Cytoplasmically male-sterile Petunia plants, intergeneric hybrids thereof, and methods for producing the same.

Abstract

Disclosed are a cytoplasmic male sterile plant of the genus Petunia that has DNA derived from the mitochondrial genome of a plant of the genus Nicotiana in the mitochondrial genome, an intergeneric hybrid plant with the cytoplasmic male sterile plant of the genus Petunia, or the posterity of the same. This cytoplasmic male sterile plant of the genus Petunia forms a stable phyletic line in which the growth of young seedlings is improved and no fertility restoration is induced. Moreover, this plant achieves cytoplasmic diversification of CMS.

Description

Technical Field

[0001] The present invention relates to a cytoplasmic male sterile plant of the genus Petunia, a hybrid plant with said cytoplasmic male sterile plant of the genus Petunia, or a progeny thereof. The present invention also relates to a method for producing the same.

Background Art

[0002] Petunia is a general term for horticultural species of the genus Petunia in the Solanaceae family, and approximately 16 species of the genus Petunia grow naturally in the Americas. The horticultural petunia is *Petunia × hybrida*, which is derived from an interspecific hybrid between *Petunia axillaris* and *Petunia integrifolia*. Since breeding of petunia began in the United Kingdom in the 1830s, a wide variety of cultivars with different flower colors, flower diameters and growth habits have been developed, and today it is one of the most important flower crops for flower beds and potted plants worldwide (Non-Patent Document 1).

[0003] Generally, plant cultivars include inbred lines and first filial generation (hereinafter referred to as "F1") cultivars, and F1 cultivars are widely used in major crops. F1 cultivars have great advantages such as vigorous growth, fast growth and increased yield due to heterosis. Furthermore, F1 cultivars can be expected to have improved resistance to pests and diseases, and improved environmental adaptability such as cold tolerance and heat tolerance due to their vigorous growth.

[0004] Furthermore, although the genotype of F1 cultivars is heterozygous, all individuals have the same genotype, so the phenotype exhibits extremely high uniformity. This increases the marketability of the product. Furthermore, useful traits controlled by dominant genes can be accumulated in the parents of F1 cultivars, enabling rapid breeding.

[0005] Due to the above advantages, F1 cultivars have become the mainstream of cultivated varieties in major crops.

[0006] When producing seeds from F1 varieties, inbreeding (self-pollination) lines are generally used as both parents, and the seed parent and pollen parent are selected from combinations that exhibit strong hybrid vigor.

[0007] Seed parents need to be emasculated to prevent self-fertilization, but manual emasculation is extremely labor-intensive. Therefore, by using cytoplasmic male sterility (CMS) lines, which are genetically male-sterile, as seed parents, manual emasculation becomes unnecessary, and F1 seeds can be produced economically and in large quantities. Commercial production systems for F1 seeds using CMS have been established for sunflowers, sugar beets, wheat, carrots, onions, leeks, cabbage, broccoli, cauliflower, radishes, and Chinese cabbage.

[0008] Cytoplasmic male-sterile lines of petunias were reportedly obtained by H.L. Everett and H.W. Gabelman by crossing petunia (P. hybrida) as the pollen parent with an unspecified wild species (presumably P. axillaris, P. integrifolia, or P. parodii) as the seed parent. This information suggests that cytoplasmic male-sterile lines of petunias are alloplasmic lines, and that the combination of nucleus and cytoplasm resulted in cytoplasmic male sterility.

[0009] However, the record of the origin of CMS in petunias is unclear, and its origin remains unknown. Furthermore, it remains possible that the mitochondrial DNA (mtDNA) encoding CMS arose not through true heterocytoplasmic male sterility, but through rearrangement induced by combining the nuclear genome of one species with the mitochondrial genome of another. Petunia CMS was introduced into petunias through backcrossing and spread among breeders, but only one type of CMS has been used to date. The causative gene for petunia CMS was sequenced by Young and Hanson in 1987 and is called the pcf gene (Petunia CMS-associated Fused gene) (Non-Patent Literature 2).

[0010] While there are many academic studies on CMS (cultivation-modifying musculoskeletal systems) using the pcf gene in petunias (hereinafter referred to as "pcf-CMS"), in practical applications, it has been reported that these systems exhibit various undesirable traits, such as stunted flower bud development, reduced flower size, and delayed flowering (Non-Patent Literature 3). These undesirable traits depend on the genotype of the line being used for CMS, and it is possible to use these systems by selecting lines in which the expression of undesirable traits is minimal. However, this has disadvantages, such as the limited number of parent lines that can be used and the need for careful prototyping before product release.

