WO2015106205A1 - Transfer of mitochondria in plant species for conferring cytoplasmic male sterility - Google Patents

Abstract

Compositions and methods for effecting the transfer of the cytoplasmic male sterility (CMS) trait in plants are disclosed. Specifically, the method involves bringing two cells in contact such that they form cell to cell channels enabling movement of male sterility causing DNA sequences. The incoming, CMS-causing DNA may incorporate into the host's mitochondrial DNA by homologous recombination, or be maintained as an episomal element.

Description

Transfer of Mitochondria in Plant Species for Conferring Cytoplasmic Male Sterility

This application claims priority to US Provisional Application Nos. 61/926,315 and 62/021,599 filed January 11, 2014 and July 7, 2014 respectively, the disclosure of each being incorporated by reference herein.

FIELD OF THE INVENTION The present invention relates to plant genetic engineering and particularly to methods for horizontal transfer of desirable traits in higher plants.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

Because male sterile maternal parental plants avoid the requirement for hand

emasculation, such plants are highly desirable in hybrid seed production. Male sterility can either be caused by mitochondrial genes or by nuclear genes alone; the resulting conditions are known as cytoplasmic male sterility (CMS) and genetic male sterility (GMS), respectively. CMS is known to be associated with mitochondrial DNA sequences which have multiple rearrangements giving rise to chimeric mitochondrial genes. The CMS maternal parent is female fertile and produces hybrid seed upon pollination by the pollen of the paternal parent. Fertility of the CMS parent is restored when a restorer gene is incorporated in the nuclear genome. CMS -causing mitochondrial genes and nuclear restorer genes have been extensively reviewed in different crop systems (Carlsson et ah, 2008; Chase, 2007; Chen and Liu, 2013; Gillman et ah, 2009).

Cultivated tomato, Solarium lycopersicum (also known as Lycopersicon esculentum and/or Lycopersicon lycopersicum) is a crop in which no cytoplasmic male sterility has been described. One approach to obtain useful forms of CMS in tomato included protoplast fusion, in which introduction of Solanum acaule or Solanum tuberosum mitochondria were introduced into tomato cells (EP 03663819 Al; Priority date October 8, 1988). The process comprises the steps of (A) fusing tomato protoplasts that contain inactivated cytoplasmic elements with Solanum protoplasts that contain inactivated nuclear elements, to obtain a plurality of fusion products; and (B) regenerating at least one fusion product of said plurality into a whole, male-sterile tomato plant.

Transgenic induction of mitochondrial DNA rearrangements for cytoplasmic male sterility was described in tomato by the manipulation of the Mshl nuclear gene that appears to be involved in the suppression of illegitimate recombination in plant mitochondria. Suppression of Mshl expression by R Ai resulted in reproducible mitochondrial DNA rearrangement and a condition of male sterility (Sandhu et al., 2007).

When chloroplast DNA moves from cell to cell over the graft junction, sequencing of the plastid genome of graft transfer events confirmed the presence of a complete, unmodified incoming ptDNA in the new host. In contrast, the mitochondrial DNA in the graft transmission plants was chimeric, consisting of segments of N. undulata mtDNA (from CMS Partner 1) and fertile mitochondrial DNA (from N. sylvestris). The plant mitochondrial DNA is present in different size sub genomic circles formed by recombination via repeated sequences (Kubo and Newton, 2008; Logan, 2007; Sugiyama et al, 2005). In somatic cells there may be more mitochondria than mitochondrial genomes and the mitochondria may contain less than a complete mitochondrial genome (Preuten et al., 2010). Plant mitochondria are known to undergo cycles of fusion (Sheahan et al., 2005). Thus, fertility- or sterility-controlling mitochondrial DNA may move from cell to cell protected in intact organelles or as naked DNA.

Transformation of mitochondria with naked DNA has not yet been accomplished in higher plants (Niazi et al, 2013). US Patent 5,530,191 (1996) entitled "Method for producing cytoplasmic male sterility in plants and use thereof in production of hybrid seed" describes production of CMS plants by the engineering of the chloroplast genome. The patent literature claims hybrid tomato, but the seed in these patents is always obtained by conventional crossing, involving manual removal of anthers and hand pollination. Claims of hybrid tomato patents focus on flavor enhancement (PCT/US2012/041478) or the benefits of seedless tomato obtained by using parthenocarpic genes (PCT/NL2000/000380; EP19990201787; EP2010000012146; US 20130189419).

