MALE STERILITY, TYPES

& USES IN HYBRID SEED

PRODUCTION

Course: Heterosis Breeding (GP-507)

PRESENTED BY-
HIRDAYESH ANURAGI
ADM. NO. 2015A29D
PhD GENETICS & PLANT BREEDING
CCS HAU HISAR, HARYANA
 Contents

 Male sterility
 Manifestation of Male sterility
 History of Male sterility
 Need of Male sterility
 Detection of Male sterility
 Creation of Male sterility
 Classification of Male sterility
 Cytoplasmic Male sterility (CMS)
 Genetic Male sterility (GMS)
 Cytoplasmic genetic Male sterility (CGMS)
 Transgenic Male sterility
 Chemical hybridizing agents (CHAs)
 Applications of Male sterility in Hybrid seed production
 Male Sterility

 Male sterility is characterized by nonfunctional pollen grains,

while female gametes function normally.
 Inability to produce or to release viable or functional pollen as
a result of failure of formation or development of functional
stamens, microspores or gametes.
 Main reason is mutation.

Sterile Fertile Sterile Fertile

 Manifestations of Male Sterility

 Absence or malformation of male organs.

 Failure to develop normal microsporogenous tissue- anther

 Abnormal microsporogenesis (deformed or inviable pollen)

 Abnormal pollen maturation

 Non dehiscent anthers but viable pollen, sporophytic control

 Barriers other than incompatibility preventing pollen from

reaching ovule
 History of Male Sterility

 J.K. Koelreuter (1763) observed anther abortion within species

& species hybrids.
 Genic male sterility has been reported in cabbage (Rundfeldt
1960), cauliflower (Nieuwhof 1961)
 Male sterility systems have been also developed through
genetic engineering (Williams et al. 1997) and protoplast fusion
(Pelletier et al. 1995)
 Male sterility were artificially induced through mutagenesis
(Kaul 1988)
 Several forms of pollination control

 Manual emasculation

 Use of male sterility

 Use of self-incompatibility alleles

 Use of male gametocides

 Use of genetically engineered “pollen killer”

genetic system
 Why Male Sterility ???

 Reduced the cost of hybrid seed production.

 Production of large scale of F1 seeds.

 Avoids enormous manual work of emasculation

and pollination.

 Speed up the hybridization programme.

 Commercial exploitation of hybrid vigour.

 Creation of Male Sterility

 Spontaneous mutations

 Interspecific hybridization

 Mutation induction (EtBr)

 Genetic Engineering

 Chemically induced male sterility (CHAs)

 Detection of Male Sterility system

Whether a particular sterile genotype belongs to which MS

system can be detected by its progeny performance on crossing
with a few normal genotypes.

 Trend-I- All progenies in all the rows may be sterile- CMS

 Trend-II- Some rows may consist all fertile
Some rows sterile and fertile in 1:1 ratio- GMS
 Trend-III- Some rows fertile. Some rows sterile and some
rows sterile and fertile in 1:1 ratio - CGMS
 Classification of Male Sterility
Kaul (1988) Classified Male Sterility in three major groups

1. Phenotypic Male Sterility (Morphological)

 Structural or Staminal Male Sterility
 Pollen Male Sterility
 Functional Male Sterility

2. Genotypic Male Sterility

 Genetic Male Sterility (GMS)
 Environmental Sensitive (EGMS)
a) Thermo sensitive genetic male sterility (TGMS)
b) Photoperiod sensitive genetic male sterility (PGMS)
 Environmental non-sensitive
 Cytoplasmic Male Sterility (CMS)
 Cytoplasmic Genetic Male Sterility (CGMS)
 Transgenic Male Sterility (TMS)

3. Chemically Induced Male Sterility (CHA)

 Phenotypic Male Sterility

 Pollen sterility: in which male sterile individuals differ from

normal only in the absence or extreme scarcity of functional
pollen grains (the most common and the only one that has played
a major role in plant breeding).

