the conditions for seed production are more favourable after selfing freshly opened flowers than after selfing the young buds. • But when such a barrier is present, as in self- incompatible varieties, bud-pollination is more effective. • Thus, the average seed-set of the self-incompatible varieties was 89% higher after bud-pollination than after selfing open flowers. M. NIEUWHOF, 1974
  • 36. Conti… • It was clearly demonstrated that early cauliflower varieties are very self-compatible. • After selfing the early varieties observed a somewhat lower seed set than after crossing. This might indicate the presence of a low degree of self-incompatibility in this group of varieties. • The autumn and winter varieties possess a distinctly higher degree of self-incompatibility than the early cauliflower varieties. M. NIEUWHOF, 1974
  • 37. RESEARCH STUDY P. K. SINGH, 2012
  • 39. Conti… • Hybrid seed production using self-incompatibility involves the development and maintenance of parents which are homozygously selfincompatible but cross-compatible, so the degree of self- incompatibility and stability in the inbred lines is of utmost importance. This is largely dependent on environmental factors, especially temperature. • Uniform, effective cross-pollination may be of great importance for obtaining a high quantity of hybrid seed in self-incompatible types. • The two inbred lines, Desi Red and Khasi Kata, can be used as parental lines to produce single cross hybrid seed. • The lines should be grown in alternate rows or with two to four rows of each, and adequate insect pollination should be ensured. The F1 populations of such hybrids will be uniform. P. K. SINGH, 2012
  • 40. RESEARCH STUDY • SI has been reported to be preferred over male sterility in crop species with entomophilous pollination since pollen-collecting bees rarely visit male-sterile plants. • Nonetheless, SI also may have disadvantages, Ex. F1 hybrids of two SI parents are also SI and this may be undesirable for crop production. • SI F1 hybrids are not a handicap for ornamental or vegetable crops, but it may hinder seed production (e.g., oilseed rape). • Consequently, breeding programs favor not only SI female lines, but also SC F1 hybrids (Kaothien-Nakayama et al., 2010) Muñoz-Sanz JV et al. 2020
  • 41. SI based systems as alternatives to androsterility for producing F1 hybrids. Self-(in)compatible parents and F1 hybrids are indicated.
  • 42. Hybrids developed using self-incompatibility CROPS HYBRIDS Cauliflower Pusa Hybrid 2, Pusa Kartik Sankar, Snow queen, Snow king Cabbage Pusa Synthetic, Pusa Cabbage 1, Meenakshi, BRH-5, H-43, H-44 Chinese cabbage Hamburg-3 Raddish Pusa Chetaki, Pusa Desi, Half Red, Chinese Pink, BDI- 689
  • 43. Need to use of self-incompatibility in the hybrid breeding of vegetable crops • Used in hybrid seed production. • Self incompatible line may be inter planted with a self compatible line. Seed from only the self-incompatible line would be hybrid. • The production of double cross and triple cross hybrids can be produced in case of Brassicas.
  • 44. Level of SI in Brassica oleracea L. Crop Level of SI Reference Kale and Round headed cabbage High Thompson, 1965 Nelson, 1927 Broccoli High to moderate Nelson, 1927 Thompson and Taylor, 1965 Autumn and winter cauliflower Moderate Watts, 1963 White sprouting broccoli and brussels-sprout Moderate to low Johnson, 1955 Green kohl rabi and purple sprouting broccoli Low Nelson, 1927 Early summer cauliflower Very low Watts, 1963,
  • 45. TEMPORARY SUPPRESSION OF SELF INCOMPATIBILITY  Bud Pollination  Increased CO2 concentration  Salt (NaCl) sprays  High temperature treatment
  • 49. -Carafa and Carratu, 1997 RESEARCH STUDY
  • 50. Conti… Self pollen grain germinate and pollen tubes penetrate the stigmatic papillae after stigma treatment with 0.5 M NaCl.
  • 53. -Dhaliwal and Malik, 1982 RESEARCH STUDY
  • 55. RESEARCH STUDY SHORT-TERM HIGH TEMPERATURE TREATMENT
  • 56. Okazaki and Hinata, 1987 RESEARCH STUDY
  • 57. Conti… Many pollen tubes penetrated the pistil in self pollination after the 40oc - 15 min treatment (x 100) Callose formed in papillae (arrows) in which selfed pollen tubes had penetrated after treatment (x 200). Abnormal pollen tubes growing in pistil treated at 50o C for 15 min ( x 200)
  • 58. Limitations in exploiting self-incompatibility • Continues selfing will lead to inbreeding depression. • Continues inbreeding may lead to complete loss of the inbred lines. • Hybrid seed would be expensive if the self-incompatible lines are difficult to maintain. • Environmental factor reduce or totally overcome SI. • Preferential visit of pollinating insects. • Transfer of S-allele is tedious and complicated.