SUMMARY OF THE INVENTION This invention provides a method for obtaining a plant cell of a multicellular plant, the mitochondria of which have acquired male sterility associated DNA sequences through a graft junction. These sequences are provided in Figure 6. The method involves bringing two cells in contact such that they form cell to cell channels enabling movement of male sterility causing DNA sequences. The incoming, CMS-causing DNA may incorporate into the host's

mitochondrial DNA by homologous recombination, or be maintained as an episomal element. The channel connections may conveniently be established by grafting the partners, one of which carries male-sterility causing DNA sequences and a second, fertile parent, the conversion of which into a male sterile form is desired. The nuclear genome of the fertile parent carries a nuclear marker gene facilitating the recovery of converted male sterile cells.

In one aspect, the creation of CMS plants entails certain steps in tissue culture. These include: (a) Marking the nucleus of the fertile partner with a marker gene via known methods of introducing heterologous sequences into recipient plants. The marker gene confers a selectable tissue culture phenotype, such as resistance to kanamycin or hygromycin, but any nuclear gene that is selectable in tissue culture can be used, (b) Marking the chloroplasts of the CMS plants with a selectable marker, such a resistance to spectinomycin, streptomycin, kanamycin, or chloramphenicol, again using methods known in the art. (c) Establishing contact between the fertile and CMS partners. The preferred embodiment involves a conventional wedge graft.

However, alternative methods of establishing contact also results in cell-to-cell movement of mitochondrial DNA, such as wounding the Partners on their stems and tying them together at the wound site, or creating a chimeric tissue by mixing cells or protoplasts, (d) In a preferred embodiment, the wedge containing the graft junction is sliced and transferred in tissue culture to select for the nuclear marker of Partner 1 and chloroplast marker of Partner 2. (e) Regenerating plants from the double-resistant cells, (f) Transferring plants into the greenhouse to visually identify mitochondrial DNA transfer events by the change of flower morphology, (g) Repeatedly regenerating plants from the Graft Transmission tissue to accelerate sorting, and screening the plants by morphology in the greenhouse, (h) In cases where the cytoplasmic male sterility causing DNA sequence is known, plants can be screened by PCR for the CMS DNA.

An alternative tissue culture-independent method relies on morphological (pigment) traits encoded by nuclear genes (Partner 1) and visual (pigment or GFP) markers encoded by the plastid genome. Such visual markers have been useful to detect plastid marker excision in greenhouse-grown plants (Tungsuchat-Huang and Maliga, 2012; Tungsuchat-Huang et ah, 2011). Graft transmission of CMS-causing mitochondrial DNA involves the following steps, (a) Graft Partner 1 (fertile, green) and Partner 2 (CMS mitochondria, visual plastid marker, such as aurea gene), (b) When the graft union has been successfully established, shoot regeneration can be forced from cells at the graft junction. This can most conveniently be achieved by

decapitating the scion, so that the graft junction is at the tip of the plants, (c) Shoots developing from the graft area should be inspected for Partner 1 morphology and the presence of visual plastid marker from Partner 2. CMS flowers on branches developing in the graft region will indicate transfer of mitochondrial DNA. (d) In cases where the cytoplasmic male sterility causing DNA sequence is known, shoots can be screened by PCR for the CMS DNA. (e) When graft transmission of CMS-causing mitochondrial sequences is achieved, the visual chloroplast marker can be removed by recombinase-mediated marker excision using established protocols

(Tungsuchat-Huang and Maliga, 2012; Tungsuchat-Huang and Maliga, 2014).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Graft transmission of mitochondrial DNA alters flower morphology. (Fig. 1A) Plant regenerated from the GT-19C graft transmission event and the close-up picture of its (Fig. IB) fertile flowers with anthers bearing pollen and (Fig. 1C) sterile flowers with anthers converted into petals.

Figure 2. Flowers of graft partners N. tabacum Nt-CMS19G (PI) and Nicotiana sylvestris Nsl37-CK2-2 (P2), and of the seed progeny obtained from fertile and CMS flowers of the GT- 19 graft plastid transmission progeny. Figure 3. The mitochondrial genome of GT-19 graft plastid transmission progeny is a chimera of the fertile N. sylvestris and CMS N. undulata mitochondrial genomes. Shown are the map positions of DNA polymorphic markers in the (Fig. 3 A) Nicotiana undulata, (Fig. 3B) Nicotiana sylvestris and (Fig. 3C) GT-19 graft plastid transmission progeny on the N sylvestris mtDNA map.