[0011] Furthermore, it is known that male sterility in "pcf-CMS" lines can be restored by a single dominant or multiple fertility restoration genes, and in some lines, it may be difficult to introduce male sterility. Moreover, even when male sterility appears stable, it is known that male sterility in "pcf-CMS" lines can be restored by environmental changes (Non-Patent Literature 3). As described above, the "pcf-CMS" lineage of petunias faces many challenges, and therefore, varieties using the "pcf-CMS" lineage are limited to Farao Seeds' Gioconda and Capri series, and there is a need for the development of new CMS lines with different origins. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Agricultural Technology System, Floriculture Edition, Volume 8, Annual and Biennial Plants: Petunias, pp. 372-4 to 372-9, published by the Association for Rural and Fishing Village Culture. [Non-Patent Document 2] JD Gillman et al, "Cytoplasmic Male Sterility and Fertility Restoration in Petunia", chapter 6, pp.107-129, Petunia, DOI (2009) [Non-Patent Document 3] MLK Kaul, Male Sterility in Higher Plants, pp.809-810, Springer-Verlag, (1998) [Overview of the project] [Problems that the invention aims to solve]

[0013] The inventors of this invention investigated the breeding and utilization of the existing "pcf-CMS" line of petunias and found that, in addition to various issues such as the cessation of flower bud development and delayed flowering that were previously known, a major problem was the reduced growth rate of seedlings.

[0014] Furthermore, it is known that male sterility in conventional pcf-CMS lines can be restored through single-factor dominance or multiple fertility restoration genes.

[0015] Furthermore, existing pcf-CMS lines utilize a wild species of the genus Petunia of unknown origin, and this is the only existing petunia CMS line that uses this species. Reliance on a single cytoplasm raises concerns about genetic vulnerability, as seen in the case of maize F1 varieties using T-type CMS that suffered extensive damage from the T-race of sesame leaf spot disease. For this reason, diversification of CMS cytoplasm has been desired.

[0016] Therefore, the present invention relates to the existing "pcf-CMS" system described above. Question issue For example, decreased growth rate or recovery of fertility in young seedlings. In light of this , new The present invention aims to provide a cytoplasmic male-sterile lineage. Furthermore, it aims to achieve diversification of the CMS cytoplasm. In addition, the present invention aims to provide a method for producing F1 seeds of petunias using this novel cytoplasmic male-sterile lineage. [Means for solving the problem]

[0017] The present inventors have now demonstrated that by performing asymmetric cell fusion using Nicotiana suaveolens as the cytoplasm donor parent and petunia having normal cytoplasm as the cytoplasm recipient parent, We have succeeded in creating a novel cytoplasmically male-sterile Petunia plant. In one aspect of the present invention, We have successfully created a novel cytoplasmic male-sterile petunia that does not experience a decrease in seedling growth, i.e., exhibits improved seedling growth. Furthermore, we have found that by using this novel cytoplasmic male-sterile petunia, F1 seeds of petunias that do not experience a decrease in seedling growth can be obtained. In addition, the obtained cytoplasmic male-sterile petunia utilizes cytoplasm derived from Tobacco plants, and it is thought that the fertility restoration genes of conventionally known Petunia plants do not function. Since there are no Tobacco-derived cytoplasmic fertility restoration genes in the Petunia plants, the cytoplasmic male sterility is considered to be stable. Therefore, we have succeeded in obtaining a stable cytoplasmic male-sterile line that does not cause fertility restoration. Furthermore, since it is different from existing cytoplasmic male-sterile lines, diversification of CMS cytoplasm is also achieved. This invention is based on these findings.

[0018] In other words, the present invention provides the following invention.

[0019] <1> A cytoplasmic male sterile Petunia plant having DNA derived from the mitochondrial genome of a Nicotiana plant in its mitochondrial genome, or a hybrid plant with said cytoplasmic male sterile Petunia plant, or a progeny thereof. <2> The cytoplasmic male sterile Petunia plant, or the hybrid plant with said cytoplasmic male sterile Petunia plant, or the progeny thereof according to <1>, wherein said Nicotiana plant is Nicotiana suaveolens.