 Structural or staminal male sterility: in which male flowers or

stamen are malformed and non functional or completely absent.

 Functional male sterility: in which perfectly good and viable

pollen is trapped in indehiscent anther and thus prevented from
functioning
 Cytoplasmic Male Sterility (CMS)

 Determined by the cytoplasm (mitochondrial or chloroplast genes).

 Result of mutation in mitochondrial genome (mtDNA)- Mitochondrial

dysfunction.

 Progenies would always be male sterile since the cytoplasm comes

primarily from female gamete only.

 Nuclear genotype of male sterile line is almost identical to that of the

recurrent pollinator strain.

 Male fertile line (maintainer line or B line) is used to maintain the

male sterile line (A line).

 CMS is not influenced by environmental factors (temperature) so is

stable.
 Utilization of CMS in Plant Breeding
 CMS can used in hybrid seed production of certain ornamental
species or in species where a vegetative part is of economic value.

 But not for crop plants where seed is the economic part because the
hybrid progeny would be male sterile.

 This type of male sterility found in onion, fodder jowar, cabbage etc.
Use of CMS lines
Transfer of CMS to new strains (Diversification)
 Genetic Male Sterility (GMS)
 Also called as nuclear male sterility.

 Mostly governed by single recessive gene (ms) but dominant gene

governing male sterility (safflower).

 Origin: Spontaneous mutation or artificial mutations (Gamma rays,

EMS) are common.

S.No. Mutagens Crops

1 Colchicine Jowar
2 Ethidium Bromide Groundnut, Maize, wheat
3 Acetone Barley

 ‘ms’ alleles may affect staminal initiation, stamen or anther sac

development, PMC formation, meiosis, pollen formation, maturation
and dehiscence.
 Types of GMS
 Environment insensitive GMS: ms gene expression is much less affected
by the environment.

 Environment sensitive GMS: ms gene expression occurs within a

specified range of temperature and /or photoperiod regimes (Rice, Tomato,
Wheat etc.).

1. TGMS: sterility is at particular temperature e.g. In rice TGMS line

(Pei- Ai645) at 23.30C (China).
 TGMS at high temperature is due to failure of pairing of two
chromosomes at metaphase was evident.
 This abnormality led to abnormal meiosis, abnormal or sterile pollens.
 Anthers were shriveled and non-dehiscence-Male sterile.

 However, these lines produced normal fertile pollen at low temp.

Sensitive period : PMC formation to Meiosis
2. PGMS: Governed by 2 recessive genes.
 Sterility is obtained in long day conditions while in short days,
normal fertile plant.
 Rice:- Sterile under Long day conditions (13 hr. 45 min + Temp. 23-
290 C) but fertile under short day conditions.
 Sensitive period: Differentiation of secondary rachis branches to
PMC formation
Inheritance & Maintenance Of male sterile line
Nuclear male sterility and hybrid seed production

P1 P2

Male sterile Male fertile

X Msms
msms

Msms msms X MsMs

Male fertile Male sterile Male fertile

F1
Msms

Male fertile
 Cytoplasmic Genetic Male Sterility (CGMS)

 CGMS is also known as nucleoplasmic male sterility.

 Case of CMS, where a nuclear gene (R) for restoring fertility in
male sterile line is known.
 R (restorer gene) is generally dominant can be transferred from
related strains or species.
 This system is known in cotton, maize, jowar, bajra, sunflower,
cotton, rice and wheat etc.
Hybrid seed production using CGMS system
 Transfer of Restorer gene ‘R’ to non restorer strain
Strain A Strain B
CMS ♀ rr
S × RR
F ♂ Restorer Restorer line R is crossed to Male sterile A

Male fertile ♀ Rr
S × rr
F ♂ Male fertile Male fertile F1 is crossed to Strain C
Non restorer (Strain-C) in which R gene is to be transferred