Figure 4. The mitochondrial genome of the GT19C seed progeny is a mosaic of the two graft parents (Figure 4A and Figure 4B). DNA sequence was obtained on the Illumina MiSeq platform, using 2x300 bp paired-end sequencing. The coverage of parental and recombinant mtDNAs was between 150-300 fold and 40-100-fold, respectively. Plotted is the fraction of undulata SNPs at every position in two recombinant fertile (Fertl, Fert2) and two recombinant CMS (Sterl, Ster2) mitochondrial genomes aligned with the parental N. sylvestris mtDNA. Alignment with the N undulata mtDNA SNPs is shown on top. The SNPs from und and sylv are on the top and the bottom in the recombinants, respectively. Black horizontal lines mark putative deletions in the N undulata mtDNA. The positions of the mitochondrial repeats are marked as Rl, R2 and R3. The general organization of the 430,597 nt N sylvestris mitochondrial genome determined by us is the same as that of the N tabacum mtDNA (Sugiyama et al., 2005) and the two genomes differ only at eight locations (6 SNPs and 2 x 1 nt indels) . Figure 5. Cytoplasmic male sterility (CMS) co-segregates with a ~ 6 kb mitochondrial DNA region unique to the CMS N undulata mitochondrial DNA marked as CMS region. Shown is the map of the mitochondrial DNA responsible for CMS in the CMS Graft Parent 1 Nicotiana tabacum CMS19G with N undulata cytoplasm (und), the fertile Graft Parent 2 Nicotiana sylvestris CK2-2 (sylv), two recombinant fertile (R- fertl, R-fert2) and two recombinant CMS (R- sterl, R-ster2) mitochondrial genomes. The CMS region comprises (a) the 1567 nt atpl gene, (b) a 1175 nt long region unique to CMS plants, and (c) a 3271 nt region that is homologous to the 389,686-393,200 nt region in the N tabacum mtDNA (NC_006581). The und and sylv SNPs are shown in blue and red as individual markers, respectively, and as a continuous line for a fragment with several SNPs (68 markers in 3515 nt fragment). Note that the N undulata mtDNA is rearranged relative to the N sylvestris mtDNA. The maps were drawn to show the N. undulata mtDNA as continuous sequence. Figure 6. DNA sequence of CMS encoding DNA region in the N. undulata mitochondrial genome (Fig. 6A; SEQ ID NO: 1) and cognate regions in fertile mitochondria (Fig. 6B; SEQ ID NO: 2, Fig. 6C; SEQ ID NO: 3), as marked in Figure 5.

Figure 7. Alignment of the N. undulata (und; SEQ ID NO: 4) and N. sylvestris (sylv; SEQ ID NO: 5) ORF102 sequences. Note four mismatches.

DETAILED DESCRIPTION OF THE INVENTION Crossing suitable maternal and paternal genetic lines yields hybrid seed of crops that favorably combine the properties of the two parents. Production of hybrid seed is labor intensive, in situations where manual removal of anthers from the maternal flowers, i.e., hand

emasculation, is required to prevent self-pollination. Genetic male sterility of the maternal parent eliminates the need for hand emasculation. The present invention provides a practical means for transfer of cytoplasmic male sterility (CMS) traits by graft transfer of mitochondrial DNA, when said mitochondrial DNA encodes sequences that confer male sterility to the flowers of the recipient plant. If a cognate fertility restorer gene is transformed into the nucleus of the pollen parent, the cross yields fertile hybrids. The example described in the present invention is creation of cytoplasmic male sterility in tomato by graft transfer of mitochondrial DNA from petunia. An alternative source of male-sterility causing mitochondrial DNA is male sterile tobacco. Tomato, petunia and tobacco are sexually incompatible. Thus, cell-to-cell movement of mitochondrial DNA, followed by recombination between the incoming and resident

mitochondrial DNAs gives rise to cytoplasmic male sterility without the transfer of nuclear genetic information.. The protocol can be applied to any graft-compatible species when the mitochondrion of one of the graft partners encodes a male sterility-causing gene. I. GENERAL METHODS FOR CONSTRUCTING PLASTID-TRANSGENIC CMS SYSTEMS AND FOR PRODUCTION OF HYBRID SEED

The transgenic CMS systems of the invention are prepared and used according to the general methods set forth below for nuclear and plastid transformation of higher plants, maintenance of parental plant lines and production of hybrid seed.