[0020] <3> The cytoplasmic male sterile Petunia plant, or the hybrid plant with said cytoplasmic male sterile Petunia plant, or the progeny thereof according to <1> or <2>, wherein said cytoplasmic male sterile Petunia plant is derived from Petunia hybrida or an interspecific hybrid plant thereof. <4> The cytoplasmic male sterile Petunia plant, or the hybrid plant with said cytoplasmic male sterile Petunia plant, or the progeny thereof according to any one of <1> to <3>, wherein said cytoplasmic male sterile Petunia plant is derived from a plant obtained by performing asymmetric cell fusion using a Nicotiana plant as a cytoplasm donor parent.

[0021] <5> The cytoplasmic male sterile Petunia plant, or the hybrid plant with said cytoplasmic male sterile Petunia plant, or the progeny thereof according to any one of <1> to <4>, wherein the hybrid plant with said cytoplasmic male sterile Petunia plant is derived from an intergeneric hybrid plant of a Petunia plant and a Calibrachoa plant. <6> The cytoplasmic male sterile Petunia plant, or the hybrid plant with said cytoplasmic male sterile Petunia plant, or the progeny thereof according to any one of <1> to <5>, comprising a mitochondrial genome derived from the plant specified by accession number FERM BP-22398.

[0022] <7> The mitochondrial genome marker identified by using one or more primers selected from the group consisting of primer sets having the nucleotide sequences shown in SEQ ID NOs. 59 and 60, and primer sets having the nucleotide sequences shown in SEQ ID NOs. 67 and 68, is of the Nicotiana suaveolens type. <1> ~ <6> Any cytoplasmically sterile Petunia plant, or a hybrid plant with the aforementioned cytoplasmically sterile Petunia plant, or their offspring.

[0023] <8> The plant having the mitochondrial genome identified by accession number FERM BP-22398, <1> ~ <7> Any cytoplasmically sterile Petunia plant, or a hybrid plant with the aforementioned cytoplasmically sterile Petunia plant, or their offspring. <9> The aforementioned, identified by accession number FERM BP-22398. <1> ~ <7> Any cytoplasmically sterile Petunia plant, or a hybrid plant with the aforementioned cytoplasmically sterile Petunia plant, or their offspring.

[0024] <10> The above is obtained by asymmetric cell fusion using a cytoplasmically male-sterile Petunia plant or a hybrid plant with a cytoplasmically male-sterile Petunia plant having the mitochondrial genome of a plant identified by accession number FERM BP-22398 as the cytoplasmic donor parent, and a Petunia plant or a hybrid plant with a Petunia plant having normal cytoplasm as the cytoplasmic recipient parent. <1> ~ <9> Any cytoplasmically sterile Petunia plant, or a hybrid plant with the aforementioned cytoplasmically sterile Petunia plant, or their offspring.

[0025] <11> The aforementioned <1> ~ <10> Any cytoplasmically sterile Petunia plant, or a hybrid plant with the aforementioned cytoplasmically sterile Petunia plant, or a part of their progeny. <12> The aforementioned <1> ~ <10> Any cytoplasmically sterile plant of the genus Petunia, or a hybrid plant with the aforementioned cytoplasmically sterile plant of the genus Petunia, or the seeds of their offspring. <13> The aforementioned <1> ~ <10> Any cytoplasmically sterile Petunia plant, or a hybrid plant with the aforementioned cytoplasmically sterile Petunia plant, or their offspring, <11> A part of the plant described above, or the aforementioned <12> The mitochondrial genome contained in the seeds described above.

[0026] <14> A method for producing cytoplasmically male-sterile Petunia plants, hybrids with said cytoplasmically male-sterile Petunia plants, or their offspring, comprising the step of performing asymmetric cell fusion using a Tobacco plant as the cytoplasm-donor parent and a Petunia plant having normal cytoplasm or a hybrid plant with a Petunia plant as the cytoplasm-receiving parent. <15> The tobacco plant is Nicotiana suaveolens, as mentioned above. <14> The manufacturing method described above.

