10. methods & problems in seed production

PROBLEMS OF SEED PRODUCTION IN VEGETABLE
CROPS
PGS-504
Submitted by:- Tapan Adhikari
Ph.D. Scholar, First Year
F-2019-15-D
Dept. Of Forest Product
Discipline: Medicinal & Aromatic
Plants
Submitted to:- Dr. Upender Singh

FOR HYBRID SEED PRODUCTION ONE
SHOULD KNOW
Principles of
seed production
Botany of
crop (period of
stigma
receptivity,
anthesis etc.,)
Techniques of
seed production

PRINCIPLES OF SEED PRODUCTION
Genetic
Mechanical admixtures
Natural crossing
Developmental variations
Minor genetic variations
Selective influence of
disease
Agronomic
Environmental
requirements
Land requirements
Pollination requirements
Isolation requirements
3

Greater productivity
Uniform produce
Better adaptability to variable environments
Better tolerance to diseases and pests
Longer harvest duration
Better market acceptability
Better nutritional quality

Methods of hybrid seed production in vegetable crops
Mechanism Commercially exploited in:
Hand emasculation + HP
Tomato, eggplant, sweet pepper, okra, hot
pepper
Male sterility + HP Tomato, hot pepper, sweet pepper
Male sterility + NP Onion, cabbage, cauliflower, carrot, hot pepper
Self incompability + NP Cole crops.
Gynoecism + NP Cucumber, muskmelon.
Remove staminate flowers + HP Most of Cucurbits
Remove staminate flowers + NP Most of Cucurbits
HP = Hand pollination NP = Natural pollination

10. methods & problems in seed production

Next day opening flower buds are selected
To be done before anther dehiscence
To be done with hand/ forceps
2.Pollen Collection
1. Emasculation:
Flower collection
Anther cones are taken
and put them in glassine
envelopes
Dry anthers at
30℃ for 24 hrs
Put the dried anther
cones in a cup then
tranfer to pollen lid cup
3. Pollination

LIMITATIONS OF EMASCULATION
AND POLLINATION
Time consuming
Labour intensive (Skill is necessary for
commercial seed production)
Increased cost of production

 Production of large scale of F1 seeds.
 Reduced cost of hybrid seed production.
 Speedup the hybridization programme.
 Commercial exploitation of hybrid vigour.

CLASSIFICATION OF MALE STERILITY
Phenotypic MS
 1. Structural male sterility : anomalies in male sex organs / missing altogether
 2. Sporogenous male sterility : stamens form, but pollen absent or rare due to
microsporogenous cell abortion before / during/ after meiosis
 3. Functional male sterility : viable pollen form, but barrier prevents fertilization
(anther indehiscence, inability of pollen to migrate to Stigma or other factors that affect
fertilization e.g., extended style, pollen is glued together in Soybean)
Genotypic MS
 i. Cytoplasmic male sterility (CMS): Non-Mendelian inheritance - cytoplasmic
 ii. Genic male sterility (GMS): Mendelian inheritance due to nuclear not cytoplasmic
genes
 iii. Cytoplasmic genic male sterility (CGMS): Both nuclear and cytoplasmic genes
are involved
Induced MS
 i. Environment Sensitive Genic male sterility or Environmental induced sterility
(EGMS)
 ii Chemically Induced Male Sterility

Figure : By microspore appearance under 100x magnification (0.5% aceti-carmine stain)

S F
S
Male sterile
Male sterile
Male fertile
F1 seeds 100% male sterile
Cytoplasmic male sterility

This was proposed by Jones, in 1908 in onion and subsequently
reported in carrot, chinese cabbage, Radish, Garlic, etc.
Advantage:-
 All progenies are male sterile. (F1 progenies)
 Longer flowering parent and longer life span of flower can be
seen in male sterile plant. That is more advantage in
ornamental plants.
 Useful to produce single cross/double cross hybrids, in crop
where the vegetative part is commercial product.
Cytoplasmic male sterility
Disadvantage:-
It is not useful to produce the hybrid seed in crop where the
fruit/seed is commercial product.

