1
2
PT = Pollen tube
2NFG = diploid female
gametophyte
EC = Egg cell
Sy =Synergids
CC =
An = Antipodals
Pemb = Parthenogenic
embryo
AI = Aposporous initials
Nemb = Nucellar embryo
3
APOMIXIS
• Two pathways of reproduction through seed exist: sexual or amphimictic, and
asexual or apomictic. (amphimictic. : capable of interbreeding freely and of
producing fertile offspring)
• Apomixis is a form of asexual reproduction that occurs via seeds in which embryos
develop without fertilization. Apomixis is associated with perrennial life cycle,
interspecific hybridization and polyploidy.
• Apomixis is a mechanism of seed formation without fertilization and is observed in
more than 300 species in 30 out of 460 angiosperm families, but it is not common
in crop species (Bashaw, 1980).
• The presence of apomixis in the species Hieracium, in which Mendel was asked to
prove the genetic principles he had developed in peas, caused him to withdraw
from the scientific world due to his failure to do so (Carneiro et al., 2006).
• Facultative apomixis is more common than obligate apomixis.
• Apomixis technology has revolutionary promise by reducing cost and breeding
time, and avoiding the complications of sexual reproduction (e.g., incompatibility
barriers) and vegetative propagation (e.g., viral transfer).
• Development of apomixis technology in agriculture requires a deeper knowledge
of the mechanisms that regulate reproductive development in plants.
• The benefits of apomixis are estimated to surpass those of the green revolution
(Grossniklaus et al., 1998). 4
• Apomixis modifies the processes of sexual reproduction and produces a
functional female gametophytic structure that precludes the sexual
assortment of genes and recombination of genes associated with meiosis.
• In apomixis the double fertilization event does not occur and the embryo
develops autonomously from the unreduced female gamete.
• In some apomicts the endosperm develops autonomously while in others
(pseudogamous apomicts) fertilization of the central cell by a sperm cell
may be required to produce a functional endosperm (the preferred
pathway).
5
Types of APOMIXIS
• Sporophytic (Adventitious embryony)
• Gametophytic (Apospory and Diplospory)
6
7
SPOROPHYTIC APOMIXIS
• Adventitious embryony (Sporophytic apomixis): The simplest pathway avoids
the production of an embryo sac, and the maternal embryo originates from
one or more somatic cells of the ovule. Among the agriculturally important
species, adventitious embryony (i.e., sporophytic apomixis) has been noted in
mango (Mangifera indica), several Citrus species, and orchids.
• In this process embryos initiate parthenogenetically outside of an embryo sac
structure. Adventitious embryony is most commonly initiated later in ovule
development from nucellar and integument tissues.
• In general, fertilization in the adjoining sexual embryo sac and subsequent
endosperm formation is necessary to form viable seeds.
• The developing embryos closest to the embryo sac grow towards it,
presumably to obtain nutrient and other developmental signals from the
embryo sac.
• Adventitious embryo formation is rapid; often multiple embryo form and these
can hinder development of the zygotic embryo.
8
9
GAMETOPHYTIC APOMIXIS
• Gametophytic apomixis : When the maternal embryo originates from a diploid egg cell
differentiated in an unreduced embryo sac, the apomictic pathway is referred to as
gametophytic apomixis (Nogler 1984).
• In gametophytic apomixis, the unreduced embryo sac may arise from a somatic nucellar cell
that acquires the developmental program of a functional megaspore, a mechanism referred
to as apospory.
• Among others, apospory has been reported in Beta, Brachiaria, Cenchrus, Chloris,
Compositae, Eriochloa, Heteropogon, Hieracium, Hyparrhenia, Hypericum, Panicum,
Paspalum, Pennisetum, Poaceae, Ranunculus, Sorghum, Themeda, and Urochloa,
• Alternatively, if the embryo sac forms from a megaspore mother cell with suppressed or
modified meiosis, the pathway is referred to as diplospory.
• Diplospory has been noted in Agropyrum, Allium, Antennaria, Boechera (formerly Arabis),
Datura, Eragrostis, Erigeron, Eupatorium, Ixeris, Parthenium, Paspalum, Poa, Taraxacum, and
Tripsacum (Table 1).
• It is worth emphasizing that apomictic plants may or may not change meiosis itself, but in any
case they do activate the gametic cell fate either in a somatic cell (apospory) or in an
unreduced megaspore (diplospory) as surrogate for meiotic products.
• Once 2n female gametophytes and gametes are formed (apomeiosis), they subsequently undergo
embryogenesis autonomously without fertilization by a male gamete (somatic parthenogenesis).
