US 2016O165825A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0165825 A1 de Jong et al. (43) Pub. Date: Jun. 16, 2016

(54) CYTOPLASMIC MALE STERILE (52) U.S. Cl. CCHORIUM CPC ...... A0IH 5/025 (2013.01); C12O I/6895 (71) Applicant: BEJO ZADEN B.V., Warmenhuizen (2013.01); C12N 15/8289 (2013.01); C12O (NL) 2600/13 (2013.01); C12O 2600/156 (2013.01) (72) Inventors: Elizabeth Rosalien de Jong, Heerhugowaard (NL); Adriana Dorien (57) ABSTRACT Haarsma, Middenmeer (NL); Witte Van Cappellen, Alkmaar (NL); Cornelis The present invention relates to cytoplasmic male sterile Glas, Tuitjenhorn (NL); Nicolaas (CMS) plants and especially to cytoplasmic male Anthonius Zutt, Avenhorn (NL). sterile (CMS) green plants; cytoplasmic male sterile Settleas Maria Schrijver, (CMS) radicchio rosso plants; cytoplasmic male sterile (CMS) red leaved chicory plants, cytoplasmic male sterile (21) Appl. No.: 14/909,935 (CMS) Treviso plants, cytoplasmic male sterile (CMS) white chicory plants, cytoplasmic male sterile (CMS) Sugar loaf (22) PCT Filed: Aug. 14, 2013 plants, cytoplasmic male sterile (CMS) Belgian endive (86). PCT No.: PCT/EP2013/067014 plants, cytoplasmic male sterile (CMS) witloof plants, cyto S371 (c)(1) plasmic male sterile (CMS) Catalogna plants, cytoplasmic (2) Date: s Feb. 3, 2016 male sterile (CMS) C. intybus var. foliosum plants, cytoplas 9 mic male sterile (CMS) C. endivia plants and cytoplasmic Publication Classification male sterile (CMS) C. intybus L. var. sativum plants. The (51) Int. Cl present invention further relates to methods for identifying E;it 5/02 (2006.01) cytoplasmic male sterile (CMS) Cichorium plants and mito CI2N 15/82 (2006.01) chondrial nucleic acid sequencesC pproviding g cytopcvtoplasmic male CI2O I/68 (2006.01) sterility (CMS) in Cichorium plants.

Patent Application Publication Jun. 16, 2016 Sheet 2 of 5 US 2016/O165825 A1

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% P153 --- 96 Cichorium % Lactuca Patent Application Publication Jun. 16, 2016 Sheet 4 of 5 US 2016/0165825 A1

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3. Patent Application Publication Jun. 16, 2016 Sheet 5 of 5 US 2016/0165825 A1

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8 US 2016/O165825 A1 Jun. 16, 2016

CYTOPLASMIC MALE STERILE 0009 Further, certation is not present in all lines of Cicho CCHORIUM PLANTS rium, thus reducing the combination possibilities to produce commercially interesting hybrids. 0001. The present invention relates to cytoplasmic male 0010 For the culture of chicory in all the described forms sterile (CMS) Cichorium plants and especially to cytoplasmic above, it will therefore be advantageous to have highly, pref male sterile (CMS) green chicory plants; cytoplasmic male erably substantially 100%, uniform F1 hybrids available. sterile (CMS) radicchiorosso plants; cytoplasmic malesterile These hybrid Cichorium varieties have several advantages (CMS) red leaved chicory plants, cytoplasmic male sterile over the now common available races. (CMS) Treviso plants, cytoplasmic malesterile (CMS) sugar 0011. The most important advantage is the superb unifor loaf plants, cytoplasmic male sterile (CMS) white chicory mity such a F1 hybrid will offer, i.e. substantially 100% plants, cytoplasmic male sterile (CMS) Belgian endive uniformity. This enables a harvest that can be performed at plants, cytoplasmic male sterile (CMS) witloof plants, cyto one time or simultaneously, in addition to resulting in a mini plasmic male sterile (CMS) Catalogna plants, cytoplasmic mal loss after Sorting the crop. male sterile (CMS) endive plants, cytoplasmic male sterile 0012 For example, radicchio rosso gets less intense in (CMS) C. intybus var. foliosum plants, cytoplasmic male color as the crop is exposed to high temperatures and intense sterile (CMS) C. endivia plants and cytoplasmic male sterile daylight for prolonged times. The possibility to simulta (CMS) C. intybus L. var. sativum plants. The present inven neously harvest this product results in a reduction of loss for tion further relates to methods for identifying cytoplasmic the grower. male sterile (CMS) Cichorium plants and mitochondrial 0013 When the Cichorium is highly or substantially nucleic acid sequences providing cytoplasmic male sterility 100% uniform with respect to the genetic composition, i.e. (CMS) in Cichorium plants. genome, of the Cichorium plant, there are several advantages, 0002 Cichorium is available in several distinct cultivated both for a producer of seeds and the grower of the crop. forms: as green chicory, radicchio rosso or red leaved chicory, 0014 When a highly uniform F1 hybrid can be provided, Treviso, white chicory, Sugar loaf, Belgian endive (witloof). the producer has the advantage that more combinations of Catalogna (all C. intybus var. foliosum), endive (C. endivia) parent lines can be used, resulting in a broader range of but also root chicory (C. intybus L. var. sativum) is known. available hybrids. The latter is cultivated for its taproot from which several 00.15 Presently, seed treatment as priming and the like or products as inulin, flavour for beer, coffee substitutes but also seed selection are of little use since uniformity in the seed lots substrates for polymer production are derived. is too low. When the high quality seeds of the desired highly 0003. Witloof is grown for its taproot initially, which then uniform hybrid are available, processes like priming and is forced in the dark to produce the well-known witloof, other seed-technological treatments of the seeds become use Belgian endive orchicon. This forcing is done hydroponically ful. or conventionally. 0016 For the grower, advantages of a highly uniform 0004 Examples of leaf (Cichorium intybus var. hybrid are a short period for harvesting and processing the foliosum) are radicchio rosso (with variegated red and green total crop, consisting of a very homogenous product without leaves, sometimes with white veins), Treviso, Catalogna and so-called off-types. For this reason, less labor is required to green hearted chicory. Typical for all chicory varieties is the harvest the crop and prepare it for sale. somewhat bitter taste. Another member of the is 0017. A highly or substantially 100% uniform hybrid has endive, Cichorium endivia. a consistent quality, especially for color when considering 0005. The Cichoriae are part of the (formerly e.g. radicchio rosso. The varieties available on the market Compositae) to which, among others, belong Sunflower (He now cannot guarantee this consistent quality. lianthus), lettuce (Lactuca), Calendula, Aster and Echinacea. 0018 Finally, it can be noted that highly uniform F1 hybrids will show a better emergence of the seeds. 0006. Using the present state of the art for commercially 0019 For Cichorium endivia (endive) production of available Cichorium seeds (like radicchio rosso, Treviso and hybrids with the current state of the art is impossible; a others) hybrids are produced using certation. mechanism as certation is not present in endive. Therefore 0007 Certation is the botanical phenomenon where there endive is 100% self-pollinating crop, albeit showing a high is competition between pollen tubes, showing a difference in level of uniformity it has a poor heterosis. growth rate. Pollen tubes of the same plant (self-pollination) 0020. To improve on the aforementioned points much grow slower through the style then pollen tubes of another, research is performed to develop other methods for hybrid cross-pollinating plant. This mechanism is exploited to ization in Cichorium. One of the methods is described in enhance the share of cross-fertilization but self-fertilization, patent applications made by Enza Zaden (NL 1001554) and resulting in impurity of the desired hybrid, cannot be Florimond Desprez (WO 97/45548) where a cytoplasmic fac excluded. In practice up to 30% of the produced seed consists tor from sunflower (Helianthus annuus) is introduced by cell of products of self-fertilization, in other words, inbred plants fusion in Cichorium species. This resulted in cytoplasmic instead of hybrid plants. Using seed cleaning methods, this male sterile (CMS) Cichorium breeding material. A similar percentage can be reduced to 10-20% but is still undesirably patent application is submitted by Sakata Seeds as WO 2007/ high. 049730 where CMS from Sunflower is transferred into let 0008. An additional drawback of using certation is that tuce, Lactuca Sativa. during maintenance of the female line (having certation), 0021. Yet another approach is described in patent applica individual plants yielding more pollen spread their genes in tion WO 2012/163389 by AZ. Agricola T&T, where a reces the population more efficiently resulting in potential loss of sive or genetically nuclear encoded male sterility is induced certation after a number of generations, i.e. an useless paren in Cichorium by mutagenesis. F1 hybrids produced with this tal line. system are, due to the recessive character of the described US 2016/O165825 A1 Jun. 16, 2016

