FIELD OF THE INVENTION

The present invention belongs to the domain of plant biology and development, and relates to a method for improving plant regeneration. BACKGROUND OF THE INVENTION

The capacity to regenerate adult fertile plants from in vitro cultured explants is well described for many species and through various developmental pathways. Multiple environmental factors have been shown to determine the in vitro responses of plant tissues. While culture conditions are usually optimized to guarantee sustained growth, protocols may also include environmental stresses to trigger organogenesis or somatic embryogenesis. In parallel, years of empirical experimentation, aiming at the regeneration of diverse cultivars or mutants, have demonstrated that in vitro development is also under strong genetic control. However, the genes or alleles that code for "regeneration traits" have been determined in only very few cases (Nishimura et al., 2005).

Despite decades of research, poor in vitro regeneration is still a lingering bottleneck for numerous plant genotypes, including elite cultivars (Altpeter et al., 2016). Classically, protocols are developed as variants of previously published methods, and rely on minor variations on common themes. For example, in vitro culture (IVC) media would be tested with ranges of phytohormone concentrations, synthetic hormones with different stability and bioactivity, and different macromineral and micronutrient concentrations.

So there is still a need for new methods towards improved plant regeneration.

The inventors have demonstrated that mutant plant tissues with an impaired mitochondrial electron transport chain (METC) produce higher numbers of somatic embryos, buds, shoots, roots, or meristems when cultured in vitro. In particular, they have developed a method comprising at least a step of cultivating such explants with inhibitors of the mitochondrial electron transport chain, and in particular with chemical inhibitors of the complex I of the mitochondrial electron transport chain. The inventors have also demonstrated that single nuclear mutations affecting plant respiration and pharmacological treatments mimicking these mutations had similar effects on growth and morphogenesis, including both organogenesis (the development of an organ) and embryogenesis (the development of an embryo), in particular mutations and chemical treatments affecting the activity of the respiratory complex I and/or complex III. They were thus able to significantly enhance the regenerative capacity of plant tissues. This link between the specific activity of mitochondrial electron transport chain complexes and morphogenesis has not been reported, in plants or in any other higher organism.

The present invention provides methods wherein alterations of respiratory functions cause a dramatic shift in regeneration capacity. Thereby, methods taking advantage of mutations that have partially deficient respiratory functions or of known mitochondrial electron transport chain inhibitors (also named 'inhibitors of METC) can be implemented to improve regeneration efficiency. In a preferred embodiment, drugs that inhibit the mitochondrial electron transport chain are used transiently to boost morphogenesis, then removed to avoid lasting growth inhibition or cell death, thus enabling the rapid development of induced somatic embryos, buds, shoots, roots, or meristems resulting in the production of true-to-type plants.

A method according to the invention can be performed in particular on crop species, to improve the clonal vegetative multiplication or the regeneration and transformation of plants.

Respiration is the fundamental process of energy production, common to all living organisms, generating ATP which is necessary for the maintenance and the growth of the cells. Mitochondria generate the major part of ATP via oxidative phosphorylation. Mitochondria are essential organelles because they host the respiratory machinery that produces the energy necessary to power the cell. They also coordinate diverse functions involving multiple metabolic and signaling pathways that control cellular activity and that regulate the homeostasis of energy. However the relationships between respiration and development have been difficult to establish because respiratory mutants generally have strong pleiotropic phenotypes and grow very slowly. Furthermore, notably in plants, the electron transport chain is remarkably flexible and alternative respiratory pathways may take over in particular growth conditions, for example under specific stresses or in respiratory mutants. Such perturbations impact cell physiology and reshape the energy metabolism. Cytoplasmic male sterility - the loss of the male gametophyte - is to date the best studied developmental defect resulting from dysfunctional mitochondria. However, most phenotypes reported for plant respiratory mutants are retarded growth, abiotic and hormonal stress responses, as well as premature or delayed flowering (Millar et ah, 2011). The internal membrane of mitochondria comprises the mitochondrial electron transport chain (METC) which is composed of four protein complexes, designated as complex I, complex II, complex III and complex IV, which interact in particular with ubiquinone (or co-enzyme Q) and cytochrome c. Complex I catalyzes the oxidation of NADH from the matrix to reduce ubiquinone. The electron flow, from NADH to oxygen is coupled to proton translocation from the matrix to the inter-membrane space, to lead to phosphorylation of ADP by the pumping of said protons. The alternative oxidase (AOX) is an enzyme that forms part of the METC in the mitochondria of different organisms. The alternative oxidase provides an alternative route for electrons passing through the electron transport chain to reduce oxygen.

Mutations in nuclear genes controlling the expression of mitochondrial genes are particularly interesting to study respiration because the routine genetic transformation of mitochondria is not yet possible. The inventors characterized Arabidopsis thaliana (hereafter referred to as Arabidopsis) and Zea mays (maize) lines that carry mutations in nuclear genes coding for pentatricopeptide repeat (PPR) proteins targeted to the mitochondria. Some of these ppr mutants have lost the ability to produce specific subunits of the mitochondrial electron transport chain and their phenotypes range from mildly retarded growth to dwarf plants that can only be rescued - and maintained for months - via in vitro culture IVC (Sosso et al, 2012; Hai ^' li et al, 2013; Dahan et al, 2014; Hai ^' li et al, 2016).

In Arabidopsis, the PPR protein MTSF1 is essential for the 3 '-end processing of the mitochondrial nad4 mRNA and mtsfl plants accumulate low amounts of a truncated form of the respiratory complex I (Hai ^' li et al, 2013); the PPR protein MTSF2 is essential for the accumulation of stable nadl mRNA (Wang et al, 2017); the PPR protein MTL1 is essential for the translation and the splicing of the mitochondrial nad7 mRNA (Haili et al., 2016); mtsfl and mill plants fail to accumulate the NAD1 and NAD7 proteins, respectively, and these mutants do not accumulate the respiratory complex I that both proteins belong to (Wang et al, 2017; Hai ^' li et al, 2016).

In maize, the protein PPR2263 is required for the editing of the mitochondrial transcripts nad5 and cob, that encode for proteins part of the respiratory complexes I and III, respectively. The ppr2263 maize mutant plants have normal complex I activity but display complex III deficiency, and their mitochondria have a compromised ultrastructure. The ppr2263 mutants are viable but have strong growth phenotypes and delayed flowering (Sosso et al, 2012). SUMMARY OF THE INVENTION

A first object of the invention relates to an in vitro method for plant regeneration comprising at least a step of cultivating an explant from a plant in which the activity of the Mitochondrial Electron Transport Chain (METC) is impaired.

As disclosed above, the METC is composed of four protein complexes, designated as complex I, complex II, complex III and complex IV, which interact in particular with ubiquinone (or co-enzyme Q) and cytochrome c. The alternative oxidase (AOX) is an enzyme that forms part of the METC in the mitochondria of different organisms.

By "impaired activity of METC" according to the invention, it refers to partial loss of function of any complex(es), enzyme(s) or other entity(ies) acting in the METC. In particular, an impaired activity of METC according to the invention may occur through a defective assembly and/or activity of anyone of the constitutive complexes, designated as complex I, complex II, complex III and complex IV, or alternative oxidase (AOX) and/or through a defective production and/or activity of functional entities acting in the METC such as the cytochrome c maturation system.

In the rest of description, we will use interchangeably 'METC or 'METC complexes' or 'METC functional entities', meaning functional complex(es), enzyme(s) or other functional entity(ies) acting in the METC and necessary for its activity(ies).

We also use interchangeably in the description the terms 'impaired' or 'partially inhibited'.

