Abstract:
The present invention relates to a novel  Eustoma  having cytoplasmic male sterility and a method for breeding the same. More specifically, the present invention relates to a novel  Eustoma  having cytoplasmic male sterility, wherein the  Eustoma  substantially lacks pollen production functions due to insufficient stamen or pollen formation, and a method for breeding the same.

Description:
TECHNICAL FIELD 
       [0001]    The present invention relates to a novel  Eustoma  having cytoplasmic male sterility and a method for breeding the same. More specifically, the present invention relates to a novel  Eustoma  having cytoplasmic male sterility, wherein the  Eustoma  substantially lacks pollen production functions due to insufficient stamen or pollen formation, and a method for breeding the same. 
       BACKGROUND ART 
       [0002]      Eustoma  is a generic name for autogamous seed-propagated plants of the genus  Eustoma  in the family Gentianaceae and grows natively in regions of southern North America to northern Central America. These plants include two species: (1)  Eustoma grandiflorum  (English name: Prairie gentian, former scientific name:  E. russellianum  (Hook) G. Don ex Sweet or  Lisianth ( i ) us russellianus  Hook.) and (2)  Eustoma exaltatum  (English name: Seaside gentian or Catchfly gentian, former scientific name:  E. selenifolium  Salisb.). The plants are also known familiarly as  Lisianthus.  In 1835, the native species was brought to Scotland and designated as  Lisianthus russellianus  Hook. The plants were introduced to Japan in the 1930s and have been actively bred since then, mainly for cut flowers or potted plants.  Eustoma grandiflorum  is mainly used in such breeding. 
         [0003]      Eustoma  is known for flowers and ornamental plants of high ornamental and market value. Particularly, varieties having diverse characters have been produced as varieties for cut flowers, and these  Eustoma  varieties are regarded as one of the principal cut flower species (see Non Patent Literature 1). 
         [0004]    In general, first filial generation (F1) plants superior in character to their parents are utilized in major horticultural crops because of the benefits from heterosis. Among  Eustoma  cultivars highly demanded as cut flowers, first filial generation varieties are also the mainstream because this allows the plants to have disease resistance or high quality. 
         [0005]    For efficient seeding in plant production, breeders are required to supply highly pure seeds. The seed production of a first filial generation variety first involves manual emasculation of a seed parent followed by pollinating the stigma of the emasculated seed parent with the pollen of a pollen parent. 
         [0006]    The period when a flower of the seed parent can be successfully emasculated is usually limited to a stage immediately before flowering, and also tends to depend on weather conditions. The emasculation therefore presents operation problems in that the possible period of emasculation is rarely predictable. Since this operation is manually performed, pollen that has remained due to insufficient emasculation may cause the emergence of unintended self-pollinated seeds. Contamination by such self-pollinated seeds results in unfavorable reduction in the quality of seeds. For these reasons, there has been a demand for the development of a method for seed production of  Eustoma  by use of male sterility that eliminates the need of the manual emasculation of seed parents. 
         [0007]    Dwarf  Lisianthus  transformed by rolC gene transfer using  Agrobacterium rhizogenes  is known (Non Patent Literature 2), which reportedly has small flowers and loses pollen fertility at the same time with the disappearance of apical dominance (rol syndrome). The technique of Non Patent Literature 2 has been applied to a disclosed method for producing an F1 variety using  Lisianthus  having male sterility, wherein the variety is maintained by adventitious shoot regeneration (Patent Literature 1). The F1 variety produced according to the method, however, possesses undesired characters. Therefore, the method cannot be used in the breeding of cut flower varieties required to have marketable plant postures or heights. 
         [0008]    Cytoplasmic male sterility (CMS), one type of male sterility, is cytoplasmically inherited. The crossing of a cytoplasmic male sterile line with a male fertile line supplies highly pure seeds. In addition, the cytoplasmic male sterile line can be crossed with a maintainer line having the same nuclear genome as that of the cytoplasmic male sterile line and a normal cytoplasm to thereby easily maintain and propagate the line. Thus, such a method for seed production of a first filial generation variety by use of cytoplasmic male sterility is very highly practical and is used in a large number of major horticultural crops. Nonetheless, a method for producing a cytoplasmic male sterile  Eustoma  has not yet been reported, though the development of the method for seed production of  Eustoma  by use of male sterility has been demanded. Accordingly, seed production that requires emasculation operation is still performed in the seed production of  Eustoma  F1 varieties due to the absence of a seed production system using practical male sterile lines. 
         [0009]    Meanwhile, good flower shelf life that achieves the prolonged ornamental period of flowers resulting from delayed petal aging has been demanded. Examples of causes of petal aging include pollination and ethylene formation caused by damage on the stigma or style of a pistil (Non Patent Literature 3). 
         [0010]      Eustoma  having a deformed pistil, the stigma of which is kept closed to thereby prevent pollination and delay flower aging is known as a  Eustoma  variety having good flower shelf life (Patent Literature 2). 
         [0011]    Improvement in flower shelf life has required breeding a line having short filaments of stamens, or using a plant line having an organ structure changed to prevent pollination on its stigma physically, as in the deformed pistil mentioned above. 
         [0012]    As one condition, the prevention of pollen dispersion has been demanded for flowers and ornamental plants of high market value. All current  Eustoma  varieties, however, have stamens that produce pollen and therefore cause, for example, pollen dispersion, which is unfavorable due to the fouling of petals or clothing. 
         [0013]    Although problems associated with flower shelf life, etc. can be solved by use of male sterility, without changing useful characters, such a practical  Eustoma  having male sterility has not yet been developed. 
       CITATION LIST 
     Patent Literature 
       [0000]    
       