×
Male sterile Male fertile
Discarded rr
S ♀ Rr
S
× rr
F ♂ Male fertile Male fertility progeny is
back crossed to strain C

×
(Strain-C)

Male sterile Male fertile

Discarded rr
S ♀ Rr
S × rr
F ♂ (Strain-C)
Male fertility progeny is
back crossed to strain C
Male fertile
6-7 Back crosses

Male sterile
Discarded

×rr
S
RR
S Male sterile
Self pollinated
Male fertile progeny is self pollinated

×
Male fertile progeny is self pollinated.
Male sterile S S S Individual plant progenies grown in next generation
rr Rr RR
Discarded and non segregating progenies are selected
1 : 2 : 1
Male fertile Self pollinated
 Production of Double cross maize hybrids using CGMS

Inbred A
♀ (Cytoplasmic ♀
rr S Single Cross –I
Male Sterile)
A×B
(Male Sterile)
Inbred B
♂ (Non restorer
Double Cross
rr f rr S (A×B) × (C×D)
male fertile)
rr S 50%

Inbred C ♂ Rr S 50%
♀ (Cytoplasmic rr S Single Cross-II (1:1 Segregation for
Male Sterile) C×D Male Fertility & Sterility)
(Male Fertile)
Inbred D
♂ (Non restorer RR S Rr S
male fertile)
 Sources of CMS & Restorer genes in some Crops

Crop species Cytoplasm Restorer Genes

CMS-CW O. spontanea

Rice CMS-bo O. Sativa boroII (single dominant)

CMS-WA O. Spontanea (WA, four genes)
CMS-W18 O. rufipogon
Wheat (T.aestivum) T. timopheevi Rf1 and rf2
A. caudata -
T. Durum Aegilops ovata -
CMS-C Rf4
Maize CMS-S Rf3
CMS-T Rf1 and Rf2
Crop species Cytoplasm Restorer Genes

N. Debneyi -
Tobaco
N. Megalosiphon -

N. bigelovii -

G. Anomalum -
Cotton
G. Arboreaum -

G. harknesii -

Sunflower PET-1 (H. petalaris) 2 polymorphic genes (Rf1, Rf2)

Jowar Milo or A1 Msc from kafir race

Bajra Tift-23A -
 Male Sterility based Hybrids in Important Crops

S.No. Crop Hybrid Variety Seed Production

1. Maize Ganga 101, Ganga 1, CMS

Deccan, Ranjit,
Trishulatha, DHM-107,
DHM-109

2. Sorgum CSH1 CMS

3. Bajra HB1 CMS
4. Sunflower BSH1 CMS
5. Rapeseed PGSH51 CMS
6. Red Gram ICPH-8 GMS
7. Rice PRH1 CMS
 Transgenic Male Sterility

 Recombinant DNA techniques for disturbing any or number of

developmental steps required for the production of functional
pollen within the microspore or for the development of any
somatic tissues supporting the microspores.
 Transgenes for male sterility are dominant to fertility.
 Also to develop effective fertility restoration system for hybrid
seed production.
 Example: Barnase/Barstar system
 Limitations of Cytoplasmic-Genetic Male Sterility

 Undesirable effects of the cytoplasm

 Unsatisfactory fertility restoration
 Unsatisfactory pollination
 Spontaneous reversion
 Modifying genes
 Contribution of cytoplasm by male gamete
 Environmental effects
 Non availability of a suitable restorer line
 Barnase/Barstar system

 Barnase is extracellular RNase; barstar is inhibitor of barnase

(Bacillus amyloliquefaciens)
 Plants with TA29 promoter-Barnase construct are male sterile
 Those with TA29-Barstar are not affected by the transgene barnase.
 Barstar is dominant over the Barnase
 Fuse the barnase and barstar genes to TA29 promoter–TA29 is a plant
gene that has tapetum specific expression.
 Cross male sterile (barnase) with male fertile (barstar) to get hybrid
seed, which now has both barnase and barstar expressed in tapetum
and, hence, is fully fertile
Hybrid seed production using Barnase/Barstar system
 Chemical Induced Male Sterility

 CHA is a chemical that induces artificial, non-genetic male

sterility in plants so that they can be effectively used as female
parent in hybrid seed production.