A. DNA Constructs and Methods for Stably Transforming Plastids With Selectable Marker Genes and Regenerating Plastid-Transgenic Plants

Methods and DNA constructs for stable, high-efficiency transformation of plastids and expression of recombinant proteins in plastids are known in the art. The methods and constructs described in the following references are preferred for practice of the present invention: Svab et al, Proc. Natl. Acad. Sci. USA, 87:8526-30 (1990); Svab & Maliga, Proc. Natl. Acad. Sci. USA, 90: 913-17 (1993); Carrer et al, Mol. Gen. Genet., 241 :49-56 (1993); Staub & Maliga, EMBO J., 12: 601-06 (1993); and U.S. patents 5,877,402, 6,138,168 and 7,667,093. All the

aforementioned disclosures describe suitable methods for stable, high-efficiency plastid transformation and expression of recombinant genes in plastids.

The following definitions will facilitate the understanding of the plastid transformation methods used in accordance with the present invention:

Heteroplasmic: refers to the presence of a mixed population of different plastid or mitochondrial genomes within a single plastid or mitochondrion in a population of plastids or mitochondria contained in plant cells or tissues.

Homoplasmic: refers to a pure population of plastid or mitochondrial genomes, either within an organelle or within cells and tissues.

Alloplasmid substitution line refers to plants in which the cytoplasm (chloroplasts and mitochondria) have been replaced by the cytoplasm of a different species (or of a genetic line). For example, an alloplasmic N. tabacum may be obtained by repeated pollination of Nicotiana undulata with Nicotiana tabacum, pollen resulting in the replacement of N. undulata

chromosomes with N tabacum chromosomes.

Transformation of plastids: stable integration of transforming DNA into the plastid genome that is transmitted to the seed progeny of plants containing the transformed plastids. The terms "selective marker" or "selectable marker" refer to a phenotype that identifies a successfully transformed organelle, cell or tissue, when a gene or allele encoding the selective marker is included in the foreign DNA used for transformation. Commonly used selective markers include resistance to antibiotics, herbicides or other compounds, which would be lethal to cells, organelles or tissues not expressing the resistance gene or allele. Selection of transformants is accomplished by growing the cells or tissues under selective pressure, i.e., on media containing the antibiotic, herbicide or other compound. If the selective marker is a "lethal" selective marker, cells which express the selective marker will live, while cells lacking the selective marker will die. If the selective marker is "non-lethal", transformants (i.e., cells expressing the selective marker) will be identifiable by some means from non-transformants, but both transformants and non-transformants will live in the presence of the selection pressure.

Several methods are available to introduce DNA into the plastids of flowering plants, including, but not limited to, Agrobacterium vectors, polyethylene glycol (PEG) treatment of protoplasts, bombardment of cells or tissues with microprojectiles coated with the plastid- transforming DNA (sometimes referred to herein as "biolistic DNA delivery") and temporary holes cut by a UV laser microbeam. Other methods include use calcium phosphate treatment of protoplasts, electroporation of isolated protoplasts and agitation of cell suspensions with microbeads coated with the transforming DNA. The biolistic method, as described by Svab & Maliga, 1993, supra is preferred for plastid transformation because it can be used on a wide variety of plants and tissues. In an alternative embodiment, useful in plant systems where protoplasts may be obtained and regenerated into intact plants, plastid transformation may be achieved by polyethylene glycol (PEG) treatment of protoplasts in the presence of the transforming DNA. Methods for stable plastid transformation in PEG-treated protoplasts are exemplified in tobacco by Golds et al, Bio/Technology, 11 : 95-97 (1993).

The term "tomato" or "tomato plant" means any variety, cultivar, or population of

Solanum lycopersicum (Lycopersicon esculentum and/or Lycopersicon lycopersicum), including both commercial tomato plants as well as heirloom varieties. In some embodiments, "tomato" may also include wild tomato species, such as, but not limited to, Solanum lycopersicum var. cerasiforme, Solanum pimpinellifolium, Solanum cheesmaniae, Solanum neorickii, Solanum chmielewskii, Solanum habrochaites, Solanum pennellii, Solanum peruvianum, Solanum chilense and Solanum lycopersicoides. As used herein, the term "plant" includes plant cells, plant protoplasts, plant cell tissue cultures from which tomato plants can be regenerated, plant calli, plant cell clumps, and plant cells that are intact in plants, or parts of plants, such as embryos, pollen, ovules, flowers, leaves, seeds, roots, root tips and the like. The term "tomato fruit" refers to the fruit produced by a tomato plant, including the flesh, pulp, meat, and seeds of the fruit.