         [Patent Literature 1] Japanese Patent Laid-Open No. 9-107829 
         [Patent Literature 2] Japanese Patent No. 4133011 
       
     
       Non Patent Literature 
       [0000]    
       
         [Non Patent Literature 1] Nogyo Gijutsu Taikei (Agricultural Technology System (in Japanese)), Flowers and Ornamental Plants, Vol. 8, addendum, No. 6, p. 387-395, 2004, issued by Rural Culture Association Japan 
         [Non Patent Literature 2] The Tissue Culture (in Japanese), Vol. 19, No. 2, p. 50-55, 1993, New Science Co., Ltd. 
         [Non Patent Literature 3] Jissen Kaki Engei Gijutsu (Actual Practice of Ornament Horticultural Techniques (in Japanese)), Cultivation Management and Blooming Adjustment of  Lisianthus,  2003, issued by Seibundo Shinkosha Publishing Co., Ltd. 
         [Non Patent Literature 4] E. S. Mousavi, M. Behbahani, E. Hadavi, S. M. Miri (2012), CALLUS INDUCTION AND PLANT REGENERATION IN  LISIANTHUS  ( EUSTOMA GRANDIFLORIUM ), ANNIVERSARY EDITION TRAKIA JOURNAL OF SCIENCES, Vol. 10, No. 1, pp. 22-25 
         [Non Patent Literature 5] J. Duminil, M.-H. PEMONGE and R. J. PETIT (2002), Molecular Ecology Notes, vol. 2, p. 428-430 “A set of 35 consensus primer pairs amplifying genes and introns of plant mitochondrial DNA” 
       