[0027] <16> A method for producing a cytoplasmically sterile petunia plant with an improved mitochondrial genome, a hybrid plant with a cytoplasmically sterile petunia plant, or a progeny thereof, comprising the step of performing asymmetric cell fusion using a cytoplasmically sterile petunia plant having DNA derived from the mitochondrial genome of a tobacco plant in its mitochondrial genome, or a hybrid plant with the said cytoplasmically sterile petunia plant, or a progeny thereof, as the cytoplasmic donor parent, and a petunia plant having normal cytoplasm or a hybrid plant with a petunia plant as the cytoplasmic recipient parent. <17> The cytoplasmically male-sterile Petunia plants are derived from Petunia hybrida or interspecific hybrids thereof. <14> ~ <16> One of the manufacturing methods. <18> The hybrid plant with the cytoplasmically male-sterile Petunia plant is derived from an intergeneric hybrid plant between a Petunia plant and a Calibrachoa plant. <14> ~ <17> One of the manufacturing methods. <19> The aforementioned <1> ~ <10> A method for producing first-generation hybrid seeds, comprising using any cytoplasmically sterile Petunia plant, or a hybrid plant with the said cytoplasmically sterile Petunia plant, or their progeny, as seed parents, crossing them with a Petunia plant capable of crossing with the said plant and an intergeneric hybrid derived therefrom as pollen parents, and collecting first-generation hybrid seeds from the seed parents after crossing. <20> The aforementioned <19> Hybrid first-generation seeds produced by the method described herein, or hybrid first-generation plants grown from such seeds, their progeny, or parts of such plants.

[0028] <21> The aforementioned <1> ~ <10> A method for producing petunia plants that express cytoplasmic male sterility and intergeneric hybrid plants derived therefrom, comprising successively backcrossing any petunia plant and an intergeneric hybrid plant derived therefrom with any cytoplasmic male sterile petunia plant, or a hybrid plant with the said cytoplasmic male sterile petunia plant, or their progeny, and replacing the cytoplasm.

[0029] <22> A primer set comprising a primer having the nucleotide sequence shown in SEQ ID NO: 59 and a primer having the nucleotide sequence shown in SEQ ID NO: 60. <23> A primer set comprising a primer having the nucleotide sequence shown in SEQ ID NO: 67 and a primer having the nucleotide sequence shown in SEQ ID NO: 68. [Effects of the Invention]

[0030] According to the present invention, A novel cytoplasmically male-sterile Petunia plant was obtained, and according to one aspect of the present invention, This invention provides a cytoplasmically sterile petunia plant that does not exhibit the reduced seedling growth seen in existing varieties, i.e., a plant with improved seedling growth. By utilizing this novel cytoplasmically sterile petunia plant, it becomes possible to efficiently collect F1 seeds of petunia plants with improved seedling growth.

[0031] While it is known that male sterility in existing pcf-CMS lines can be restored through single-factor dominance or multiple fertility restoration genes, the cytoplasmic male sterility line according to the present invention utilizes cytoplasm derived from Tobacco plants. Therefore, conventionally known fertility restoration genes from Petunia plants are not thought to function, and cytoplasmic fertility restoration genes derived from Tobacco plants are also absent in Petunia plants. Consequently, the present invention provides a stable cytoplasmic male sterility line that does not induce fertility restoration.

[0032] Furthermore, existing pcf-CMS lines utilize a wild species of the genus Petunia of unknown species, and this is the only type of CMS line currently used for petunias. Reliance on a single cytoplasm raises concerns about genetic vulnerability, as seen in the case of maize F1 varieties using T-type CMS that suffered extensive damage from the T-race of sesame leaf spot disease. For this reason, diversification of CMS cytoplasm has been desired. According to the present invention, a new petunia cytoplasmically male-sterile line is provided that is different from existing petunia cytoplasmically male-sterile lines, thereby realizing "diversification of CMS cytoplasm." This makes it possible to develop a wider variety of new varieties than before.