As used herein, the term "variety" or "cultivar" means a group of similar plants within a species that, by structural features, genetic traits, performance, and/or content of volatile compounds, sugars, and/or acids, can be identified from other varieties/cultivars within the same species.

The method described is not restricted to creating CMS in tomato, because cell-to-cell movement of sterility causing DNA can be used to convert any fertile plant into a CMS form. Such male-sterility causing mitochondrial genes have been described in a number of species, including without limitation, brassica, carrot, common bean, maize, pepper, petunia, radish, rice, sorghum, sugar beet, sunflower, tobacco, and wheat (Carlsson et ah, 2008; Chen and Liu, 2013).

A "plant sector" refers to a region or a full leaf of a plant that is visually identifiable due to expression of a selectable marker gene or the excision of a selectable marker gene in accordance with the present invention.

"Operably linked" refers to two different regions or two separate genes spliced together in a construct such that both regions will function to promote gene expression and/or protein translation.

"Nucleic acid" or a "nucleic acid molecule" as used herein refers to any DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its

complementary sequence in either linear or circular form. In discussing nucleic acid molecules, a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5' to 3' direction. With reference to nucleic acids of the invention, the term "isolated nucleic acid" is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated. For example, an "isolated nucleic acid" may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism. When applied to RNA, the term "isolated nucleic acid" refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues). An isolated nucleic acid (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.

The term "functional" as used herein implies that the nucleic or amino acid sequence is functional for the recited assay or purpose.

The phrase "consisting essentially of when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ ID No:. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel characteristics of the sequence.

A "replicon" is any genetic element, for example, a plasmid, cosmid; bacmid, phage or virus, that is capable of replication largely under its own control. A replicon may be either RNA or DNA and may be single or double stranded.

A "vector" is a replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element.

The term "probe" as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method.

The terms "transform", "transfect", "transduce", shall refer to any method or means by which a nucleic acid is introduced into a cell or host organism and may be used interchangeably to convey the same meaning. Such methods include, but are not limited to, transfection, electroporation, microinjection, PEG-fusion, biolistic bombardment and the like.

A "clone" or "clonal cell population" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell or cell population that is capable of stable growth in vitro for many generations.

The following examples are provided to illustrate certain embodiments of the invention. They are not intended to limit the invention in any way. EXAMPLES

We describe here a novel approach for generating CMS tomato plants by the graft transmission of male-sterility causing mitochondrial DNA sequences from graft compatible solanaceous species, such as tobacco or petunia. The method is based on co-transmission of chloroplasts and mitochondria through a graft junction, normally without the transfer of any nuclear (chromosomal) genetic information. If nuclear DNA from the CMS parent is transferred, it can be removed by repeated pollination with the fertile partner. The feasibility of the approach was shown by (a) marking the nucleus of a Nicotiana tabacum plant with a transgenic kanamycin or hygromycin resistance gene; (b) transforming the chloroplasts of a second species, Nicotiana sylvestris, with a selectable spectinomycin resistance gene; (c) grafting one species as the rootstock and the second species as scion, so that the organellar DNA (organelles) can traverse through the graft junction; (d) slicing up the graft junction and selecting in tissue culture for the nucleus of N. tabacum by the kanamycin or hygromycin resistance gene and the chloroplasts of N. sylvestris by spectinomycin resistance; (e) regenerating plants from the double-resistant cells and (f) and transferring the regenerated plants to the greenhouse to identify the CMS plants by flower morphology. The CMS in the plants is due to partial or full

substitution of N. tabacum or N. sylvestris mitochondria with the Nicotiana undulata

mitochondria, that causes homeotic transformation of anthers into petals or stigma-like structures.

In Experiment 1 , when chloroplast graft transmission events were selected grafting fertile N tabacum (Graft Partner 1) onto CMS N sylvestris carrying spectinomycin resistant plastids (Graft Partner 2), no co-transfer of chloroplasts and mitochondrial DNA was apparent (Thyssen et al., 2012). However, in Experiment 2, when graft transmission of chloroplasts was studied grafting CMS N tabacum (Graft Partner 1; gentamycin resistant Nt-CMS92G) and fertile N sylvestris (Graft Partner 2; spectinomycin resistant chloroplasts; Nsl37-CK2-2 fertile plant), co- transfer of mitochondria with the selected chloroplasts was readily obvious by the appearance of male fertile flowers in one of the three regenerated plants of event GT19-1C. No co-transmission of mitochondria with chloroplasts was found in two other events in Experiment 2. Co-transfer of chloroplasts and mitochondria must have occurred at some frequency in both experiments. We surmise that detection of the transfer of male fertility-encoding DNA was facilitated by the dominant nature of male fertility over CMS in Experiment 2.