     
       SUMMARY OF INVENTION 
     Technical Problem 
       [0021]    In light of the problems such as cumbersome emasculation during the F1 seed production of conventional  Eustoma  varieties, the problem of reduced seed quality, and challenges to flower shelf life or pollen dispersion as described above, the present invention is to provide a novel  Eustoma  having cytoplasmic male sterility and a method for producing the same. 
       Solution to Problem 
       [0022]    The present inventors have conducted diligent studies to attain the objective and consequently completed a breeding method which involves producing a novel  Eustoma  plant having cytoplasmic male sterility, breeding the  Eustoma  line having cytoplasmic male sterility, and obtaining a first filial generation variety using the cytoplasmic male sterile  Eustoma.    
         [0023]    Specifically, the present invention relates to a novel  Eustoma  having cytoplasmic male sterility and a method for breeding the same. More preferably, the present invention relates to a novel  Eustoma  having cytoplasmic male sterility, wherein the  Eustoma  substantially lacks pollen production functions due to insufficient stamen or pollen formation, and a method for breeding the same. 
         [0024]    Specifically, the present invention provides the following (1) to (34):
   (1) A  Eustoma  plant having cytoplasmic male sterility, or progeny thereof.   (2) The  Eustoma  plant or progeny thereof according to (1), wherein the  Eustoma  plant or progeny thereof has the nucleotide sequence represented by SEQ ID NO: 1 or 2.   (3) The  Eustoma  plant or progeny thereof according to (1) or (2), wherein the  Eustoma  plant or progeny thereof is a cytoplasmic male sterile plant produced from a cross pollination whose seed parent is a  Eustoma  plant having the nucleotide sequence represented by SEQ ID NO: 1 or 2 and whose pollen parent is an arbitrary  Eustoma  plant.   (4) The  Eustoma  plant or progeny thereof according to any of (1) to (3), wherein the  Eustoma  plant or progeny thereof is a cytoplasmic male sterile plant produced from a cross pollination whose seed parent is the  Eustoma  plant having the nucleotide sequence represented by SEQ ID NO: 1 or 2 and whose pollen parent is  E. grandiflorum.      (5) A partial plant body of the  Eustoma  plant or progeny thereof according to any of (1) to (4).   (6) A seed of the  Eustoma  plant or progeny thereof according to any of (1) to (4).   (7) A callus comprising cells of the  Eustoma  plant or progeny thereof according to any of (1) to (4).   (8) A  Eustoma  plant that is induced from the callus according to (7) and asexually propagated by tissue culture, or progeny thereof.   (9) A partial plant body of the  Eustoma  plant or progeny thereof according to (8).   (10) A method for preparing a  Eustoma  plant that is induced from the callus according to (7) and asexually propagated by tissue culture.   (11) A cytoplasm contained in the  Eustoma  plant or progeny thereof according to any of (1) to (4) and (8), the partial plant body according to any of (5) and (9), the seed according to (6), or the callus according to (7).   (12) A mitochondrion contained in the  Eustoma  plant or progeny thereof according to any of (1) to (4) and (8), the partial plant body according to any of (5) and (9), the seed according to (6), or the callus according to (7).   (13) A  Eustoma  plant having cytoplasmic male sterility which is designated by Deposition No. FERM BP-11506, or progeny thereof.   (14) A partial plant body of the  Eustoma  plant or progeny thereof according to (13).   (15) A seed of the  Eustoma  plant or progeny thereof according to (13).   (16) A cytoplasm contained in the  Eustoma  plant or progeny thereof according to (13), the partial plant body according to (14), or the seed according to (15).   (17) A mitochondrion contained in the  Eustoma  plant or progeny thereof according to (13), the partial plant body according to (14), or the seed according to (15).   (18) A callus of a  Eustoma  plant having cytoplasmic male sterility which is designated by Deposition No. FERM BP-11507.   (19) A  Eustoma  plant that is induced from the callus according to (18) and asexually propagated by tissue culture, or progeny thereof.   (20) A partial plant body of the  Eustoma  plant or progeny thereof according to (19).   (21) A method for preparing a  Eustoma  plant that is induced from the callus according to (18) and asexually propagated by tissue culture.   (22) A cytoplasm contained in the callus according to (18), the  Eustoma  plant or progeny thereof according to (19), or the partial plant body according to (20).   (23) A mitochondrion contained in the callus according to (18), the  Eustoma  plant or progeny thereof according to (19), or the partial plant body according to (20).   (24) A method for producing a first filial generation seed, comprising crossing the  Eustoma  plant or progeny thereof according to any of (1) to (4) and (8) as a seed parent with a  Eustoma  plant capable of being crossed with the seed parent plant as a pollen parent, and producing a first filial generation seed from the seed parent thus crossed.   (25) A method for producing a first filial generation seed, comprising crossing the  Eustoma  plant or progeny thereof according to (13) as a seed parent with a  Eustoma  plant capable of being crossed with the seed parent plant as a pollen parent, and producing a first filial generation seed from the seed parent thus crossed.   (26) A method for producing a first filial generation seed, comprising crossing a  Eustoma  plant regenerated from the callus according to (18), or progeny thereof as a seed parent with a  Eustoma  plant capable of being crossed with the seed parent plant as a pollen parent, and producing a first filial generation seed from the seed parent thus crossed.   (27) A first filial generation seed produced by the method according to any of (24) to (26).   (28) A first filial generation plant grown from the first filial generation seed according to (27).   (29) A method for producing a  Eustoma  plant, comprising successively back-crossing a  Eustoma  plant having the nucleotide sequence represented by SEQ ID NO: 1 or 2 in its cytoplasm with a  Eustoma  plant having a useful character to produce a  Eustoma  plant that has the useful character and expresses cytoplasmic male sterility.   (30) A method for producing a  Eustoma  plant, comprising successively back-crossing a  Eustoma  plant having cytoplasmic male sterility which is designated by Deposition No. FERM BP-11506, or progeny thereof with a  Eustoma  plant having a useful character to produce a  Eustoma  plant that has the useful character and expresses cytoplasmic male sterility.   (31) A method for producing a  Eustoma  plant, comprising successively back-crossing a  Eustoma  plant that is induced from a callus of a  Eustoma  plant having cytoplasmic male sterility which is designated by Deposition No. FERM BP-11507 and asexually propagated by tissue culture, or progeny thereof with a  Eustoma  plant having a useful character to produce a  Eustoma  plant that has the useful character and expresses cytoplasmic male sterility.   (32) The method for producing a  Eustoma  plant according to any of (29) to (31), wherein the  Eustoma  plant having a useful character is derived from  E. grandiflorum.      (33) A  Eustoma  plant produced by the method according to any of (29) to (32), or progeny thereof.   (34) A partial plant body of the  Eustoma  plant or progeny thereof according to (33).   
 
       Advantageous Effects of Invention  
       [0059]    Use of the novel  Eustoma  having cytoplasmic male sterility, provided by the present invention, achieves breeding a  Eustoma  plant and its F1 variety with excellent seed productivity, flower shelf life, and/or ornamental characteristics and producing high-quality seeds of its F1 variety. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0060]      FIG. 1  shows the cytoplasmic male sterile  Eustoma  of the present invention and a male fertile  Eustoma.    FIG. 1(A)  shows a male fertile line,  FIGS. 1(B) and 1(C)  show a cytoplasmic male sterile line having imperfectly developed stamens.  FIG. 1(D)  shows a cytoplasmic male sterile line having no stamen. 
           [0061]      FIG. 2  shows results of electrophoresis after PCR using, as templates, the total DNAs of the cytoplasmic male sterile  Eustoma  line (SSE-CMS cytoplasm: lane 1), wild  Eustoma  species (lanes 2 to 8) retained by Sakata Seed Corporation, and  Eustoma  cultivars (lanes 9 to 15) bred by Sakata Seed Corporation. The lane M indicates a molecular weight marker reference.  FIG. 2(A)  shows results obtained using Marker 1 which is a marker specific for the SSE-CMS  Eustoma  line.  FIG. 2(B)  shows results obtained using Marker 2 which is a marker specific for the SSE-CMS  Eustoma  line.  FIG. 2(C)  shows results obtained using nad5/4-5 which is a marker common to all  Eustoma  plants. 
           [0062]      FIG. 3  shows the nucleotide sequences of Marker 1 and Marker 2. 
       