[0033] Furthermore, the asymmetric reversal cell fusion method of the present invention makes it possible to improve the cytoplasm, particularly the mitochondrial genome, of plants of the genus Petunia and its intergeneric hybrid plants. [Brief explanation of the drawing]

[0034] [Figure 1] The figure shows the morphology of the flowers of the new petunia cytoplasmic male-sterile line "P4". [Figure 2] The figure shows a comparison of seedling growth between the progeny (BC3) of the new cytoplasmic male-sterile petunia line "P4" (group on the left) and its repeating parent line (group on the right). In the figure, the progeny of the "P4" progeny from repeated backcrosses show reduced seedling growth compared to the parent line. [Figure 3]The image shows a comparison between "Pt3," a multiflora petunia parent line with red flowers, and "Q15," a new cytoplasmic male-sterile petunia line selected through repeated backcrossing using "Pt3" as the pollen parent (repetitive parent). [Figure 4] The figure shows photographs taken with a stereomicroscope of the anther morphology of "Pt3" with normal cytoplasm, "pcf-CMS" (a cytoplasmic male-sterile line obtained by sequential backcrossing of "Pt3" (BC7)), and "Q15". [Figure 5] The figure is a photograph showing the differences in growth when seedlings were grown in an artificial climate chamber set to a daytime temperature of 22°C, a nighttime temperature of 15°C, and 16 hours of lighting, using "Pt3", a new CMS line "Q15" which was created by backcrossing "Pt3" seven times in succession (BC7), and the existing CMS line "pcf-CMS". [Figure 6] The figure shows photographs taken with a stereomicroscope of the anther morphology of an intergeneric hybrid plant having the same cytoplasm as a novel cytoplasmically male-sterile line, and an intergeneric hybrid plant having normal cytoplasm. [Modes for carrying out the invention]

[0035] The present invention will be described in detail below.

[0036] Novel cytoplasmic male-sterile petunia plants, hybrid plants with the aforementioned cytoplasmic male-sterile petunia plants, or their offspring As described above, the present invention relates to a cytoplasmically sterile Petunia plant having DNA derived from the mitochondrial genome of a Tobacco plant in its mitochondrial genome, or a hybrid plant with the said cytoplasmically sterile Petunia plant, or their progeny. The cytoplasmically sterile Petunia plant according to the present invention, or a hybrid plant with the said cytoplasmically sterile Petunia plant, or their progeny, exhibit improved seedling growth compared to existing cytoplasmically sterile Petunia plants.

[0037] The cytoplasmically male-sterile Petunia plant according to the present invention has DNA derived from the mitochondrial genome of a Tobacco plant within its mitochondrial genome. Here, "Tobacco plants," that is, Nicotiana plants, can be used as cytoplasmic donor parents in the present invention. Preferably, Nicotiana plants are N. suaveolens, N. debneyi, N. acuminata, and N. longiflora, with Nicotiana suaveolens being more preferred.

[0038] Whether or not a cytoplasmically male-sterile Petunia plant according to the present invention has DNA derived from the mitochondrial genome of a Tobacco plant in its mitochondrial genome can be confirmed, for example, by the method described in the examples of this application, using a predetermined primer described later as an indicator.

[0039] In the cytoplasmically male-sterile Petunia plants according to the present invention, "Petunia plants" include P. hybrida, P. axillaris, P. integrifolia, P. alpicola, P. altiplana, P. bajeensis, P. bonjardinensis, P. exserta, P. guarapuavensis, P. helianthemoides, P. humifusa, P. inflata, P. interior, P. ledifolia, P. littoralis, P. mantiqueirensis, P. occidentalis, P. patagonica, P. pubescens, P. reitzii, P. riograndensis, P. saxicola, P. scheideana, P. variabilis, and P. villadiana. Furthermore, "Petunia plants" may also be derived from interspecific hybrid plants of species belonging to the Petunia genus. Among them, "petunia plants" are preferred, specifically P. hybrida, a cultivated variety of petunia.

[0040] Here, "interspecific hybrid plants" refers to plants obtained through interspecific hybridization, cell fusion, or grafting between different species of the Petunia genus, as exemplified above.

[0041] Therefore, according to one preferred embodiment of the present invention, the cytoplasmically male-sterile Petunia plant is derived from Petunia hybrida or an interspecific hybrid of Petunia plants.

[0042] In this specification, "hybrid plants with Petunia plants" means plants derived from intergeneric hybrids obtained by crossing Petunia plants with closely related genera. Here, closely related genera include, for example, plants of the genera Calibrachoa, Pineapple, and Brunfelsia. Preferred closely related genera are Calibrachoa and Pineapple, and more preferably Calibrachoa.

[0043] Therefore, according to one preferred embodiment of the present invention, hybrids of cytoplasmically male-sterile Petunia plants are derived from intergeneric hybrids of Petunia and Calibrachoa plants, and include plants obtained by cell fusion or grafting.