Endosperm formation may be fertilization-independent (autonomous endosperm) or may require
fertilization (pseudogamous endosperm).
10
Apospory
• In apospory, the nucellar cells that give rise to the apomictic embryo
sac, termed aposporous initials, are distinct from the mmc.
• Once the aposporous initial cells differentiate they immediately
enter mitosis to produce an embryo sac.
• Some ovules can contain multiple embryo sacs and, depending on the
species, the structure of the embryo sac may be quite different from
that seen in the sexual process.
• The initiation of the aposporous embryo sac can occur together with
a sexual one or it can displace or inhibit sexual embryo sac
formation.
• Termination of the sexual process is most complete if the
aposporous initial cells differentiate early, around the time of mmc
meiosis, producing an unreduced apomictic embryo sac.
11
12
Diplospory
• In diplospory, the unreduced embryo sac is derived from the megaspore
mother cell either directly by mitotic division or by aborted meiotic
events.
• In the Taraxacum type, meiotic prophase is initiated but then the process
is aborted resulting in two unreduced dyads one of which gives rise to the
embryo sac by mitotic division.
• In the Ixeris type, two further mitotic divisions of the nuclei to give rise
to an eight-nucleate embryo sac follow equational division following
meiotic prophase.
• The Taraxacum and Ixeris types are known as meiotic diplospory
because they involve modifications of meiosis.
• By contrast, in the Antennaria type, referred to as mitotic diplospory, the
MMC does not initiate meiosis and directly divides three times to
produce the unreduced embryo sac.
13
14
• How to identify apomictic plant ??
15
Comparison between apomixis and conventional method
16
Apomixis Conventional hybrid
Features of apomictic plant
• Many apomictic plants belong to genera in which sexual members
predominantly exhibit physiological self-incompatibility, dioecy, or
heterostyly.
• Apomictic species are almost perennials, and often use a vegetative
reproduction.
• Apomicts are found in frequently disturbed habitats or where barriers
operate to inhibit the successful crossing of compatible individuals.
• Gametophytic apomixis is more common in herbaceous than tree species.
e.g. Poaceae and the Asteraceae, mostly composed of herbaceous species.
• It is probably true that there are as many mechanisms of apomixis as there
are plant taxa that express the trait.
• Most apomicts produce viable pollen. Even in diplosporous apomicts, defects
in the meiotic events of female gametophyte development are not
automatically extended to male gametophyte formation or function.
• From an evolutionary point of view, apomixis may be regarded as a
consequence of sexual failure rather than as a recipe for clonal success.
17
Ideal apomictic system
• Obligate apomixis : All progeny should be apomictic (Clones of
maternal parent).
• Apomictic genotype should be male fertile and self
incompatible and reproduce via apogamy.
• In diplospory, chromosome should not recombine during
meiosis (i.e. to prevent genetic variation).
• In diplospory, restitution must occur at or just after first
meiosis to prevent genetically variable progeny.
• Apomixis must be dominant over sexual reproduction.
Usually, apomixis is governed by two or more genes.
• Apomixis should not be affected by environmental changes.
18
APPLICATIONS
1. Fixation of heterosis
2. If apomixis were to be available as a controlled tool where it could be
switched on and off as required in plant breeding, hybrid vigour could be
fixed, enabling indefinite multiplication of the hybrid of uniform quality
without decrease in the yield advantage.
3. In this way apomixis could eliminate dependency on current inbred lines
(maintenance of parental lines) and enable us to expand our current
agricultural genetic base.
4. Apomixis would also remove the need for pollination for fruit and seed
production.
5. Adverse environmental conditions such as drought and cold inhibit pollen
formation and result in crop losses in sexually reproducing systems; in
apomictically grown plants these losses could be avoided.
19
6. A significant proportion of the assimilate of the growing plant is
channelled into the production of the male reproductive apparatus and
gametes; these could be redirected towards increasing yield.
7. Apomixis will also facilitate uniform clonal seed propagation of many
plants that are currently vegetatively propagated, including fruit trees,
plantation timbers, and potatoes.
8. Little is known about the genes controlling apomixis. Research is
currently being pursued to isolate these genes and to understand how
the process of apomixis can be regulated to result in high yields of clonal
seed. The value of apomixis will only be realized if it can be efficiently
controlled under conditions of commercial production.
9. Production of vybrids : Vybrids are obtained by crossing two facultative
apomicts followed by selection of F1 like plants.