trait, male fertile. A further draw-back of this system is that 0033 Briefly, protoplasts from both species were isolated. male sterile mother lines (denoted as ms/ms) have to be Protoplasts from Cichorium (the acceptor) were treated with propagated vegetatively by in vitro or in vivo methods. iodoacetamide (IOA) or iodoacetate (IA) for inactivation of 0022 Considering the above, it is an object of the present the cytoplasm. Protoplasts of Lactuca sp. (the donor) were invention, amongst other objects, to provide cytoplasmic treated with ionizing radiation to inactivate or destroy the male sterile Cichorium plants obviating the above drawbacks nuclear genome but maintaining active genetic elements in of the prior art. the cytoplasm (chloroplasts and mitochondria). 0023 The above object, amongst other objects, has been 0034. After a PEG/Ca" mediated cell fusion, one or more met by the present invention, by providing plants as described novel cells are formed with the nuclear genome (and thus in the appended claims. horticultural characteristics) of Cichorium but with the cyto 0024 Specifically, the above object, amongst other plasm of Lactuca. This fusion can be performed either in test objects, has been met by the present invention, according to a tubes or petridishes. Here PEG acts as a means to promote the first aspect, by cytoplasmic male sterile Cichorium plants association ofcells, a high pH and Ca" ions are a prerequisite comprising mitochondria of Lactuca, wherein said mitochon for the actual fusion of cells. A FDA staining procedure is dria of Lactuca comprise at least one mitochondrial nucleic used to ascertain the resulting cells are viable. acid sequence selected from the group consisting of SEQID 0035. A suitable medium comprising water, salts, vita No. 1, SEQID No. 2, SEQID No. 3, SEQID No. 4, SEQID mins, Sugars and plant hormones was used to promote the No. 5, SEQID No. 6, SEQID No. 7, SEQID No. 8, SEQID formation of calli. Subsequently, the regeneration of plants No. 9, and SEQID No. 10. was performed by promoting the advent of shoots which 0025 A research program was initiated to identify plant finally are rooted; also these stages are dependent of the species which might serve as a Suitable donor for the intro application of Suitable plant hormones. duction of cytoplasmic male sterility (CMS) in Cichorium. In 0036. After regeneration, the plants obtained are checked this context, “suitable' also means that the disadvantages of for a correct DNA content using flowcytometry. Diploid the prior art are obviated. plants resulting from this check then are subject to a molecu 0026. In lettuce, Lactuca sp., no CMS is present. Lactuca lar test to ascertain the presence of the Lactuca cytoplasm. To and Cichorium belong to the family of the Asteraceae and this enda RAMP technique was used to discriminate between within this family to the tribe Cichorieae or Lactuceae, the Cichorium and Lactuca cytoplasm. respectively. 0037. Further research on plants containing the Cichorium nuclear material combined with the Lactuca cytoplasm 0027. The combination Lactuca and Cichorium was stud showed that between the regenerated fusion plants, individual ied to determine whether a combination of Cichorium nuclei plants occur which are male sterile while retaining female with the cytoplasm of Lactuca would result in a cytoplasmic fertility, i.e. plants according to the present invention. The male sterile (CMS) Cichorium. frequency with which these plants were identified was about 0028. In lettuce, Lactuca sp., no CMS is present. Lactuca 1 in 21 original fusion plants. is related to Cichorium, both belong to the family of the 0038. The plant obtained had flowers with visible pollen Asteraceae and within this family to the tribe Cichorieae or grains on the anthers but these are smaller than wild type Lactuceae; therefore this plant was chosen as a candidate both pollen grains and using a FDA staining technique, it was for crosses and asymmetric cell fusions with Cichorium. shown that pollen of the hybrid plant were not viable. Accord 0029. With the Lactuca mentioned above, the combina ingly, the F1 hybrid plants are male sterile. Thus, from the tion with Cichorium was studied to determine whether a above fusion plants, the present Cichorium plants are combination of Cichorium nuclei with the cytoplasm of Lac obtained which are suitable to produce 100% pure hybrids of tuca would result in a cytoplasmic male sterile Cichorium. Cichorium. This research was aimed especially at several Lactuca species 0039. A representative sample of a plant according to the since initial research learned that crossing of Cichorium with present invention was deposited on Mar. 12, 2013 at NCIMB Lactuca resulted in a -limited- amount of seeds. Plants grown Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aber from these seeds were recognized as hybrid both by flowcy deen, AB219YA, UK with deposit number NCIMB 42125. tometrical as molecular studies. 0040. According to a preferred embodiment of this first 0030. After obtaining this hybrid between Cichorium and aspect, the present invention relates to cytoplasmic malester Lactuca it will be necessary to perform several backcrosses to ile Cichorium plants, wherein the cytoplasmic male sterile regain the genetic setup of Cichorium. With a crop like Cicho Cichorium plants are further identifiable by a molecular rium it will take at least 8 years to reconstruct the complete marker of 1592 bp using SEQID No.31 and SEQID No. 32. genome of this species in the product of the crossings. Next to 0041 According to another preferred embodiment of this that, a cross between distinct species is very inefficient, espe first aspect, the present invention relates to cytoplasmic male cially in the first generations, so the amount of seeds yielded sterile Cichorium plants, wherein the cytoplasmic male Ster will be very limited initially. ile Cichorium plants are further identifiable by a molecular 0031. After several generations the amount of Lactuca marker of 293 bp using SEQID No. 33 and SEQID No. 34. genome is diluted Sufficiently but an inbred generation, made 0042. According to an especially preferred embodiment, by self-pollination, is required to select the required geneti the present invention relates to cytoplasmic male sterile cally stable candidate parent line for hybrid production. How Cichorium plants having at least two, at least three, at least ever, since this material will be male sterile, this final self four, at least five, at least six, at least seven, at least eight, at pollination step is impossible. least nine, or ten of the present mitochondrial nucleic acid 0032 To provide a solution to the drawbacks mentioned Sequences. before Bejo Zaden B.V. performed asymmetric cell fusions 0043. The plants according to the present invention are between Cichorium species and Lactuca sp. preferably selected from the group consisting of green US 2016/O165825 A1 Jun. 16, 2016