S F
S
S F
S
P1
P2
Male sterile Male Fertile
Male sterile
F1
Male sterile Male Fertile
Male sterile
Back cross
♀ ♂
♂♀

2. GENIC MALE STERILITY (GMS)
Genic or Genetic male sterility is ordinarily governed by a single
recessive genes ‘msms’
Male sterility alleles arise spontaneously or may be artificially
induced.
A male sterile line may be maintained by crossing it with
heterozygous male fertile plants (Msms). Such a mating
produces 1:1 male sterile and male fertile plants.
Marker genes which are linked to male sterility/fertility can be
used to identify the male fertile plants before flowering stage.
Male sterile line will be having white translucent anthers, while
fertile line will be having yellow, plumpy anthers
At the time of flowering, each and every plant is to be examined for
identification of sterile and fertile lines

The A line is sterile because it has ‘ms ms’ gene
The B line is similar to A line but for the heterozygous
condition (Ms ms)
The R line is completely fertile (Ms Ms)
Maintenance of GMS line
msms (A- line) X Msms (B- line) = msms (50 % plants Male
sterile)+ Msms (50 % plants Male fertile)
Production of hybrid seed
msms (A- line) X MsMs (R- line) = Msms ( Male fertile F1
hybrid)

The difficulties in the use of GMS are
Maintenance of GMS requires skilled labour to identify fertile and
sterile lines
Labeling is time consuming
In hybrid seed production, spot identification of fertile line and
removing them is expensive
Use of double the seed rate of GMS line is also expensive
High temperature leads to break down of male sterility in some crops

This CGMS was first reported in onion by Jones and Davis- 1944. It
is also reported in Barley, Sorghum, Bajra, Rice, Maize, Brassica, Sugar
beat etc.
There are two steps are involved:
Maintenance or multiplication of ‘A’ line (Male sterlie) ‘B’ line and ‘R’
line parents are multiplied in separate isolated blocks independently.
So this stage is called Foundation seed production stage.
Cytoplasmic genetic male sterility
•‘A’ line can be maintained by crossing the homozygous male sterile
line having sterile cytoplasm with homozygous male fertile pollinator
parent having fertile cytoplasm.

Male sterile line (A- line- MS Line)- Smsms
Male fertile line (B- line- Maintainer line )-
Fmsms
Restorer line (R- line- Restore fertility through
nuclear genes)
SMsMs
FMsMs

Maintenance of CGMS Male Sterile Line or A line:
 Since A line does not produce pollen, seed is not formed for
maintaining A line. It has to be crossed with its fertile counter part
having similar nuclear genes with fertile cytoplasm which is known as
B-line.
Application CGMS for production of Hybrid seed:
 CGMS is used in Maize, Jowar, bajra, sunflower, rice, wheat, onion
etc
 For production of hybrid seed, A-line has to be kept as female parent
and the pollen parent should posses the restorer genes in order to
induce fertility and seed development in the next generation.
 Such line is known as restorer line and denoted as ‘R'line.
 The A line & R line should be of different genetic constitution and
should be able to give maximum heterosis.

ENVIRONMENTAL GENIC MALE STERILITY
(EGMS)
EGMS is more popularly termed as “Two line Hybrid Breeding” as
against “Three Line Hybrid Breeding” in case of CMS system.
Certain GMS lines are conditional mutants, meaning thereby, in
particular environment male sterile mutant plants turn into
male fertile.
After determination of critical environment (Usually temperature
or photoperiod) for sterility and fertility expression, such GMS
mutants are classified under Environmental Genic Male
Sterility (EGMS) lines.
EGMS lines have been reported in several vegetable crops.

