This document discusses two methods for producing hybrid rice: two-line and three-line breeding systems. The two-line system uses environmentally sensitive genetic male sterility (EGMS) or chemically induced male sterility (CIMS) to produce hybrid seed. EGMS lines are male sterile under certain temperature or photoperiod conditions. Two-line hybrids have advantages over three-line such as lower production costs and greater genetic diversity of parents. China has had success adopting two-line hybrid rice, which now covers over 2 million hectares. Further research aims to develop more stable EGMS lines and higher-yielding two-line hybrids with stress tolerance and quality traits.

1. TWO LINE METHOD
BREEDING IN RICE
SUBMITTED BY,
SANDESH,G.M
2016610811
TNAU, MADURAI

2. WHAT IS HYBRID RICE?
The first generation offspring of a rice cross between two genetically diverse parents.

3. How Hybrid Rice?
Normal Rice Spikelet
(self pollinated crop)
Sterile Rice Spikelet
(Male Sterility)
Hybrid Seed Production
(Male Sterile x Normal Rice)

4. WHY HYBRID RICE?
Heterosis (hybrid vigor) application to increase:
• Productivity (yield/unit/time, 15-20% of yield advantage).
• Economic returns
Heterosis :
• A universal phenomenon that F1 generation shows superiority to both
parents in agronomic traits or yield.
• It presents in all biological systems and has been exploited commercially in
many agricultural crops.

5. Two Commercial Systems for Hybrid Rice

6. Why there is switch over from three line to
two line breeding ??
• Cytoplasmic male sterility-fertility restoration system based on three line
breeding is often difficult to maintain.
• Problems in the maintenance of A line.
• Lack of diversity in A and A lines, and the presence of minor fertility genes in B
lines, lead to low heterotic potential and high seed production costs.
• Hence two line breeding emerged.

7. TWO LINE METHOD OF HYBRID RICE
• Crop scientists have been engages in developing an effective and viable two line
approach to exploit hybrid vigour in rice since more than a decade.
• Two major approach have emerged as a result and they are as follows,
1. Environment Sensitive Male Sterility (EGMS).
2. Chemically Induced Male Sterility (CIMS).

8. Environment Sensitive Genetic Male Sterility (EGMS)
• At a particular critical range of temperature, certain genotypes become male
sterile and those can be used as a female parent in development of hybrids.
• While at some other ranges of temperature or photoperiod same become fertile.
Hence can be multiplied without a maintainer line unlike in other systems.
• Those genotypes which exhibit sterility/fertility with respect to temperature are
called Thermo-sensitive Genetic Gale Sterile (TGMS) lines.
• Those showing similar response to photoperiod are termed as Photoperiod
Sensitive Genetic Male Sterile (PGMS) lines.

9. Temperature sensitive genetic male sterility
(TGMS)
• These are male sterile lines whose male sterility/fertility alteration is considered
by different temperature regimes.
• For ex: most of the TGMS lines remain sterile at high temperature (23.3 oC or
higher for rice TGMS line pei-ai645 and they revert back to partial sterility at
lower temperature (<23.3 oC )
• The critical sterility/fertility points vary from genotypes to genotypes.

10. TGMS and two-line hybrid
• Based on thediscovery of
P(T)GMS mutant
• Male sterility controlled by
1 or 2 pairs of recessive
gene(s) Fertile
S-line
Multiplication
Critical Fertility Point
Critical Sterility
Point
Reproductive Upper Limit
Reproductive Lower Limit
Sterile
F1 Seed
Production
Partial Sterility
Model of Sterility / Fertility Expression for TGMS Rice
Temperature
Lo
w
Hig
h

11. Desirable characteristics of TGMS / PGMS lines
• Population of EGMS lines should consist of more than 1000 individual plants and their
morphological characters should be uniform.
• The percentage of male sterile plants should be 100% and the degree of male sterility
(percentage of abortive pollen grains should be over 99.5%).
• The change of fertility to sterility and vice-versa should be quite clear and distinct.
• Male sterile period should be last for more than 30 consecutive days.
• Seed setting during fertile period should be over 30%.
• Out crossing percentage during sterile phase should be lower than check ‘A’ lines.

12. Development of two-line Experimental Hybrids
• Making several test crosses between the TGMS/PGMS line and several
promising genotypes.
• Evaluation of the test cross to identify promising two line hybrid combinations.
• The subsequent procedures of observational yield trials, regional or national
trials, multilocational trials, on-farm verification trials etc., For identification of
best possible hybrid remains same, as in three-line system.

13. EGMS lines can be developed by any of the following
methods
• Screening of existing varieties.
• Induced mutagenesis.
• Hybridization followed by pedigree selection,
• Anther culture, backcrossing.
• Marker-aided selection (MAS).