chicory, radicchio rosso, red leaved chicory, Treviso, white 0053 FIG. 2: shows result of a molecular characterization chicory, Sugar loaf, Belgian endive, witloof. Catalogna, C. of nuclear DNA using the RAMP technique. From left lane to intybus var. foliosum, C. endivia, and C. intybus L. var. Sati right lane, Cichorium intybus (P155); Lactuca sp. (P157); iFi. hybrids 1 to 3 (H1-H3, i.e. hybrid plants of a cross between 0044 According to most preferred embodiments of the P155 and P157; discriminating bands are indicated: present invention, the present plants comprise Lactuca mito 0054 FIG.3: shows agraphic representation of pollen size chondria derived from a plant according to deposit NCIMB of Cichorium, Lactuca and the selected fusion plant P153. 41985 and/or the present plants comprise a cytoplasm mater Legend to this graph: nally derived from a plant according to deposit NCIMB 0.055 X axis: diameter of pollen grains in um2 42125. 0056 Y axis: percentage of pollen grains present in the 0045. According to a second aspect, the present invention distinct sizes; relates to methods for identifying a cytoplasmic male sterile 0057 FIG. 4: shows TOP: Photograph of a flower of P153, Cichorium plant, the method comprises establishing in a a selected plant from the described fusion products obtained. sample of said cytoplasmic male sterile Cichorium plant one BOTTOM: detail of the anthers showing (non-functional) or more of the present mitochondrial sequences, i.e. nucleic pollen grains; acid sequences selected from the group consisting of SEQID 0058 FIG.5: shows TOP: Photograph of a fertile flower of No. 1, SEQID No. 2, SEQID No. 3, SEQID No. 4, SEQID Cichorium intybus L. BOTTOM: detail of the anthers. No. 5, SEQID No. 6, SEQID No. 7, SEQID No. 8, SEQID No. 9, and SEQID No. 10. EXAMPLES 0046 According to a third aspect, the present invention relates to methods for identifying a cytoplasmic male sterile Example 1 Cichorium plant, the method comprises establishing in a sample of said cytoplasmic male sterile Cichorium plant a Sterilization and Sowing of Seeds molecular marker of 1592 bp using nucleic acid amplification 0059. Seeds from both the donor (Lactuca sp., deposited primers SEQID No.31 and SEQID No.32 and/or a molecu on Jun. 1, 2012 at NCIMB Ltd, Ferguson Building, Craib lar marker of 293 bp using nucleic acid amplification markers stone Estate, Bucksburn, Aberdeen, AB21 9YA, UK as SEQID No. 33 and SEQID No. 34. deposit NCIMB 41985, and the acceptor (Cichorium intybus 0047 According to a fourth aspect, the present invention L.) were sown in vitro. To this end 100 seeds of each species relates to mitochondrial nucleic acid sequences selected from were sterilized by a rinse in 70% ethanol in water, succeeded the group consisting of SEQID No. 1, SEQID No. 2, SEQID by a treatment with a bleach solution (1% (w/v) NaOCl+0. No. 3, SEQID No. 4, SEQID No. 5, SEQID No. 6, SEQID 0.1% (v/v) Tween80 indemineralized water). After a thorough No. 7, SEQID No. 8, SEQID No. 9, SEQID No. 10, SEQID rinse with sterile demineralized water, seeds were placed on a No. 11, SEQID No. 12, SEQID No. 13, SEQID No. 14, SEQ suitable medium as /2MS15 and placed in a growth room at ID No. 15, SEQID No. 16, and SEQID No. 17. 25°C., 16/24 hrs. light. After 4 weeks plants were suitable for 0048. According to a fifth aspect, the present invention protoplast isolation. relates to cytoplasmic malesterile hybrid plants of Cichorium and Lactuca, wherein the cytoplasmic male sterile hybrid Example 2 plants comprise mitochondria of Lactuca and said mitochon dria comprise at least one mitochondrial nucleic acid Isolation of Protoplasts sequence selected from the group consisting of SEQID No. 1, SEQID No. 2, SEQID No. 3, SEQID No. 4, SEQID No. 5, 0060 Young, surface-sterilized leaves of 4 week old SEQID No. 6, SEQID No. 7, SEQID No. 8, SEQID No. 9, plants were collected. These leaves were cut in pieces of 1-2 and SEQID No. 10. mm in TC quality petri dishes (Greiner Bio-One, article 0049 According to a preferred embodiment of this fifth 664160) in plasmolysing solution WS9M. After collecting all aspect of the present invention, the present cytoplasmic male material, this solution was replaced by 20 ml of enzyme sterile hybrid plants are a cytoplasmic male sterile hybrid Solution containing cell wall degrading enzymes like pecti plant according to deposit NCIMB 42125 and/or comprise nase and/or cellulase. After an overnight incubation (dark, mitochondria derived from a Lactuca plant according to 25°C.) on an orbital shaker with a speed of 30 rpm, material deposit NCIMB 41985. was sieved on respectively a 110 and a 53 um filter; these 0050. According to a sixth aspect, the present invention filters were rinsed twice with 10 ml wash solution. relates to the use of mitochondria of a Lactuca plant for 0061 The collected filtrate was centrifuged for 5 minutes obtaining a cytoplasmic male sterile Cichorium plant, the at 60xg. After resuspending the pellet in 10 ml wash solution, present use preferably comprises a protoplast fusion of a this centrifugation step was repeated. Protoplasts were kept nuclear genome inactivated protoplast of Lactuca as donor on ice until further processing. and a mitochondrial genome inactivated protoplast of Cicho rium as acceptor. Example 3 0051 Below, the present invention will further be described in examples of preferred embodiments of the Viability Control present invention. In the examples, reference is made to fig 0062. A check for viability of the isolated protoplasts and ures wherein: pollen grains was performed with FDA. To this end, samples 0052 FIG. 1: shows flowcytograms of: (A) Cichorium of stained protoplasts, from both donor and acceptor, or pol intybus L. (upper left); (B) Lactuca sp. (upper right); (C) len grains collected from donor or selected fusion plants, Hybrid 1 (according to the present invention; lower left); and were checked with a fluorescence-microscope using the (D) a mixed sample of A, B and C (lower right); appropriate filters, magnification 10x. US 2016/O165825 A1 Jun. 16, 2016

