This document provides information on various methods of inducing male sterility in plants, especially rice, for the purpose of hybrid seed production. It discusses chemical, genetic, and transgenic approaches. Specifically, it describes cytoplasmic male sterility (CMS), nuclear male sterility (NMS), and cytoplasmic-genetic male sterility (CGMS). It also discusses the use of marker-assisted selection (MAS) to more efficiently select for male sterility genes and introgress them into adapted varieties through techniques like marker-assisted backcrossing (MAB). Overall, the document outlines methods for inducing and tracking male sterility that can facilitate efficient hybrid rice breeding programs.

1. Welcome
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2. 2

3. RICE FLOWER
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4. VIPIN MOHAN
2011-09-112
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5. Rice–thegrain
Second in production & consumption
Staple food
90% of world’s rice is produced and
consumed in Asia
Wide adaptability
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6. but….
• Growth in rice production :
• 2.5 – 3.0 % during 1970 – 80
• 1.5 % during 90’s
• Population growth :
• by 2025 - 8 billion
• Required rice production :
• 40 % more
6Nas et al., 2013

7. What is Male Sterility?
 Failure of plants to produce functional anthers, pollen,
or male gametes.
 Agronomically important for the hybrid seed
production
 Occurs mainly in bisexual plants.
 Pollen sterility/structural sterility/functional sterility
Flower of male-fertile chilly Flower of male-sterile chilly
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8. Based on its inheritance or origin
 Chemically induced male sterility
 Transgenic male sterility
 Cytoplasmic male sterility (CMS)
 Nuclear male sterility (NMS)
 Cytoplasmic-genetic male sterility
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9. Biochemical means of
producing male sterile plants
 Feminizing hormones
 Inhibitors of anther or pollen development
a. acting on sporophytic tissue
b. acting on gametophytic tissue (gametocides)
 Inhibitors of pollen fertility
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10. Advantages of Chemical
hybridization
 High degree of efficacy and developmental selectivity
 Persistence during the development of flower or spikes
 Low cost
 Acceptable levels of toxicity
 Low general phytotoxicity
 Agronomic performance of hybrid seed
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11. Barnase/Barstar system for engineered male
sterility (Mariani et al., 1992)
Male sterility through modification of
biochemical pathways (Marc et al., 2000)
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MALE-STERILITY THROUGH
RECOMBINANT DNA
TECHNOLOGY

12. Barnase/Barstar system
 Targeting the expression of a gene encoding a
cytotoxin by placing it under the control of an anther
specific promoter (Promoter of TA29 gene)
 Expression of gene encoding ribonuclease (chemical
synthesized RNAse-T1 from Aspergillus oryzae and
natural gene barnase from Bacillus
amyloliquefaciens)
 Success in oilseed rape, maize and several vegetative
species
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13. 13

14. Male sterility through
modification of biochemical
pathways
Carbohydrates :-
 Play a critical role in the anther and pollen development by sustaining
growth as well as signal pathways.
 Their transportation from photo synthetically active source tissues to
developing sinks is regulated by extra cellular invertase
 The extracellular invertase Nin 88 of tobacco shows expression pattern in
developing anther
 The tissue specific antisense repression of nin88 under the control of
Nin88 promoter in plant caused male sterility .
 Exogenous supply of carbohydrates able to partially overcome blocking
of pollen development so it maintaining the male sterility.
 Restoration by crossing this GMS system with plants expressing
distantly relate invertase
(Marc et al.,2000)
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15. Cytoplasmic male-sterililty
 Structural changes in the cytoplasmic organelle
genome
 Maternally inherited
 Three lines of plants must be maintained(A,B and R
lines)
 It is divided into:
a. Autoplasmic
b. Alloplasmic
Eg.: Pusa6A and IR262829A 15

16. Drawbacks:
 insufficient or unstable male sterile
 Difficulties in restoration system
 Difficulties with seed production
 Undesirable pleiotropic effect
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17. 17

18. Genetic male sterility
Genic/genetic/Mendelian
 Inheritable
Controlled by a number of nuclear genes (a
pair of recessive alleles “msms”)
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19.  Temperature
- Changing the optimal temperature can induce sterility
eg.: EC720903, C815S
Photoperiod
- It has a strong influence (Photoperiod sensitive)
Changing the growth habit can stimulate the sterility
eg.: Nongken58S
Determining factor
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20. 20