EXAMPLE 1

CMS Tomato by Graft Transmission of

Tobacco CMS92 Mitochondrial DNA

Graft transmission of tobacco CMS92 mitochondrial DNA into tomato can be

accomplished via performance of the following steps.

(1) Transform the tomato nucleus with a selectable gentamycin or kanamycin resistance gene. Agrobacterium binary vectors with a number of different marker genes have been described, including those conferring resistance to gentamycin and kanamycin (Hajdukiewicz et al., 1994; Miki and McHugh, 2004). A suitable tissue-culture responsive tomato cultivar, such as IPA64 (Ruf et al., 2001) can be used for this purpose, but other cultivars are available, such as

Dorothy's Green and Green Pineapple (Ruf and Bock, 2014).

(2) Create a tobacco plastid genome that is compatible with the tomato nuclear background in the tobacco CMS92 background (TV. tabacum or TV. undulata plastids and the CMS sequence from TV. undulate in the mitochondrial genome). This can be achieved by converting codon 264 of the atpA gene from Pro (cCc) to Leu (cUc) in a CMS92 plant. It is known that Atropa belladonna (nightshade), a related solanaceous species, has no capacity to edit the tobacco atpA gene. The tobacco plastid genome, when introduced into the Atropa nuclear background, yielded pigment deficient plants. Mutation of the cCc codon to cUc restored normal greening (Schmitz-

Linneweber et al., 2005). Tomato, as Atropa, has a T nucleotide at the critical position in the atpA gene, thus it is unlikely to have a capacity to edit the tobacco atpA site (Kahlau et al, 2006). The problem can be pre-empted by replacing the Pro codon with a Leu codon using standard plastid engineering methods. The point mutation can be introduced into the atpA by making the mutant atpA gene part of the vector targeting sequence, and screening for the incorporation of the mutation in the transformed chloroplasts (Kanevski et al, 1999; Sinagawa- Garcia et al, 2009). A second tobacco codon that needs to be pre-edited is rpsl4 codon 50 (Kahlau et al, 2006). As part of step 2, the spectinomycin resistance {aadA) gene is introduced into the plastid genome. Incorporation of target sites for site-specific recombinases to flank aadA facilitates post-transformation excision of the marker gene.

(3) Graft IPA64-G (gentamycin resistant) plants and the engineered Nt-CMS-92 (carrying a spectinomycin resistance gene in its chloroplast genome).

(4) Slice up the graft junction and select for the transfer of CMS92 chloroplasts on gentamycin and spectinomycin medium. (5) Regenerate plants from double-resistant tissue, and inspect the flowers for homeotic transformation. Analyze mitochondrial DNA to identify recombination events. This may be by PCR amplification and sequencing of polymorphic regions, DNA gel blot (Southern) analyses of polymorphic regions or sequencing entire mitochondrial genomes to detect SNPs and insertions and deletions in the mitochondrial genome. (6) Repeat the plant regeneration multiple times to accelerate sorting of mitochondrial DNA.

As an alterative to tobacco chloroplasts for the co-transfer of CMS-causing mitochondrial DNA, we may construct an intermediate source of CMS (the bridge plant) by transferring the tomato chloroplasts into the tobacco CMS92 background. The rational is that, if the requirement for editing is eliminated by a mutation at the DNA level, the requirement for editing is no longer there. Thus, the tomato plastid genome should be fully compatible with the CMS92 tobacco background. Accordingly, as an alternative to Step 2 above, plastids may be transformed in tomato with the aadA gene, then transferred by graft transmission into the tobacco CMS92 background where they will be combined with the tobacco CMS gene. The tobacco CMS mitochondrial sequence can subsequently be introduced by graft transmission into tomato. When the desired tomato line is obtained, the aadA gene can be removed by site-specific recombinases, as described (Kittiwongwattana et al, 2007; Lutz and Maliga, 2007; Lutz et al, 2006). The advantage of using tobacco bridge plants is protection against any unknown form of plastid- nucleus incompatibility that may be encoded in the tobacco ptDNA in the final product, the CMS tomato, which will have its native, unmodified chloroplast genome and minimal input of the tobacco mitochondrial DNA, preferably restricted to the CMS-causing sequence.

