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\DOI{10.5802/crbiol.125}
\datereceived{2023-09-03}
\daterevised{2023-10-04}
\dateaccepted{2023-10-05}
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\dateposted{2024-01-11}
\begin{document}

\begin{noXML}

\RubricEF{Review Article}{Article de revue}

\title{Clonal reproduction by seed of a cultivated hybrid plant: a new
perspective for small-scale rice growers}

\alttitle{Reproduction clonale par grain d'une plante hybride
cultiv\'{e}e~: une nouvelle perspective pour les petits riziculteurs}

\author{\firstname{Aurore} \lastname{Vernet}\CDRorcid{0009-0006-6811-9376}\IsEqualContrib}
\address{CIRAD, UMR AGAP Institut, 34398 Montpellier, France}
\address{Universit\'{e} de Montpellier, CIRAD, INRAE, Institut Agro,
Montpellier, France}

\author{\firstname{Donaldo} \lastname{Meynard}\CDRorcid{0009-0000-7951-3287}\IsEqualContrib}
\addressSameAs{1}{CIRAD, UMR AGAP Institut, 34398 Montpellier, France}
\addressSameAs{2}{Universit\'{e} de Montpellier, CIRAD, INRAE, Institut
Agro, Montpellier, France}

\author{\firstname{Emmanuel} \lastname{Guiderdoni}\CDRorcid{0000-0003-2760-2864}\IsCorresp}
\addressSameAs{1}{CIRAD, UMR AGAP Institut, 34398 Montpellier, France}
\addressSameAs{2}{Universit\'{e} de Montpellier, CIRAD, INRAE, Institut
Agro, Montpellier, France}
\email[E. Guiderdoni]{emmanuel.guiderdoni@cirad.fr}

\shortrunauthors

\keywords{\kwd{Apomixis}\kwd{Clonal reproduction}\kwd{F1
hybrid}\kwd{Plant breeding}\kwd{Rice}}

\altkeywords{\kwd{Am\'{e}lioration des
plantes}\kwd{Apomixie}\kwd{Hybride F1}\kwd{Reproduction
clonale}\kwd{Riz}}

\def\thanksname{Note}
\thanks{Aurore Vernet received the Grandes Avanc\'ees en Biologie 2022
award.}

\thanks{\textbf{Funding.} This work was conducted thanks to a grant
from the France-Berkeley exchange program to V.~Sundaresan and
E.~Guiderdoni.}

\begin{abstract} 
Transferring an asexual mode of reproduction by seeds (apomixis) to
cultivated plants would enable clonal reproduction of heterozygous
genotypes such as F1 hybrids with hybrid vigor (heterosis),
facilitating their access and multiplication by small-scale growers.
Although sources of apomixis and the genetic loci controlling its
constituent elements have been identified in wild species, their
transfer by crossing to cultivated species has so far been
unsuccessful. Here, we have introduced synthetic apomixis in hybrid
rice to produce a high (95--100\%) frequency of clonal seeds, via the
inactivation of three meiotic genes---resulting in unreduced,
non-recombined gametes---and the addition of an egg cell
parthenogenesis trigger. The genotype and phenotype, including grain
quality, of the F1 hybrid are reproduced identically in the clonal
apomictic progenies. These results make synthetic apomixis compatible
with future use in agriculture.
\end{abstract} 

\begin{altabstract} 
Le transfert d'un mode de reproduction clonale asexu\'{e}e par grain
(apomixie) aux plantes cultiv\'{e}es permettrait de reproduire de
fa\c{c}on g\'{e}n\'{e}tiquement identique des g\'{e}notypes
h\'{e}t\'{e}rozygotes comme ceux des hybrides~F1 dot\'{e}s d'une
vigueur hybride (heterosis), facilitant ainsi leur acc\`{e}s et leur
multiplication par les petits cultivateurs. Bien que des sources
d'apomixie et les loci g\'{e}n\'{e}tiques contr\^{o}lant ses
\'{e}l\'{e}ments constitutifs aient \'{e}t\'{e} identifi\'{e}s chez les
esp\`{e}ces sauvages, leur transfert par croisement aux esp\`{e}ces
cultiv\'{e}es a jusqu'\`{a} pr\'{e}sent \'{e}t\'{e} infructueux. Ici,
nous avons introduit chez un riz hybride une apomixie synth\'{e}tique
produisant une haute fr\'{e}quence de grains clonaux (95--100\%), via
l'inactivation de trois g\`{e}nes m\'{e}iotiques --- permettant
d'obtenir des gam\`{e}tes non r\'{e}duits et non recombin\'{e}s --- et
l'apport d'un d\'{e}clencheur de la parth\'{e}nogen\`{e}se. Le
g\'{e}notype et le ph\'{e}notype, incluant la qualit\'{e} de grain, de
l'hybride~F1 sont reproduits \`{a} l'identique dans les descendances
apomictiques clonales. Ces r\'{e}sultats rendent compatible l'apomixie
synth\'{e}tique avec une future utilisation en agriculture.
\end{altabstract} 