0063 Viable cells can be recognized by a bright yellow amount of light was increased to approx. 1700 lux. Calli green color, non-viable cells do not fluoresce. without shoots were transferred to fresh CRM medium every three weeks. Example 4 0074 Emerging shoots were cut from the calli, leaving behind as much callus as possible, and placed on MS30 Pretreatment of Isolated Protoplasts gelrite medium either in jars or petridishes. To promote root 0064. Isolated protoplasts from the donor (Lactuca) were ing, shoots were cut—to remove any residual callus- and treated with a suitable source of ionizing radiation (e.g. a transferred to fresh MS30-gelrite medium. Source of Roentgen or gamma radiation). A Suitable dose is in the range of 250-500 Gray as measure of absorbed energy. Example 6b From the protoplasts isolated from the acceptor (Cichorium intybus var. foliosum), cellular organelles were inactivated by Embedded in Alginate a treatment with a final concentration IOA of 10 mM (Sup plied from a 60 mM IOA stock solution) during 10 minutes at 0075 Starting with the fused protoplasts as described in 4°C. After this treatment cells were washed twice with wash example 5, protoplasts were resuspended in Solution A; per Solution and centrifuged at 60xg during 5 minutes. tube containing 1x10° protoplasts. To each tube, 2.5 ml solu 0065. Fusion and successive regeneration of the proto tion A was added; after carefully re-suspending the cells, 2.5 plasts obtained can be performed in two alternative ways as ml Sodium alginate Solution was added. Drops of 1 ml Sus described in the examples 5 and 6a (culture in liquid medium) pension were placed on solid medium B. After 1 hour the and 5 and 6b (embedded in Solid medium using sodium algi solidified medium including the protoplasts were rinsed with nate) respectively. PM1 medium and transferred to dishes containing 3 resp. 6 ml PM1 medium (petri dishes of 6 or 9 cm diameter, respec Example 5 tively). Further media changes were similar as described in example 6a. Fusion of Protoplasts 0076. As soon as the calli reach a diameter of 2 mm, the alginate matrix was dissolved by replacing the culture 0066 Per 10 ml tube (round bottom, Greiner, catalogue medium by 50 mM sodium citrate solution. Each alginate disc number 163189) 1x10° protoplasts (in a ratio of acceptor: was cut in 8 pieces and the dishes are placed on an orbital donor of 1:1 or 2:1) in wash solution were added. shaker for 2 hours (30 rpm). 0067. After centrifugation (5 minutes at 60*g) each pellet was resuspended in 0.3 ml wash solution. Then, 0.4 ml 0077 Microcalli were collected using a pipette and trans PEG 1500 solution per test tube was added by letting separate ferred to a test tube. To this tube PM2 medium was added to drops fall into the tubes; no further mixing was allowed here. a final volume of 10 ml and then the tube is centrifuged for 5 After standing still for 30 minutes, 0.8 ml PEG diluting solu minutes at 60*g. tion was added, by slowly flowing down the test tube wall. (0078. After washing with PM2 medium twice, calli were Without further mixing the cells are left for 10 minutes. placed on CRM medium at 1000 lux; calliwhich turned green 0068. After this, tubes are topped up with CPW-Ca" solu were grown further at 1700 lux. Calli were kept on this tion to a final volume of 10 ml, without further mixing the medium and transferred, if necessary, to fresh CRM medium cells are centrifuged for 5 minutes at 60*g. After three repeti every three weeks. tive washing with CPW-Ca" solution pellets were resus 0079 Shoots which emerged on these calli were cut from pended in PM1 culture medium providing a final density of the callus and placed on MS30-gelrite medium, either in petri 20.000 protoplasts/ml. dishes or glassjars, shoots were transferred on fresh medium of the same composition until roots were formed. Rooted Example 6 plants were placed on MS30 medium. Culture of Fused Protoplasts Example 7 Example 6a Determination of Ploidy Level Using Liquid Regeneration Media 0080 From plants resulting from cell fusion or crosses 0069 Culturing was performed in dark at 25°C., every between Cichorium and Lactuca plants, the relative ploidy week the culture medium is partly replaced: level was determined. To this end, a small piece of leaf tissue (0070) 6 cm dish: 3 ml (out of 4.5 ml) is replaced by 3 ml 1 cm was taken from the plants examined and chopped with fresh PM1 medium a razorblade in CyStain(R) UV Ploidy buffer (Partec, Munster, (0071 9 cm dish: 5 ml (out of 9 ml) is replaced by 5 ml Germany). fresh PM1 medium I0081. After filtration, the ploidy level of the sample was 0072. As soon as the first cell divisions were observed (2 to determined using a Partec PA flow cytometer and compared 4 cell stage) the culture medium was replaced by PM2 to the result of samples from Cichorium and Lactuca plants. medium, also this medium is partly replaced on a weekly Only diploid plants, resulting from cell fusion were retained. basis as described above. I0082 Plants resulting from crosses between Cichorium 0073 Calli of 2 mm diameter were placed on solid CRM and Lactuca sp. possess a ploidy level intermediate between medium at 1000 lux. As soon as the calli turn green the Cichorium and Lactuca sp. (FIG. 1). US 2016/O165825 A1 Jun. 16, 2016

Example 8 nylon mesh (75 um). The residue was homogenized further using a precooled mortar and pestle and Subsequently filtered Molecular Characterization Genomic DNA as described. 0091 Filtrates were pooled and centrifuged for 10 minutes 0083) Resulting from the cross between Cichorium at 1.000xg; the supernatant was re-centrifuged at 2.000xg. (ID=P155) and Lactuca (ID=P157), several seeds were har Finally mitochondria were collected by centrifugation at Vested, of which three germinated seeds were analyzed as 16.000xg. The resulting pellet was resuspended using a paint described in example 7 and identified as hybrid plants. To brush in 10 ml DNAse storage buffer. further characterize these plants, a molecular analysis was 0092 Following centrifugation at 2.000xg, the resulting performed. supernatant was pipetted to a centrifuge tube, 50 ul 2M 0084. From these putative hybrid plants and both parents MgCl, and 200 lul 5 mg/ml DNAse were added. After incu DNA was isolated, following a standard isolation procedure, bation for one hour, 20 ml of shelf buffer was pipetted under this mixture. starting with leaf explants of +0.3 cm. 0093. After centrifugation at 14.000xg, the resulting pellet I0085. On this isolated DNA, a RAMP analysis was per was resuspended in 10 ml shelf buffer. The suspension was formed with two primers generating polymorphisms between centrifuged for 10 minutes at 16.000xg, the pellet was dis Cichorium and Lactuca. The result showed unequivocally solved in 600 ul Lysis Buffer and transferred to a 1.5 ml that the hybrid plant harbored genomic DNA of both parents, Eppendorf test tube. In this lysate, containing the mitochon thus demonstrating this plant was indeed a genomic hybrid drial DNA, 1 mg proteinase K is dissolved and 10 ul between Cichorium and Lactuca. RNAse-A solution was added. Incubation was first 60 min Primers used for the reaction were: utes at 37° C. followed by 30 minutes incubation at 65° C. 0094. After this incubation, DNA was precipitated by add ing 200 lul 5 MKAc and mixed by turning over the test tube, (SEQ ID No. 37; RAPD primer) leaving it then for maximum 15 minutes on ice. Next is a Bejop O9631 AGTCGGGTGG centrifugation during 10 minutes at 20.000xg at room tem (SEQ ID No. 38; iSSR primer) perature. Finally, the DNA is subsequently extracted with Bejop 01751 CCAGGTGTGTGTGTGT phenol/chloroform/isoamylalcohol and chloroform/isoamy lalcohol. The remaining water phase was mixed with 400 ul PCR conditions: isopropanol and 27 Jul 7.5 Mammoniumacetate. 2 minutes 93° C. (0095. After centrifugation for 10 minutes at 20,000xg (20°C.) the remaining pellet was washed with 500 ul 70% 40 cycles of 30 s.93° C-30 s. 35° C.-90 s. 72° C. ethanol in water and re-centrifuged for 2 minutes at 20.000xg 5 minutes 72° C. (20°C.) After drying the resulting DNA is dissolved in 200 ul Heating is performed with 0.3°C./sec. TE buffer. 0096. A second DNA precipitation was performed by add Example 9 ing 20 ul 7.5 M Na-Acetate and 150 ul isopropanol, the mixture was mixed gently for a few seconds. Polyacrylamide Gel Electrophoresis (0097. The next centrifugation is 10 minutes at 20,000xg (20° C.). The pellet was washed with 500 ul 70% ethanol in I0086 To analyze the RAMP patterns a “Gene Read IR water and re-centrifuged shortly (2 minutes at 20.000xg (20° 4200 DNA analyzer' (Licor Inc.) was used. Based on an C.). The pellet was dried and dissolved in 22 ul sterile ultra optimal concentration of 6.5% acrylamide DNA fragments pure water. Storage was at 4°C. differing 1 base pair in size can be separated. To envisage (0098 Purity and concentration of the DNA was deter these fragments labeled (IRDye labels) primers were used, mined using a Nanodrop 2000 Spectrophotometer, a Qubit one third of the amount of forward primer was replaced by a 2.0 Fluorometer and standard agarose gel electrophoresis labeled, identical primer. techniques. 0087. The result showed unequivocally that the obtained (0099. The sequence of the isolated mitochondrial DNA plants harbored genomic DNA of both parents, thus demon was determined by Next Generation sequencing using strating these three plants (H1 to H3) were indeed hybrids sequencing techniques generally known to the person skilled between Cichorium and Lactuca (FIG. 2). in the art. 0100. This sequencing resulted in the identification of sev Example 10 eral regions where the mitochondrial DNA (mtDNA) of the selected CMS Cichorium plant equals either the original fer Characterization of Mitochondrial DNA of the tile Cichorium plant or the Lactuca donor, proving the mito Selected Fusion Plant chondrial DNA of the selected CMS Cichorium plant to be a rearrangement of the mtDNA of both the original fertile 0088 Mitochondrial DNA was characterized to demon Cichorium plant and the Lactuca donor. strate that indeed the cytoplasm of Cichorium was replaced 0101 To this end, 4 sets of PCR primers were developed to by the Lactuca cytoplasm. identify the unique rearranged mtDNA of the selected fusion 0089 Buffers used were essentially as described. How plant leading to the CMS phenotype. Also a set of 17 SNPs ever, some buffers were adjusted as denoted in the section were developed as a further characterization of the selected “Solutions and chemicals'. All experimental steps were per fusion product (Table 2). From this data it is concluded that formed with precooled buffers and at 4° C., unless stated the selected CMS Cichorium plant contains an unique rear otherwise. ranged mitochondrial DNA with fragments of both parent 0090 100 grams of fresh leaves were homogenized on ice lines being responsible for the CMS character of the with a blender in 250 ml GB buffer. The homogenate was described plant. Experimental details are shown in the tables vacuum filtered on 4 layers of cheesecloth and 2 layers of mentioned above. US 2016/O165825 A1 Jun. 16, 2016

