Topic: Heterosis Seed Production

Introduction

Heterosis describes the phenomenon in which hybrids formed between individuals of

the same or closely related species are more robust or vigorous than their parents. Thus,
the terms heterosis and hybrid vigor are often used interchangeably. There are two
broad classes of hypotheses for the mechanisms underlying heterosis: dominance and
over dominance. Under the dominance hypothesis, detrimental recessive alleles
accumulate in the homozygous state during inbreeding and cause a reduction in vigor,
or inbreeding depression. When inbred individuals are mated, their offspring become
heterozygous at these loci and the detrimental alleles are covered by dominant alleles,
thus relieving inbreeding depression and restoring vigor. Under the over dominance
hypothesis, the products of heterozygous loci are more robust than those of either
homozygous locus. Thus, increased homozygosity reduces vigor, and heterozygosity
introduced by outbreeding leads to enhanced robustness. These two hypotheses make
similar predictions at the level of genetics, but may be distinguished by combined
molecular and genetic analysis. They are not mutually exclusive; both mechanisms may
be active and important in heterosis in different organisms under different conditions.

Selection of parents for the generation of heterosis

Heterosis refers to the superior phenotypes observed in hybrids relative to their inbred
parents with respect to traits such as growth rate, reproductive success, and yield.
Heterosis was discovered in maize about a century ago and has subsequently been found
to occur in many crop species. The increase in yield as a result of the cultivation of
hybrid offspring ranges from 15 to 50%, depending on the crop. The theory of
quantitative genetics predicts a positive correlation between parental divergence and the
estimated degree of heterosis. However, the data obtained to date in support of this
correlation are not conclusive, and the ability to predict levels of heterosis based on the
genetic distance between parents varies with different traits and crops.

Heterosis Breeding
Heterosis breeding is here to stay as a potent genetic tool for exploiting the
predominantly non-additive gene action. In self-pollinated crops, utilization of hybrid
vigour is dependent upon a system for economically producing F 1 seeds. Hybrid vigour
in cowpea, as in other crops, is dependent on the specific parents used in hybrid
combination. Some hybrids exhibit marked pod yield increase over the parents. Among
the pod yield components, manifestation of heterosis for pod number per plant, pod
length and seed weight was also reported. When actual heterosis was considered against
the corresponding genetic divergence between the parents of the cross, a one-to-one
correspondence was not found owing to balancing or even cancellation of various
components of heterosis. Seed protein content of the F1 hybrids but were unable to
demonstrate encouraging results. In this situation, enhancement of protein yield per
plant through the increase in yield should be given priority, and heterosis breeding for
protein should concentrate on the sulfur-containing amino acids. Heterosis for stomata
frequency has also been reported. The self-pollinating behavior of cowpea due to
cleistogamous flower structure and the small number of seeds produced in cross-
pollination reduce the prospects of hybrid seed production and utilization of hybrid
vigour in increasing yield potential in cowpea.

Hybrid breeding versus inbreeding

Heterosis (or hybrid vigour) is a major reason for the success of the maize industry as
well as for recent advances in yield potential in rice. Switching from inbreeds to hybrids
provides a one-time boost to primary production and yield potential of about 10%.
There is relatively little information on the physiological basis of this phenomenon.
Development of a reliable method for predicting hybrid performance without testing
hundreds of thousands of single crosses would therefore be of great value. Hybrid wheat
production may not be economically feasible in many cases due to higher seed costs and
because heterosis may in theory be fixed in polyploidy plants, giving hybrids no
advantage over inbred lines.