\maketitle
\vspace*{-.5pc}

\twocolumngrid

\end{noXML}

\section{Rice improvement and apomixis}\label{sec1}

With 500~million tons of milled rice produced over more than
160~million hectares, rice is the leading cereal for human consumption
and the staple food for more than half of humanity. It is mainly a
self-consumption food crop, with only 10\% of production entering
international trade, compared with 20\% for bread wheat. The varieties
of rice (\textit{Oryza sativa}~L.) grown throughout the world are the
\mbox{result} of \mbox{domestication} events in Asia, the number of
which is still debated, and which led to two partially interfertile
subspecies---\textit{japonica} and \textit{indica}. The varieties show
a wide range of adaptations to a diversity of climates, cropping
systems, practices and consumer preferences. Independent domestication
took place in Africa to produce the cultivated species \textit{Oryza
glaberrima} Steud, grown mainly in West Africa. The challenges of
feeding the world's population (expected to reach 10~billion in 2050)
in a changing climate, while reducing the environmental impact of rice
cultivation (saving water and fertilizer resources and reducing
greenhouse gas emissions, such as methane in paddy fields), require the
co-design of new diversified varieties to meet both specific local
constraints and demands and these global adaptation
efforts~\cite{bib1}.

The creation of new varieties mobilizes accessions present in
cultivated species and their wild relatives (over 100,000 in the case
of rice) by crossing them to obtain genetically variable progenies,
particularly for agronomic traits, from which the breeder will choose
the most interesting plants and then lines through selection cycles
carried out over subsequent generations. The return to varietal
homogeneity is facilitated in rice, as in wheat, by its mode of sexual
reproduction by self-fertilization (the plant is said to be
autogamous): the gametes present in the male gametophyte (pollen) and
the female gametophyte (embryo sac) carried respectively by the stamen
and the ovary, most frequently belonging to the same flower, will unite
to form the embryo and the endosperm (reserves) of the seed in a
process called double fertilization (Figure~\ref{fig1}A). The gametes
are the end product of a first specialized cell division---meiosis---of
an initial diploid cell (two parental chromosome sets), during which
the parental chromosomes recombine and are halved in number to become
haploid. The cells resulting from meiosis---the functional megaspore
and the microspores on the female and male sides---undergo divisions
(mitoses) to form \mbox{gametophytes}.

\begin{figure*}
\includegraphics{fig01a}
\caption{\label{fig1}\fontsize{9.73}{12}\selectfont
(A)~Double fertilization in rice:
as in over 70\% of angiosperm species, the female gametophyte (embryo
sac) of rice is of the \textit{Polygonum} type, composed of 7 cells
resulting from cellularization of an 8-nucleus coenocyte produced by 3
divisions of a single functional megaspore (left). The functional
megaspore is one of the four spores produced by female meiosis, the
other three spores degenerating. During meiosis, the homologous
parental chromosomes of the diploid mother cell, after having
replicated their DNA into two sister chromatids, pair up for reciprocal
or non-reciprocal exchanges of DNA constituting genetic recombination,
then migrate towards opposite poles during the first division of
meiosis. During the second meiosis division, the recombined or
unrecombined sister chromatids separate and are distributed among four
spores. The number of chromosomes is thus divided by two in recombined
spores, which are haploid. 
%%%
(Continued on next page.)}
\end{figure*}
\def\thefigure{1 (cont.)}
\begin{figure*}
\ContinuedFloat
\includegraphics{fig01}
\caption{ 
%%%
The male gametophyte, the pollen, is
trinucleated, and forms after two divisions of a product of meiosis,
the microspore (right). The two sperm cells of the pollen grain are the
male gametes, which fuse with the nucleus of the egg cell of the embryo
sac, the female gamete, and the two nuclei of the central cell to form
the embryo and the albumen of the grain, respectively. As shown in the
central figure, rice self-pollinates, i.e. gametes from the same plant
unite to form offspring. This process of self-fertilization makes or
maintains the two parental batches identical throughout the plant's
cycles (homozygous). (B)~During diplosporic gametophytic apomixis
(right), the diploid mother cell undergoes simple mitosis to form
spores and then gametophytes bearing gametes. Parthenogenesis of the
egg cell allows a diploid embryo to develop without fertilization
(parthenogenetic), while fertilization of the central cell may or may
not take place to form the albumen.}
\end{figure*}
\def\thefigure{\arabic{figure}}
\setcounter{figure}{1}