    
    
     DESCRIPTION OF EMBODIMENTS 
       [0063]    Hereinafter, the present invention will be described in detail. 
       1. Cytoplasmic Male Sterile  Eustoma    
       [0064]    A method for breeding the  Eustoma  having male sterility of the present invention comprises screening  Eustoma  plant hybrids for a  Eustoma  having cytoplasmic male sterility. 
         [0065]    The “ Eustoma  plant” or the “ Eustoma ” according to the present invention refers to a plant of the genus  Eustoma  in the family Gentianaceae. This plant is a horticultural crop known familiarly as  Lisianthus  in Japan. 
         [0066]    Flowers, including  Eustoma  flowers, usually have sepals and petals within which stamens and pistils are present. Typical stamens consist of pollen-containing anthers and filaments. The “male sterility” according to the present invention refers to the state where imperfectly developed stamens cannot sufficiently produce pollen. The male sterility is exhibited by, but not limited to, for example, a  Eustoma  having no stamen or imperfectly developed stamens (see  FIG. 1 ). The “cytoplasmic male sterility” refers to a male sterile character maternally inherited by an organ-derived gene of the cytoplasm. 
         [0067]    In the present invention, the “progeny of a  Eustoma  plant having cytoplasmic male sterility” means a next-generation or later  Eustoma  plant that is obtained by the crossing of a female parent (seed parent)  Eustoma  plant having cytoplasmic male sterility with a male parent (pollen parent)  Eustoma  plant capable of being crossed with the plant and possesses maternally inherited male sterility. In the present invention, the “partial plant body” contains one or more cell(s) of the plant or a cytoplasm from one or more cell(s) of the plant and specifically means any of organs or tissues such as flowers, leaves, stems, and roots, cells (including protoplasts prepared from the cells) or cytoplasms from these organs or tissues, and a population of the cells or the cytoplasms. 
         [0068]    In the present specification, the scope of the “ Eustoma  plant which is designated by Deposition No. FERM BP-11506” also includes plants that have the character of cytoplasmic male sterility and are substantially equivalent to the plant. Specifically, the scope of the “ Eustoma  plant which is designated by Deposition No. FERM BP-11506” also includes, for example, mutants or gene recombinants of the  Eustoma  plant designated by Deposition No. FERM BP-11506 as long as the mutants or gene recombinants maintain cytoplasmic male sterility. 
         [0069]    In the present specification, the scope of the “callus of a  Eustoma  plant which is designated by Deposition No. FERM BP-11507” also includes calluses from which plants having the character of cytoplasmic male sterility are regenerated and which are substantially equivalent to the callus. Specifically, the scope of the “callus of a  Eustoma  plant which is designated by Deposition No. FERM BP-11507” also includes, for example, mutants or gene recombinants derived from the callus of a  Eustoma  plant which is designated by Deposition No. FERM BP-11507 and calluses derived from mutants or gene recombinants (maintaining cytoplasmic male sterility) of the  Eustoma  plant which is designated by Deposition No. FERM BP-11506, as long as plants regenerated therefrom maintain cytoplasmic male sterility. 
         [0070]    The cytoplasmic male sterile  Eustoma  according to the present invention has the following features:
   (1) This line eliminates the need for the emasculation of seed parents for the production of a first filial generation variety and therefore achieves economically efficient seed production.   (2) The crossing of this line with a male fertile line supplies highly pure seeds because of its male sterile character.   (3) This line can be crossed with a maintainer line having the same nuclear genome as that of the line and a normal cytoplasm to thereby easily maintain and propagate the line.   (4) This line, which is free from self-pollination, can suppress flower aging caused by pollination, resulting in good flower shelf life.   (5) This line has no pollen and therefore, does not foul petals or clothing by pollen dispersion.   
 