TABLE 1. Data for PCR reactions discriminating between both fusion parents and the selected CMS Cichorium Primer Forward Rewerse combi – primer primer Fertile CMS Fragment nation sequence sequence Lactuca Cichorium Cichorium size

1. SEQ ID 29 SEQ ID 30 -- -- 611 bp 2 SEQ ID 31 SEQ ID 32 -- -- 1592 bp 3 SEQ ID 33 SEQ ID 34 -- -- 293 bp 4. SEQ ID 35 SEQ ID 36 -- -- 107 bp

SEO ID 29 : GCCTCCAGGGTATGATCCTTAA SEO ID 3 O: CGAGCACTTATTTGACCTGTGT SEO ID 31: TGCTAACGAGGTTCAATGATG SEO ID 32 : TTCGATTCAGGATCAAGCCCAG SEO ID 33: TCGATATTCTTTTCGCGACAGG SEQ ID 34 : TTAGGTTATTTCGTTGGTCGCC SEO ID 35 : TTTATAGACAGCGACTCCCTCC SEO ID 36: ACCTGAAGGGAGTTATGGCATT

PCR conditions for this reaction were as follows: 1 minute 94° C. total DNA was isolated from young leaf tissue as described 30 cycles of 15s. 94° C.; 15 s. 58° C.; 1 minute 72° C.) and treated with RNAseA/T1. DNA integrity was confirmed 5 minutes 72° C. by agarose gel electrophoresis. DNA from all samples was diluted to 15 ng/ul for PCR reactions. The amount oftemplate Store at 4° C. DNA was 15 ng per PCR reaction. PCR for both marker combinations was carried out using Biomix Red (GC Bio- 0102 Five ul of the PCR reactions were loaded on a 1% tech) in 20 ul reactions with 10 pmol of each primer men- agarose gel and separated using standard techniques, known tioned above. PCR parameters were: to the person skilled in the art. TABLE 2 Sequence of the 17 SNP markers developed to discriminate between both fusion parents and the selected fusion plant. Denoted are the sequences used and between brackets ( N/N ) is the single nucleotide differing between the plants as elucidated in the three columns right of this sequence. Analysis was performed bp LGC Genomics, see WWW ldccenomics CO

SNP Fertile CMS SEQ ID Sequence Lactuca Cichorium Cichorium

1. CCGAACCGCGCGAAATGGTCGCCTATTACACGGCTCACTAACTCTGCCTGG/T G T G AGTGGTGGTACCTATTATTCGTCGGCGGGGGCGGTCCGGCGATAC

2 CICTTAAGGTTAGTTCTATGCCTCACAACAACTACCTCATAGGTGATTCTA/C A. C A. AATCCGTCTTTAGATCAGAAGTAGATGCCTCTTCCAGGGCTCTGT

3 GTACCAAATGCTTGGCAATCCTTGGTAGAGCTTATTTATGATTTCGTGCTG/T T G T AACCTGGTAAACGAACAAATAGGGGGICTTTCCGGAAATGTTAAA

4 TATTGATTACACTCCTTCTCCTACTGCTTCTCCTTATACCCACTAATGTTA/C C A. C TTCTTCGTCCTACCTTGACTATTGGGAAATTTCCATTCGAGAAAG

5 TGCAGCGTAAACTCCTGCTAAATGAATGCCTTTGCTTTGGCTTTGAAATTA/C A. C A. TGTTCCTGATTCAACTCCTTCATGCCATTGATTGATGCAAGTCTT

6 CTGAATGACCTCTCTCATCTAATCGATCGGTGAAGGATGTCTTTGAAGATA/C A. C A. ATCTGGTCTGGCTCGTTACCATTAGTTCCTCTCTCTATAGTCGAG

7 CTCTTGTTCTCTTTTCTTAGTTAGGCCCAAGAAAGTACAAGATATAGCTTG/T G T G ACTAATATACTAAGATGATAGCTAGAGACTAGAGATGAGAGTGCA

8 TIGGATATTGTACGACAGGATCCCCATCCGTGTTTTCGTTTCGCTAACGAIG/T G T G AAAGGCCGGAAAGCGTGGGACCTATTTAAGTGACCTGGCCGATGG

9 TTTTTCGTTCCTTCTCTTCGATAAGAATATCGATTTGAAATGATAAAAATG/T G T G CCTCTTTATTCTCTTGTGCTCAGCGAAAGAACTGCCTAAGCATAC

10 GTCCGATCGTCGCTAGAGGCCCGTCGACTATAATACACTGTTCGAAATTTA/C A. C A. TTCGTTTCTGCCGAAACAAAACGGGATTGGCTATCTTAACCTTAG US 2016/O165825 A1 Jun. 16, 2016

TABLE 2 - continued Sequence of the 17 SNP markers developed to discriminate between both fusion parents and the selected fusion plant. Denoted are the sequences used and between brackets ( N/N ) is the single nucleotide differing between the plants as elucidated in the three columns right of this sequence. Analysis was performed bp LGC Genomics, see WWW ldcdenomics Col