20
Advantages of apomixis
1. Obligate apomixis permits fixation of heterosis.
2. Reduced cost and price of hybrid seed production.
3. Farmers can produce their own hybrid seeds which is not possible with
conventional approach.
4. No need to identify cms and restorer sources.
5. The shorter procedures and lower costs involved in apomictic hybrids will
make it possible to adapt a “boutique breeding” approach to develop
specific hybrids for microproduction areas.
6. Crossing is to be attempted only once, special advantage in case of
strictly self pollinating species.
7. Parental lines are not to be maintained.
8. Minimum isolation distance (3m) is required to avoid mechanical mixture
irrespective of mode of pollination.
21
9. Flowering synchronization of parental lines is not required.
10. Nucleus seed can be maintained conveniently.
11. Pollen dispersal is not relevant during hybrid seed production.
12. Emasculation of any kind is not required for hybrid seed production.
13. Rouging is not required except in case of mechanical mixture.
14. No scope for creation of variability except due to mutation.
15. A major advantage of apomixis for plant breeding is that it will increase
the survival of interspecific crosses since chromosomal irregularities in
meiosis, and consequently hybrid sterility, is not a problem in apomictic
plants. It will facilitate gene transfer from secondary and tertiary gene
pools.
16. In vegetatively propagated crops, apomict seeds can reduce the cost of
seed storage and transportation required in handling of bulky
propagules. It also reduces viral contamination in these crops as bulky
propagules are not used.
17. Virus free clonal propagation through seed is possible without utilizing
costly tissue culture techniques and also in the crops where such
protocols are not available. 22
Problems associated with apomixis
1. Unfortunately, barring a few exceptions in some forage grasses and fruit trees,
apomixis is not a common feature among crop species.
2. A complicated mechanism. Breeding methods and seed certification procedures
are to be modified.
3. Level of apomixis is difficult to judge in case of facultative apomixis.
4. In case of facultative apomixis, proportion of sexual progenies is influenced by
environmental factors.
5. Maintenance of facultative apomixis is difficult.
6. Genetic and molecular base of apomixis is not clear.
7. Good theory, No practical utility ?? = Functional genomics tools, transgene
technology and genome editing.
8. This technology will cripple the seed industry by allowing farmers to produce their
own seeds, jeopardizing future yield increases ?? = Area specific breeding and
replacement within 5 years due to mechanical mixture and variation. Increase in
area under hybrid seed production.
9. Another concern about apomictic crops is that apomixis genes could escape into
wild relatives and cause genetic erosion. Maternal inheritance by placing the
transgene in chloroplast, will easily obliterate these theoretical risks. 23
10. In fact, none of the major crop plants have been bred for apomixis, and only some
features of apomixis have been genetically engineered in model species.
Consequently, even in the era of genomics, achieving an understanding of the
genetic control and molecular regulation of apomixis appears much more
complicated than expected.
11. Large amounts of cytological and ecological information, along with genetic and
molecular data, have been collected mainly from model species (i.e., Boechera
holboellii, Hieracium spp., Hypericum perforatum, Paspalum spp., Poa pratensis,
Ranunculus spp., and Taraxacum officinale) and have often been tested in
Arabidopsis thaliana (Arabidopsis) to elucidate the mechanisms of apomeiosis,
parthenogenesis, and apomixis.
24
Mechanisms of apomixis
• The generation of a cell capable of forming an embryo without prior
meiosis (apomeiosis);
• The spontaneous, fertilization-independent development of the embryo
(parthenogenesis);
• The capacity to either produce endosperm autonomously or to use an
endosperm derived from fertilization
• Two main subgroups of mechanisms are apparent. In sporophytic
apomixis, or adventitious embryony, embryos arise spontaneously from
ovule cells late in the temporal sequence of ovule maturation.
• Gametophytic apomixis operates through the mediation of an unreduced
embryo sac. Endosperm development in these plants may be either
spontaneous (autonomous) or fertilization induced (pseudogamous)
(Koltunow, 1993).
25
Genetics of apomixis
• Genetic basis of apomixis and its components (i.e., apomeiosis and
parthenogenesis).
• Dominance of apomixis over sexuality.
• Genetic analysis demonstrated a simple inheritance system involving a few
Mendelian genes controlling the expression of apomixis or its
components.
• In contrast, molecular and cytogenetic analyses of chromosomal region(s)
carrying the determinants of apomixis have revealed a complex genetic
control mechanism.
• Gametophytic apomixis is thought to rely on three genetically
independent Mendelian loci, each exerting control over a key
developmental component, including apomeiotic megaspores,
parthenogenic unreduced egg cells, and modified endosperms.