       2. Method for Producing Cytoplasmic Male Sterile  Eustoma    
       [0076]    The method for producing a male sterile  Eustoma  according to the present invention involves crossing a wild  Eustoma  plant species as a female parent with  Eustoma grandiflorum  as a male parent, screening individuals that exhibit male sterility among the progeny. An individual having no stamen or imperfectly developed stamens is screened for as the individual that exhibits male sterility. In order to further confirm that the male sterility is a character that is cytoplasmically inherited, the individual is back-crossed with the male fertile  Eustoma  line, and their progeny plants are confirmed to exhibit male sterility. 
       3. Method for Producing First Filial Generation Seed 
       [0077]    The cytoplasmic male sterile  Eustoma  produced by the method of the present invention can be successively back-crossed with a  Eustoma  plant having a preferred character to thereby obtain a preferred line having cytoplasmic male sterility as progeny. This preferred line having cytoplasmic male sterility can be used as a seed parent for obtaining a first filial generation seed (F1 seed). 
       4. Flower Shelf Life Test 
       [0078]    The cytoplasmic male sterile  Eustoma  according to the present invention can be evaluated for its flower shelf life as shown below. In the present specification, the “flower shelf life” means the “duration of flowering”. Accordingly, the “good flower shelf life” means a “relatively long flowering period from the start of flowering to the finish of flowering”. Whether the flowering period of a  Eustoma  is relatively long is determined by the comparative evaluation of a plurality of respective individuals of the normal male fertile  Eustoma  and the cytoplasmic male sterile  Eustoma  cultivated under the same environmental conditions. 
         [0079]    As an example of the flower shelf life test, a method for evaluating the shelf life of a cut flower will be described. In this method, appropriate numbers of normal male fertile individuals and male sterile individuals of  Eustoma  cultivated under the same conditions are first prepared as test materials. The flowers of these  Eustoma  individuals are collected almost simultaneously on the day of flowering (day when the opening of petals is observed) with their peduncles on. The peduncles are adjusted to a constant length (e.g., approximately 4 to 6 cm). Subsequently, the adjusted test materials are left standing in a temperature-controlled room having a constant temperature (preferably 18 to 22° C.), a constant humidity (preferably 55 to 65%), and 12-hour light/12-hour dark cycles such that the cut sections of their stems are dipped in water. Change in the appearance of the flowers is observed. 
         [0080]    The “start of flowering” is defined as the time when petals have opened. The “finish of flowering” is determined from the appearance of the petals (shriveling or withering of the petals) to measure the flowering period of each flower (period from the start of flowering to the finish of flowering). The averages of the respective measured flowering periods can be determined and compared between the normal male fertile individuals and the male sterile individuals to evaluate the flower shelf life of the male sterile  Eustoma  of the present invention. 
       5. Development of Molecular Markers to Distinguish the Cytoplasm 
       [0081]    Mitochondrial genomic sequences are compared between the cytoplasmic male sterile  Eustoma  line prepared by the present invention and a conventionally known  Eustoma  plant. A region specific for the cytoplasmic male sterile  Eustoma  line can be identified to thereby prepare a molecular marker serving as a distinguishing factor. The molecular marker can be detected according to a method well known to those skilled in the art, such as PCR. Use of the molecular marker can demonstrate that the cytoplasmic male sterile  Eustoma  line according to the present invention distinctly differs not only in morphological feature but in molecular biological feature from conventionally known  Eustoma  plants. 
         [0082]    The nucleotide sequences represented by SEQ ID NOs: 1 and 2 can be used as molecular markers to distinguish the cytoplasmic male sterile  Eustoma  line of the present invention. Alternatively, a  Eustoma  line having a nucleotide sequence derived from the nucleotide sequence represented by SEQ ID NO: 1 or 2 by the deletion, substitution, or addition of one or several base(s) can also be regarded as substantially the same line as the cytoplasmic male sterile  Eustoma  line of the present invention, as long as the nucleotide sequence is amplified and detected in the same way as in the nucleotide sequence of SEQ ID NO: 1 or 2. For example, a nucleotide sequence having 80%, preferably 90%, more preferably 95% or higher homology to the nucleotide sequence represented by SEQ ID NO: 1 or 2 can be used as a molecular marker to distinguish the cytoplasmic male sterile  Eustoma  line. 
       6. Callus Induction, Propagation, and Redifferentiation of Cytoplasmic Male Sterile  Eustoma  Line 
       [0083]    The cytoplasmic male sterile  Eustoma  line prepared by the present invention may be asexually propagated by tissue culture. The cytoplasmic male sterile  Eustoma  line can be asexually propagated by, for example, a method for callus induction and redifferentiation disclosed in Non Patent Literature 4. 
         [0084]    Specifically, the surface of the leaf disc of the cytoplasmic male sterile  Eustoma  line cultivated in a greenhouse is sterilized, then placed to a callus induction medium, and cultured to induce a callus. Then, the formed callus is transferred to a redifferentiation medium and cultured to induce a shoot. Subsequently, the formed shoot is transferred to a rooting medium to induce rooting. In this way, a plant is regenerated. Those skilled in the art can appropriately set culture conditions for callus induction, propagation, and redifferentiation according to a technique known in the art. 
         [0085]    The contents of all patents and references explicitly cited herein are incorporated herein by reference in their entirety. Also, the contents described in the specification and drawings of Japanese Patent Application No. 2012-213296 (filed on Sep. 27, 2012) on which claim for the priority of the present application is based are incorporated herein by reference in their entirety. 
       EXAMPLES 
       [0086]    The present invention will be described specifically with reference to Examples below. However, the present invention is not limited to these Examples by any means. 
       Example 1 
     Production of Novel  Eustoma  Plant Having Male Sterility 
       [0087]    The  Eustoma  having male sterility of the present invention was produced at the Misato Research Station of Sakata Seed Corporation by screening from hybrids between a native  Eustoma  plant of unidentified species name introduced from the United States of America and  Eustoma grandiflorum.    
       Breeding Process of  Eustoma  Plant According to the Present Invention  