SNP Fertile CMS SEQ ID Sequence Lactuca Cichorium Cichorium

1 AAGGAAATGATCTTTACATGGAAATGAAAGAATCTGGAGTAATTAATGAAAAC A. C C AAAATATTCCAGAATCAAAAGTAGCTCTAGTTTACGGTCAGATGA

2 GGGGCGTCCAAAGAGTCGATCATTTCTGCATGATTTTTTTCTCGTGCACTA/G A. G G CCCCTTCCAATGGGTTTCGAAGATAAAAATCCTTTATTGGCCCAT

3 CATACGCTAAAATTTGTCCCTTTTTAATGCATTTACCCCGCTGAACCTGGA/G G A. A. GTTTTGATGCATACAAGTATTTTTGTTGGAACGTTGATACATAA

4 AGATAAAAATAACAAGGAATAGAATTTGATTAGTTGGTATTCAAAATATAC/T T C C GATTCAAGTAGTCAAGTCGAGAAAGAGATGGTTGAATCAAAATAA

5 TAAGCAATATATTGATTTTCTTCTCCAGGAACAGGCTCGATTCCATAGCAIG/T T G G CGCCCTTTGTAACGATCAAGGCTCGTAAGTCCATCGGICCACACA

6 ACTTTTTATTGGCATTGGAAGCACATTACATTATGGCAGGGTAATGTTTCA/G G A. A. CAGTTTAATGAATCTTCCACTTATTTGATGGGCTGGTTAAGAGAT

7 GGTATGAATAGTTTATCAGTCTGGGCATGGATGTTTTTATTTGGACATCTG/T G T T GTTTGGGCTACTGGATTTATGTTTTTAATTTCCTGGCGTGGATAT

Example 11 Solutions and chemicals. Characterization of Pollen Grains Unless stated otherwise all solutions were autoclaved (20 minutes at 120° C.) 0103) To illustrate the difference between the original fer tile chicory plant and the selected fusion plant, a study was /MS15 MS30 MS30-gelrite made of the available pollen grains. Studying the selected fusion plant P153, putative pollen grains were visible, how l/MS medium Duchefa, MO233 2.28 g/l ever, both an attempted self-fertilization and a stain with FDA MS medium Duchefa, MO222 4.40 g/l 4.40 g/l (Example 3) showed this putative pollen was not functional. Sucrose 15 g/l 30 g/l 30 g/l 0104 Further testing of the pollen of Cichorium and the Agar Duchefa; M1002 7 g/l 7 g/l selected fusion plant learned that from the latter pollen grains Gelrite Duchefa, G1101 7.5 g/1 were significantly smaller. Pollen grains, Suspended in pH 5.8 5.8 5.8 WS9M, were imaged using regular bright field techniques on an inverted microscope with a 10x objective lens and recorded with a 3 MB CCD camera. Digital images of pollen grains were processed and analyzed using Image.J Software (Rasband, W. S., Image.J., U.S. National Institutes of Health, WS9M medium Bethesda, Md., USA, http://image.nih.gov/i/, 1997-2012). KH2PO O.20 nM CaCl2HO 10 mM 0105 Images were converted into 8-bit binary images. KNO. 1 mM Holes were closed, dark outliers were removed (radius=10 pixels), and pollengrain projections touching each other were MES (monohydrate) 2.8 mM separated using the “watershed command. The resulting Mannitol O.SM images were analyzed with the “analyze particles' command pH 5.6; 580 mOsm/kg to obtain the Surface areas of the pollen grain projections. Objects touching the edge of the image were excluded. Using Microsoft Excel statistical functions, the measurements were converted intoum and averaged. enzyme solution: 0106 For the selected fusion plants pollen surface varied WS9M medium supplemented with: between 1150 and 1850 um (SD=180), for the original Yakult Cellulase Onozuka R10 0.2% (w/v) Cichorium plant pollen grains, the determined surface was Pectinase (Sigma, P4300) 0.2% (w/v) between 1550 and 2550 um (SD-226). Furthermore, student pH 5.6-5.7, filter sterile T-test analysis indicated that the chance that both populations of pollen grains are equal is below 0.01 (FIG. 3). US 2016/O165825 A1 Jun. 16, 2016

wash solution: CRM medium

13.6 mM MS medium (Duchefa, MO222) 4.40 gram CaCl2HO Sucrose 30 mM KCI O.34M Zeatin 4.56 M MES (monohydrate) 2.8 mM BA 2.22 LM pH 5.8: 640 mOsm/kg caseinhydrolysate (Duchefa; C1301) 300 mgram/l agar (Duchefa; M1002) 8 graml pH 5.7

60 mM IOA stock solution: Sodium citrate 50 mM:

0.222g IOA per 20 ml wash solution Solution A, Sodium alginate Solution, medium B: ref 5 Gamborg B5 media:

PCR mix: PEG1500 solution: Tris-HCl (pH 8.8) 75 nM PEG1500 40% (w/v) NHSO 20 nM Glucose anhydrous (w/v) O.3M Tween2O 0.01% (v/v) CaCl2.H2O 50 mM MgCl2 2.8 mM filter sterile dNTPs O.25 mM forward primer 0.15 M reverse primer 0.2 M Red Hot (R DNA Polymerase 0.04 unitsul (ABgene, Epsom U.K.) genomic plant DNA ~0.2 ngful PEG diluting solution Glycine 50 mM glucose anhydrous O.3M CaCl2.H2O 50 mM pH 10.5: 520 mOsm/kg: filter sterile GB (Grinding buffer) sorbitol O3SM mannitol O.1M Tris-HCl pH 7.6 50 nM Na-EDTA 5 mM CPW-Cat solution 200 ml BSA 0.2% (w/v) PVP 1.0% (w/v) 10x CPW salts stock (ref. 3) 20 ml spermidine 0.025% (w/v) CaCl2HO 50 mM mercaptoethanol 0.125% (v/v) Mannitol O.36SM DIECA 10 mM pH 5.7:530 mOsm/kg: filter sterile

DNase storage buffer: Culture media PM1 PM2 Sodiumacetate SM KNO. 7.42 mM CaCl2.H2O 1M MgSO4·7H2O O.69 mM O.69 mM pH 4.5 using acetic acid; CaCl2.H2O 1.5 mM 1.5 mM prior to use, an equal volume glycerol is added KH2PO O.62 mM O.62 mM KC 5 mM Gamborg B5 micro 100x stock 5 mill 5 mill KI 0.73 M 0.73 M FeNaFDTA O.11 mM O.11 mM Gamborg B5 vitamins 11.2 mgram 11.2 mgram DNase Buffer (DB) (4° C.) (Duchefa; G0415) Glutamine 2.57 nM 5.14 mM DIECA (add just prior to use) 10 mM Sucrose 14.6 mM 14.6 mM Mannitol O41M O3OM NAA 10.2 M 2.5 M BA 4.44 M Caseinehydrolysate 150 mgram? Shelf Buffer (SB) (Duchefa; C1301) pH S.S.-S.6 S.S.-S.6 Sucrose O.6M oSmolarity 530 mOsm/kg 400 mC)Sm/kg Tris HCl pH 7.2 10 mM filtersterile filtersterile Na-EDTA 20 mM DIECA (add just prior to use) 10 mM US 2016/O165825 A1 Jun. 16, 2016