Released rice varieties are generally pure lines, with two largely
identical chromosomal sets, ensuring varietal homogeneity. However,
there is an advantage in directly using progenies, also homogeneous,
formed from hybrid seeds resulting from the crossing of two pure lines,
because they exhibit hybrid vigor. Hybrid vigor, also known as
``heterosis'' already highlighted by Darwin~\cite{bib2} and
rediscovered by Shull in maize~\cite{bib3}, concerns a whole range of
traits, the best known of which is grain yield, which is more
stable and increased by 20 to 30\%. The genetic and molecular bases of
this vigor phenomenon, resulting from the presence and interactions of
the two parental genomes in a single hybrid genome, have been
extensively studied~\cite{bib4}. Following the example of maize, F1
hybrid rice varieties are used, but as rice plants do not cross
spontaneously with high frequency, the use of complex genic or
geno-cytoplasmic male sterility systems is necessary to produce hybrid
seeds~\cite{bib5}. Fertilization of the female parent by pollen from
the male parent often has to be mechanically assisted. The production
of F1 seeds is therefore costly: this limits the number of combinations
tested and distributed, and above all access to these seeds for
small-scale rice growers in the South, all the more so as they have to
be renewed with each planting. In fact, F2 seeds harvested from F1
hybrid plants, if sown, will form heterogeneous offspring, particularly
in terms of essential agronomic traits (plant height, crop cycle, grain
quality, etc.), making them difficult to use by rice growers. This
divergence is linked to allele shuffling occurring during meiosis,
which recombines homologous chromosomes from the different genomes of
the two parents. In order to reproduce an identical hybrid through
seeds over successive generations, clonal asexual reproduction would
have to be set up in rice. This mode of reproduction could be achieved
if the initial cell of the female gamete divided by simple mitosis,
without recombination or reduction of the parental chromosomes, forming
two identical daughter cells, followed by the formation of an embryo
from the egg cell of the embryo sac, without fertilization by the male
gamete.

A mode of asexual reproduction by seeds known as ``Apomixis'' exists in
nature in over 400 species of flowering plants, mainly wild but also
cultivated, such as forages and Citrus, where it takes place in
different ways depending on the origin of the cell giving rise to the
clonal embryo (either sporophytic or gametophytic origin)~\cite{bib6}.
In its most advanced form (diplosporic gametophytic), apomixis bypasses
the allele shuffling and chromosome number reduction involved in sexual
reproduction by transforming meiosis into mitosis (apomeiosis),
followed by the development of the egg cell by parthenogenesis, without
fertilization by the male gamete carried by the pollen
(Figure~\ref{fig1}B). Fusion of the central polar nuclei with the male
gamete may or may not be necessary for the formation of grain reserves
(if not, then we speak of autonomous development of the endosperm).

Because of its interest in the production of ``immortalized'' hybrid
formulas, the search for a source of apomixis in cultivated species and
their wild relatives has been conducted for many years. Genetic loci
involved in the determinism of apomeiosis and parthenogenesis have been
identified in wild species~\cite{bib7}, but their transfer to
cultivated species has so far failed to convert them to apomixis.
No~source of apomixis has been found in rice~\cite{bib8}.{\break} An
alternative is therefore to engineer synthetic apomixis in cultivated
plants, which has mobilized research over the last 20 years.

\section{Development of synthetic apomixis in rice} \label{sec2}

As described above, three elements are required to mimic diplosporic
gametophytic apomixis: (i)~conversion of meiosis into mitosis;
(ii)~development of the egg cell into an embryo without fertilization
by the male gamete; and (iii)~formation of the endosperm with (or
without) fertilization of the polar nuclei by the male gamete.