 Also called as Male gametocides, male sterilants, selective male

sterilants, pollen suppressants, pollenocide, androcide etc.

 The first report was given by Moore and Naylor (1950), they
induced male sterility in Maize using maleic hydrazide (MH).
 Properties of an Ideal CHA

 Must be highly male or female selective.

 Should be easily applicable and economic in use.
 Time of application should be flexible.
 Must not be mutagenic.
 Must not be carried over in F1 seeds.
 Must consistently produce >95% male sterility.
 Must cause minimum reduction in seed set.
 Should not affect out crossing.
 Should not be hazardous to the environment.
 Some important CHAs

S.No. CHAs Critical stage Crop species

1. Zink Methyl Arsenate 5 days before heading Rice
Sodium Methyl Arsenate

2. Ethephon/ Ethrel Depends on crop Barley , oat, bajra,

rice

3. Mendok Depends on crop Cotton, sugarbeet

4. Gibberellic acid 1-3 days before meiosis Maize, Barley,

Wheat, Rice,
Sunflower

5. Maleic Hydrazide Early microsporogenesis Maize, wheat,

cotton, onion
 Hybrid Seed Production based on CHAs

Conditions required:-

 Proper environmental conditions (Rain, Sunshine, temp, RH etc.)

 Synchronisation of flowering of Male & Female parents.

 Effective chemical emasculation and cross pollination

 CHA at precise stage and with recommended dose

 GA3 spray to promote stigma exertion.

 Supplementary pollination to maximise seed set

 Avoid CHA spray on pollinator row.

 Advantages of CHAs

 Any line can be used as female parent.

 Choice of parents is flexible.

 Rapid method of developing male sterile line.

 No need of maintaining A,B&R lines.

 Hybrid seed production is based on only 2 line system.

 Maintenance of parental line is possible by self pollination.

 CHA based F2 hybrids are fully fertile as compared to few sterile

hybrids in case of CMS or GMS.

 Limitations of CHAs

 Expression and duration of CHA is stage specific.

 Sensitive to environmental conditions.

 Incomplete male sterility produce selfed seeds.

 Many CHAs are toxic to plants and animals.

 Possess carryover residual effects in F1 seeds.

 Interfere with cell division.

 Affect human health.

 Genotype, dose application stage specific.

 Significance of male Sterility in Plant Breeding

 Male sterility a primary tool to avoid emasculation in hybridization.

 Hybrid production requires a female plant in which no viable

pollens are borne. Inefficient emasculation may produce some self

fertile progenies.
 GMS is being exploited (Eg.USA-Castor, India-Arhar).

 CMS/ CGMS are routinely used in Hybrid seed production in corn,

sorghum, sunflower and sugarbeet, ornamental plants.

 Saves lot of time, money and labour.
 Limitations in using Male Sterile line

 Existence and maintenance of A, B & R Lines is laborious and

difficult.

 If exotic lines are not suitable to our conditions, the native/adaptive

lines have to be converted into MS lines.

 Adequate cross pollination should be there between A and R lines for

good seed set.

 Synchronization of flowering should be there between A & R lines.

 Fertility restoration should be complete otherwise the F1 seed will be

sterile Isolation is needed for maintenance of parental lines and for
producing hybrid seed.
 Applications
of
Male Sterility
in
Hybrid Seed Production
 Male sterility system in Rice hybrid seed production

 Male sterility: a condition in which the pollen grain is unviable or

cannot germinate and fertilize normally to set seeds.

 Male Sterility Systems (genetic and non-genetic):

 Cytoplasmic genetic male sterility (CMS)
Male sterility is controlled by the interaction of a genetic factor
(S) present in the cytoplasm and nuclear gene (s).