However, from practical view point, it is necessary to identify
critical temperature or photoperiod for the fertility/sterility
expression in temperature and photoperiod sensitive genetic
male sterility, respectively and may not be hundred per cent.
Seeds of EGMS line can be multiplied in an environment where it
expresses male fertility trait, while hybrid seed can be produced
in other environment, where it expresses male sterility trait.
Initially EGMS lines were thought to be of very less practical value,
as they were unstable. But now they are considered to represent
most efficient system for hybrid seed production.

CHEMICALLY INDUCED MALE STERILITY

 Many chemicals affect the function of male reproductive organs
in plants.
 These compounds are called male gametocides, androcides,
pollenocides, male sterilants, pollen suppressants etc.
 McRae (1985) suggested the use of single term i.e. Chemical
Hybridizing Agents (CHAs) to avoid confusion.
 A CHA is a chemical which induces artificial, non-genetic male
sterility in plants so that they can be used a female parent in
hybrid seed production.

CHA TECHNOLOGY
The purpose of a CHA is to facilitate the production of hybrid seed.
The technology for making hybrids with a CHA is essentially
identical to cytoplasmic male-sterile (CMS) system but not
inherited.
The only difference is that functional male sterility in the female
parent is obtained with a chemical rather than by genetic
manipulations.

CHARACTERS OF AN IDEAL CHA ARE:
Selectively induce male sterility i.e. Induction of male but not
female sterility.
Uniform effect in inducing male sterility i.e. Complete inhibition of
pollen development
No side effects on other traits of plant growth and development i.e.
Absence of phyto-toxicity or other adverse effects.
Should not be carried over to F1 seeds.
Non mutagenic
Environmental safety
Independent from environmental conditions.
Independent from genotypic differences.
Cost effectiveness.

DISADVANTAGES OF CHAS
Appropriate developmental stage for application i.e. use of CHAs
are stage specific
Effect of environment is hard to predict i.e. CHA induced male
sterility is affected by environment.
Many CHAs are toxic to plants and animals
Some CHAs like arsenicals and WL 84811 produce carry over
residue effects in F1 hybrids.
Difficult to have uniform conditions.
CHAs are generally genotypic, dose and application stage specific
Weather can prevent application at optimal stage

SELF
INCOMPATIBILITY
Inability to set seed from application of pollen produced on same
plant or it refers to failure of viable pollen of a given plant to
fertilise the ovules of the same plant, but it capable of fertilising
effectively the ovules of the most other plant of the same
variety.
Self incompatibility occurs in more than 3000 sp belonging to 250
genera, spread in about 70 families.

CLASSIFICATION OF SELF
INCOMPATIBILITY
According to Lewis (1954) the self incompatibility is classified as follows:

1. Gametophytic.
SI reaction of a pollen is determined by its own genotype
not by the genotype of the plant on which the pollen is
produced
2. Sporophytic.
SI reaction of pollen is governed by the genotype of the
plant on which the pollen is produced and not by the
genotype of the pollen.
37

GAMETOPHYTIC SYSTEM
Gametophytic incompatibility was first described by East and
Mangelsdorf in 1925 in Nicotiana sanderae.
The incompatibility reaction of pollen is determined by its own
genotype, and not by the genotype of the plant on which it is
produced.
Generally, incompatibility reaction is determined by a single gene
having multiple alleles, e.g.,Trifolium, Nicotiana, Lycoperscion,
Solanum, Petunia etc.
If same allele as that of Pollen is present in the stylar tissues, it
opposes the growth of pollen tube in the style, so Gametophytic
incompatibility is also called as ‘oppositional factor system’.
Pollen tube grows very slowly in the style containing the same S
allele as the pollen, and fails to effect fertilization. Therefore, all
the plants are heterozygous at the S locus.