The CMS tomato plants will be male sterile due to the homeotic transformation of anthers, but female fertile. The CMS tomato plants can be propagated by pollination with any fertile tomato that will serve as the maintainer line. Repeated pollination with different maintainer lines will yield isogenic pairs of CMS and fertile lines.

Hybrid seed can be obtained by pollination with a suitable pollen parent. In the absence of pollen, the hybrid plants normally will not set seed. However, in tomato, seedless fruits develop if parthenocarpic genes are incorporated in the genetic lines (Gorguet et al., 2005;

Medina et al., 2013). If restoration of male sterility is required, the restorer gene can be isolated from Nicotiana undulata by standard molecular biology techniques and transformed into the nucleus of tomato to be used as a fertility restorer line.

Cultivated tomato and related wild species can be crossed. Thus, it may be advantageous to transfer the CMS92 male sterility gene first into a related wild species with good tissue culture regeneration potential, and then subsequently introduce the mitochondrial CMS trait with the engineered chloroplasts by graft transmission into cultivated tomato. Wild species with shooting response in tissue culture are L. chilense, L. peruvianum var. humifusum, L. esculentum x L. peruvianum, L. esculentum cv. MsK, L. hirsutum f. hirsutum (Peres et al, 2001).

Introduction of the Cytoplasmic Male Sterility trait is facilitated by information about the male sterility causing DNA sequences. This information has been obtained by the analyses of fertile and sterile recombinant mitochondrial genomes (Figure 4), which differ in a 3kb-region between the fertile and sterile plants. The map position of CMS-causing sequences is shown in Figure 5. The DNA sequence of CMS-causing N. undulata mitochondrial DNA (~ 6-kb) and the cognate sequence in the fertile N. sylvestris is given in Figure 6. This sequence encodes mitochondrial ORF102, which has four predicted amino acid exchanges in the N. undulata ORF relative to the N. sylvestris ORF (Figure 7). The presence of CMS-causing mtDNA can be tracked by sequencing PCR-amplified mitochondrial DNA. A convenient visual marker is the PCR fragment obtained with primers 5'- TTGCTTTGCCTCCTTCCTTCTTC-3 ' (mt390702F; SEQ ID NO: 6) and 5 '- TCTGTAAGCCCCGAAACAGACTC-3' (mt390864R; SEQ ID NO: 7), amplifying a 163nt fragment from N. sylvestris and a 142nt fragment from N. undulata the mtDNA. This fragment is amplified from a region located at ~1.8kb from ORF 102. Similar to plastids, mitochondrial R As also undergo extensive mR A editing

(Takenaka et al., 2013). The lack of R A editing or partial R A editing of heterologous mitochondrial mR As can also be the source of reduced plant viability. Incompatibility due to problems with editing of heterologous mitochondrial mRNA can be reduced or eliminated by replacement of the heterologous (tobacco) mtDNA with tomato mtDNA during repeated cycles of horizontal mtDNA transfer.

EXAMPLE 2

CMS tomato by Graft Transmission of Petunia mitochondrial DNA

Cytoplasmic male sterility in Petunia is associated with Pcf, a fused mitochondrial gene (Young and Hanson, 1987). The petunia fused gene is expressed at the protein level, and the abundance of the 25 -kd protein is much lower in fertile plants carrying the dominant nuclear fertility restorer gene (Nivison and Hanson, 1989). The fertility restorer gene is a

pentatricopeptide repeat-encoding gene (Bentolila et al., 2002) (US Patent 20030177535). For a review of CMS and fertility restoration in Petunia, see reference (Gillman et al., 2009).

The mechanism of male sterility is different in Petunia and the CMS92 tobacco line. In Petunia, CMS is due to the expression of a toxic protein rather than homeotic transformation of the anthers as in tobacco. Therefore, it may be also beneficial to introduce the Petunia Pcf gene into tomato mitochondria. The engineering steps required to introduce the Pcf gene into tomato are the same as described for the CMS92 tobacco mitochondrial DNA sequence. The plastid- nucleus compatibility problems are also the same, since Petunia plastids can replace tobacco plastid in the tobacco nuclear background (Glimelius and Bonnett, 1986). Protocols for plastid transformation to provide a marker for the selection of cell-to-cell movement of Petunia plastids are available (Zubko et al., 2004). Because the Pcf DNA sequence is known, introduction of the male-sterility causing gene can be tracked by PCR. If necessary, male sterility can be restored by introducing the fertility restorer gene into the tomato nucleus. EXAMPLE 3