21. Advantages
2 line system
Stability
100% male sterile progenies
Inheritable
Restoration
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22. Cytoplasmic - genetic male
sterility
 Controlled by - nuclear (with Mendelian inheritance)
and cytoplasmic (maternally inherited) genes
 Restorers of fertility (Rf) genes present
 Rf genes - no expression of their own unless the sterile
cytoplasm is present
 Plants with:
• N cytoplasm are fertile
• S cytoplasm with genotype Rf- leads to fertile
• S cytoplasm with rfrf produces only male steriles
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23. Limitations of CGMS system
Undesirable effects of the cytoplasm
Unsatisfactory fertility restoration
Unsatisfactory pollination
Spontaneous reversion
Modifying genes
Contribution of cytoplasm by sperm
Environmental effects
Non- availability of suitable restorer line
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24. 24

25. MARKER ASSISTED
SELECTION
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26. MARKER
A marker (morphological, biochemical or one
based on DNA/RNA variation) is used for
indirect selection of a genetic determinant or
determinants of a trait of interest (e.g.
productivity, disease resistance, abiotic stress
tolerance, and quality). This process is used in
plant and animal breeding.
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27. 27
MARKERS
Morphological Biochemical DNA based
Hybridization based e.g. RFLP PCR based Chip based
Arbitrary primers e.g. RAPD,ISSR,AFLP Specific primers SNP based
Repeat based
e.g. SSRs
Sequence based
e.g. SCAR, CAPs, SNP

28. F2
P2
F1
P1 x
large populations consisting of
thousands of plants
PHENOTYPIC SELECTION
Field trialsGlasshouse trials
DonorRecipient
CONVENTIONAL PLANT BREEDING
Salinity screening in phytotron Bacterial blight screening
Phosphorus deficiency plot
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29. Molecular Marker Assisted Breeding
 Conventional screening difficulties
 Molecular marker indicates the presence or absence of gene at an
early stage
 A molecular marker very closely linked to the target gene can act
as a “tag” which can be used for indirect selection of target gene
(Jena et al., 2003)
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30. F2
P2
F1
P1 x
large populations consisting of
thousands of plants
ResistantSusceptible
MARKER-ASSISTED SELECTION (MAS)
MARKER-ASSISTED BREEDING
Method whereby phenotypic selection is based on DNA markers 30

31. Markers must be polymorphic
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
Primer A Primer B
P1 P2
P1 P2
Not polymorphic Polymorphic!
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32. Markers must be
tightly-linked to target loci!
• Ideally markers should be <5 cM from a gene or QTL
• Using a pair of flanking markers can greatly improve
reliability but increases time and cost
Marker A
QTL
5 cM
RELIABILITY FOR
SELECTION
Using marker A only:
1 – rA = ~95%
Marker A
QTL
Marker B
5 cM 5 cM
Using markers A and B:
1 - 2 rArB = ~99.5%
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33. 33
Sl.
No
TGMS
gene
Linked Marker Chromosome
location
References
1 tms1 OPB-19(RAPD) 8 wang et al., 1995
2 tms2 RM11,RM2 9
Lopez et al., 2003,
Pitnjam et al., 2008,
Yamaguchi et al., 1997
3 tms3 OPF-18,OPAC-3(RAPD) 6 Lang et al.,1999,
Subudhi et al. 1997
4 tms4 RM27 2 Dong et al., 2000
5 tms5 RM174,RM5862,RM5897 2 Nas et al., 2005
6 tms6 RM3351 5 Lee et al., 2005
7 tms7 RM224,RM21 11 Hussain et al.,2012
8 tms9 Indel37,Indel57 2 Sheng et al. 2013
TGMS genes and their linked
markers in rice

34. (1) LEAF TISSUE
SAMPLING
(2) DNA EXTRACTION
(3) PCR
(4) GEL ELECTROPHORESIS
(5) MARKER ANALYSIS
Overview of
‘marker
genotyping’
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35. Advantages of MAS
Simpler method compared to phenotypic
screening
Selection at seedling stage
Increased reliability
More accurate and efficient selection of
specific genotypes
More efficient use of resources
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36. Marker-assisted
backcrossing (MAB)
Method to introgress a single locus
controlling a trait of interest while retaining
the essential characteristics of recurrent
parent.
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37. Marker-assisted backcrossing
(MAB)
MAB has several advantages over conventional
backcrossing:
– Effective selection of target loci
– Minimize linkage drag
– Accelerated recovery of recurrent parent
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38. P1 x F1
P1 x P2
CONVENTIONAL BACKCROSSING
BC1
VISUAL SELECTION OF BC1 PLANTS THAT
MOST CLOSELY RESEMBLE RECURRENT
PARENT
BC2
MARKER-ASSISTED BACKCROSSING
P1 x F1
P1 x P2
BC1
USE ‘BACKGROUND’ MARKERS TO SELECT PLANTS
THAT HAVE MOST RP MARKERS AND SMALLEST %
OF DONOR GENOME
BC2
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39. Gene
pyramiding
• MAS helps to identify the desired
resistant genes
• Resistance GENES can be
pyramided to make a line having
multi – race resistance
Samis et al. (2002)
• MAS has provided an approach to
pyramid beneficial alleles 39

