The objective of any hybridization system is to enforce cross-pollination between inbred lines. Most crop plants are hermaphroditic, that is, possess male and female organs on the same flower, and, therefore, undergo some degree of self-pollination during flowering. To make hybrids, you must eliminate self-pollination on the female parent line and force pollination by the selected male parent. One way to do this is to make the female line male-sterile, which can be accomplished mechanically or genetically.

1. HYBRID SEED
PRODUCTION:
TWO LINE SYSTEM
AND THREE LINE
SYSTEM
By Dr. Shweta Tiwari

2. WHAT IS LINE?
• Pants are fertilized by their own pollen, even before they
leave the flowering stage OR a line have group of
genetically similar plants.
WHAT IS LINE DEVELOPMENT?
• Production or development of particular type of desired
line
• When we talk about development of line in hybrid seed
production , then there are majorly three type of lines: A
Line, B Line and R Line
• And two type of line system: Three line system and two
line system

3. WHAT IS A, B AND R LINE?
• Pants are fertilized by their own pollen, even before they
leave the flowering stage OR a line have group of
genetically similar plants.
MALE STERILE
A LINE
1. FEMALE PARENT
2. DESIRED
CHARACTERSTICS
MAINTAINER LINE
B LINE
1. MALE PARENT
2. MALE FERTILE LINE
3. ISOGENIC LINE
RESTORER LINE
R LINE
1. MALE PARENT
2. MALE FERTILE

4. WHAT IS THREE LINE DEVELOPMENT?
• Based on CGMS
• A, B, R line used
• CYTOPLASM: Main controller of sterility
• NUCLEUS: Contain R gene for restoration of fertility
• R: Male fertility; r: inactive allell
• RR: Male fertility
• Rr: Male fertility
• rr: Favour
Cytoplasm effect
CYTOPLASM
NUCLEUS

5. WHAT IS THE USE OF B LINE?
Sterile
cytoplasm
rr
Fertile
cytoplasm
rr
Sterile
cytoplasm
rr
A LINE B LINE
A LINE PRODUCE : Characters of A
line is similar with B line, so progeny
also similar

6. MAINTAINENCE IN FIELD?
• Planting ratio 4:2
• Seed collected from A line
A-line row (4) B-line row (2)

7. HYBRID SEED PRODUCTION?
• A LINE X R LINE = HYBRID SEEDS (Hybrid Plants obtain are male fertile)
• In hybrids seed production field, the plantation ratio is 4:2 and seed must
be collected from A line
A-line row (4) R-line row (2)
• Now , this is the Three line system used for hybrid seed production
• After having understanding of difficulties in this line we can proceed
further for two line system

8. WHY TWO LINE SYSTEM?
• difficult to maintain CGMS system
• Problems in the maintenance of A line
• high seed production costs
 Use of the environment genic male sterility (EGMS) system in two-line
breeding is simple, efficient, inexpensive and eliminates the limitations
associated with the cytoplasmic-genetic male sterility (CGMS) system.
 The EGMS system advantage includes no need of maintainer line, no need of
restorer line and does not have any negative effect of cytoplasm. EGMS system of
hybrid seed production is more economic and efficient than to the CGMS
method.
TWO APPROCHES?
1. Environment Sensitive Male Sterility (EGMS)
2. Chemically Induced Male Sterility (CIMS)

9. ENVIRONMENT SENSITIVE MALE
STERILITY (EGMS)
• 1) PGMS: Photoperiod-Sensitive Genic Male Sterility
• 2) TGMS: Temperature-Sensitive Genic Male Sterility
• 3) PTGMS: Photothermosensitive Genic Male Sterility
 TGMS lines are delicate to the temperature for the expression of male
sterility or fertility. For example, most TGMS lines stay male sterile at high
temperature (day temperature >30ºC/night >24ºC) and they revert
back to partial fertility at a lower temperature (day<24ºC>16ºC night)
(Virmani et al., 2003)
 PGMS lines are responsive to the spell of day length for the expression of
sterility or fertility. For example, most PGMS lines show male sterility under
long-day (>13.75 h) conditions and turn back to fertility under short-day
(<13 h) conditions (Virmani et al., 2003; Sindhu and Kumar, 2018).
 PTGMS lines are sensitive to both temperature and photoperiod. longer
photoperiod will enhance male sterility at lower temperatures vis a
shorter photoperiod (14 h at 30ºC will make the PTGMS line sterile in
comparison with 13 h at 30ºC)