Regarding the first component, three major mechanisms differentiate
meiosis from mitosis: (i)~the pairing of homologous parental
chromosomes and their recombination; (ii)~the monopolar migration of
sister chromatids resulting from DNA replication in the first division
of meiosis; (iii)~the existence of the second meiosis division, which
distributes the recombined chromatids into four haploid spores
(Figure~\ref{fig2}A). The \textit{MiMe} (\textit{Mitosis instead of
Meiosis}) triple mutation in \textit{Arabidopsis} abolishes these
differences~\cite{bib9}: firstly, recombination and pairing are
suppressed by inactivating a member of the recombination initiation
complex (e.g. \textit{SPO11-1} or \textit{PAIR1}); next, the joint
migration of sister chromatids during meiosis is replaced by their
separation through inactivation of the \textit{REC8} cohesin; finally,
the second meiotic division is suppressed by inactivation of the cell
cycle regulator \textit{OSD1} (Figure~\ref{fig2}A). In collaboration
with INRAE Versailles, we have successfully introduced these three
mutations into rice~\cite{bib10}, and \textit{MiMe} rice forms
unrecombined diploid male and female gametes (containing $2 \times 12$
chromosomes), i.e.~with the genotype of the parent plant. The diploid
egg cell is then fertilized by a diploid male gamete and forms a
tetraploid clonal sexual embryo, while the endosperm formed after
fusion of diploid polar nuclei and a diploid male gamete is assumed to
be hexaploid (Figure~\ref{fig2}B). Apomeiotic tetraploid rice plants
can grow but are sterile.

\begin{figure*}
\includegraphics[scale=1.25]{Figure 2.png}
\caption{\label{fig2}Synthetic apomixis in rice. (A)~The \textit{MiMe}
(\textit{Mitosis instead of Meiosis}) triple mutation transforms
meiosis into mitosis in \textit{Arabidopsis}~\cite{bib9} and
rice~\cite{bib10}. \textit{MiMe} abolishes the three essential
differences distinguishing meiosis from mitosis namely (i)~pairing of
homologous chromosomes and recombination, (ii)~migration of sister
chromatids towards the same pole during the first division of meiosis,
and (iii)~existence of a second division separating sister chromatids.
(B)~The combination of \textit{MiMe} and parthenogenetic initiation of
embryogenesis in the egg cell without fertilization, by expression of
the \textit{BABYBOOM} transcription factor in this cell, enables the
development of a clonal embryo~\cite{bib13}. Grain formation is
completed by fertilization of the central cell nuclei to form the
albumen.}
\end{figure*}

The second component is the parthenogenetic development of the egg cell
without fertilization: the study of genes transcribed in isolated rice
gametes has identified a candidate transcription factor of the
APETALATA~2 family, \emph{BABYBOOM} \emph{(OsBBM1)}~\cite{bib11}. The male allele of
OsBBM1 is expressed in sperm cells, but the female allele remains
repressed in egg cells until fertilization occurs. An ortholog of this
gene discovered in rapeseed (\textit{Brassica napus}) has been
previously shown to induce somatic embryogenesis when constitutively
expressed in \textit{Arabidopsis} plant cells~\cite{bib12}. Similarly,
constitutive accumulation of OsBBM1 in rice triggers the formation of
somatic embryos on various organs~\cite{bib13}. These data, added to
the fact that a putative ortholog of \emph{OsBBM1} is also present in the
locus responsible for facultative apomixis in the wild grass
\textit{Pennisetum}~\cite{bib14}, designated OsBBM1 as a good candidate
to successfully trigger parthenogenesis by its ectopic expression
specifically in the egg cell of the embryo sac, which indeed led to
haploid embryos in wild-type rice seeds~\cite{bib13}. Combined with the
\textit{MiMe} triple mutation, egg cell-specific expression of \emph{OsBBM1}
enabled synthetic apomixis to be obtained, resulting in the production
of diploid clonal apomictic plants with a frequency of up to 30\%, the
rest of the offspring being tetraploid plants resulting from sexual
fertilization (Figure~\ref{fig2}B)~\cite{bib13}.

Finally, the third component is the formation, with or without
fertilization, of grain endosperm. In the previous study, the endosperm
accompanying the parthenogenetic embryo formed in an apparently normal
manner, but its ploidy or quality has not been analyzed~\cite{bib13}.