40. F2
F1
Gene A + B
P1
Gene A
x P1
Gene B
MAS
Select F2 plants that have
Gene A and Gene B
Genotypes
P1: AAbb P2: aaBB
F1: AaBb
F2
AB Ab aB ab
AB AABB AABb AaBB AaBb
Ab AABb AAbb AaBb Aabb
aB AaBB AaBb aaBB aaBb
ab AaBb Aabb aaBb aabb
Process of combining several genes, usually from 2
different parents, together into a single genotype
x
Breeding plan
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41. Studies…
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46. CONCLUSION
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With the assistance of Biotechnology(marker assisted
breeding), we can produce rice hybrids very
effectively with in a short time. It will be a big
solution for meeting demands of growing population.

47. Reference
s:
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1. Dong NV, Subudhi PK, Luong PN, Quang VD, Quy TD, Zheng HG, Wang B,
Nguyen HT (2000). Molecular mapping of a rice gene conditioning
thermosensitive genic male sterility using AFLP, RFLP and SSR techniques.
Theor. Appl. Genet. 100: 727-734.
2. Jing J., Mou T., Yu H., and Zhoru F. 2015. Molecular breeding of TGMS lines of
rice for blast resistance using Pi2 gene. Rice 8:11.
3. Nas, T. M. S., Sanchez, D. L., Diaz Ma. G. Q., Mendioro M. S. and Sant S.
Virmani S. S. 2005. Pyramiding of thermosensitive genetic male sterility
(TGMS) genes and identification of a candidate tms5 gene in rice. Euphytica
145:67-7.
4. Khora P., Priyadarshi R., Singh A., Mohan R., Gangashetti M. G., Singh B. N.,
Kole C. and Shenoy V. 2012. Molecular characterization of different cytoplasmic
male sterility lines using mitochondrial DNA specific marker in rice. J. biosci.
98: 56-78.

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5. Lang, N. T., Subudhi, P. K., Virmani, S. S.,Brar, D. S., Khush, G. S. andli, Z.
1999. Development of PCR-based markers for thermosensitive genetic male
sterility gene tms3(t) in rice (Oryzasatiua L.) heredital 131:121-127.
6. Ngangkham, U., Parida, S. K., De, S., Kumar, A. R., Singh, A. K., Singh, N.
K., and Mohapatra, T. 2010. Genic markers for wild abortive (WA) cytoplasm
based male sterility and its fertility restoration in rice. Mol. Breed. 26:275-292
7. Niya Celine V. J., Roy Stephen, Manju R.V. and Shabana R. 2014. Evaluation
of thermosensitive genic male sterile lines in rice suitable to Kerala through
marker assisted selection J. Trop. Agric. 52 (1) : 74-78.
8. Wang, B., Wang, J. Z., Wu, W., Zheng, H. G., Yang, Z. Y., Xu, W. W., Ray, J.
P. and Nguyen, H. T. 1995. Tagging and mapping the thermo-sensitive genic
male-sterile gene in rice (Oryzasativa L.) with molecular markers. Theor.
Appl. Genet. 91:1111-1114.

49. 49
9. Wang YG, Xing QH, Deng QY, Liang, FS, Yuan LP, Weng ML, Wang
B(2003). Fine mapping of the rice thermo sensitive genic male sterile gene
tms5. Theor. Appl. Genet. 107: 917-921.
10. Yamaguchi, Y., Ikeda, R., Hirasawa, H., Minami, M. andUjihara, P. 1997.
Linkage analysis of thermosensitive genic male sterility gene, tms-2 in rice
(Oryzasativa L.). Breed Sci. 47:371-373.
11. Yang, R. C. and Wang, N. Y. 1988. 5460S Indica photosensitive genic male-
sterile rice. Int. Rice Res. Newsl. 13:6-7.
12. Zhonghua Sheng, Xiangjin Wei, Gaoneng Shao, Mingliang Chen, Jian Song,
Shaoqing Tang, Ju Luo, Yichao Hu, Peisong Hu, Liyun Chen.2013.
Plant.Breed,http://www.bionity.com/en/publications/527796/genetic-analysis-
and-fine-mapping-of-tms9-a-novel-thermosensitive4-genic-male-sterile-gene-
in-rice-oryza-sativa-l.html(30/9/2014)

50. Thank you….!
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