In a first embodiment, the activity of the Mitochondrial Electron Transport Chain (METC) is 'genetically impaired', which means that the impairment is related to the use of an explant from a plant carrying mutation(s) in nuclear gene(s) resulting in the partial loss of function of complex(es), enzyme(s) or other entity(ies) acting in the METC. We also use the expression 'mutant resulting in METC impairment' to designate such mutation.

So a particular embodiment of the invention relates to an in vitro method for plant regeneration comprising:

a) obtaining an explant from a plant carrying at least one mutation in nuclear gene(s) coding for protein(s) involved in the assembly and/or in the activities of the mitochondrial electron transport chain (METC), and b) cultivating said explant in a culture medium and obtaining somatic embryos, buds, shoots, roots or meristems from said explant to generate plantlets.

The term 'and' between step a) and step b) means that step a) is followed by step b), ie sequential process.

In a preferred embodiment of the invention, the mutation(s) in nuclear gene(s) coding for a protein(s) involved in the assembly and/or the activities of METC result(s) in partial loss of function of complex(es), enzyme(s) or other entity(ies) acting in the METC, in particular loss of function of anyone of complex I, complex II, complex III, complex IV, alternative oxidase (AOX) or cytochrome C maturation system.

In a second embodiment, the activity of the Mitochondrial Electron Transport Chain (METC) is 'chemically impaired', which means that the impairment is related to the use an inhibitor of METC in the culture medium.

So another particular embodiment of the invention relates to an in vitro method for plant regeneration comprising:

a) obtaining an explant from a plant,

b) cultivating said explant in a culture medium with at least one inhibitor of the mitochondrial electron transport chain (METC) selected in the group consisting of inhibitor of complex I, inhibitor of complex II, inhibitor of complex III, inhibitor of complex IV, inhibitor of AOX and inhibitor of cytochrome C maturation system, preferably of inhibitor of complex I and/or inhibitor of complex III, more preferably inhibitor of complex I, and

c) transfering to a culture medium without said inhibitor of METC for obtaining somatic embryos, buds, shoots, roots, or meristems from said explant to generate plantlets.

According to the invention, step a) is followed by step b), then followed by step c), ie sequential process.

Preferably, the inhibitor of METC is used at a concentration and for a time period that partially inhibits cell proliferation within the explants without terminating its growth. The man skilled in the art will define the adapted concentration and time period in function of the nature of explant and plant as usually performed when fine tuning a regeneration protocol. In particular, the potential delay in growth and development resulting from a genetic or chemical impairment of the METC can be corrected by culturing the impaired explants for longer periods of time.

The present invention also relates to the use of at least a plant mutant carrying at least one mutation in nuclear gene(s) coding for protein(s) involved in the assembly and/or the activities of the mitochondrial electron transport chain (METC) and/or at least an inhibitor of METC, preferably an inhibitor of complex I and/or an inhibitor of complex III, for improving in vitro method of plant regeneration. DETAILED DESCRIPTION OF THE INVENTION

Definitions

The expression "in vitro method for plant regeneration" designates an in vitro method for the production of a plant from an explant under controlled environmental conditions, wherein said method comprises the generation of a plantlet from an explant, and wherein said plantlet can further give rise to a plant.

The term "meristem" encompasses buds, shoot and/or root meristems.

The term "explant" designates a material extracted from a plant, wherein said material comprises viable cells, and wherein the term "material" designates in particular a cell, an ovule, a microspore, an organ, a fragment of an organ, a somatic or zygotic embryo, a fragment of a somatic or zygotic embryo, a piece of tissue, a fragment of tissue, a cotyledon, a fragment of a cotyledon, a leaf, a fragment of a leaf, a root, a fragment of a root, a protoplast, a callus, a callus obtained from an organ or a fragment of an organ, a callus obtained from a somatic or zygotic embryo or a fragment of a somatic or zygotic embryo, a callus obtained from a tissue or a piece of tissue, or a fragment of tissue, a callus derived from a fragment of a cotyledon, a callus derived from a fragment of a leaf , a callus obtained from a protoplast, a callus derived from an ovule, a somatic embryo derived from an ovule, a callus derived from a microspore, or a somatic embryo derived from a microspore.

The term "cultivating an explant" designates fostering the growth of said explant, and encompasses in vitro culture conditions and components in the medium, which are well known from a man skilled in the art.

The term "culture medium" designates medium for in vitro plant culture, whose composition is well known from a man skilled in the art and easily adaptable depending of the nature of explant and/or plant. The culture medium may be liquid, gelled or solid, and encompasses culture media for different steps of the regeneration protocol, such as pre-culture medium, organogenesis or embryogenesis inducing medium and regeneration medium.

The expression "inhibitor of the mitochondrial electron transport chain (METC)" designates an agent that has been demonstrated, by appropriate biochemical assays such as described in the present application or such as known by a man skilled in the art, to inhibit the mitochondrial electron transport chain, when taken as a global function.

The expression "mutant of the mitochondrial electron transport chain METC" according to the invention designates mutation in the nuclear genome of the plant that has been demonstrated, by appropriate methods such as described in the present application or such as known by a man skilled in the art, to result in partial loss of function of any complex(es), enzyme(s) or other entity(ies) acting in the METC, in particular loss of function of anyone of complex I, complex II, complex III, complex IV, alternative oxidase (AOX) or cytochrome C maturation system. Examples of mutants of METC are disclosed further in the description.

Explant and derivatives thereof, and plant species

In a particular embodiment, an in vitro method for plant regeneration according to the invention comprises a step of obtaining an explant from a plant, wherein said explant or derivative thereof is selected in the group consisting of : a cell, an ovule, a microspore, an organ, a fragment of an organ, a somatic or zygotic embryo, a fragment of a somatic or zygotic embryo, a piece of tissue, a fragment of tissue, a cotyledon, a fragment of a cotyledon, a leaf, a fragment of a leaf, a root, a fragment of a root, a protoplast, a callus, a callus obtained from an organ or a fragment of an organ, a callus obtained from a somatic or zygotic embryo or a fragment of a somatic or zygotic embryo, a callus obtained from a tissue or a piece of tissue, or a fragment of tissue, a callus derived from a fragment of a cotyledon, a callus derived from a fragment of a leaf , a callus obtained from a protoplast, a callus derived from an ovule, a somatic embryo derived from an ovule, a callus derived from a microspore, or a somatic embryo derived from a microspore. We use interchangeably Obtained from' or 'derived of .

Fragments and callus derived from a fragment are also named 'derivatives of the said explant' or 'derivatives thereof in the following description.

In particular, the explant or derivative thereof is preferably selected in the group consisting of: a protoplast, a callus or a protoplast-derived callus,

an ovule, or a callus derived from an ovule, or a somatic embryo derived from an ovule,

a microspore, or a callus derived from a microspore, or a somatic embryo derived from a microspore,

a cotyledon, a fragment of a cotyledon or a callus derived from a fragment of a cotyledon,

a leaf, a fragment of a leaf or a callus derived from a fragment of a leaf,

a somatic or zygotic embryo, a fragment of a somatic or zygotic embryo or a callus derived from a fragment of a somatic or zygotic embryo, and

a root, a fragment of a root or a callus derived from a fragment of a root,

and more preferably:

a protoplast, a callus or a protoplast-derived callus,

a cotyledon, a fragment of a cotyledon or a callus derived from a fragment of a cotyledon, and

a somatic or zygotic embryo, a fragment of a somatic or zygotic embryo or a callus derived from a fragment of a somatic or zygotic embryo.

The explant may be haploid, diploid, or polyploid.

In a particular embodiment, the explant is haploid.

In another particular embodiment, the explant is diploid.

In another particular embodiment, the explant is polyploid.