\section{High-frequency synthetic apomixis in hybrid rice} \label{sec3}

In a collaboration with UC Davis and the Max Planck Institute in
Cologne, we transferred a single construct carrying the elements to
engineer in a single step both apomeiosis via the \textit{MiMe} triple
mutation (by \textit{CRISPR/Cas9} mutagenesis) and parthenogenesis
(\textit{Arabidopsis} or rice egg cell-specific promoter controlling
the \textit{OsBBM1} gene) into a commercial rice hybrid (BRS-CIRAD~302)
(Figure~\ref{fig3}A)~\cite{bib18}. All the constructs and methods have
been reported and are detailed in~\cite{bib18}. The
\textit{indica/indica} F1 hybrid BRS-CIRAD~302, released in Brazil in
2010 has been chosen because of its high performance and outstanding
grain quality. Fertile regenerated plants exhibited the \textit{MiMe}
triple mutation~\cite{bib18}. The progenies of plants transformed with
the construct without the parthenogenetic trigger are all tetraploid as
expected and possess the hybrid diagnostic microsatellite markers, and
are therefore apomeiotic. When the parthenogenetic trigger is present
in the \textit{MiMe} context, a certain frequency of diploid plants is
expected in the progenies: in our case, 80\% of the 28 triple mutants
observed had a diploid progeny frequency of over 80\%. A frequency of
95 to 100\% was observed in 4 selected events over the following two
generations, indicating that \emph{OsBBM1} stably induces parthenogenesis with a
high frequency~\cite{bib18} (Figure~\ref{fig3}B). Clonal reproduction
is supposed to maintain the F1 hybrid genome identical over successive
generations: we therefore sequenced at low depth regenerated plants
over two generations and observed a sequence rigorously identical to
that of the hybrid~\cite{bib18}. This shows that synthetic apomixis,
induced by the combination of \textit{MiMe} mutations and \textit{OsBBM1}
expression, enables highly efficient clonal propagation of F1 hybrids
through seeds. Apomixis should also enable transmission of the F1
hybrid phenotype over generations: Phenotypes were further examined in
second-generation progenies of four apomictic hybrids grown with
BRS-CIRAD~302 F1 control plants (Figure~\ref{fig3}C). No significant
differences in plant traits were observed between the apomictic
progenies and the hybrid control, which are therefore faithfully and
homogeneously recapitulated via synthetic apomixis~\cite{bib18}.

\begin{figure*}
\includegraphics{fig03}
\bcaption{\label{fig3}}{2}{High frequency
synthetic apomixis in one step in hybrid rice~\cite{bib18}.
(A)~Synthetic apomixis is achieved in hybrid rice by introducing into
its genome a single construct, whose variations are schematically shown
here. The construct carries the \textit{CAS9} gene and specific RNA
guides (gRNAs) targeting each of the 3 genes (\textit{PAIR1},
\textit{REC8} and \textit{OSD1}) to be inactivated, as well as a
parthenogenesis-triggering cassette to be expressed specifically
in the egg cell (here governed by either an \textit{Arabidopsis} (T314)
or a rice (T315) promoter with egg cell-specific activity). The first
construct (T313) is a control construct to produce only apomeiotic
\textit{MiMe} plants, whose progenies are 100\% tetraploid. The other
two constructs (T314 and T315) designed to convert the hybrid to
apomixis lead to the production of \textit{MiMe} triple mutant plants
($n = 28$), 80\% of which generate diploid clonal seeds with a
frequency of over 80\%. (B)~The ploidy of the progenies of four
selected apomictic plants reaches 95--100\% of diploid clonal plants
over the following two generations. (C)~The phenotype of the
second-generation progenies of the four apomictic plants (plant growth
trays 2--5) is identical to that of the starting hybrid (plant growth
tray~1), as is their genomic sequence, verified elsewhere.}
\end{figure*}

An important trait to examine was grain size and quality. The grain
size of apomictic hybrids is comparable to that of grain harvested from
F1 hybrids. Analysis of the ploidy of the endosperm during formation
showed that, as expected, it was initially hexaploid---and not
triploid---and thus a product of the fusion of three diploid nuclei.
Finally, grain quality was analyzed, an important factor in a variety's
acceptability to consumers. Apomictic grains showed starch and amylose
contents comparable to those of grains harvested from the BRS-CIRAD~302
F1 hybrid, known for its excellent levels of these traits~\cite{bib18}.

\section{Bringing apomictic hybrids to the field} \label{sec4}

The very next steps are aimed at improving the fertility of apomictic
hybrids, which is still reduced by 20 to 40\% compared with F1 hybrids.
Main possible causes of this defect are currently being explored:
insufficient penetrance of \textit{MiMe} or defects in the temporality,
level and/or territorialization of \textit{OsBBM1} expression. The
stability of parthenogenetic induction will have to be tested in the
field under real and also unfavorable environmental conditions, which
are not represented in the greenhouse. These field trials will also
confirm the maintenance of hybrid vigor through apomixis over many
generations. While the stability of the genome, transcriptome and
methylome has been demonstrated in the first greenhouse generations of
synthetic apomixis~\cite{bib18, bib26}, it will be important to further
investigate this under field conditions and to assess longer-term
consequences of this mode of reproduction on the epigenome. We have
recently applied synthetic apomixis to other hybrids, notably distant
\textit{indica}/\textit{japonica} hybrids, which are expected to show
even greater heterosis of the order of 30\%.