Methods of hybrid seed production

Hybrid is produced by crossing between two genetically dissimilar parents. Pollen from
male parent (Pollen parent) will pollinate, fertilize and set seeds in female (seed parent)
to produce F1 hybrid seeds. For production of a hybrid CROSSING between two parents
is important, the crossing process will results in heterosis. In self pollinated cross it is
difficult to cross but in cross pollinated crops it is easier.
In nature to create genetic variability and for its wider adaptation in different
environmental conditions, flowering plants has adopted many mechanisms for cross
pollination. Cross-pollination results in genetic heterogeneity and show wider
adaptations. Flowering plants have evolved a number of devises to encourage cross-
pollination. Those mechanisms are;
1. Dicliny: Flowers are unisexual. In monoecious plants male and female flowers are
borne on the same plant e.g., cucurbits, maize, castor and coconut. In dioecious
plants male flowers are borne on different plants e.g., papaya, cannabis,
mulberry.
2. Dichogamy: Time of anther dehiscence and stigma receptivity are different
forcing them for cross-pollination. The time gap between the two may vary from
one day to many days. In protoandry anthers dehisce earlier than the stigma
receptivity e.g.; maize, sunflower. In protogyny stigma become receptive earlier
than the anther dehisce e.g., Pearl millet mirabilis.
3. Self-incompatibility: self-fertilization in avoided by recognizing the self pollen by
the stigma. E.g., Brassica, Petunia, Lilium.
4. Herkogamy: there is spatial separation of the anthers and stigma. Their relative
position is such that self fertilization cannot occur. The stigma projects beyond
the anthers and therefore pollen cannot land on stigma. E.g., Lucerne stigma is
covered with a waxy film. The stigma does not become receptive until this waxy
membrane is broken by visit of honeybees resulting in cross-pollination.
5. Male sterility: Absence or atrophy or miss or malformed of male sex organ
(functional pollen) in normal bisexual flower. Male sterility is of three types:
genetic male sterility, cytoplasm sterility and cytoplasmic- genetic male sterility.
6. A combination of two or more of the above mechanisms may occur in some
species. This improves the efficiency of the system in promoting cross-pollination

Requisites of hybrid seed production:

1. Breeders responsibilities:
o Develop inbred lines
o Identification of specific parental lines
o Develop system for pollen control
2. Major problems for breeders & producers
o Maintenance of parental lines
o Separation of male and female reproductive organs
o Pollination
 3. Basic procedures for hybrid seed production
• Development and identification for parental lines
• Multiplication of parental lines
• Crossing between parental lines and production of F1

Characteristics of parental lines.

Male Parent Female Parent

High seed yield Good pollen production

Good seed characteristics Long shedding period

Male sterility Plant height

Lodging resistant Fertility restoration

Commercial hybrid seed production demands crossing technique which is easy and also
economic to maintain parental lines. Only few crossing mechanisms have been adopted
for commercial hybrid seed production they are;
1. Hand emasculation and pollination
2. Self-incompatibility
3. Dicliny : monoecious and dioecious
4. Male sterility
These techniques are specific to crop floral biology and flowering behavior. These
techniques have their own advantages and disadvantages. Based on the crop behavior
and crossing technique have been adapted for production of hybrid seeds commercially.
Among different techniques self-incompatibility and sex expression have significance
particularly in vegetable and flower hybrid seed production.
1. Hand emasculation and pollination:
Hybrid seeds are produced manually by modifying the plant structure by removal
of male organ from female plant before an thesis. This system is possible only
when the male and female parts of a single flower or plants are separate. This is
 being adopted in bisexual perfect flowers where the androecium is removal with
case. By removing the anther column / or male part from female line, the sterility
of female line is created and is dusted with the pollen of desired male parent.
2. Self Incompatibility:
Self-incompatibility is a mechanism which avoids self fertilization through
recognition of self pollen in or on stigma on the female pistil. But when pollen
from other plant carried by wind or insects are accepted and sets seeds.
Self-incompatibility will prevents self pollination (inbreeding) and promotes
crosspollination (out breeding) and creates genetic variability. SI are seen in
hermaphrodite and homomorphic flowers. Self-incompatibility is a widespread
mechanism in flowering plants that prevents inbreeding and promotes
outcrossing. The self-incompatibility response is genetically controlled by one or
more multi-allelic loci, and relies on a series of complex cellular interactions
between the self-incompatible pollen and pistil.