10. Multiplication of TGMS lines
PROCEDURE OF TWO-LINE (TGMS)
HYBRID RICE SEED PRODUCTION
1) Multiplication of TGMS lines
2) Hybrid rice seed production
Multiplication of TGMS lines
1) Nucleus seed production of a TGMS line starts within the fertility- inducing
environment. Seeding of TGMS lines is organized in such the simplest way
that the sensitive stage occurs when the temperature is good for a higher
seed set.
2) At the duration of flowering, approximately hundred typical plants are
chosen from the population of a TGMS line and their panicles are bagged.
The selection method should be done within 1 week.
3) After the harvest, the chosen plants are scored for spikelet fertility (based
on the main panicle) and 50 plants with higher spikelet fertility (>30%) are
selected.

11. Multiplication of TGMS lines
PROCEDURE OF TWO-LINE (TGMS)
HYBRID RICE SEED PRODUCTION
4) Progenies of the chosen plants are grown within the sterility-inducing
atmosphere. About thirty seeds are taken from every of the chosen plants
to grow single-row progenies and the remaining seeds are put away
carefully. The balance of the seeds of the progenies that are uniform and
completely male sterile must be distinct and bulked to create the nucleus
seed.
5) Nucleus seed of the TGMS line is utilized for producing breeder seed
under strict isolation. Breeder seed for the TGMS line is produced in the
fertility-inducing environment.
6) The breeder seed produced under the oversight of the plant breeder
has high genetic purity and utilized for producing foundation seed of
parental lines, which will be used for producing hybrid seed.
II. Hybrid rice seed production

12. Multiplication of TGMS lines
PROCEDURE OF TWO-LINE (TGMS)
HYBRID RICE SEED PRODUCTION
1. In two-line hybrid seed production, the two safe-periods refers to
the useful atmospheric conditions that support first the induction of
absolute male sterility and second the facilitation of proper pollen
movement from the pollen parent and fertilization of the TGMS
parent So, the first safe-period determines seed purity, while the
second safe-period determines hybrid seed yield
2. A multidisciplinary approach in developing superior TGMS lines and
pollen parents can help to develop two-line rice hybrids suitable
for the distinct ecological situations in which rice is grown
3. Despite the promise that two-line hybrid rice technology holds, it
would be wise to have a harmonious balance in using three-line
and two line hybrids and conventional rice varieties in an
appropriate manner in national rice production programs

13. Multiplication of TGMS lines
POLLEN PARENT DEVELOPMENT
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.

14. Multiplication of TGMS lines
PROCEDURE
• Make a cross between TGMS Line or PGMS line with restorer line.
• The temperature maintain at range in which the A line will show
sterility
• Now, Secondly for the maintenance of A line, place it in location
which maintain or revert the fertility of the genotype.
• And for further utilization in future, again shift the TGMS line to
temperature sensitive to male sterility or temperature inducing
sterility

15. Multiplication of TGMS lines
GENETIC PURITY STANDARD
• Use nucleus seeds
• Apply anther culture
• 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

16. Multiplication of TGMS lines
CHEMICAL INDUCED MALE
STERILITY(CIMS)
1. This non-genetic method of including male sterility involves the use
of chemical called hybridizing agents (cha) or gametocides.
2. This method is useful for plants with bisexual flowers in which it is
difficult to obtain genetic or cytoplasmic genetic male sterility.
3. 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.

17. Multiplication of TGMS lines
1. For developing hybrids using gametocides:
 The female line should have a synchronous flowering habit.
 The line should respond to chemical treatment.
 The parent should possess good out crossing trait.
2. Important factors that decide the efficiency of chemical
gametocides are,
 1. The correct dosage of the chemical.
 2. Appropriate stage of treatment

18. Multiplication of TGMS lines
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.

19. Multiplication of TGMS lines
Future research priorities
1. Development of stable EGMS lines.
2. 2. Use of anther culture to develop and/or purify elite EGMS
lines.
3. 3. Breeding for super high-yielding two-line hybrids.
4. Incorporation of hybrids with resistance to biotic stress.
5. Abiotic stress tolerance.
6. 6. Quality

20. Multiplication of TGMS lines
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