In a single gene system, there are three types of mating;
 i) Fully incompatible, e.g., S1S2 x S1S2
 ii) Fully compatible, e.g., S1S2 x S3S4
 iii) Partially (i.e., 50% of the pollen) compatible, e.g., S1S2 x S2S3
In some cases, an allele for self-fertility, Sf, is found (East and
Yarnel). Pollen carrying the Sf alleles does not show
incompatibility reaction.
The gametophytic system is found in families like Solanaceae,
Rosaseae, Graminae, Leguminoseae, Chenopodiaceae,
Ranunculaceae
Red clover, white clover, rye, potato, tomato are important crop
plants where this system of SI exists

SPOROPHYTIC SYSTEM
In the sporophytic system also, the self -incompatibility is governed
by a single gene, S, with multiple alleles ; more than 30 alleles
are known in Brassica oleracea.
In general, the number of S alleles is considerably larger in the
gametophytic than in the sporophytic system.
The incompatibility reaction of pollen is governed by the genotype
of the plant on which the pollen is produced, and not by the
genotype of the pollen.
It was first reported by Hughes and Babcock in 1950 in Crepis
foetida, and by Gerstel (1950) in Parthenium argentatum.
In Sporophytic system, the S alleles may exhibit dominance, i.e. S1
is dominant over S2 and S2 over S3 and so on (S1>S2>S3>S4……..
So on).
In this system crosses between different genotypes are fully fertile
or sterile.

RESULT OF GAMETOPHYTIC AND SPOROPHYTIC INCOMPATIBILITY SYSTEMS
FROM DIFFERENT CROSSES

 For hybrid seed production,
(1) two self-incompatible, but cross-compatible, lines are interplanted ;
On both the lines hybrid seed would be produced.
(2) Alternatively, a self-incompatible line may be interplanted with a self-
compatible line. From this scheme, seed from only the self incompatible line
would be hybrid.
(3) Schemes for the production of double cross and triple cross hybrids have
also been proposed and their feasibility has been demonstrated in the case of
brassicas.

Line A Line B
Parents S11 S22
Propagation Bud pollination
S11 Χ S22
SCH S12
Two season and two parents are required.
Single cross hybrid

• Parents A X B C X D
S11 S22 S33 S44
• 1st season propagation through propagation through
bud pollination bud pollination
• 2nd season S11 X S22 S33 X S44
SCH S12 S34
Double cross hybrid

• 3rd season S12 X S34
(SCH) (SCH)
DCH - S13, S14, S23, S24 [S1234]
The seeds are given to farmer for commercial cultivation for
production maintain 1:1 or 2:4 ratio and here 4 parents and
3 season are required.

• Inbred A B C D E F
S11 S22 S33 S44 S55 S66
• transplant selfed are produced by
inbreed in to small isolation
block and cross AXB.
Self in bud pollination
SCH S12 (AXB) S33 (C) SCH S45 (DXE) S66 (F)
After that sow the seeds in proportion of 3:1 ratio in both cross.
Triple cross hybrid seed production

AB: C DE:F
S12 X S33 S45 X S66
s13: s23 S46:S56
We get the 3 way cross We get the 3 way cross
3 way cross X 3 way cross
Seeds of two 3 way crosses are mixed and distributed commercial seed
firm for final cross.
 Raise the crop and left for natural pollination. Then harvest the seeds
of TCH for commercial cultivation.
 Here 4 seasons and 6 parents are required.

 Stable self incompatibility.
 High seed set of self pollination at bud stage.
 Favorable and uniform economic characters.
 Desirable combination ability.
48

USE OF GYNOECIOUS LINES IN
HYBRID SEED PRODUCTION

GYNOECY OR THE USE OF LINES WITH ONLY
FEMALE FLOWERS AS FEMALE PARENTS OF
HYBRIDS.
This method has been used in cucumber.
Hybrid seed is produced by growing the gynoecious female line
in the same field as the male line producing the pollen.
Pollination is performed by bees.