In a particular and preferred method according to the present invention, the explant is a protoplast, a callus or a protoplast-derived callus. A "protoplast" is a plant cell that had its cell wall completely or partially removed with mechanical and/or enzymatic means, a protoplast therefore comprises a cell nucleus surrounded by cytoplasmic material. Plant protoplasts are prepared by using protocols known by a man skilled in the art, in particular according to a protocol described in Chupeau et al. (2013).

In a more particular embodiment, an in vitro method for plant regeneration according to the invention comprises a step of obtaining an explant from a plant, wherein said explant is a callus obtained from a protoplast.

Calli are obtained from protoplasts by a method known by a man skilled in the art. Conventional methods for the propagation of plants through tissue culture are generally described in the authoritative review of Murashige (1974). More specifically, methods for plant regeneration from cultured protoplasts are described by Evans and Bravo (1983) and Binding (1985).

In a more particular embodiment, the plant is chosen among eudicotyledons and monocotyledons.

In a more particular embodiment, the plant is an angio sperm chosen among eudicotyledons and monocotyledons.

In a particular embodiment, said eudicotyledon is chosen among Brassicales. In a more particular embodiment of the invention, said Brassicales is a Brassicaceae. In an even more particular embodiment of the invention, said Brassicaceae is Arabidopsis thaliana or Brassica napus.

In another particular embodiment, said eudicotyledon is chosen among Cucurbitales. In a more particular embodiment of the invention, said Cucurbitales is a Cucurbitaceae. In an even more particular embodiment of the invention, said Cucurbitaceae is Cucumis melo.

In another particular embodiment, said eudicotyledon is chosen among Solanales. In a more particular embodiment of the invention, said Solanales is a Solanaceae. In an even more particular embodiment of the invention, said Solanaceae is Solanum lycopersicum.

In another particular embodiment, said monodicotyledon is chosen among Poales. In a more particular embodiment of the invention, said Poales is a Poaceae. In an even more particular embodiment of the invention, said Poaceae is Zea mays or Sorghum bicolor or Saccharum officinarum or Triticum aestivum or Triticum durum or Hordeum vulgare or Avena sativa or Brachypodium distachyon.

In a more particular embodiment, said plant is selected from the group consisting of

Arabidopsis thaliana, Brachypodium distachyon, Brassica napus, Solanum lycopersicum, Triticum aestivum and Zea Mays.

Mutants resulting in METC impairment

As first alternative, the present invention relates to an in vitro method for plant regeneration based on the induction of organogenesis or somatic embryogenesis in an explant from a plant carrying a nuclear gene mutation resulting in METC impairment, as defined above as "impaired activity of METC".

In a first alternative of the invention, the in vitro method for plant regeneration comprises:

a) obtaining an explant from a plant carrying at least one mutation in nuclear gene(s) coding for protein(s) involved in the assembly and/or in the activities of the mitochondrial electron transport chain (METC), and

b) cultivating said explant in a culture medium and obtaining somatic embryos, buds, shoots, roots, or meristems from said explant to generate plantlets.

In a preferred embodiment of the invention, the mutation(s) in nuclear gene(s) coding for protein(s) involved in the assembly and/or the activities of METC result(s) in the partial loss of function of any complex(es), enzyme(s) or other entity(ies) acting in the METC and thereby an impaired plant respiratory activity.

In particular, the explant is obtained from a plant in which the mitochondrial respiratory complex I, II, III and/or IV, alternative oxidase (AOX) or complex involved in cytochrome c maturation system, more preferably mitochondrial complex I and/or mitochondrial complex III has been impaired. In particular, the mutation in nuclear gene(s) results in a defective assembly and/or activity of anyone of the constitutive complexes, designated as complex I, complex II, complex III and complex IV, or alternative oxidase (AOX) and/or through a defective production and/or activity of functional entities acting in the METC such as the cytochrome c maturation system

In a particular embodiment, the explant is obtained from a plant carrying at least one mutation in nuclear gene(s) involved in the stability, splicing, editing, and/or translation of transcripts encoded in the mitochondrial genome, themselves encoding for proteins assembled in the different complexes of the METC.

In a preferred embodiment, the explant is obtained from a plant carrying at least one mutation in nuclear gene(s) selected from genes coding for pentatricopeptide repeat (PPR) proteins targeted to the mitochondria and involved in the assembly and/or the activities of METC, preferably PPR proteins involved in the assembly and/or the activities of complex I and/or complex III of the METC. Genes coding for PPR proteins have been found in all eukaryotic genomes. PPR proteins are involved in organellar gene expression. Multiple plant ppr mutant lines have been characterized in several species leading to the identification of the causal nuclear gene mutation and to the understanding of how that mutation results in the impairment of one or several mitochondrial functional complexes essential for the functioning of the respiratory chain (Table I, ppr mutants).

Examples of ppr mutants resulting in the impairment of the METC, which may be used in the in vitro method for plant regeneration according to the present invention, are listed in the following Table 1 :

(*) ccm system, cytochrome C maturation system

Table 1

As they associate with specific RNA sequences, different PPR proteins have been involved a wide range of RNA-related regulations: RNA transcription, RNA stabilization, RNA end processing, RNA translation, RNA cleavage, RNA splicing, RNA site-specific editing. Their functions are also dictated by the sub-cellular compartment(s) in which they are targeted.

The identification of relevant target nuclear genes to enhance regeneration phenotypes in one species by reproducing mutations of interest observed in another is relatively straightforward for several reasons. (1) Most PPR orthologues can be paired by analyzing phylogenetic trees (O' Toole et al, 2014). (2) Conserved PPR proteins have conserved targets and orthologues may complement each other, for example between maize and Arabidopsis (Manavaski et al, 2012). (3) Transit peptide sequence analysis is a useful - although not perfect - predictor of the targeted organelle(s) and can be used as a pre-screen to select proteins likely functioning in the mitochondrion (Tanz et al, 2013). (4) Based on our observation, different ppr mutants in a given species, that target the same mitochondrial transcripts, result in similar growth defect and morphogenesis enhancement. (5) By extrapolation, a mutant in one species carrying a defect in a PPR gene homologous to a PPR gene known to cause METC impairment in another species is likely to have the same positive effect on morphogenesis if it shows similar growth retardation.

Consequently, the man skilled in the art is able to define the appropriate mutation(s) in nuclear gene(s) coding for protein(s) involved in the assembly and/or activities of METC to be used in the in vitro method of the invention, taken into account his knowledge on available methods for having mutation(s) in nuclear genes of the plant. Mutants of interest can be identified by systematically sequencing natural accessions that may show diversity in PPR gene sequences (Stoll et al, 2015). Alternatively, they can be isolated from mutagenized populations (e.g. insertional mutants such as T-DNA lines in Arabidopsis or Mutator lines in maize; EMS-induced TILLING populations available for various species), in which the homogeneity of the original genotype facilitates the identification of lines with a retarded growth phenotype. Finally, targeted loss-of-function mutant alleles can be generated through the expression of site-specific nucleases, such as TALENs or CRISPR/Cas9, in plant tissues (Baltes and Voytas, 2015).

In a particular embodiment, a plant mutant carrying at least one mutation in nuclear gene(s) coding for protein(s) involved in the assembly and/or activities of complex I and/or complex III is used according to the invention. As illustrated in the example 3 further in the description, the inventors demonstrated that there is a correlation between the alteration of a nuclear gene (mutant allele) and the in vitro response (plant regeneration). In particular, the stronger is the resulting growth retardation phenotype, the better is the in vitro response. As a consequence, in a preferred embodiment, the plant mutants carrying at least one mutation in nuclear gene(s) coding for protein(s) involved in R A transcription, R A stabilization, R A end processing, R A translation, R A cleavage, RNA splicing, or RNA site-specific editing of transcripts encoded in the mitochondrial genome, involved in particular in the assembly and/or activities complex I and/or complex III, may be advantageously chosen in the in vitro method of the invention.