14. Characterization of EGMS lines
under field conditions
1. Identify 3–4 distinct periods of high and low temperatures during the year.
Likewise ,determine the longer and shorter daylength durations during the year and
over locations.
2. Seed/plant EGMS lines at 15–25-day intervals in such a way that their heading
coincides with the high temperature or longer photoperiod.
3. Study pollen fertility of the EGMS lines from the top five spikelets of primary
panicles under the microscope.

15. CONTD..,
4. Relate the pollen sterility data to temperatures/daylengths prevailing during the
period of 15–25 days before heading (“tracking technique”).
5. Determine the critical fertility point (i.E., The lowest temperature at which
maximum pollen fertility is achieved) by using the tracking technique.

16. Developing pollen parents for two-line hybrids
The following are the characteristic features of and ideal elite pollen parent:
• Strong fertility-restoring ability : when a cross is made with the EGMS line, the
hybrids have a normal seed setting percentage (>80%) and are less affected by changes
in environmental conditions.
• Good general combining ability : when crossed with different egms lines, the F1
hybrids from many crosses perform well.
• Good agronomic characters : the pollen parent should be a high-yielding inbred line
with favorable traits for outcrossing. Eg:, good anther dehiscence, good anther
protrusion, large anther size, high pollen load, etc.
• Genetic distance : Considerable genetic distance from the EGMS lines will be the key
to enhanced heterosis.

17. Procedure of utilization of TGMS/PGMS line in
seed production
X
OR
TGMS ANY RESTORER LINE
PGMS
HYBRID SEED

18. Exploitation of TGMS in rice
• Consider the temperature/thermosensitive genetic male sterile line 5460S.
TGMS LINE TEMPERATURE EXPRESSION
5460S
< 28 oC
Complete male
Fertility
>30 oC
Complete male
Sterility

19. Maintenance of TGMS in rice
LOCATION A
(<28 oC Temperature)
TGMS line (5460S)
TGMS line
(Complete Fertile)

20. Utilization of TGMS line in Hybrid seed production
LOCATION B
(> 30 oC temperature)
TGMS line (5460S) x Male fertile line
Hybrid Seed
(Produced on TGMS line)

21. Two-line hybrid rice seed production
• Multiplication of EGMS lines.
• Hybrid rice seed production.

22. SOURCE NURSERY Elite lines from different sources
TGMS Line Breeding To evaluate parents and make testcross B & R line Breeding Program
Pollinator line Breeding Progam
Breeder Seeds TESTCROSS NURSERY
To identify TGMS & P lines Hybrid Seed Production for OYT
Core Seeds Premarily heterosis evaluation, 2 rows w/ parent Isolation Bags or hand-crossing
Foundation Seed RETESTCROSS NURSERY (OYT)
Re-evaluate F1 hybrids Hybrid Seed Production for PYT
Certified Seeds Stage 1, 1 rep, 3 rows Isloated Net or bags
TGMS Line Release Preliminary Yield Trial (PYT)
Stage 2, 1 rep, plot Hybrid Seed Production for AYT & NYT
Isolation Block
Advanced Yield Trial (AYT)
Stage 3, 3 reps, plot
Hybrid Pilot Seed Production
National Yield Trial Isolation Block
Stage 4, 3-4 reps, muti-location, 2-years
Hybrid and R line Release
On-Farm Trial (Strip Trial)
Flowchart of 2-Line Hybrid Rice Evaluation and Seed Production

23. High-yielding techniques for PGMS line
multiplication
• Autumn-season multiplication:
In autumn, the daylength is getting shorter and a high percentage of seed setting can
be achieved if the sensitive stage occurs under this condition. However, seed quality
becomes inferior, especially when the temperature falls sharply in late autumn.
• Winter-season multiplication :
In lower-latitude areas for PGMS self seed multiplication, short daylength and low
temperature in lower-latitude areas in the winter season are the most suitable
environmental conditions.

24. High-yielding techniques for TGMS line
multiplication
• Spring-season multiplication :The fertility of TGMS lines is mainly influenced
by temperature. TGMS lines should be sown in early spring so that the sensitive
stage matches the temperature for fertility induction based on local
meteorological data.
• Autumn-season multiplication : the temperature is low enough for fertility
induction in the autumn season. The TGMS lines should be sown at the proper
time to make the sensitive stage coincide with the low-temperature period in
order to obtain higher yields.

25. CONTD..,
• High-altitude multiplication : In high-altitude areas (with altitudes from 800 to
1,000 m), moderate temperature and longer daylength conditions are suitable for
the short growing period for PGMS and TGMS line self seed multiplication.
• Chilled-water irrigation : Chilled-water (>17 ºc) irrigation can induce TGMS
lines to produce fertile pollen similar to ambient low temperatures. The
advantage of chilled-water irrigation is that it provides flexibility for adjusting
the sensitive stage

26. Maintenance of genetic seed purity standards
The following methods are recommended for purification of EGMS lines:
1. Use nucleus seeds directly from the research center for seed reproduction of
EGMS lines.
2. Apply anther culture for the purification of EGMS lines.
3. Introduce a recessive morphological marker into the EGMS line or a dominant
morphological marker gene into the pollen parent line by gene transformation or
conventional backcross breeding methods. The pseudo hybrids can be identified and
eliminated in the nursery, thereby increasing the purity of hybrid rice in the field.