       [0088]    Approximately 60 sets in total of hybrids were previously prepared between dozen lines of native  Eustoma  plants of unidentified species name retained by Sakata Seed Corporation and parent lines ( E. grandiflorum ) of different origins also retained by Sakata Seed Corporation to obtain F1 seeds. These F1 seeds were sown, and the phenotypes of the F1 plants were checked. Then the F1 populations were screened for two or three appropriate individuals, which were then subjected to mass crossing to obtain progeny (hereinafter, referred to “F2 generation”) seeds. 
         [0089]    The F2 generation seeds were sown and approximately 50 to 100 individuals were cultivated. Phenotypic characters included in the F2 populations were checked. As a result, the segregation of the diameters, colors, and types of flowers, and earliness was seen in most of the F2 populations. Among these populations, an F2 population derived from F1 seeds (hereinafter, referred to as Set A) obtained by the crossing of a native  Eustoma  plant E-1 of unidentified species name retained by Sakata Seed Corporation as a female parent with a parent line G-1 ( E. grandiflorum ) as a male parent offered, unexpectedly, a plurality of individuals that exhibited a male sterile character that had previously been unknown about  Eustoma  plants. One individual was selected from among these individuals and crossed with 4 parent lines G-2, G-3, G-4, and G-5 retained by Sakata Seed Corporation as pollen parents to obtain progeny seeds (F2BC1 generations; referred to as Sets B, C, D, and E, respectively). The generations are indicated by BC1, BC2, . . . , with respect to the number of backcrosses to  E. grandiflorum  with a CMS (Cytoplasmic male sterile) line in order to avoid confusion. 
         [0090]    These progeny seeds (F2BC1 generations) were sown and the characters of 12 individuals per line were checked. As a result, all lines and individuals exhibited a male sterile character with no stamen or imperfectly developed stamens. Four F2BC1-generation individuals derived from Set C (hereinafter, referred to as ms-1), two F2BC1-generation individuals derived from Set D (hereinafter, referred to as ms-2), three and four F2BC1-generation individuals derived from Set E (hereinafter, referred to as ms-3 and ms-4) were selected and crossed with pollen parents, i.e., parent lines G-3 and G-6 for ms-1, parent lines G-4 and G-7 for ms-2, parent lines G-5 and G-8 for ms-3, and parent lines G-5 and G-9 for ms-4 to obtain their respective progeny seeds (F2BC2 generations). 
         [0091]    The progeny seeds (F2BC2 generations) were sown, and the phenotypic characters of the progeny were checked. As a result, progeny populations of lines obtained by the crossing of ms-1 with G-3 and G-6 had some male sterile individuals having imperfectly developed stamens, while the other combinations exhibited a male sterile character with no stamen. These male sterile individuals were crossed with approximately 100 parent lines to obtain progeny seeds (F2BC3 generations). 
         [0092]    The progeny seeds (F2BC3 generations) were sown and the phenotypic characters of the progeny was examined. As a result, the progeny was confirmed to have a male sterile character. This demonstrated that a cytoplasmic male sterile line exhibiting stable maternal inheritance was successfully produced. Thus, its cytoplasm was designated as an SSE-CMS cytoplasm. The F2BC1 seed exhibiting stable cytoplasmic male sterility was internationally deposited with National Institute of Technology and Evaluation (NITE) International Patent Organisms Depositary (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan) on Jul. 20, 2012 (indication given by the depositor for identification: 08P-81S; Deposition No. FERM BP-11506). 
       Example 2 
     Validation of Reproducibility of Male Sterility 
       [0093]    In order to confirm the stable emergence of the male sterile character, the  Eustoma  plant E-1 was crossed as a female parent with the parent line G-10 ( E. grandiflorum ) as a male parent to produce F1 seeds (hereinafter, referred to as Set F). The seeds of Sets A and F were sown, and 6 individuals per set were selected and subjected to mass crossing. As a result, the individuals derived from Set A produced F2-generation seeds at a yield of 0.22 g for 56 pods, while the individuals derived from Set F produced F2-generation seeds at a yield of 0.47 g for 59 pods. The F2-generation seeds of each set were sown and examined for the rate of emergence of male sterile individuals. As a result, 19 out of 92 F2-generation individuals derived from Set A and 14 out of 88 F2-generation individuals derived from Set F exhibited the male sterile character. 
         [0094]    These results showed that the male sterile character emerges stably. 
       Example 3 
     Flower Shelf Life Test on  Eustoma  Variety Having Male Sterility 
       [0095]    Male sterile lines and fertile lines were subjected to a flower shelf life test. 
       (1) Test Materials 
       [0096]    Two types of  Eustoma  plants were used as test materials: F1 individuals of normal male fertile  Eustoma  and F1 individuals of cytoplasmic male sterile  Eustoma.  The flowers (which all appeared to have started flowering) of these two types of  Eustoma  individuals were collected from a  Eustoma  cultivation field with their peduncles on. The peduncles were adjusted to a length of 4 cm. The stigmas of the male fertile  Eustoma  individuals were hand-pollinated with pollen on the assumption that these individuals would be transported. Also, the cytoplasmic male sterile  Eustoma  individuals were or were not hand-pollinated with the pollen of a male fertile line, and these individuals were both subjected to the test. 
       (2) Test Method 
       [0097]    The flower shelf life test was conducted with each test material put into a test tube filled with tap water and carried out in a temperature-controlled room having a temperature of 20° C., a humidity of 60% (±5%), and 12-hour on/12-hour off cycles of a general fluorescent lamp (I-line White Rapid 40 W). During this test, water was not replaced because pollution, etc., was not particularly observed in the water. 
       (3) Evaluation and Determination 
       [0098]    The “start of flowering” is defined as the time when petals have opened. The “finish of blooming” was determined on the basis of petal appearance (shriveling or withering of flowers) by a breeder. 
       (4) Test Results 
       [0099]    The cytoplasmic male sterile  Eustoma  pollinated with the pollen of a fertile line did not much differ in flower shelf life from the male fertile  Eustoma.  This demonstrated that the test conditions were conditions under which the genetic background of the line used did not influence flower shelf life. Since the cytoplasmic male sterile  Eustoma  produces no pollen, its flower shelf life was as much as 7 days longer on average than that of the male fertile line (Table 1). 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Flower shelf life test 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 The number of individuals at finish of flowering 
               