Inhibitors of METC

In another alternative, the present invention relates to an in vitro method for plant regeneration based on the induction of organogenesis or somatic embryogenesis in an explant cultivating in the presence of at least an inhibitor of METC.

So the present invention also concerns an in vitro method for plant regeneration comprising:

a) obtaining an explant from a plant,

b) cultivating said explant in a culture medium with at least one inhibitor of the mitochondrial electron transport chain (METC) selected in the group consisting of inhibitor of complex I, inhibitor of complex II, inhibitor of complex III, inhibitor of complex IV, inhibitor of AOX and inhibitor of cytochrome C maturation system, preferably of inhibitor of complex I and/or inhibitor of complex III, more preferably inhibitor of complex I, and c) transfering to a culture medium without said inhibitor of METC for obtaining somatic embryos, buds, shoots, roots, or meristems from said explant to generate plantlets.

A culture medium without inhibitor of METC is also named inhibitor of METC-free culture medium.

Methods for testing the ability of an agent to inhibit the mitochondrial electron transport chain (METC), and, more precisely the ability of an agent to inhibit a function of the mitochondrial electron transport chain, are known by a man skilled in the art. More particularly, said methods include the morphological study of the mitochondria in situ; the analysis of mitochondrial components, and particularly the analysis of mitochondrial proteome, transcriptome and/or genome, and the analysis of mitochondrial activities (or mitochondrial functions) including the analysis of oxygen consumption, ATP synthesis, glucose uptake, and/or reactive oxygen species [ROS] production, wherein said analyzed mitochondria have previously been isolated from disrupted cells. Methods for testing the ability of an agent to inhibit the function of the mitochondrial electron transport chain also include magnetic resonance spectroscopy and live cell microscopy, in combination with probes of mitochondrial activities, in particular with fluorescent probes of mitochondrial activities (Martin et ah, 2011; Perry et ah, 2013).

Inhibitors of the mitochondrial electron transport chain are very diverse and do not correspond to a unified structural category. Said inhibitors are described in the publications of Degli Espositi (1998) and Orme-Johnson (2008). Inhibitors of the mitochondrial electron transport chain are represented in the following Table 2, which comprises: inhibitors of the complexes of the mitochondrial electron transport chain, including complex I, complex II, complex III, complex IV, adenine nucleotide translocator, AOX, as well as uncoupling reagents dissipating the proton gradient across the inner-mitochondrial membrane.

Inhibitor Site of action

Atractyloside and carboxyatractyloside Adenine nucleotide

translocator

Bongkrekic Adenine nucleotide

translocator

n-Propylgallate Alternative oxidase (AOX)

Salicylhydroxamic acid (SHAM) AOX

Amytal (Amobarbital) Complex I

Annonaceous acetogenins, includes annonin, annonacin, Complex I

asimicin, bullatacin, gigantetrocins, molvazarin, otivarin,

rolliniastatins, squamocin, trilobactin

b-Carboline derivatives Complex I

Capsaicin Complex I

Flavonoids, includes flavone Complex I

Isoquino lines or Isoquinoliniums Complex I

MPP+ (l-methyl-4-phenylpyridinium) and analogues Complex I

Piericidin Complex I

Rhein Complex I

Rotenoids, includes: deguelin, rotenone, analogues of deguelin Complex I

and analogues of rotenone

Diphenylene-Iodonium Complex I (AOX)

Malonate Complex II

3-Nitropropionic acid Complex II

TTFA (Thenoyltrifluoroacetone) Complex II

Table 2

In a particular embodiment, an in vitro method for plant regeneration according to the invention comprises a step of cultivating an explant in the presence of an inhibitor of the mitochondrial electron transport chain selected in the group consisting of : an inhibitor of complex I, an inhibitor of complex II, an inhibitor of complex III, an inhibitor of complex IV, an inhibitor of the adenine nucleotide translocator, an inhibitor of the AOX of the mitochondrial electron transport chain, and an uncoupling reagent dissipating the proton gradient across the inner-mitochondrial membrane.

In a more particular embodiment, the inhibitor of the mitochondrial electron transport chain is chosen among an inhibitor of complex I, an inhibitor of complex III, and mixtures thereof. In a preferred embodiment, the said inhibitor is added to the in vitro culture medium at a concentration and for a time period that partially inhibits cell proliferation within the explants without terminating its growth. The man skilled in the art will adapt the concentration and the time period in function of the nature of the explant and the plant species, as disclosed above.

In a particular embodiment, an in vitro method for plant regeneration according to the invention comprises a step of cultivating an explant in the presence of at least one inhibitor selected in the group consisting of rotenoids, Salicylhydroxamic acid (SHAM), flavonoids, Thenoyltrifluoroacetone (TTFA), Cyanide, Azide, preferably rotenoids.

In a particular embodiment, an in vitro method for plant regeneration according to the invention comprises a step of cultivating an explant in the presence of at least an inhibitor of the complex III of the mitochondrial electron transport chain.

In a more particular embodiment, an in vitro method for plant regeneration according to the invention comprises a step of cultivating an explant in the presence of at least an inhibitor of the complex I of the mitochondrial electron transport chain.

Inhibitors of the complex I of the mitochondrial electron transport chain are also very diverse and do not correspond to a unified structural category. According to Degli Espositi (1998), complex I inhibitors can broadly be classified as:

inhibitors acting like rolliniastatin-2 or piericidin A at one of the two inhibition sites of complex I,

inhibitors acting like rotenone binding at the most specific inhibition site of complex I,

a group of inhibitors including compounds chemically unrelated to rotenoids or piericidins, such as quinol analogues, especially Q2H, which are particularly potent for the membrane potential generation of complex I and the redox activity with hydrophilic substrates such as Q-l, that promote inefficient proton pumping presumably because these quinones are protonated in the aqueous phase and not within the membrane like natural ubiquinone. Myxothiazol, stigmatellin, NP, capsaicin, demerol and some cationic MPP ⁺ analogues also appear to interact with a hydrophilic site in complex and share the inhibitory properties of quinols. In a more particular embodiment, an in vitro method for plant regeneration according to the invention comprises a step of cultivating an explant with an inhibitor of the respiratory complex I, wherein said inhibitor is chosen in the group consisting of the rotenoids. The term "rotenoids" designates naturally occurring substances containing a cis-fused tetrahydrochromeno[3,4-b]chromene nucleus. Rotenoids are related to isoflavones and include the molecules listed in the Table 3 below.

In a particular embodiment, an in vitro method for plant regeneration according to the invention comprises a step of cultivating an explant in the presence of an inhibitor of the function of the respiratory complex I, wherein said inhibitor is rotenone.

In a particular embodiment, an in vitro method for plant regeneration according to the invention comprises a step of cultivating an explant in the presence of an inhibitor of METC and in particular rotenone, wherein the inhibitor of METC and in particular rotenone is added in the culture medium at a final concentration superior to 0.1 μΜ and inferior to 40 μΜ, said concentration being preferably comprised between 0.5 and 20 μΜ, preferably between 1 and 10 μΜ, preferably between 5 and 10 μΜ, and more preferably at a rotenone concentration chosen among: 1.25 μΜ, 2.5 μΜ, 5 μΜ, 10 μΜ and 20 μΜ. In a particular embodiment, an in vitro method for plant regeneration according to the invention comprises a step of cultivating an explant in the presence of an inhibitor of METC and in particular rotenone, wherein the inhibitor of METC and in particular rotenone is added to a culture medium which may be liquid or semi- liquid, solid or gelled culture medium depending the nature of the explant and/or the plant.