 Environment-sensitive genic male sterility (EGMS)

Male sterility system is controlled by nuclear gene expression,
which is influenced by environmental factors such as temperature
(TGMS), daylength (PGMS), or both (TPGMS).

 Chemically induced male sterility

Male sterility is induced by some chemicals (gametocides)
Two Commercial MS Systems for Hybrid Rice
 Flowchart of 2-Line Hybrid Rice Evaluation and Seed Production

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)
 TGMS and two-line hybrid

Temperature
Reproductive Upper Limit
high
 Based on the Sterile
discovery of F1 Seed
P(T)GMS mutant Production

Critical Sterility Point

 Male sterility Partial Sterility
controlled by 1 or 2 Critical Fertility Point
pairs of recessive
Fertile
gene(s) S-line
Multiplication
Reproductive Lower Limit
low

Model of Sterility / Fertility Expression for TGMS Rice

 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
 Requirements for 3 Lines in CMS System

A-line
Stable Sterility
Well developed floral traits for outcrossing
Easily, wide-spectum, & strongly to be restored
B-line
Well developed floral traits with large pollen load
Good combining ability
R-line
Strong restore ability
Good combining ability
Taller than A-line
Large pollen load, normal flowering traits and timing
 Flowchart of 3-Line Hybrid Rice Evaluation and Seed Production

Elite CMS line SOURCE NURSERY Elite lines from different sources

To evaluate parents and make testcross B & R line Breeding Program

P line Breeding Progam

CMS BACKCROSS NURSERY TESTCROSS NURSERY

BC2- BC4, CMS Evaluation To identify B, R & P lines R & P Line
Backcross CMS pairs (BC1)
Premarily heterosis evaluation, 2 rows w/ parent Hybrid Seed Production for OYT
Isolation Bags or hand-crossing
AxB Paircross RETESTCROSS NURSERY (OYT)
Breeder Seeds Re-evaluate F1 hybrids
Stage 1, 1 rep, 3 rows Hybrid Seed Production for PYT
Isloated Net or bags
AxB Increase Preliminary Yield Trial (PYT)
Core Seeds Stage 2, 1 rep, plot
Hybrid Seed Production for AYT & NYT
AxB Seed Production Advanced Yield Trial (AYT) Isolation Block
Foundation Seeds Stage 3, 3 reps, plot

AxB Seed Production National Yield Trial Hybrid Pilot Seed Production
Certified Seeds Stage 4, 3-4 reps, muti-location, 2-years Isolation Block

A & B Line Release On-Farm Trial (Strip Trial) Hybrid and R line Release
Advantage & Disadvantage of 3-line hybrid rice system

Advantages
 Stable male sterility.

Disadvantages
 Limit germplasm source (CMS, Restorer)
 Dominant CMS cytoplasm in large area (WA)
 One more step for parental seed production
 Time consuming of CMS breeding
Male sterility system in Maize hybrid seed production

Different ways of inducing male sterility in maize

I. Manual/mechanical emasculation (detasselling)
II. Genic male sterility
III. Cytoplasmic genetic male sterility
IV. Gametocides
1. Genetic Male sterility
Male sterility determined by single recessive gene
40 loci involved have been identified (ms1 to ms52)
ms5 –cloned
Problem : impossible to maintain male sterile inbred
detasselling required
 2. Cytoplasmic Male sterility

1. CMS-T (Texas) (Rogers and Edwardson, 1952)

 Highly stable under all environmental conditions
 Characterized by failure of anther exertion and pollen abortion
 Susceptible to race T of the southern corn leaf blight - (Cochliobolus
heterostrophus = Bipolaris maydis)
 Widespread use of T-cytoplasm for hybrid corn production led to
epidemic in 1970 with the widespread rise of Race T.
 Toxin produced by C. heterostrophus = T-toxin.
 Fertility restoration is sporophytic
 Rf1 (chr. 3) & Rf2 (chr.9) are responsible for fertility restoration
T-urf13 gene in T cytoplasm maize
 Mitochondrial gene T-urf13 is a unique chimeric sequence
Effect of URF13 protein-
 Degeneration of the tapetum during microsporogenesis
 Disruption of pollen development leading to male cell abortion