Types of Self-Incompatibility (SI)

1. Heteromorphic self-incompatibility:
In this system flowers are of different morphology of the reproductive
parts. The morphological differences can be seen visibly in flowers this will
coincide with crossibility. The characters affecting this type of SI are style
length, filament length, pollen size, exine sculpturing. The presence or
absence of other SI mechanisms will not affect cross pollination in
heteromorphic SI.
 Heteromorphic distyle : e.g., Primula
The flowers of Primula have style at two different heights. Has two
types of flowers
1. Thrum flower: short style and long anther
2. Pin flower: has short anthers and long style
But these two are cross compatible.
Pin x Pin (ss x ss): Incompatible mating
Pin x Thrum (ss x Ss): Compatible mating
Thrum x Pin (Ss x sass): Compatible mating
Thrum x Thrum (Ss x Ss): Incompatible mating
 Heteromorphic Tristyle: e.g., Lithium
Three types of flowers;
1. Short style and stamens are mid and long
2. Mid style and stamen are short and long
 3. Long style and stamen are short and mid
When homomorphic flowers are crossed it results in incompatible
mating and when heteromorphic flowers are crossed it results in
compatible mating.
2. Homomorphic flowers
Flowers morphological are same, so mating types cannot be recognized by
morphological features. This types of self-incompatibility is controlled by
same alleles. For crossing parental same alleles should be different, then
only fertilization takes places and seed sets.
Two types of self-incompatibility;
 Sporophyte self-incompatibility
 Gametophyte self-incompatibility

Sporophyte self-incompatibility (SSI)

SSI is less common or rare when compared to GSI. The rejection of pollen
is controlled by S loci which is dominant. The dominance relationship are
like S1> S2>S3>……. This type of self-incompatibility is controlled by
diploid genotypes of the sporophyte (pollen). Pollen will not germinate on
the stigma of the flower that contains either of the two alleles in the pollen
so, rejected.

Gametophyte self-incompatibility (GSI)

GSSI is more common when compared to SSI. The rejection and

acceptance of pollen is controlled by ‘S’ loci. Unlike SSI,
incompatibility is controlled by haploid genotype of pollen itself.
S1 pollen can germinate on pistil of S1S2 but due to common S1 allele the
pollen tube growth seizes. Similarly in S2 the pollen tube growth seizes.
When S3 pollen come in contact with pistil of S1S2 there will be normal
growth of pollen tube and fertilization takes place, this is called as partial
compatibility. Whereas, S3 and S4 pollen can pierce in to the style (S1 S2)
and cause fertilization, called as complete compatibility.
In single gene system there are three types of mating systems they are;
S1S2 X S1S2: 0% compatibility
S1S2 X S2S3: 50% compatibility
S1S2 X S3S4: 100% compatibility
Pollen rejection and acceptance of cross pollen is a mechanism of complex
interactions.
There are three main interactions;
 Pollen –stigma interaction seen in SSI
 Pollen- style interaction seen in GSI
 Pollen tube- ovule interaction seen in GSI
Pollen tube- ovule interaction is very rare and seen in some cases of
cocoa, pollen tube reaches ovule and effect fertilization but
incompatible combination will degenerate the embryo at early stage
development only resulting in no seed set.

 Pollen-stigma interaction

This type of interaction is seen in SSI. It includes dry stigma. The

stigma has a hydrated layer of proteins know as pellicle. This
pellicle is involved in incompatible reaction. Within a few minutes
of pollen reaching stigmatic surface, the pollen releases an exine
exudates which is glycoprotein. Due to same allele proteins
interactions induces immediate callous formation in the papillae,
this is in direct contact with pollen. Also this calluses is deposited
on the protruding pollen tube preventing further germination of the
pollen. Here stigma is the site of incompatibility reaction. If once
pollen cross this stigmatic barrier, there is no further rejection of
pollen.
In case of wet stigma seen in GSI, when pollen sits on the stigma,
pollen coat stimulate an unusual form of stigmatic reaction focused
beneath the areas of coating. This results in the deposition of fibro
granular electron opaque layer by the extracellular vesicles in the
outer layer of the papillae wall. This results in blockage of water
passage or loss of osmotic competence by pollen resulting in drying
of pollen.

Pollen tube - style interaction:

This is common in gametophyte self-incompatibility. The pollen can

germinate and pollen tube growth will pierce the stigma. But the
rate of the pollen tube growth is very slow when compared to
 compatible pollen. This slow growing pollen tube will stops due to
exhaustion of reserve materials and deposition of ‘S’ allele
polysaccharide at the tip of the pollen tube which blocks the growth
of tube. This deposition is limited to the tip and thus will not affect
the other compatible pollen tube growth.