Transitory addition of the inhibitor of METC in the culture medium

In a particular embodiment, in a method according to the invention for plant regeneration, the step of cultivating said explant in the presence of an inhibitor of the mitochondrial electron transport chain is limited in time ('transitory' or 'intermittent'), wherein the contact of said explant with said inhibitor is initiated by a step consisting of, for example, the addition of said inhibitor to the culture medium, the transfer of said explant from a first culture medium not containing said inhibitor to a second culture medium containing said inhibitor, and wherein the contact of said explant with said inhibitor is terminated by a step consisting of the transfer of said explant from a second culture medium containing said inhibitor to a third culture medium not containing said inhibitor.

So in a particular and preferred embodiment, the in vitro method for plant regeneration comprises the following steps:

a) obtaining an explant from a plant,

b) cultivating said explant in a culture medium (first culture medium) without any inhibitor of METC but containing advantageously at least one phyto hormone, c) cultivating said explant in said culture medium (second culture medium) with an inhibitor of METC, preferably inhibitor of complex I and/or inhibitor of complex III, in particular with rotenone, optionally in combination with at least one phytohormone, and

d) transfering said explant in a culture medium (third culture medium) without any inhibitor of METC but containing optionally at least one phytohormone, and obtaining somatic embryos, buds, shoots, roots, or meristems to generate plantlets.

The in vitro method for plant regeneration according to the invention generally comprises a step of proliferation/de-differentiation of the cells in the explant followed by a step of differentiation after transfer to another culture medium, for obtaining somatic embryos, buds, shoots, roots, or meristems to generate plantlets.

In a particular and preferred embodiment, the transitory addition of the inhibitor of METC as defined above is realized before the step of differentiation.

The methods for adding an inhibitor to a culture medium or for transfering an explant from a second culture medium to a third culture medium are well known by a person skilled in the art.

The first, second and third culture media generally have similar composition excepting the presence/absence of the said inhibitor of METC and optionally the presence/absence of growth substance. Advantageously, all culture media contain at least one growth substance. Examples of growth substance are disclosed further in the description.

In a more particular embodiment, a method according to the invention for plant regeneration comprises a step of cultivating for at least one hour said explant in the presence of an inhibitor of the mitochondrial electron transport chain.

In a more particular embodiment, a method according to the invention for plant regeneration, comprises a step of cultivating for at least one day said explant in the presence of an inhibitor of the mitochondrial electron transport chain.

In a more particular embodiment, a method according to the invention for plant regeneration comprises a step of cultivating for at least one week said explant in the presence of an inhibitor of the mitochondrial electron transport chain.

In a more particular embodiment, a method according to the invention for plant regeneration comprises a step of cultivating for at least two weeks said explant in the presence of an inhibitor of the mitochondrial electron transport chain.

In a method for in vitro plant regeneration according to the invention, the step of

"cultivating said explant in the presence of at least one inhibitor of the mitochondrial electron transport chain" comprises the contact of said explant with said inhibitor, preferably by the addition of said inhibitor to the culture medium wherein said explant is cultivated.

In another particular embodiment, the present invention relates to a method for plant regeneration comprising the step of obtaining an explant, and subsequently preparing a derivative of said explant, and cultivating an explant, or derivative thereof, with at least one inhibitor of the function of the mitochondrial electron transport chain in said explant, wherein said explant or derivative thereof is cultivated in a culture medium containing at least one plant growth substance.

The appropriate culture medium may be liquid or gelled and is easily chosen by a man skilled in the art.

Culture medium and additional plant growth substance

In a particular embodiment, a method for plant regeneration according to the invention comprises cultivating an explant having an impaired activity of the mitochondrial electron transport chain (inhibitor of METC or mutation resulting in METC impairment), wherein said culture medium is added with at least one plant growth substance. In a preferred embodiment, the said at least one growth substance is chosen in the group of phytohormones. In a more particular embodiment of a method for plant regeneration according to the invention, said at least one phytohormone is a natural or a synthetic phytohormone. In another more particular embodiment of a method for plant regeneration according to the invention, said phytohormone is chosen among auxins and cytokinins.

In a more particular embodiment, a method for plant regeneration according to the invention comprises cultivating an explant having an impaired function of the complex I and/or complex III of the mitochondrial electron transport chain, wherein said culture medium is added with at least one plant growth substance, wherein said at least one plant growth substance is a phytohormone chosen among natural and synthetic phytohormones, and/or said phytohormone being chosen among auxins and cytokinins.

In an even more particular embodiment, a method for plant regeneration according to the invention comprises cultivating an explant having impaired function of METC, in particular with rotenone, wherein said culture medium is added with at least one

phytohormone chosen among auxins. Auxins commonly used for the in vitro culture of plant tissues are summarized in the following Table 4:

Name CAS number

2,4,5-Trichlorophenoxy acetic acid 37785-57-2

2,4-Dichlorophenoxyacetic acid (2,4-D) 94-75-7

Dicamba 1918-00-9

Indole-3 -acetic acid (IAA) 87-51-4

4-[3-Indolyl]butyric acid (IBA) 133-32-4

p-Chlorophenoxyacetic acid (4-CPA) 122-88-3 Picloram 1918-02-1

2-(l-Naphthyl) acetic acid (NAA) 86-87-3

(2-Naphthoxy) acetic acid 120-23-0

Table 4

In another more particular embodiment, a method for plant regeneration according to the invention comprises cultivating an explant having impaired function of METC, in particular with rotenone, wherein said culture medium is added with at least one

phytohormone chosen among cytokinins. Cytokinins commonly used for the in vitro culture of plant tissues are summarized in the following Table 5:

Table 5

In a more particular embodiment, a method according to the present invention relates to a method for plant regeneration comprising cultivating an explant, or derivative thereof, with at least one inhibitor of the function of the mitochondrial electron transport chain in said explant, wherein said inhibitor is rotenone and wherein rotenone is added in the culture medium at a concentration of 1.25, 2.5, 5 or 10 μΜ.

In a more particular embodiment, a method for plant regeneration according to the invention, comprises cultivating an explant with rotenone, wherein rotenone is added in the culture medium at a concentration of 1.25, 2.5, 5 or 10 μΜ and wherein said culture medium is added with at least one plant growth substance chosen among natural or synthetic auxins and cytokinins.

In a more particular embodiment, a method according to the present invention relates to a method for plant regeneration comprising cultivating an explant, or derivative thereof, with at least one inhibitor of the function of the mitochondrial electron transport chain in said explant, wherein said inhibitor is rotenone and wherein rotenone is added in the culture medium before the transfer of the explant to a gelled medium.

In a more particular embodiment, the present invention relates to a method for plant regeneration comprising the step of cultivating an explant obtained from said plant with at least one inhibitor of the function of the mitochondrial electron transport chain in said explant, wherein said inhibitor of the function of the mitochondrial electron transport chain inhibits the function of complex I.

In another particular embodiment, a method for plant regeneration according to the invention comprises cultivating an explant in the presence of an inhibitor of the mitochondrial electron transport chain, wherein said culture medium is added with at least one plant growth substance before the addition to the culture medium of an inhibitor of the mitochondrial electron transport chain.

In another particular embodiment, a method for plant regeneration according to the invention comprises cultivating an explant in the presence of an inhibitor of the mitochondrial electron transport chain, wherein said culture medium is added with at least one plant growth substance after the addition to the culture medium of said inhibitor of the mitochondrial electron transport chain.

In another particular embodiment, a method for plant regeneration according to the invention comprises cultivating an explant in the presence of an inhibitor of the mitochondrial electron transport chain, wherein said culture medium is added with at least one plant growth substance before, during and/or after the addition to the culture medium of said inhibitor of the mitochondrial electron transport chain.

In a more particular embodiment, a method according to the present invention for plant regeneration comprises cultivating an explant, in the presence of at least one inhibitor of the mitochondrial electron transport chain, wherein said plant is chosen among eudicotyledons and monocotyledons.