27. List of PGMS lines in Rice
GENETIC
CONTROL
LINES ORIGIN EXPRESSION
OF MALE
STERILITY
Controlled by two
recessive genes
PGMS Lines
Nongken 54S Spontaneous
mutation
Day length shorter
than 13.75 hr
X88 Hybridization Day length shorter
than 13 hr
MG 201 Ems mutagenesis Day length 12 hr

28. List of TGMS lines in Rice
GENETIC
CONTROL
LINES ORIGIN EXPRESSION OF
MS
Controlled by
single recessive
gene
Annong-1S Spontaneous mutation Temperature of 27 oC
Hennong S Hybridization Temperature <29 oC
5460S Irradiation Temperature >30 oC
SM-38 Spontaneous mutation Temperature <22 oC
SM-5 Spontaneous mutation Temperature <22 oC
JP-2 Spontaneous mutation Temperature <26 oC
JP-38S Spontaneous mutation Temperature >30 oC
IR 32 364 (IRRI), IR 68945 (IRRI), IR 68949 (IRRI), Norin PL 12 (JAPAN)

29. Two-line hybrid production in China
Two Line Hybrid Rice In China
6200
6400
6600
6800
7000
7200
7400
7600
7800
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
Year
Yield(kg/ha)
0
200
400
600
800
1000
1200
1400
1600
Area(1000ha)
2-line Hybrid Yield All Hybrid Yield Area

30. CHEMICAL INDUCED MALE STERILITY(CIMS)
This non-genetic method of including male sterility involves the use of chemical called
hybridizing agents (cha) or gametocides. This method is useful for plants with bisexual
flowers in which it is difficult to obtain genetic or cytoplasmic genetic male sterility.
The Ideal Gametocide-
• Selectively induce male sterility without adversely affecting the female sterility.
• Must not be mutagenic.
• Have a broad range of affectivity in order to withstand adverse environmental
conditions.
• Should consistently produce complete (>95%) male sterility.
• Should not be hazardous to the environment.

31. Important gametocides found useful in Rice
CHEMICAL CONCENTRATION GROTH STAGE FOR
APPLICATION
Ethrel 800-1000 ppm Prior to anthesis
Zinc methyl arsenate 0.02% or 2000 ppm Uni-nucleate pollen stage
Sodium methyl aresenate 0.02% or 2000 ppm Five days before heading

32. CONTD..,
• For developing hybrids using gametocides:
1. The female line should have a synchronous flowering habit.
2. The line should respond to chemical treatment.
3. The parent should possess good outcrossing trait.
• Important factors that decide the efficiency of chemical gametocides are,
1. The correct dosage of the chemical.
2. Appropriate stage of treatment.

33. Operational flowchart for
hybrid rice breeding using
CIMS

34. Rice hybrids based on CHA’s
• Qing – Hua-Fu-Gwi
• Gang-Hua-Qing-Lan
• Gang-Hua-Lan
• Gang-Hua-2
• You-Za-1
• Ya-You-2
These are produced in china using arsenical CHA’s.

35. Advantage & Disadvantage of 2-line hybrid rice
system
Advantages :
• Simplified procedure of hybrid seed production .
• Multiple and diverse germplasm available as parents,
• Any line could be bred as female.
• 97% (2-line) vs 5% (3-line) of germplasm as male.
• Increased chance of developing desirable & heterotic hybrids.
• Multiple cytoplasm courses as female parents.
Disadvantages :
• Environmental effect on sterility could cause seed purity problem.

36. Conclusion
• There is a continuous need to reduce the cost and increase the efficiency of
hybrid rice seed production.
• The discovery of the EGMS system in China and later in Japan, at IRRI, and in
India has improved the chances of substantially reducing the cost of seed
production by using two-line rice hybrids.
• These hybrids also help to increase heterosis beyond the level of three line rice
hybrids.

37. Contd..,
• The two-line hybrids have already created an impact in china, with their area
reaching 2.6 million ha. Among the several two line rice hybrids, liangyou peijiu
(peiai64s/9311) gave the highest average yields of 11 t ha–1 on 10-ha farms in two
successive years.
• Outside China, two-line rice hybrids are also being developed at IRRI and in
Vietnam, India, Korea, the Philippines, Thailand, and Egypt.

38. Future research priorities
1. Development of stable EGMS lines.
2. Use of anther culture to develop and/or purify elite EGMS lines.
3. Breeding for super high-yielding two-line hybrids.
4. Incorporation of hybrids with resistance to biotic stress.
5. Abiotic stress tolerance.
6. Quality.
A multidisciplinary approach in developing superior egms lines and pollen
parents can help to develop two-line rice hybrids suitable for the different
ecological situations in which rice is grown.

39. THANK YOU..,