 Three-way hybrid
Two SI lines are crossed to get a heterozygote and again crossed
with self-compatible parent which has suppressor’s locus and give
F1 hybrid which is self-compatible
 Double cross hybrid.
Hybrid that is produced when two different single-cross hybrids are cross-
pollinated. As the name implies, producing a double-cross hybrid requires
two stages of crossing involving two pairs of inbreeds.

 Triline hybrid
Hybrid seed production is done by crossing of two inbreed lines.
Have to maintenance these inbreed lines which are self-
incompatible. For success of self pollination need to eliminate SI.
There are many methods to overcome.
1. Bud pollination
2. Washing stigma surface/ pollen grains with organic solvents
3. Mechanical and electric methods is CO2 Treatment
4. High temperature treatment.

 Modification of sex
Hermaphrodite flowers has both male and female reproductive organ in a
single flower.
o Complete flower; flowers containing all the four whorls viz., sepals,
petals, androecium and gynoecium.
o Incomplete flower: flowers missing any of the one whorl.
o Imperfect Flowers- Sexual distinctness: Monoecious (Maize) vs.
Dioecious (Hollies, Poplars)
o Perfect Flowers- Gamete Maturation Time (Dichogamy):
Production of this unisexual/ imperfect flowers will naturally leads;
  Outcrossing avoids the deleterious effects of inbreeding depression
 Promotes heterozygosity, genetic variability, and genetic exchange,
 Advantageous to the long-term survival and adaptation of a species.
11. Monoecious:
Flowers are unisexual and are present at different position on the same plant.
E.g. Cucumber. Terminal flowers are male flower. In the middle of the plant is
female favoring crosspollination.
Dioecious:
Male flowers and female flowers are in different plant. So called as male plant
and female plant.
12. Sex modification through hormones and chemicals
Sex expression in dioecious and monoecious plants is genetically determined and
can be modified to a considerable extent by environmental and introduced
factors such as mineral nutrition, photoperiod, temperature, phytohormones.
Amongst these, phytohormones have been found to be most effective agents for
sex modification and their role in regulation of sex expression in flowering plants
has been documented. The morphological differences in various sex types and
their specific metabolic characteristics result from the possession of specific
patterns of proteins, enzymes and other molecules. Modification of sex
expression in cucurbits has been induced both by changing the environmental
conditions and by applying treatments with growth regulators. Auxin treatments
increase the female sex tendency while gibberellins cause a shift towards
maleness.
Hormones & Chemicals inducing Femaleness: Auxin- NAA, Ether, Ethephon,
Cytokinis- BA, Brassinosteriods etc.
Hormones & Chemicals inducing Maleness: GA3, AgNO3, ABA Thio porpinic
acid, Pthalimide, Paclobutrazol etc.
In cucumber AgNO3 found to be potent inhibitors of ethylene action leading to
femaleness.It should be sprayed when first true leaf is fully expanded. Gibberlic
acid spray will leads to excessive elongation and weakening of plants and there
will be increased number of mall formed male flowers with less pollen. In
gynoecious cucumber there will be increased number of male nodes when
sprayed with silvernitrate and gibberlic acid, which made possible for
multiplication of gynoecious in hybrid seed production.
13. Environmental sex modification.
Environment has greater influence on the sex modification. But due to
introduction of photosensitive varieties or hybrids in modern era of agriculture it
 has gained less importance. However in seed production it has its influence on
sex expression. In cucumber, high temperature and long day length (> 14 hours)
favors male flowers. High temperature will extends the flowering of female
flowers. As the temperature increases form 19 0C to 230 C the node for first
female flowers has also increased from 9.6 to 16.5 number. This clearly indicates
that high temperature favors male and delays female flowering.
Male sex expression of several plant species is favored by high temperatures and
female sex expression by low temperatures. Male sterile mutant of tomato
developing male sterile flowers at a minimum temperature of 30°C and normal
flowers at lower temperatures. In Brussels sprouts of low temperature effect on
the development of the androecium. In onions a slight production of viable pollen
by normally male sterile plants above 20 °C.