In a more particular embodiment, a method according to the present invention for plant regeneration comprises cultivating an explant in the presence of at least one inhibitor of the mitochondrial electron transport chain in said explant, wherein said plant is an angiosperm chosen among eudicotyledons and monocotyledons. In a more particular embodiment, a method according to the present invention for plant regeneration comprises cultivating an explant from a eudicotyledon chosen among Brassicales, in the presence of at least one inhibitor of the mitochondrial electron transport chain. In a more particular embodiment of the invention, said Brassicales is a Brassicaceae. In an even more particular embodiment of the invention, said Brassicaceae is Arabidopsis thaliana or Brassica napus.

In a more particular embodiment, a method according to the present invention for plant regeneration comprises cultivating an explant from a eudicotyledon chosen among Cucurbitales, in the presence of at least one inhibitor of the mitochondrial electron transport chain. In a more particular embodiment of the invention, said Cucurbitales is a Cucurbitaceae. In an even more particular embodiment of the invention, said Cucurbitaceae is Cucumis melo.

In a more particular embodiment, a method according to the present invention for plant regeneration comprises cultivating an explant from a eudicotyledon chosen among Solanales, in the presence of at least one inhibitor of the mitochondrial electron transport chain. In a more particular embodiment of the invention, said Solanales is a Solanaceae. In an even more particular embodiment of the invention, said Solanaceae is Solanum lycopersicum.

In a more particular embodiment, a method according to the present invention for plant regeneration comprises cultivating an explant from a eudicotyledon chosen among Poales, in the presence of at least one inhibitor of the mitochondrial electron transport chain. In a more particular embodiment of the invention, said Poales is a Poaceae. In an even more particular embodiment of the invention, said Poaceae is Zea mays or Sorghum bicolor or Saccharum officinarum or Triticum aestivum or Triticum durum or Hordeum vulgare or Avena sativa or Brachypodium distachyon.

In a more particular embodiment, a method according to the present invention relates to a method for plant regeneration comprising cultivating an explant, or derivative thereof, with at least one inhibitor of the function of the mitochondrial electron transport chain in said explant, wherein said plant is selected from the group consisting of Arabidopsis thaliana, Brachypodium distachyon, Brassica napus, Solanum lycopersicum, Triticum aestivum and Zea Mays.

In another particular embodiment, a method according to the present invention for plant regeneration comprising cultivating a protoplast-derived callus in a culture medium with an inhibitor of the complex I of the mitochondrial electron transport chain, wherein said inhibitor is rotenone at a concentration superior to 0.1 μΜ and inferior to 40 μΜ, and wherein said plant is selected from the group consisting of: Arabidopsis thaliana, Brachypodium distachyon, Brassica napus, Solarium lycopersicum and Triticum aestivum.

In another particular embodiment, a method according to the present invention for plant regeneration comprising cultivating a protoplast-derived callus in a culture medium with an inhibitor of the complex I of the mitochondrial electron transport chain, wherein said inhibitor is rotenone at a concentration superior to 0.1 μΜ and inferior to 40 μΜ, in the presence of at least one phyto hormone chosen among auxins and cytokinins, and wherein said plant is selected from the group consisting of: Arabidopsis thaliana, Brachypodium distachyon, Brassica napus, Solanum lycopersicum and Triticum aestivum. Conventional methods for the propagation of plants through tissue culture are generally described in the authoritative review of Murashige (1974). More specifically, methods for plant regeneration from cultured protoplasts are described by Evans and Bravo (1983) and Binding (1985).

In a more particular embodiment, a method according to the present invention relates to a method for plant regeneration comprising cultivating an explant, or derivative thereof, with at least one inhibitor of the function of the mitochondrial electron transport chain in said explant, said method also comprising a step of obtaining buds.

In another particular embodiment, the present invention relates to the use of an inhibitor of the function of mitochondrial electron transport chain, and particularly an inhibitor of complex I, for the regeneration of a plant.

In a particular embodiment, a method according to the present invention relates to a method for plant regeneration comprising cultivating a cotyledon, a cotyledon fragment or a callus derived from a cotyledon fragment from a plant, with at least one inhibitor of the METC, preferably at least one inhibitor of complex I.

In a particular embodiment, a method according to the present invention relates to a method for plant regeneration comprising cultivating a cotyledon, a cotyledon fragment or a callus derived from a cotyledon fragment from Solanaceae, preferably from Solanum lycopersicum, with at least one inhibitor of the METC, preferably at least one inhibitor of complex I.

In a particular embodiment, a method according to the present invention relates to a method for plant regeneration comprising cultivating a cotyledon, a cotyledon fragment or a callus derived from a cotyledon fragment from Solanaceae, preferably from Solanum lycopersicum, with at least rotenone, preferably in a concentration ranging from 1 to 10 μΜ, preferably 5 μΜ.

In a particular embodiment, a method according to the present invention relates to a method for plant regeneration comprising cultivating a cotyledon, a cotyledon fragment or a callus derived from a cotyledon fragment from Solanaceae, preferably from Solanum lycopersicum, with at least one inhibitor of complex I, wherein the culture medium contains at least one phytohormone.

In a particular embodiment, a method according to the present invention relates to a method for plant regeneration comprising cultivating a cotyledon, a cotyledon fragment or a callus derived from a cotyledon fragment from Solanaceae, preferably from Solanum lycopersicum, with at least rotenone, preferably in a concentration ranging from 1 to 10 μΜ, preferably 5 μΜ, wherein the culture medium contains at least one phytohormone. In a particular embodiment, a method according to the present invention relates to a method for plant regeneration comprising cultivating a protoplast from a plant, or derivative thereof, said plant carrying a mutation in a nuclear gene coding for a protein involved in the assembly and/or activities of the METC.

In a particular preferred embodiment, a method according to the present invention relates to a method for plant regeneration comprising cultivating a protoplast, a callus or a protoplast derived-callus from Brassicaceae, preferably Arabidopsis thaliana, carrying a mutation in a nuclear gene (MTL1, MTSF1 or MTSF2) coding for a protein involved in the assembly and/or activities of the METC, preferably in the assembly and/or activity of complex I.

In a particular preferred embodiment, a method according to the present invention relates to a method for plant regeneration comprising cultivating a protoplast, a callus or a protoplast derived-callus from Brassicaceae, preferably Arabidopsis thaliana, carrying a mutation in a nuclear gene (MTL1, MTSF1 or MTSF2) coding for a protein involved in the assembly and/or activities of the METC, preferably in assembly and/or activity of complex I, wherein the culture medium contains at least one phytohormone.

In a particular embodiment, a method according to the present invention relates to a method for plant regeneration comprising cultivating an (immature) zygotic embryo from a plant, said plant carrying a mutation in a nuclear gene coding for a protein involved in the assembly and/or activities of the METC, preferably in assembly and/or activity of complex I and/or III.

In a particular embodiment, a method according to the present invention relates to a method for plant regeneration comprising cultivating an (immature) zygotic embryo from Poaceae, preferably from Zea mays, carrying a mutation in a nuclear gene (PPR2263) coding for a protein involved in the assembly and/or activities of the METC, preferably in assembly and/or activity of complex I and/or III.

In a particular embodiment, a method according to the present invention relates to a method for plant regeneration comprising cultivating an (immature) zygotic embryo from Poaceae, preferably from Zea mays, carrying a mutation in a nuclear gene (PPR2263) coding for a protein involved in the assembly and/or activities of the METC, preferably in assembly and/or activity of complex I and/or III, wherein the culture medium contains at least one phytohormone.

The present invention also relates to the use of at least a plant mutant carrying at least one mutation in nuclear gene(s) coding for protein(s) involved in the assembly and/or the activities of the mitochondrial electron transport chain (METC) and/or at least an inhibitor of METC, preferably of Complex I and/or III, for improving in vitro method of plant regeneration.