Graft Transmission of CMS by Transient Selection for Nuclear Transfer

When transfer of CMS is carried out by selection for a plastid marker, the probability of co-transfer of CMS depends on how much cytoplasm is co-transferred with the plastids. The likelihood of success can be significantly enhanced when graft transmission is used first to obtain nuclear hybrids (Fuentes et al., 2014), in which case more complete mixing of the cytoplasm is likely by the movement of the larger nucleus through the graft junction. Indeed, three out of five nuclear graft transmission evens was accompanied by formation of recombinant mitochondria (Fuentes et al., 2014). In Example 3 of the present invention both graft parents carry a different nuclear gene, such as the fertile Parent 1 (tomato) a gentamycin resistance gene and CMS Parent 2 (tobacco) a kanamycin resistance gene. Parent 2 also carries a selectable plastid marker, such as spectinomycin resistance. The two parents are grafted as in Example 1 and Example2, and then the graft junction is sliced up and the tissue slices are selected in tissue culture for gentamycin-kanamycin resistance to recover nuclear hybrids. Nuclear hybrids of species such as tobacco and tomato are likely to be unstable. Thus initial double-selection should be followed by selection for the nuclear marker of Parent 1 (gentamycin resistance) and plastid marker of Parent 2 (spectinomycin resistance). In the absence of selection for the chromosomes of Parent 2, the tobacco chromosomes of Parent 2 are likely to be preferentially lost in the hybrid during cultivation in culture. The result is recovery Parent 1 (tomato) nucleus with chloroplasts of Parent 2 and recombinant mitochondria. Tobacco chromosomes retained in the regenerated tomato plants can be eliminated by repeated pollination of the plants with wild-type tomato pollen.

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While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for effecting intercellular transfer of mitochondria in plants for the creation of cytoplasmic male sterile plants, comprising:

a) joining the cells of the first fertile plant and a second CMS -mitochondria plant, said first and second plants cells comprising distinct plastid and nuclear genetic markers; or

b) joining a first fertile plant and a second CMS- mitochondria plant, said first and second plants comprising distinct plastid and nuclear genetic markers; or

c) joining a root stock of a first fertile plant and a scion from a second

CMS-mitochondria plant, said first and second plants comprising distinct plastid and nuclear genetic markers; and

d) culturing said plants for a suitable period for grafting to occur;

i) fragmenting or slicing the graft region and

ii) transferring said fragment or slice to a plant regeneration medium and selecting for cells expressing the nuclear and plastid genetic markers from said first and second plants; or, e) forcing shoot formation from the graft junction and

i) identifying mitochondria gene transfer events by altered plant morphology and/or visually detectable plastid-specific markers, wherein said transfer confers a CMS phenotype to said fertile plant.

2. The method of claim 1, wherein said plant is selected from the group consisting of tomato, brassica, carrot, soybean, common bean, maize, pepper, petunia, radish, rice, sorghum, sugar beet, sunflower, tobacco, and wheat.

3. A plant regenerated from the method of claim 1 or claim 2.

4. Progeny and seed from the plant of claim 3.

5. A recombinant isolated nucleic acid of SEQ ID NO: 1, conferring male sterility operably linked to regulatory sequences suitable for expressing said nucleic acid in a target plant of interest.

6. A method for graft transmission of CMS comprising; a) providing a fertile parent plant comprising a first nuclear selectable marker conferring resistance to a first selection agent; b) providing a CMS parent plant comprising a second nuclear selectable marker conferring resistance to , said CMS parent plant further comprising a third plastid selectable marker conferring resistance to a third selectable marker; c) grafting said first and second parents such that a graft junction is formed between said first and second parent plants; d) growing cells harvested from said graft junction in the presence of said first and second selection agents, thereby selecting nuclear hybrid cells; e) culturing the cells of d) in the presence of said first and third selection agents, thereby selecting cells having nuclei from said fertile parent plant and chloroplasts and mitochondria from said CMS parent plant.

7. A CMS plant obtained from the cells of claim 6.

8. The method of claim 6, further comprising repeated pollination of said sterile plants obtained from step e) with wild type pollen from said parent plant, thereby removing any chromosomes remaining from said CMS parent plant.

9. The method of claim 6, wherein said sterile plant is a tomato plant and said CMS plant is a tobacco plant.

10. A CMS tomato plant obtained from the method of claim 9.