0111 DIECA Na-diethyldithiocarbamate TE buffer O112 EDTA ethylene diaminetetra acetic acid FDA fluorescein diacetate Tris-HCl pH 7.5 50 mM 0113 Na-EDTA 10 mM 0114 IOA iodoacetamide 5 mg/ml DNase in DNase storage buffer 0115 IA iodoacetate 0116 MES 2-(N-morpholino)ethanesulfonic acid Proteinase K mtDNA mitochondrial DNA RnaseA solution: Thermo Scientific productnir. ENO551 0117 SMKAc 0118 NAA 1-naphthaleneacetic acid Phenol/chloroformisoamylalcohol (25:24:1) 0119) ng nanogram chloroformisoamylalcohol (24:1) 0120 PCR polymerase chain reaction 7.5M NH-Acetate 0121 PEG polyethylene glycol 7.5M Na-Acetate pH 4.5-5.2 0122) PVP polyvinylpyrrolidone (0123 RAMP Random Amplified Microsatellite Polymor phism Abbreviations: 0.124 SD standard deviation 0107 BA 6 benzylaminopurine 0.125 SNP single nucleotide polymorphism 01081 bp basepairs 0126 KG or T) 0109 BSA bovine serum albumin O127 MA or C) nucleotide ambiguity codes 0110 CMS cytoplasmic male sterile cytoplasmic male 0128 RG or A) according to the rules of IUPAC sterility 0129 YT or C) US 2016/O165825 A1 Jun. 16, 2016 10

Form PCT/RO/134 (SAFE) Indications Relating to Deposited Microorganism(s) or Other Biological Material (PCT Rule 13bis) O-1-1 Prepared Using PCT Online Filing Version 3. 5. OOO. 235 MT/FOP 2002O701/O. 20.5. 20 International Application No. PCTIEP2013,067014 0-3 Applicant's or agent's file reference 4/2OL49/18

The indications made below relate to the deposited microorganism(s) or other biological material referred to in the description on: 1-1 page 10 line 32 1-3 Identification of deposit -3-1 Name of depositary institution NCIMB NCIMB Ltd. -3-2 Address of depositary institution Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom -3-3 Date of deposit O1 June 2012 (01.06.2012) -3-4 Accession Number NCIMB 41985 1-5 Designated States for Which indications are Made All designations The indications made below relate to the deposited microorganism(s) or other biological material referred to in the description on: 2-1 page 7 2-2 line 24 2-3 Identification of deposit 2-3-1 Name of depositary institution NCIMB NCMB Ltd. 2-3-2 Address of depositary institution Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom 2-3-3 Date of deposit 12 March 2013 (12.03.2013) 2-3-4 Accession Number NCIMB 421.25 2-5 Designated States for Which indications are Made All designations

FOR RECEIVING OFFICE USE ONLY

0-4 This form was received with the international application: yes (yes or no) O-4-1 Authorized officer Annik Appelen

FOR INTERNATIONAL BUREAUUSE ONLY

This form was received by the international Bureau on: O-5-1 Authorized officer US 2016/O165825 A1 Jun. 16, 2016 11

SEQUENCE LISTING

< 16 Os NUMBER OF SEO ID NOS: 38

SEQ ID NO 1 LENGTH: 96 TYPE: DNA ORGANISM: Lactuca FEATURE: NAMEAKEY: source LOCATION: 1.96 OTHER INFORMATION: /mol type= DNA" /organism=Lactuca."

SEQUENCE: 1 cc.galaccgcg cgaaatggtc gcct attaca C9gct cacta act ctgcct g gagtggtggit 6 O acct attatt C9tcggcggg ggcggtc.cgg catac 96

SEQ ID NO 2 LENGTH: 96 TYPE: DNA ORGANISM: Lactuca FEATURE: NAMEAKEY: source LOCATION: 1.96 OTHER INFORMATION: /mol type= DNA" /organism=Lactuca."

SEQUENCE: 2

Ctcttaaggt tagttctat cotcacaa.ca actacct cat aggtgattct aaatc.cgtct 6 O ttagat Caga agtagatgcc tict tccaggg Ctctgt 96

SEQ ID NO 3 LENGTH: 96 TYPE: DNA ORGANISM: Lactuca FEATURE: NAMEAKEY: source LOCATION: 1.96 OTHER INFORMATION: /mol type= DNA" /organism=Lactuca."

SEQUENCE: 3 gtaccalaatig cittggcaatic cittgg tagag cittatttatg attitcgtgct taacctggta 6 O aacgaacaaa tagggggtct titc.cggaaat gttaaa 96

SEQ ID NO 4 LENGTH: 96 TYPE: DNA ORGANISM: Lactuca FEATURE: NAMEAKEY: source LOCATION: 1.96 OTHER INFORMATION: /mol type= DNA" /organism=Lactuca."

SEQUENCE: 4 tattgattac act cottct c ctact.gctitc. tcc titat acc cactaatgtt cittctitcgt.c 6 O ctaccttgac tattgggaaa titt coatt.cg agaaag 96

SEO ID NO 5 LENGTH: 96 TYPE: DNA ORGANISM: Lactuca FEATURE:

US 2016/O165825 A1 Jun. 16, 2016 14

- Continued

FEATURE: NAME/KEY: Solice LOCATION: 1.96 OTHER INFORMATION: /mol type= DNA" /organism=Lactuca."

SEQUENCE: 14 agataaaaat aacaaggaat agaatttgat tagttggitat tcaaaatata yogattcaagt 6 O agt caagt cq agaaagagat ggttgaatca aaataa 96

SEQ ID NO 15 LENGTH: 96 TYPE: DNA ORGANISM: Lactuca FEATURE: NAME/KEY: Solice LOCATION: 1.96 OTHER INFORMATION: /mol type= DNA" /organism=Lactuca."

SEQUENCE: 15 taagcaat at attgattitt c ttct coagga acaggct cqa titc catagoa kcgcc ctittg 6 O taacgatcaa gogotcgtaag to catcggtc. cacaca 96

SEQ ID NO 16 LENGTH: 96 TYPE: DNA ORGANISM; Lactuca FEATURE: NAME/KEY: Solice LOCATION: 1.96 OTHER INFORMATION: /mol type= DNA" /organism=Lactuca."

SEQUENCE: 16 acttitt tatt ggcattggaa goa cattaca ttatggcagg gtaatgtttic roagtttaat 6 O gaatct tcca cittatttgat gggctggitta agagat 96

SEQ ID NO 17 LENGTH: 96 TYPE: DNA ORGANISM: Lactuca FEATURE: NAME/KEY: Solice LOCATION: 1.96 OTHER INFORMATION: /mol type= DNA" /organism=Lactuca."

SEQUENCE: 17 gg tatgaata gtttat cagt ctgggcatgg atgtttitt at ttgga Catct kgtttgggct 6 O actggattta totttittaat titcctgg.cgt gigatat 96

SEQ ID NO 18 LENGTH: 96 TYPE: DNA ORGANISM: Cichorium FEATURE: NAME/KEY: Solice LOCATION: 1.96 OTHER INFORMATION: /mol type= DNA" /organism= Cichorium"

SEQUENCE: 18

US 2016/O165825 A1 Jun. 16, 2016 17

- Continued

22 Os. FEATURE: <221 > NAMEAKEY: source &222s. LOCATION: 1.96 <223> OTHER INFORMATION: /mol type= DNA" /organism= Cichorium" <4 OOs, SEQUENCE: 28 gtc.cgatcgt. c9ctagaggc ccgt.cgacta taatacactg titcgaaattt citt cqtttct 6 O gcc.gaaacaa aacgggattg gct at Cttaa cct tag 96

<210s, SEQ ID NO 29 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: artificial sequences 22 Os. FEATURE: <221 > NAMEAKEY: source &222s. LOCATION: 1. .22 <223> OTHER INFORMATION: /mol type= DNA" /note= primer" /organism=artificial sequences"