Advantageously, the said plant mutant carrying a mutation in a nuclear gene coding for a protein involved in the assembly and/or the activities of the mitochondrial electron transport chain (METC) or at least an inhibitor of METC, preferably an inhibitor of complex I and/or an inhibitor of complex III, according to the invention, replaces commonly used explant stress treatments or conditions well known by the man skilled in the art to induced organogenesis or embryogenesis in plant regeneration methods.

The present invention will be further described by the illustrative and non-limitative examples and figures. FIGURES

Figure 1 : Comparative vegetative phenotypes of plants (from left to right): Col-0, mtll-1 and mtll-2. The photography was taken after 8 weeks of culture in long-day conditions. Homozygous mtll mutants grow much slower than wild-type plants on soil, and produce twisted rosette leaves.

Figure 2: Schematic representation of the successive stages of the Arabidopsis protoplast regeneration process. The method is adapted from Chupeau et al. (2013). In the three first stages, dividing protoplasts, micro-colonies and micro-calli are cultivated in liquid medium, at the bottom of Petri dishes. In the last stage, calli are transferred onto gelled medium. Under the timeline, the green bar illustrates the only addition of an exogenous synthetic auxin (2,4- dichlorophenoxyacetic acid [2,4-D]) in the regeneration protocol, at the onset of protoplast cultivation. The maroon and violet bar underneath represents the four successive additions of exogenous synthetic cytokinins: thidiazuron (TDZ) in the liquid medium and meta-topolin in the gelled medium. Shoots appear after transfer from liquid medium onto gelled medium as shown in Figure 3.

Figure 3: Regeneration of shoot meristems in calli derived from Arabidopsis protoplasts, In vitro response after cytokinin induction as described in Chupeau et al. (2013). Left panel: wild type (Col-0); right panel: mtll-1 mutant (in Col-0 genetic background).

Figure 4: Growth retardation is associated with enhanced organogenesis in complex I deficient Arabidopsis mutants. A, Growth defect of complex I mutants cultivated in greenhouse. B, Mean callus size after transfer (T) and two weeks after transfer (T+2 weeks). Size was estimate as a 2D-projection of imaged Petri dishes with the Image J software. Data represent mean size of at least 31 calli per genotype. Error bars indicate standard errors. C, Relative distribution of calli producing shoots for each complex 1 mutant (mean ± SE). In the upper panel, asterisks mark genotypes with significantly higher organogenesis rate compared to wild type (Student's t-test; p < 0.05). Bottom panel provides a pie chart representation of classes of calli with 1, 2 and 3-or-more buds for all five genotypes analyzed.

Figure 5 : Chemical structure of rotenone.

Figure 6: Rotenone treatment schemes, in view of the time-sequence from protoplast culture to transfer.

Figure 7: Histogram representing the medium size of calli (in mm ² ) as a function of rotenone concentration (in μΜ). Callus size was measured just after transfer on gelled medium (white bars) then 2 weeks later (solid black bars) after growing in contact or not with rotenone at the indicated concentration (n=20-40). Callus size was estimated on 2-D scans of Petri dishes with the Image J software package. A star indicates conditions in which calli have significantly grown in size after 15 days of in vitro culture. Letters distinguish rotenone concentrations that cause significantly different effects on callus growth as observed after 15 days of treatment.

Figure 8: Histogram representing the relative frequency of wild-type regenerative Arabidopsis calli as a function of rotenone concentration (in μΜ) in different treatment schemes (Scheme 1 , 2 or 3; untreated control (NT), from left to right) or of the mtll-1 mutant. All samples were mixed with the same concentration of DMSO (0.5%) in which rotenone was pre-dissolved, except for the control sample "without DMSO". Shoot formation was quantified 30 days after transfer (n=19-21). One shoot per callus, white bar; two shoots per callus, grey bar; three shoots per callus, black bar.

Figure 9: Enhanced regeneration of shoots on tomato cotyledon explants treated with rotenone. Cotyledons sampled from 8-days old tomato plantlets were sectioned in 6 explants, incubated on preculture-medium (PM medium), treated for 24 hours on regeneration-medium (RM medium) containing rotenone or a mock control, and further incubated on fresh rotenone-free RM medium. The graph in the top panel shows the average number of buds counted on mock-treated explant (explant number=176) or treated with 5 μΜ rotenone (explant number=169), 15 days after the transfer on fresh rotenone-free RM medium. The asterisk highlights the statistically significant difference between in vitro regeneration response from the mock or rotenone-treated explants (a= 0.05 Student t test). The bottom panel illustrates cotyledon explants cultivated for 15 days on rotenone-free RM medium after either mock treatment or a 24 h-treatment with the indicated amount of rotenone.

Figure 10: Enhanced induced somatic embryogenesis in calli derived from maize immature zygotic embryos. Ear of a self-fertilized plant carrying the heterozygous ppr2263 Mutator insertional recessive allele.

EXAMPLES

Example 1: Mutations affecting the activity of the complex I in the mitochondrial electron transport chain in Arabidopsis thaliana result in reduced growth.

The search for ppr mutants revealed that the homozygous mtll-1 mutant plants displayed significantly retarded growth on soil compared to wild type (Figure 1) (Ha ^' ili et ah, 2016). The affected PPR gene in this line corresponded to the At5g64320 gene and encoded an 82- kDa protein comprising 16 PPR repeats according to predictions. A second T-DNA insertion line affecting the same gene was subsequently identified. This second allelic mutant, named mtll -2, displayed similar growth defects as mtll -I, confirming that the developmental phenotype observed in these lines was effectively associated with inactivation of the At5g64320 gene.

The growth phenotype of the mtll mutants was also assessed through the quantification of callus growth in order to measure cell proliferation, instead of the development of complex organs. The inventors observed that, similarly to what was observed for plants grown in soil, microcalli obtained from mtll-l mutant protoplasts grew more slowly than from wild-type protoplasts.

Protoplasts were prepared according to Chupeau et al. (2013) (Figure 2). In brief, in vitro grown plantlets were macerated overnight in a cocktail of cell wall degrading enzymes. Tissue debris were filtered out and the recovered protoplasts were cultivated for 10 days in liquid medium added with a synthetic auxin (2,4-D; 1 mg/L) and a synthetic cytokinin (TDZ; 0.2 mg/L). The liquid medium was then diluted in a 1 :2 ratio with the addition of fresh TDZ, and again three weeks later at a 1 :3 ratio with another dose of TDZ. Finally, 2-3 mm calli were transferred onto gelled medium containing another synthetic cytokinin, meta-topolin. For this example, six independent protoplast cultures were prepared from 3-week-old wild- type plants and two from ppr mutant plants, which grew more slowly.

Example 2: Mutations affecting the activity of the complex I in the mitochondrial electron transport chain in Arabidopsis thaliana result in improved organogenesis.

Calli obtained in vitro in the presence of phytohormones may be induced to form shoot meristem by reducing the ratio of auxin vs. cytokinin, leading to shoot initiation and growth. This developmental reprogramming has been achieved in many plant species and with various explants, wherein in this particular case, starting with protoplast culture (see dilutions 1 and 2, Figure 2). Yet, certain species or genotypes cannot be regenerated through such hormonal treatments, or only with very low efficiency. This ability is controlled by genetic factors, but the mechanisms that determine regeneration efficiency via-caulogenesis are essentially unknown.