In addition to grain yield and stability, which are shared with
sexually propagated F1 hybrids, hybrids propagated by synthetic
apomixis offer additional advantages: the grain endosperm presents a
single, predictable F1 genotype, rather than a more randomly segregated
F2 genotype, to which the variable grain quality harvested from
commercial hybrids has been attributed, at least in part. As the
parthenogenetic egg cell of the apomictic hybrid cannot be fertilized,
seed purity is maintained during multiplication, which is important for
countries in the South without an adequate seed distribution system.
The creation and multiplication of hybrids should eventually be
facilitated, so that small-scale rice growers can acquire and multiply
themselves seeds supplied by national technical institutes.
Facilitating the creation of hybrid formulas should make it possible to
diversify the locally adapted hybrid combinations made available after
co-design with users. It will also avoid the large-scale use of a
single male sterile cytoplasm, which can be a source of sensitivity.
Finally, the diversification of the tested hybrid combinations should
enable effective targeting of tolerance to abiotic constraints and
biotic pressures as breeding \mbox{objectives}.

Intellectual property (e.g. of the \emph{CRISPR/Cas} system) and regulatory
bottlenecks will have to be lifted to facilitate the optimization and
unleash the implementation of synthetic apomixis in crops by
laboratories and National Agricultural Research Systems. Regarding
regulatory issues, several strategies, applied before or after
generation of the synthetic apomictic events, should allow the
elimination of unwanted superfluous T-DNA sequences (i.e. the
\emph{CRISPR/Cas} system and the selectable gene cassette) in the released
apomictic hybrid. This will leave only the parthenogenesis cassette
integrated, which can be made only with rice elements~\cite{bib18},
thereby making apomictic hybrids considered intragenic and not
transgenic. If this cassette is transferred to the egg cell of
conventional rice via the apomictic diploid pollen sperm cell, it would
lead to an unviable triploid \mbox{embryo}.\looseness=-1

Management of agrobiodiversity also has to be taken into account: on
the one hand, as mentioned above, synthetic apomixis should facilitate
the diversification of released hybrid combinations by custom tailoring
them to meet the local needs and constraints of farmers and consumers,
contrasting in that with the usual release of a few mega F1 hybrids of
rice cultivated on dozens of thousands of hectares. In that sense it
should favor the maintenance of genotypic diversity in the landscapes.
On the other hand, apomictic hybrids cannot be used for crossing
anymore by construction, thereby locking their further use for
generating new diversity by the farmer and the breeder: if synthetic
apomictic hybrids become popular to a point of replacing pure lines it
will be crucial to enforce a system to make their parental lines
compulsorily accessible to breeders and farmers in public genetic
resource repositories.

\section{Conclusion} \label{sec5}

We have shown that a high frequency of clonal seed production is
possible by synthetic apomixis, making this mode of reproduction
compatible with agricultural use. It enables faithful transmission of
the hybrid sequence and phenotype in the \mbox{progenies,} while preserving
grain quality. More fundamentally, our work shows that the integration
of an additional component inducing autonomous development of the
endosperm (without fertilization) is not \mbox{necessary} to achieve a
high frequency of apomixis. Moreover, it demonstrates that the
epigenetic \mbox{contribution} made by the male gamete does not appear
to be necessary for the manifestation of heterosis in an obligate
sexual species. Taken together, these results pave the way for
international research to improve the system right up to its
application in the field~\cite{bib28}. Ultimately, apomixis should
enable breeders to exploit more effectively the potential of F1 hybrids
that are tolerant to abiotic constraints and biotic pressures, and thus
better equipped to meet the global challenges posed by climate change
and increasing food demand.

\section*{Declaration of interests}

The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

\section*{Acknowledgements}

The authors would like to thank their collaborators Pr.~Rapha\"{e}l
Mercier (INRAE Versailles, France then MPIPZ, Cologne, Germany) and
Pr.~Venkatesan Sundaresan (UC Davis, Davis, USA), as well as the other
co-authors who contributed to the success of the study.

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