2. CMS-C (Charrua) (Beckett, 1971)

 Mutations in three genes viz atp6, atp 9 and cosII- confer CMS
phenotype
 Fertility restoration is Sporophytic
 Rf4, Rf5, Rf6 are responsible for fertility restoration

3. CMS-S (USDA) (Jones,1957)

 Sterility associated with orf355-orf77 chimeric mt gene
 Fertility restoration is Gametophytic
 Rf3 (chr. 2) are responsible for fertility restoration
 Plasmid like element S1 & S2
 Reversion to fertility

 The reversion of CMS

strain to male fertility is
based on genetic change

 Reversion can be
spontaneous or mutagen
induced

 S-cytoplasm revert rather

frequently to male fertility
(than T & C). Maize-CMS Restoration of fertility system:
different classes of pollen grains are produced,
but not all of them are viable
Triple Cross Hybrid Double Cross Hybrid
A X B A X B C X D
(frfr) (frfr) (frfr) (frfr) (frfr) (FrFr)
ms mf ms mf ms mf

AB AB X CD
X C
(frfr) (frfr) (Frfr)
(FrFr)
ms mf ms mf

ABCD
ABC 1 : 1
(Frfr)
(frfr) : (Frfr)
mf
ms : mf
Male sterility system in Bajra hybrid seed production

Types of Hybrids

 Single cross hybrid (A×B)

 Double cross hybrid (A×B)×(C×D)
 Three way cross Hybrid (A×B)×C
 Top cross (C×OPV)
 Hybrid blends
 Inter-population hybrids
 Chance hybrids
 Hybrid seed production using CGMS
 Depends on the cytoplasm that produce male sterility and gene that
restores the fertility.

 Steps:
 Multiplication of CMS (A) line
 Multiplication of Maintainer (B) line and Restorer (R) line
 Production of Hybrid seed (A×R)

 Maintenace of A & B lines:

 Grow A line and its corresponding B line together in an isolated
plots.
 Isolation distance for A×B production fields is at least 1000m.
 A ratio of 1A:1B row is maintained.
 Pollens produced by the B line fertilize the male sterile plant (A)
and seeds produced thus
 Give rise to A line again.
 Maintenance of R line:
 Pearl millet R line could be either an inbred line or an Open
pollinated variety which can be multiplied as variety.
 Seeds of R lines are produced by multiplying seeds in isolated
plots having distance 1000m.
 Any plant in the R line plot appearing different from true R
type should be uprooted or rogued out before anthesis.
 Purity of the parental seed is very important because it affects
the quality of the hybrid seeds that is generated.
 Scheme of hybrid seed Layout of hybrid seed
production in pearl millet production plot
 Identification of potential hybrid parents (A,B and R lines)

 Potential male and female parents for hybrid seed production are
identified by crossing male fertile parent (Inbreds, variety,
germplasm, breeding stocks in advanced generations) to a male sterile
line (A line) and observing their corresponding hybrids in small plots
of an observation nursery.