In a particular embodiment of regeneration protocol of the invention, the treatment of undifferentiated calli with an auxin (2,4-D) and a cytokinin is initiated in liquid culture, and pursued with another cytokinin (meta-topolin) phytohormone together with the switch from liquid medium onto gelled medium (Chupeau et ah, 2013) (Figure 2). The inventors observed that calli derived from mill respiratory mutants with no functional respiratory complex I produced large amounts of shoot meristems (SM) (up to 2-3 shoot primordia per callus, counted on its upper side) whereas their wild-type counterpart produced much fewer shoots (0.2 meristem per callus on average) (Figure 3). In one experiment whose results have been replicated multiple times, 10.1 % of the wild-type control calli produced shoots compared to 72.9% of the calli obtained from mtll-1 protoplasts. Example 3: In ppr mutants resulting in complex I impairment, growth penalty correlates with improved organogenesis.

To investigate whether the favorable in vitro response of mill mutants is specific to mutations in this particular gene or a more general characteristic of complex I deficiency, the inventors compared the response of calli (bud regeneration from calli) derived from protoplasts prepared from the wild type and four Arabidopsis mutants that were chosen because they display a range of growth retardation phenotypes: bir6, mtsfi, mill and mtsfl in increasing phenotype strength order (Figure 4A; see above and Table 1 for references and associated mitochondrial defects). All mutant calli displayed reduced growth in vitro (Figure 4B). Despite the high variability inherent to in vitro plant explant response experiments, the mutants' organogenesis responses ranked in the same order as their greenhouse growth defects (Figure 4C), indicating that the mechanisms or signals induced by complex I impairment leading to a growth defect also promote organogenesis with similar quantifiable potency.

Example 4: Treatments of Arabidopsis calli with rotenone, an inhibitor of the respiratory complex I, result in reduced growth.

The complex I mutant IVC phenotypes establish a link between respiration deficiency and shoot organogenesis, an observation that has not been previously reported. The inventors sought to confirm this causal link by comparing the in vitro response of mtll mutant calli with that of wild-type calli exposed to a complex I inhibitor, rotenone (Figure 5). Rotenone is a potent inhibitor of the mitochondrial electron transport chain that appears to be specific to complex I at concentrations below 40 μΜ in Arabidopsis (Garmier et al., 2008). Rotenone is a natural chemical extracted from plants. The inventors wished to investigate whether the effect of complex I inhibition on shoot meristem initiation may occur before, at or after the transfer from liquid to gelled medium (Figure 6). As a first step, different rotenone treatment schemes were tested: during one week prior transfer (scheme 1), starting one week before transfer and continuing after transfer (scheme 2), starting at transfer (scheme 3).

Rotenone (Sigma Aldrich) was pre-dissolved in dimethyl sulfoxide (DMSO) prior to addition into plant culture medium. A range of rotenone concentrations were tested for all three schemes because the toxicity of the inhibitor on protoplast-derived calli was unknown: 1.25, 2.5, 5, 10, 20, 40 μΜ and more. In all biological repeats, concentrations of 40 μΜ and over lead to the death of the cultured wild-type Arabidopsis calli. Rotenone was not fully soluble in the culture medium at 40 μΜ and above. However, calli treated with less than 10 μΜ of rotenone were viable following treatment according to scheme 1 (Figure 7). Interestingly, the growth defect observed 2 weeks after transfer, thus three weeks after the start of the one-week rotenone treatment, increased with the inhibitor concentration.

Example 5: Treatments of Arabidopsis calli with rotenone result in improved organogenesis.

The potential impact of rotenone on organogenesis was measured in all three treatment schemes (Figure 8). In schemes 2 and 3, the fraction of regenerative calli counted 30 days after transfer was lower than for control calli. But, in scheme 1 , more regenerative calli were counted (up to 5-fold) compared to control samples.

Example 6: Treatments of tomato cotyledon explants with rotenone result in improved organogenesis.

The inventors also demonstrated that rotenone enhances in vitro response in other species and in other regeneration protocols. In a particular embodiment of regeneration protocol of the invention, tomato (S. lycopersicum, cv. WVA106) cotyledon fragments were shown to yield significantly higher numbers of regenerated shoots following treatments with rotenone in at least three biological replicates. According to a previously described protocol for the induction of organogenesis in tomato (Cortina and Culianez-Macia, 2004), cotyledons of sterile in vitro cultured 8-day plantlets were sectioned in six to sixteen sectors, then placed upside down, in the dark, on a preculture medium (PM) containing MS salts, sucrose (30 g/L), a synthetic auxin (NAA; 1 mg/L), a synthetic cytokinin (BAP; 1 mg/L) and a gel (agargel; 8 g/L). The cotyledon explants were then transferred onto a regeneration medium RM containing MS salts, sucrose (30 g/L), a synthetic auxin (IAA; 0.5 mg/L), a synthetic cytokinin (zeatin riboside; 0.5 mg/L) and a gel (agargel; 8 g/L). After testing a range of rotenone concentrations (2.5, 5, 10, 20 and 40 μΜ) and various treatment schemes, the inventors determined that, in their hands, a high enhancement of the tomato explant in vitro response was obtained with a 24 h treatment at a rotenone concentration of 5 μΜ (Figure 9).

Example 7: A mutation affecting the activity of the mitochondrial electron transport chain in Zea mays results in improved somatic embryogenesis.

In addition to the proofs that complex I defects (caused either by an inhibitor or a mutation) enhance organogenesis, the inventors also show that a mutation resulting in a dysfunctional mitochondrial electron transport chain enhances somatic embryogenesis in maize. The ppr2263 maize mutant plants have normal complex I activity but display complex III deficiency, and their mitochondria have a compromised ultrastructure (Sosso et al, 2012). In a particular embodiment of regeneration protocol of the invention, maize immature zygotic embryos were sampled from ears of self-fertilized ppr2263 heterozygous plants (insertional allele identified in active Mutator stock and backcrossed into inbred line F252, Sosso et al, 2012) and cultivated in vitro for the production of embryogenic calli and regenerated plants (according to Ishida et al, 2007, but without Agrobacterium co-cultivation).

Seeds on the self-fertilized ears are distributed in two easily distinguishable size classes, in a ratio of approximately 3 : 1 : wild-type size seeds corresponding to wild-type or heterozygous ppr2263 embryo and endosperm; reduced size seeds, corresponding to homozygous ppr2263 mutant embryo and endosperm (Figure 10 and Table 6 related to the distribution of sampled seed size).

Table 6 The growth impairment characterizing the ppr2263 homozygous maize seeds and developing embryos is reminiscent of the growth retardation associated with the Arabidopsis mtll plants and protoplast-derived calli. Because the developmental stage and size of the maize zygotic immature embryos upon transfer to in vitro culture medium is key to the successful regeneration of plantlets, immature embryos were extracted 12 days after pollination from wild-type size seeds and 20 days after pollination from smaller ppr2263 homozygous maize seeds. Only embryogenic calli derived from the homozygous ppr2263 immature zygotic embryos reproducibly resulted in the regeneration of plantlets, at an average rate of approximately 9% (Table 7 related to the count of the immature zygotic embryos put in IVC and count of plantlets regenerated from the derived calli).

Table 7 The majority of today's maize lines are considered as recalcitrant to regeneration and despite multiple attempts, in the hands of the inventors and with this protocol, no regenerant of European lines such as F252, Fv2 or F03802 were ever obtained with this protocol, in contrast to experiments with the A188 genotype, which is known for its excellent in vitro response. The ability to recover plantlets via somatic embryogenesis in ppr2263 mutant tissues is thus a remarkable progress as it expands the range of maize genotypes amenable to regeneration.

Conclusion: Defects in complex I activity results in enhanced shoot formation, at least in calli derived from Arabidopsis protoplasts or from tomato cotyledons explants induced by phytohormones. Furthermore, enhanced morphogenesis has been observed in particular after genetic inactivation of complexes in the mitochondrial electron transport chain, in particular complex I and complex III, at least in Arabidopsis via organogenesis and in maize via somatic embryogenesis.

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