             
          
           
               
                   
                   
                 4 days 
                 5 days 
                 6 days 
                 7 days 
                 8 days 
                 9 days 
                 10 days 
                 11 days 
               
               
                 Material 
                 Treatment method 
                 later 
                 later 
                 later 
                 later 
                 later 
                 later 
                 later 
                 later 
               
               
                   
               
               
                 Male fertile line 
                 Hand-pollination 
                 12 
                 17 
                 15 
                 9 
                 5 
                 2 
                 1 
               
               
                 Male sterile line 
                 Hand-pollination with 
                 3 
                 11 
                 14 
                 10 
                 11 
                 8 
                 3 
               
               
                   
                 pollen derived from 
               
               
                   
                 fertile line 
               
               
                 Male sterile line 
                 Without pollination 
                   
                   
                   
                 3 
                 3 
                 4 
                 6 
                 7 
               
               
                   
               
             
          
           
               
                   
                   
                   
                 The total 
                 The average 
               
               
                   
                   
                 The number of individuals at finish of flowering 
                 number of 
                 days until 
               
             
          
           
               
                   
                   
                 12 days 
                 13 days 
                 14 days 
                 15 days 
                 16 days 
                 17 days 
                 18 days 
                 examined 
                 finish of 
               
               
                 Material 
                 Treatment method 
                 later 
                 later 
                 later 
                 later 
                 later 
                 later 
                 later 
                 individuals 
                 flowering 
               
               
                   
               
               
                 Male fertile line 
                 Hand-pollination 
                   
                   
                   
                   
                   
                   
                   
                 61 
                 5.8 
               
               
                 Male sterile line 
                 Hand-pollination with 
                   
                   
                   
                   
                   
                   
                   
                 60 
                 6.9 
               
               
                   
                 pollen derived from 
               
               
                   
                 fertile line 
               
               
                 Male sterile line 
                 Without pollination 
                 6 
                 8 
                 5 
                 5 
                 2 
                 4 
                 7 
                 60 
                 12.8 
               
               
                   
               
             
          
         
       
     
       Example 4 
     Development of Molecular Markers to Distinguish SSE-CMS   Eustoma  Line 
       [0100]    The SSE-CMS  Eustoma  line produced by the present invention exhibited a phenotypic feature that was not found in conventional cultivars or wild species. Meanwhile, a molecular marker to distinguish the SSE-CMS  Eustoma  line was prepared in order to demonstrate that this line also differed from conventionally known  Eustoma  plants from the standpoint of molecular biology. 
         [0101]    Consensus primers for mitochondrial DNA described in Non Patent Literature 5 were used to carry out PCR analysis with the SSE-CMS  Eustoma  line and an existing  Eustoma  line as templates. As a result, markers differing in the sizes of the amplified DNA fragments were found. Two types of markers, i.e., nad4L/orf25 and nad7/4-5, were selected from among such polymorphic markers, and the nucleotide sequences of the amplified fragments were analyzed. As a result, regions specific for the SSE-CMS  Eustoma  line were identified. In order to use these regions as distinctive factors, primers shown in Table 2 were designed, and PCR analysis (30 repetitive cycles each involving thermal denaturation at 94° C. for 1 minute, annealing at 65° C. for 1 minute, and elongation reaction at 72° C. for 1 minute) was carried out. As a result, 2 types of molecular markers, “Marker 1” and “Marker 2”, which allowed amplification of DNA fragments only from the SSE-CMS  Eustoma  line were successfully developed. Marker 1 is a marker that allows amplification of the 323-bp DNA fragment represented by SEQ ID NO: 1 using primers orf25-F and orf25-R ( FIG. 3 ). Marker 2 is a marker that allows amplification of the 492-bp DNA fragment represented by SEQ ID NO: 2 using primers nad7-F and nad7-R ( FIG. 3 ). 
         [0102]    The markers prepared by the approach described above were used to carry out a PCR test using, as templates, 190 existing  Eustoma  lines ( E. grandiflorum ) bred by Sakata Seed Corporation and 34 lines of wild  Eustoma  species retained by Sakata Seed Corporation. As a result, the line carrying these two types of nucleotide sequences was confirmed to be only the SSE-CMS  Eustoma  line. The part of the test results are shown in  FIG. 2 . The experiments of  FIGS. 2(A) to 2(C)  were conducted under the following conditions: 
       FIG. 2(A): Marker 1: Marker Specific for SSE-CMS  Eustoma  Line 
       [0103]    The nucleotide sequence (323 bp) represented by SEQ ID NO: 1 was amplified by PCR (30 cycles each involving thermal denaturation at 94° C. for 1 minute, annealing at 65° C. for 1 minute, and elongation reaction at 72° C. for 1 minute) using primers orf25-F and orf25-R. 
       FIG. 2(B): Marker 2: Marker Specific for SSE-CMS  Eustoma  Line 
       [0104]    The nucleotide sequence (492 bp) represented by SEQ ID NO: 2 was amplified by PCR (30 cycles each involving thermal denaturation at 94° C. for 1 minute, annealing at 65° C. for 1 minute, and elongation reaction at 72° C. for 1 minute) using primers nad7-F and nad7-R. 
         [0000]      FIG. 2(C) : nad5/4-5: Marker Common to All  Eustoma  Plants 
         [0105]    DNA fragments of approximately 1.5 kb were amplified from all lines by PCR (30 cycles each involving thermal denaturation at 94° C. for 1 minute, annealing at 60° C. for 1 minute, and elongation reaction at 72° C. for 1 minute) using primers nad5/4 and nad5/5. 
         [0106]    Consequently, the SSE-CMS  Eustoma  line produced by the present invention was shown to differ from conventionally known  Eustoma  plants. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Primers used in the present invention 
               