 A few plants of each cross are subjected to the bagging test i.e.
covering the few panicles with the paper bags before anthesis and
observing the seed set under the bag after few weeks.
 CGMS
A1 Tift 23 A (Most of the world hybrids contains A1 Blood),
Burton,1958
A2, A3 Not stable cytoplasm
A4 Derived from P. glacum subspecies monodii
Does not have effective restorer
Used in forage hybrid production
 Male sterility system in Brassica hybrid seed production

Cytoplasmic male-sterile
 Stamen (anther and filament) and pollen grains are affected
 It is divided into:
a. Autoplasmic
 Arisen within a species as a result of spontaneous
mutational changes in the cytoplasm, most likely in the
mitochondrial genome
b. Alloplasmic
 Arisen from intergeneric, interpecific or occasionally
intraspecific crosses and where the male sterility can be
interpreted as being due to incompatibility or poor co-
operation
between nuclear genome of one species and the organellar
genome.
 Another CMS can be a result of interspecific protoplast fusion
 Various CMS systems
 Raphanus or ogu system
 Polima or pol system
 Shiga-Thompson or nap system
 Diplotaxis muralis or mur system
 Tournefortii (tour) system
 Moricandia arvensis or mori system
 Chinese juncea or jun system

17 systems are available, only difference is the use of male sterile

cytoplasmic sources differs for each system

 Nap system– B.napuus cross b/w winter & spring var.

 pol system – B.napus var polima
 mur system--Diplotaxis muralis x B.campestris cv Yukina
 tour system– B.juncea collections
Ogu system:-

 First discovered in Japanese radish (Raphanus sativus) by Ogura, 1968

 B.napus genome was transferred into the back round of R.sativus (mst)
through intergeneric crosses followed by back crossing with B.napus.

 CMS seedling under low temperature showed chlorosis , because

chloroplast of R.sativus is sensitive to cold, it is governed by cp-DNA ,
but mst is governed by mt DNA.

 Protoplast fusion of R.sativus with B.napus carried out to have normal

green plants with ogu CMS characterisitics

 This system now has been used for developing alloplasmic male sterile
line in B.juncea and B.campestris.
 Genetic Male Sterility
 GMS is governed by two genes either recessive or dominant
genes(Kaul,1988)

 One more dominant gene is associated with development of male

sterility in B.napus type by means of transgenic male sterility

Chemical Male sterility

 Enthrel – Brassica juncea
 Zinc methy arsenate- B.napus
 GA- B.oleracea var capitata
 Development of Male sterile B. napus from R. sativus

B.napaus
Rhapanus sativus G-Rs
x G-Bn
C-Rs N-Bn

mst mft

F1 interspecific cross 1/2G-Rs

1/2G-Bn
F1 Sterile

C-Rs
Doubling by colchince
Fertile amphidiploid

1/2G-Rs
1/2G-Bn

C-Rs
mst
 1/2G-Rs
1/2G-Bn x G-Bn B.napus

N-Bn
C-Rs

mst mft

BC3

G-Bn

C-Rs

Male sterile B.napus

 Development of Alloplasmic Male sterile Brassica campestris

Male sterile B.napus B.campestris

G-Bn
x G-Bct
S-Rs N-Bc

mst mft

1/2G-Bn G-Bc
F1 interspecific cross
1/2G-Bc x G-Bc
S-Rs N-Bc

BC4

G-BC

S-Rs
 Male sterility system in Safflower hybrid seed production

Presently genetic male sterility (GMS), cytoplasmic male sterility

(CMS) and thermo sensitive genetic male sterility (TGMS) lines are
available in India.

Development of agronomically superior genetic male-sterile lines in

safflower in India have resulted in the development and release of
spiny safflower hybrids DSH-129 and MKH-11 in 1997 and NARI-
H-15 in 2005, the first non-spiny hybrid safflower NARI-NH-1 in
2001.
 Male sterility system in Sunflower hybrid seed production

Genetic Male sterility (GMS)

 Complete male sterility

 ms1-ms5 = male sterility in sunflower recessive gene

 Two types of g-mst

 Type 1-gmst-Bloomington type

 Type 2-gmst-Modern type

 Cultivated Sunflower variety Karlik-68(Dwarf 68)- two recessive

genes msi1,msi2 (Stable and complete male sterile)