Male Sterility
Hybrid production requires a female plant in which no viable male gametes are borne.
Emasculation is done to make a plant devoid of pollen so that it is made female. Another
simple way to establish a female line for hybrid seed production is to identify or create a
line that is unable to produce viable pollen. This male sterile line is therefore unable to
self-pollinate and seed formation is dependent upon pollen from the male line.
In hermaphrodite flowers pollens are non-functional or inactive or sterile while, female
gametes functions normally. It is the inability of plant to produce or to release
functional pollen as a result of failure of formation or development of functional
stamens, microspores or gametes. Male sterility can be either genetic or cytoplasmic or
cytoplasmic-genetic. This prevents autogamy and permits crosspollination. Promotes
heterozygosity. Sterility is due to nuclear genes or Cytoplasmic gene or both.
In hybrid seed production process female is a male sterile line crossed with male fertility
restorer line to get F¬1 hybrid.
Cytoplasmic male sterility.
Cytoplasmic male sterility, as the name indicates, is under extra nuclear genetic control
mainly mitochondrial genome. They show non-Mendelian inheritance and are under the
regulation of cytoplasmic factors. In this type, male sterility is inherited maternally. In
general there are two types of cytoplasm: N (normal) and the aberrant S (sterile)
cytoplasm. These types exhibit reciprocal differences. Cytoplasmic male sterility (CMS)
is caused by the extra nuclear genome (mitochondria or chloroplast) and shows
maternal inheritance. Manifestation of male sterility in CMS may be either entirely
controlled by cytoplasmic factors or by the interaction between cytoplasmic and nuclear
factors.

 Stamen (anther and filament) and pollen grains are affected

 It is divided into:
o Auto-plasmic
CMS has arisen within a species as a result of spontaneous mutational
changes in the cytoplasm, most likely in the mitochondrial genome
o Alloplasmic
CMS has 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. Cytoplasmic male sterility is used in hybrid
seed production. In this case, the sterility is transmitted only through the
female and all progeny will be sterile. This is not a problem for crops such
as onions or carrots where the commodity harvested from the F1
generation is produced during vegetative growth. These CMS lines must be
maintained by repeated crossing to a sister line (known as the maintainer
line) that is genetically identical except that it possesses normal cytoplasm
and is therefore male fertile.
Disadvantages
1. insufficient or unstable male sterile
2. Difficulties in restoration system
3. Difficulties with seed production
Cytoplasmic-genetic male sterility
Male sterility is controlled by an extranuclear genome and often nuclear genes
can have the capability to restore fertility. When nuclear restorations of fertility
genes are available for CMS system in any crop, it is cytoplasmic-genetic male
sterility; the sterility is manifested by the influence of both nuclear (Mendelian
inheritance) and cytoplasmic (maternally inherited) genes. There are also
restorers of fertility genes, which are distinct from genetic male sterility genes.
The genes do not have any expression of their own unless the sterile cytoplasm is
present. Genes are required to restore fertility in S cytoplasm which causes
sterility. Thus N cytoplasm is always fertile and S cytoplasm with genotype
produces fertile; while S cytoplasm with produces only male sterile. Another
feature of these systems is that mutations (i.e., mutations to or no fertility
restoration) are frequent, so N cytoplasm with is best for stable fertility.
Cytoplasmic-genetic male sterility systems are widely exploited in crop plants for
hybrid breeding due to the convenience to control the sterility expression by
manipulating the cytoplasm combinations in any selected genotype.
Incorporation of these systems for male sterility evades the need for emasculation
in cross-pollinated species, thus encouraging cross breeding producing only
hybrid seeds under natural conditions.
In cytoplasmic-genetic male sterility restoration of fertility is done using restorer
lines carrying nuclear restorer genes in crops. The male sterile line is maintained
by crossing with a maintainer line which has the same genome as that of the MS
line but carrying normal fertile cytoplasm.

Genetic Male sterility:

Male sterility is controlled by mutations in nuclear genes in the single recessive
genes affect stamen and pollen development, but it can be regulated also by
dominant genes. MS alleles are generally recessive. A male sterile line is
maintained by crossing with heterozygous male fertile line.
Male sterile plants of monoecious or hermaphrodite crops are potentially useful
in hybrid program because they eliminate the labor intensive process of flower
emasculation Constraint of the use of genetic male sterility
o The maintenance of the male sterile line. Normally, a GMS line (A-line) is
maintained by backcrossing with the heterozygote B-lines (Maintainer
lines), but the progeny produced are 50% fertile and 50% male sterile