  • 60. REFERENCES 1. Bateman, A. J. (1955) Heredity 9, 52–68 2. Watanabe M, Suwabe K, Suzuki G. 2012. Molecular genetics, physiology and biology of self-incompatibility in Brassicaceae. Proc. Jpn. Acad., Ser. 88(10): 519-535. 3. Hee-Jeong Jung , Nasar Uddin Ahmed, Jong-In Park , Mi-Young Chung, Yong-Gu Cho, Sup Nou. 2013. Molecular Genetic Aspects of Self-incompatibility in Brassicaceae. Plant Breed. Biotech, 1(3):205-217. 4. Gulzar S. Sanghera, Waseem Hussian, Gurpreet Singh and G .A. Parray. 2012. Molecular Perspective to Understand the Phenomenon of Self-Incompatibility and Its Utilisation in Crop Plants. INROADS, Vol. 1, No. 2. 5. Anubhav Thakur1 and K.C. Dhiman. 2021. Self-Incompatibility in Vegetable Crops – A Review. Just Agriculture, Vol.1 Issue-10: 2582-8223. 6. Suwabe K, Nagasaka K, Windari EA, Hoshiai C, Ota T, Takada M, Kitazumi A, Masuko-Suzuki H, Kagaya Y, Yano K, Tsuchimatsu T, Shimizu KK, Takayama S, Suzuki G and Watanabe M (2020) DoubleLocking Mechanism of Self- Compatibility in Arabidopsis thaliana: The Synergistic Effect of Transcriptional Depression and Disruption of Coding Region in the Male Specificity Gene. Front. Plant Sci. 11:576140. doi: 10.3389/fpls.2020.576140. 7. June B Nasrallah, 2017. Plant mating systems: self-incompatibility and evolutionary transitions to self-fertility in the mustard family. Current Opinion in Genetics & Development, 47:54–60. 8. June Nasrallah B. Recognizing self in the self incompatibility response. Plant Phy. 1987; 125:105-108. 9. June Nasrallah B. Cell Cell signaling in the self incompatibility response. Current Opinion in Plant Biology. 2000; 3:368- 373. 10. Nasrallah, J. B. 1997. Evolution of the Brassica self-incompatibility locus: A look into S-locus gene polymorphisms. Proceedings of the National Academy of Sciences, 94: 9516-9519.
  • 61. Conti… 11. Verma, S. K., Anokhe, A., Singh, A., Kumar, R., Kumari, S. and Debbarma, R. (2018). Self-incompatibility a Mechanism for Controlled Pollination in Vegetable Crops. International Journal of Current Microbiology and Applied Sciences, 7: 4602 – 4607. 12. Muthuselvi, R. and Praneetha, S (2019). Molecular basis of self-incompatibility in vegetable crops. International Journal of Chemical Studies, 7(4): 506-510. 13. Dresselhaus, T. and Franklin-Tong, N. (2013). Male–Female Crosstalk during PollenGermination, Tube Growth and Guidance, andDouble Fertilization. Molecular Plant, 6(4):1018-1036. 14. Robert J. Cabin, Ann S. Evans, Diane L. Jennings, Diane L. Marshall, Randall J. Mitchell, and Anna A. Sher. (1996). Using bud pollinations to avoid self-incompatibility: implications from studies of three mustards.. Can. J. Bot. 74: 285-289. 15. Yong Yang ID , Zhiquan Liu, Tong Zhang, Guilong Zhou, Zhiqiang Duan, Bing Li, Shengwei Dou, Xiaomei Liang, Jinxing Tu, Jinxiong Shen, Bin Yi, Tingdong Fu, Cheng Dai and Chaozhi Ma. (2018). Mechanism of Salt-Induced Self-Compatibility Dissected by Comparative Proteomic Analysis in Brassica napus L. International Journal of Molecular Sciences, 19, 1652; doi:10.3390/ijms19061652. 16. Carafa, A. M. and Carratu, G. (1997). Stigma treatment with saline solutions: Anew method to overcome self-incompatibility in Brassica oleracea L. Journal of Horticultural Science, 72(4): 531-535. 17. Xintian Lao,, Keita Suwabe, Satoshi Niikura, Mitsuru Kakita, Megumi Iwano, and Seiji Takayama. (2014). Physiological and genetic analysis of CO2-induced breakdown of self-incompatibility in Brassica rapa. Journal of Experimental Botany, Vol. 65, No. 4, pp. 939–951. 18. Dhaliwal, A. S. and Malik, C. P. (1982). Effect of CO2 in ovecroming self-incompatibility bar¡ in Brassica campestris L. var. toria . Proe. Indian Ac, ad. Sci. (Plant Sci.), Voi. 91: 227-234. 19. A. Palloix, y. Herve, R. B. Knox and C. Dumas. (1985). Effect of carbon dioxide and relative humidity on self incompatibility in cauliflower, Brassica oleracea. Theor Appl Genet, 70:628-633 . 20. Okazaki, K. and Hinata, K. (1987). Repressing the expression of self-incompatibility in crucifers by short-term high temperature treatment. Theor Appl Genet, 73: 496- 500.
  • 62. Conti… 21. Dresselhaus, T.and Franklin-Tong, N. (2013). Male–Female Crosstalk during Pollen Germination, Tube Growth and Guidance, and Double Fertilization. Molecular Plant, 6(4): 1018-1036. 22. Narayanapur, V. B., Suma, B. and Minimol, J. S. (2018). Self-incompatibility: a pollination control mechanism in plants. INTERNATIONAL Journal Of Plant Sciences, 13(1): 201-212. 23. Johnson, A. 0. 1955. Ann. Rep. Nat. Veg. Res. Sta. for 1955, p. 14. 24. ThompsoN, K. F. 1957. Self-incompatibility in marrow-stem kale. 1. Demonstration of a sporophytic system. 3'. Genet., 55, 45-60. 25. ThompsoN, K. F., AND TAyLOR, j. s'. 1965. Identical S alleles in different botanical varieties of Brassica aleracea. Nature, 208, 306-307. 26. Watts, L. N. 1963. Investigations into the breeding System of cauliflower Brassica aleracea var. batrytis (L.). I. Studies of self-incompatibility. Euphytica, 12, 323-340. 27. Nelson, A. 1927. Fertility in the Genus Brassica. 3'. Genet., 18, 109-136.