Cucumber is the species most extensively studied in the
Cucurbitaceae for the production of hybrid seed
Among the many types of cucumber cultivars, it is possible to
find gynoecious genotypes, i.e. plants that only have female
flowers.
Parthenocarpy, or the development of fruits without fertilization
and seed formation, is another important trait available for
cucumber breeding.
Gynoecious cultivars with parthenocarpic fruits are usually
preferred for greenhouse production because of their higher
yields and ease in crop management.

USE OF GYNOECIOUS LINES IN HYBRID
SEED PRODUCTION
The gynoecious trait is determined by a single dominant gene: “F”.
Because these plants have only female flowers, hybrid seeds may be
produced using gynoecious maternal lines without the
requirement for male flower emasculation.
In the field, the ratio between female and male line plants is usually
3:1 and pollination is performed by bees.

 There are different systems that have been proposed to produce
hybrid cucumber seed using a gynoecious line (Robinson, 2000).
 They differ in the flower type of the line used as pollen donor.
Gynoecious x Monoecious hybrids
Gynoecious x Gynoecious hybrids
Gynoecious x Hermaphroditic hybrids, and
Gynoecious x Andromonoecious hybrids.
 From these systems, the Gynoecious x Monoecious and Gynoecious
x Gynoecious hybrids are most commonly used

GYNOECIOUS X MONOECIOUS
HYBRIDS
This system became popular for the development of stable inbred lines
for the gynoecy trait during the late 1950s.
The hybrids produced by the cross of a gynoecious and monoecious
line resulted in hybrid vigor and a high degree of female sex
expression, with uniform and concentrated fruit formation, which
was especially advantageous for mechanical harvest.
Because of its tendency to produce mostly female flowers, seed from
this type of hybrid usually is blended with seed from a monoecious
cultivar added in a proportion of about 10%. This practice
improves pollination, which is required for fruit set in genotypes
that are not parthenocarpic.
However, it has the disadvantage of affecting uniformity, which is one
of the principal advantages of hybrid cultivar production.
When the hybrid is parthenocarpic, blending is not required because
fruits can develop without pollination. This is the case for many
cucumber hybrids used in greenhouse production.

GYNOECIOUS X GYNOECIOUS
HYBRIDS
 When two gynoecious inbred lines homozygous for the gene F are
crossed, the resulting F1 hybrid is homozygous for F.
 These hybrids are more stable for gynoecious sex expression
compared to hybrids produced by crossing gynoecious and
monoecious lines.
 In the case of hybrids heterozygous for the gene F, some
environments such as high temperature and long days may promote
the development of male flowers, which is less likely in gynoecious x
gynoecious hybrids.
 The stability of gynoecious sex expression in these types of hybrids
is especially important for parthenocarpic cultivars used for
greenhouse production. These cultivars produce long, seedless fruits
in the absence of pollination.
 However, when female flowers are pollinated, the formation of
seeds enlarges the fruits at the blossom end, affecting their shape
and quality.

Use of growth regulators to promote maleness for
maintenance of gynoecious lines
Self-pollination is required to maintain inbred gynoecious lines, but these genotypes
do not produce pollen.
The solution to this problem came with the discovery of growth regulators able to
induce formation of male flowers in these lines.
The first compound used to induce maleness in gynoecious lines was GA. Some
recommendations were three applications of GA3 at 1000 ppm or GA4/7 at 50
ppm beginning at the two leaf plant stage and spraying bi-weekly
A problem observed with GA use is that different gynoecious lines vary in response
to GA application and, in some cases, the number of induced male flowers was
not sufficient for hybrid seed production.
Additionally, GA applications typically cause excessive stem elongation or
malformed male flowers.
Because silver ions inhibit ethylene action, an alternative to GA is silver nitrate.
This compound induces male flowering in gynoecious lines for extended periods
and is often more effective than GA
Two to three applications of silver nitrate 250-500 ppm at 2-3 true leaf stage has
been recommended.
Silver thiosulfate is another alternative and appears to be less toxic than silver

10. methods & problems in seed production

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