26
• A close relationship between apomictic mechanisms and
heterochromatic regions of the genome that are rich in retrotransposons
has raised the intriguing possibility that DNA structure and/or RNA
interference could play a role in regulating the expression of apomixis-
related genes.
• A well-characterized class of small regulatory RNAs that are widespread in
eukaryotes appears to regulate gamete function and fertilization in plants
by altering gene expression through post-transcriptional gene silencing,
translational inhibition, and heterochromatin modification.
• Pupilli and Barcaccia (2012) have recently hypothesized that a relatively
simple genetic system controls apomixis in terms of the number of genes
involved in the expression of its components (i.e., genes controlling
apomeiosis and parthenogenesis and eventually autonomous endosperm
development).
• However, elements within the chromosome block carrying the apomixis
genes (e.g., transposable elements, repetitive elements, and
pseudogenes) make it a complex genetic system, with loci that vary from
elementary and primitive to evolutionarily advanced.
27
Genes controlling natural apomixis
• The main approaches that have been followed to study the molecular basis of
apomixis address the isolation of genes that prime the expression of apomixis in
natural apomicts and/or the identification of genes that mime the features of
apomictic pathways when they are deregulated in model sexual systems.
• Majority of apomicts do not constitute agriculturally important crops and, with a
few exceptions (e.g., Tripsacum and maize), do not have agriculturally important
relatives.
• Currently, basic inheritance is usually thought to depend on a single master
regulatory gene or a few dominant key genes, which allow a megaspore mother
cell or a somatic nucellar cell to form an embryo sac without meiotic reduction and
an embryo to develop from an unreduced egg cell without fertilization.
• Once apomictic genes initiate embryo development and the initial cell forms and
divides, the genes controlling embryo cell formation and patterning are most
likely the same as those required for sexual embryo development.
• Currently, a number of researchers support the hypothesis that zygotic
embryogenesis and apomictic parthenogenesis follow similar pathways during
embryo and seed production.
28
• in Brachiaria species, among the 12 candidates isolated by Leblanc et al.
(1997), only two proved to be specifically expressed in mature ovaries
containing unreduced (aposporic) embryo sacs.
• Rodrigues et al. (2003) searched for candidates expressed in both
genotypes of the same species and identified 11 genes that were
differentially expressed between apomictic and sexual genotypes.
29
Work in model species
Gianni Barcaccia and Emidio Albertini, 2013
30
Genes miming (mimic) apomixis in sexual
model plants
• If it is true that apomixis is a consequence of sexual failure, rather than a
means for clonal success, from an evolutionary point of view.
• apomixis is a result of the spatial and temporal deregulation of sex related
gene expression as a consequence of heterochronic expression due to
hybridization.
• The idea that apomixis is an altered form of sexuality that results from
temporal and spatial alterations in the action of the sexual program
suggests that a synthetic apomixis system using variant alleles of genes
isolated from sexual model species, such as Arabidopsis, can be
developed.
31
Putative mutation in crop plants related to apomixis
32
Breeding apomixis
• Selection for change over a parental type would work against a mechanism such
as apomixis that acts to maintain uniformity.
• There also are few apomictic species of significant relatedness available for use in
introgression programs, which may explain at least some of the difficulties
experienced when attempts have been made to introduce apomixis into crops
through hybridization. For example, major programs aimed at introducing
apomixis into maize (Sokolov et al., 1998; Savidan, 2000a, 2001) from the wild
relative Tripsacum dactyloides have been under way now for decades, yet they
have proven unsuccessful in terms of generating apomictic plants with
agronomically acceptable levels of seed set.
• Current breeding efforts with apomictic crop species, such as the forage grasses
Brachiaria and Panicum, are frustrated by the need to use complex breeding
strategies to accommodate the inaccessibility of the female gamete to generate
hybrid progeny.
• the best solution would be the introduction of apomixis into crops in an
inducible format, permitting its use during seed increase but allowing for its
silencing during hybridization.
• To achieve this, information will be required concerning the genes that control the
trait, their interrelationship with sexual processes, and the impact the trait might
have on seed yield, viability, and quality for a given plant. 33
Following strategies may be adopted to date:
1. Direct introgression of apomixis into crop plants by
means of conventional breeding schemes;
2. Genetic transformation of crop plants by transferring
exogenous genes that control the expression of
apomixis; and
3. Genetic transformation of crop plants by deregulating the
endogenous genes that trigger the expression of
apomixis.
4. Genome editing
34