<4 OOs, SEQUENCE: 29 gcct coaggg tatgat cott aa 22

<210s, SEQ ID NO 3 O &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: artificial sequences & 22 O FEATURE; <221 > NAMEAKEY: source &222s. LOCATION: 1. .22 <223> OTHER INFORMATION: /mol type= DNA" /note= primer" /organism=artificial sequences"

<4 OOs, SEQUENCE: 30 cgagcactta tittgacctgt git 22

<210s, SEQ ID NO 31 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: artificial sequences 22 Os. FEATURE: <221 > NAMEAKEY: source &222s. LOCATION: 1. 21 <223> OTHER INFORMATION: /mol type= DNA" /note= primer" /organism=artificial sequences"

<4 OOs, SEQUENCE: 31 tgctaacgag gttcaatgat g 21

<210s, SEQ ID NO 32 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: artificial sequences 22 Os. FEATURE: <221 > NAMEAKEY: source &222s. LOCATION: 1. .22 <223> OTHER INFORMATION: /mol type= DNA" /note= primer" /organism=artificial sequences"

<4 OOs, SEQUENCE: 32 titcgatticag gat caa.gc.cc ag 22 US 2016/O165825 A1 Jun. 16, 2016 18

- Continued

SEQ ID NO 33 LENGTH: 22 TYPE: DNA ORGANISM: artificial sequences FEATURE: NAMEAKEY: source LOCATION: 1. .22 OTHER INFORMATION: /mol type= DNA" /note= primer" /organism=artificial sequences"

SEQUENCE: 33 tcqatatt ct titt cqcgaca gg 22

SEQ ID NO 34 LENGTH: 22 TYPE: DNA ORGANISM: artificial sequences FEATURE: NAMEAKEY: source LOCATION: 1. .22 OTHER INFORMATION: /mol type= DNA" /note= primer" /organism=artificial sequences"

SEQUENCE: 34 ttaggittatt togttggit cq cc 22

SEQ ID NO 35 LENGTH: 22 TYPE: DNA ORGANISM: artificial sequences FEATURE: NAMEAKEY: source LOCATION: 1. .22 OTHER INFORMATION: /mol type= DNA" /note= primer" /organism=artificial sequences"

SEQUENCE: 35 tittatagaca gcgact coct c c 22

SEQ ID NO 36 LENGTH: 22 TYPE: DNA ORGANISM: artificial sequences FEATURE: NAMEAKEY: source LOCATION: 1. .22 OTHER INFORMATION: /mol type= DNA" /note= primer" /organism=artificial sequences"

SEQUENCE: 36 acctgaaggg agittatggca tt 22

SEO ID NO 37 LENGTH: 10 TYPE: DNA ORGANISM: artificial sequences FEATURE: NAMEAKEY: source LOCATION: 1. ... 10 OTHER INFORMATION: /mol type= DNA" /note= RAPD primer" /organism=artificial sequences" US 2016/O165825 A1 Jun. 16, 2016

- Continued

<4 OO > SEQUENCE: 37 agtcgggtgg 10

<210s, SEQ ID NO 38 &211s LENGTH: 16 &212s. TYPE: DNA <213> ORGANISM: artificial sequences 22 Os. FEATURE: <221 > NAMEAKEY: source &222s. LOCATION: 1. 16 <223> OTHER INFORMATION: /mol type= DNA" /note= iSSR primer" /organism=artificial sequences"

<4 OOs, SEQUENCE: 38 cCaggtgttgt gtgttgt 16

1. Cytoplasmic male sterile Cichorium plant comprising mitochondrial nucleic acid sequences selected from the group mitochondria of Lactuca, wherein said mitochondria of Lac consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, tuca comprise at least one mitochondrial nucleic acid SEQID No. 4, SEQID No. 5, SEQID No. 6, SEQID No. 7, sequence selected from the group consisting of SEQID No. 1, SEQID No. 8, SEQID No. 9, and SEQID No. 10. SEQID No. 2, SEQID No. 3, SEQID No. 4, SEQID No. 5, 9. Method for identifying a cytoplasmic malesterile Cicho SEQID No. 6, SEQID No. 7, SEQID No. 8, SEQID No. 9, rium plant, said method comprises establishing in a sample of and SEQID No. 10. said cytoplasmic male sterile Cichorium plant a molecular 2. Cytoplasmic male Sterile Cichorium plant according to marker of 1592 bp using nucleic acid amplification primers claim 1, wherein said cytoplasmic male sterile Cichorium SEQID No.31 and SEQID No.32 and/or a molecular marker plant is further identifiable by a molecular marker of 1592 bp of 293 bp using nucleic acid amplification markers SEQID using SEQID No. 31 and SEQID No. 32. No. 33 and SEQID No. 34. 10. A nucleic acid sequence selected from the group con 3. Cytoplasmic male Sterile Cichorium plant according to sisting of SEQID No. 1, SEQID No. 2, SEQID No. 3, SEQ claim 1, wherein said cytoplasmic male sterile Cichorium ID No. 4, SEQID No. 5, SEQID No. 6, SEQID No. 7, SEQ plant is further identifiable by a molecular marker of 293 bp ID No. 8, SEQID No. 9, SEQID No. 10, SEQID No. 11, SEQ using SEQID No. 33 and SEQID No. 34. ID No. 12, SEQID No. 13, SEQID No. 14, SEQID No. 15, 4. Cytoplasmic male Sterile Cichorium plant according to SEQID No. 16, and SEQID No. 17. claim 1, wherein said at least one mitochondrial nucleic acid 11. A cytoplasmic male sterile hybrid plant of Cichorium sequence is at least two, at least three, at least four, at least and Lactuca, said cytoplasmic male Sterile hybrid plant com five, at least six, at least seven, at least eight, at least nine, or prises mitochondria of Lactuca and said mitochondria com ten mitochondrial nucleic acid sequences. prise at least one mitochondrial nucleic acid sequence 5. Cytoplasmic male Sterile Cichorium plant according to selected from the group consisting of SEQID No. 1, SEQID claim 1, wherein said Cichorium plant is selected from the No. 2, SEQID No. 3, SEQID No. 4, SEQID No. 5, SEQID group consisting of green chicory, radicchio rosso, red leaved No. 6, SEQID No. 7, SEQID No. 8, SEQID No. 9, and SEQ chicory, Treviso, white chicory, Sugar loaf, Belgian endive, ID No. 10. witloof. Catalogna, C. intybus var. foliosum, C. endivia, and 12. Cytoplasmic male sterile hybrid plant according to C. intybus L. var. sativum. claim 11, wherein said cytoplasmic male sterile hybrid plant 6. Cytoplasmic male Sterile Cichorium plant according to is a plant according to deposit NCIMB 42125. claim 1, wherein said Cichorium plant comprises Lactuca 13. Cytoplasmic male sterile hybrid plant according to mitochondria derived from a plant according to deposit claim 11, wherein said mitochondria are derived from a Lac NCIMB 41985. tuca plant according to deposit NCIMB 41985. 7. Cytoplasmic male Sterile Cichorium plant according to 14. Use of mitochondria of a Lactuca plant for obtaining a claim 1, wherein said Cichorium plant comprises a cytoplasm cytoplasmic male sterile Cichorium plant. maternally derived from a plant according to deposit NCIMB 15. Use according to claim 14, wherein said use comprises 42125. a protoplast fusion of a nuclear genome inactivated protoplast 8. Method for identifying a cytoplasmic malesterile Cicho of Lactuca as donor and a mitochondrial genome inactivated rium plant, said method comprises establishing in a sample of protoplast of Cichorium as acceptor. said cytoplasmic male sterile Cichorium plant one or more k k k k k