 Partial male sterility –p mst

 CGMS
H.petiolaris × H.annuus Repeated backcross of H.annuus
results in cms1 which is extensively
used mst in hybrid seed production of
sunflower all over the world
H.giganteus× H.annuus Cms3( S cytoplasm source)
H.annuus subspp lenticularis Indiana 1
× H.annuus CV commander
 Male sterility system in Cotton hybrid seed production

All three types of male sterility occurs (g mst,c mst,gc mst) in cotton
 Genetic Male Sterility (GMS):
 Reported in upland, Egyptian and arboreum cottons.
 In tetraploid cotton, male sterility is governed by both
recessive and dominant genes.
 However, male sterility governed by recessive genes is used in
practical plant breeding
 Sixteen different genes in tetraploid cottons (13 in G. hirsutum
and 3 in G. barbadense) and two in G. arboreum have been
identified for genetic male sterility.
 Sterility is conditioned by dominant alleles at five loci viz, MS4,
MS7, MS10, MS11 and MS12 by recessive allele at other loci
viz. msl, ms2, ms3, ms13, ms14 (Dong A), ms15 (Lang A) and ms16
(81 A).
 G. hirsutum line Gregg (MS 399) from USA is the basic
source of GMS possessing ms5 ms6 gene for male sterility.
Genetic Male Sterility
 CMS System
In case of CMS, the originally discovered CMS sources involving G.
arboreum and G. anomalum cytoplasmic systems having interaction
with ms3 locus were not found effective or stable under different
environments.

The only stable and dependable CMS source under varied environment
was developed through the utilization of G. harknessii. The complete
genome of G.hirsutum was transferred into the G. harknessii cytoplasm.

A single dominant gene ‘Rf’ from G.harknessii is essential for fertility

restoration.

Fertility enhancer factor 'E' for this CMS restorer system was obtained
from a G.barbadense stock.

The harknessii system is reported to contribute to good agronomic

properties and attraction to honey bees.
 Sources of Male sterility in Cotton

Source of ms cytoplasm Nuclear genome

G. anomalum, G. hirsutum
G. arboreum, G. harknessii
G. anomalum, G. arboreum Heat sensitive , less stable
G. harknessii × G. hirsutum Stable cms all over the environment
New sources of CMS
G. aridum Skovt. × G. hirsutum (D4)
G. trilobum × G. hirsutum CMS 8 (D-8)
G. sturtianum × G. hirsutum CMS-C1
New sources of CGMS
G. anomalum x G. thurberi Cg-mst
Mutation
 G. arboreum, the first spontaneous male sterility mutant was identified
in variety DS-5

Chemical based male sterility

 FW 450(Sodium B-Dichloro-iso-butyrate)
 MH-30 (Maleic hydrazide)
 Ethidium bromide

Male sterility based hybrid Production

 GMS system. CPH2 (Suguna), First hybrid based on GMS released at
CICR, RS, Coimbatore
 G. harknessii based cms with fertility restoration gene sources were
used in developing the hybrid CAHH 468 (PKV Hy-3).
 Male sterility system in Potato hybrid seed production

Inter-specific Hybridization

Cytoplasm Nuclear genome Reference

S.acaule (4X) S.tuberosum Lamm,1953
S.chacoense(4X) S.tuberosum Rammanna and Hersmen(1974)
S.phureja(2x) S.tuberosum Magoon et al.,1958b
S.stoloniferum(4x) S.tuberosum Ross (1961)
S.Verrucosum(2X) S.tuberosum Abdalla (1970)
 Chemical mutagens
 FW 450(Sodium B-Dichloro-iso-butyrate)
 MH-30 (Maleic hydrazide)
 Ethidium bromide
Development of Male sterility

Genome transfer
S cytoplasm is in the genome of fr genes

Unreduced Gamete Production

S.tuberosum (2x) × S.tuberosum (4x)

Protoplast Fusion
S cytoplasm is retained
Di haploid

S.tuberosum (4x) × S.phureja (4x)

(2x) (2x)
F1 (4x)

Anther culture

DiHaploid (2x)