               
                 and their nucleotide sequences 
               
             
          
           
               
                 Marker 
                 Primer 
                 Nucleotide sequence 
               
               
                 name 
                 name 
                 (5′→3′) 
               
               
                   
               
               
                 nad4L/ 
                 orf25 
                 CTGTYTTTTCGCACTTAGGC 
               
               
                 orf25 
                 (SEQ ID NO: 7) 
                   
               
               
                   
                 nad4L 
                 GTCCGRGGTACTATTGCTGT 
               
               
                   
                 (SEQ ID NO: 8) 
                   
               
               
                   
               
               
                 nad7/ 
                 nad7/4 
                 TGTCCTCCATCACGATVTCG 
               
               
                 4-5 
                 (SEQ ID NO: 9) 
                   
               
               
                   
                 nad7/5 
                 CCAAATTCTCCTTTAGGTGC 
               
               
                   
                 (SEQ ID NO: 10) 
                   
               
               
                   
               
               
                 Marke1 
                 orf25-F 
                 CTACTGAATCCAAGCGAGTGG 
               
               
                 (SEQ ID 
                 (SEQ ID NO: 3) 
                   
               
               
                 NO: 1) 
                 orf25-R 
                 TAGGTAGAGCTGGGCGAGAA 
               
               
                   
                 (SEQ ID NO: 4) 
                   
               
               
                   
               
               
                 Marker2 
                 nad7-F 
                 AACCACTAACACCTTCCTCGT 
               
               
                 (SEQ ID 
                 (SEQ ID NO: 5) 
                   
               
               
                 NO: 2) 
                 nad7-R 
                 GCTCTATCTACGTATCTTG 
               
               
                   
                 (SEQ ID NO: 6) 
                 AAGTTTC 
               
               
                   
               
               
                 nad5/ 
                 nad5/4 
                 CCAATTTTTGGGCCAATTCC 
               
               
                 4-5 
                 (SEQ ID NO: 11) 
                   
               
               
                   
                 nad5/5 
                 CATTGCAAAGGCATAATGAT 
               
               
                   
                 (SEQ ID NO: 12) 
               
               
                   
               
             
          
         
       
     
       Example 5 
     Callus Induction, Growth, and Redifferentiation of SSE-CMS  Eustoma  Line 
       [0107]    In order to asexually propagate, by tissue culture, the SSE-CMS  Eustoma  line prepared by the present invention, the leaf disc of the SSE-CMS  Eustoma  line cultivated in a greenhouse was collected. The surface of leaf disc was sterilized for 10 minutes using a 1% sodium hypochlorite solution and rinsed with sterile water. The sterilized leaf disc was placed to an MS medium supplemented with 1.5 mg/l NAA to induce a callus. The callus was transferred to a B5 medium supplemented with 0.5 mg/l GA3 and 1.5 mg/l BA to induce a shoot. The formed shoot was transferred to a plant hormone-free B5 medium for rooting to regenerate a plant, which was then confirmed to be male sterile. 
         [0108]    The callus prepared by the method described above was internationally deposited with National Institute of Technology and Evaluation (NITE) International Patent Organisms Depositary (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan) on Jul. 26, 2012 (indication given by the depositor for identification: 12S-134C; Deposition No. FERM BP-11507). 
       INDUSTRIAL APPLICABILITY 
       [0109]    Use of the novel  Eustoma  having cytoplasmic male sterility, provided by the present invention, achieves breeding a  Eustoma  plant and its F1 variety with excellent seed productivity, flower shelf life, and/or ornamental characteristics and the producing high-quality seeds of its F1 variety.