[0044] In this specification, "progeny of cytoplasmically sterile petunia plants" means the next generation of cytoplasmically sterile petunia plants that inherit the cytoplasm through cytoplasmic inheritance, obtained by crossing a cytoplasmically sterile petunia plant with pollen from a petunia plant that is compatible with it. Therefore, the progeny includes progeny using cytoplasmically sterile petunia plants that have DNA derived from the mitochondrial genome of tobacco plants in their mitochondrial genome, as well as progeny using hybrid plants with the aforementioned cytoplasmically sterile petunia plants, and further includes hybrids obtained by crossing a cytoplasmically sterile petunia plant or its hybrid plant according to the present invention with a petunia plant that is compatible with it. Therefore, "progeny of cytoplasmically sterile petunia plants" also include, for example, those obtained by crossing a cytoplasmically sterile petunia plant according to the present invention as the seed parent (female parent) with a petunia plant capable of crossing with it as the pollen parent (male parent). Furthermore, "progeny of cytoplasmically sterile petunia plants" also include, for example, somatic hybrid plants or grafted hybrid plants resulting from cell fusion between a cytoplasmically sterile petunia plant according to the present invention and a petunia plant.

[0045] In this specification, "asymmetric cell fusion" refers to a method in which one of the isolated protoplasts used for cell fusion is destroyed beforehand, and then the cell fusion is performed using that destroyed protoplast. In this asymmetric cell fusion, the cell whose nuclear genome is destroyed during fusion and which donates its cytoplasm to the fused cell is called the cytoplasm donor parent. Conversely, the cell whose nuclear genome is maintained without destruction during fusion and which accepts the cytoplasm from the cytoplasm donor parent is called the cytoplasm receptor parent.

[0046] Furthermore, "asymmetrical re-cell fusion" here refers to performing one or more (preferably one) further asymmetrical cell fusions, using the plant obtained by asymmetrical cell fusion or its progeny as the cytoplasmic donor parent, while using one of the plants used in the initial asymmetrical cell fusion as the cytoplasmic receptor parent. In other words, asymmetrical re-cell fusion involves performing two or more asymmetrical cell fusions, including the initial one.

[0047] In this invention, "normal cytoplasm" is typically used to mean that it is normal and does not exhibit sterility, as opposed to the cytoplasm of a plant exhibiting male sterility, i.e., male-sterile cytoplasm.

[0048] In obtaining the cytoplasmically male-sterile Petunia plants of the present invention, it is desirable to use a Tobacco plant as the cytoplasm-donating parent in asymmetric cell fusion. Furthermore, it is desirable to use a Petunia plant with normal cytoplasm as the cytoplasm-receiving parent.

[0049] According to a preferred embodiment of the present invention, a cytoplasmically male-sterile Petunia plant, or a hybrid plant with the said cytoplasmically male-sterile Petunia plant, or their progeny, has at least one mitochondrial DNA region identified by a mitochondrial genome marker using one or more primers selected from the primer set having the nucleotide sequences shown in SEQ ID NOs. 59 and 60 (primer No. 30) and the primer set having the nucleotide sequences shown in SEQ ID NOs. 67 and 68 (primer No. 34), which is of the Nicotiana suaveolens type.

[0050] In other words, cytoplasmic male-sterile Petunia plants, or hybrid plants with the aforementioned cytoplasmic male-sterile Petunia plants, or their progeny, have at least one mitochondrial DNA region identified by a mitochondrial genome marker using one or more primers selected from those having the nucleotide sequences shown in SEQ ID NOs. 59, 60, 67, and 68, that is of the Nicotiana suaveolens type.

[0051] According to a more preferred embodiment of the present invention, the cytoplasmic male-sterile Petunia plant according to the present invention, or a hybrid plant with the cytoplasmic male-sterile Petunia plant, or their progeny, contains a mitochondrial genome derived from a plant identified by accession number FERM BP-22398 (details below), and more preferably, is identified by accession number FERM BP-22398.

[0052] In this specification, "part of a plant" of a cytoplasmic male-sterile Petunia plant, a hybrid plant with such cytoplasmic male-sterile Petunia plant, or their offspring includes one or more cells or cytoplasm from one or more cells of the plant, specifically meaning organs or tissues such as flowers, leaves, stems, and roots, or cells (including protoplasts prepared from cells) or cytoplasm from such organs or tissues, or aggregates of such cells or cytoplasm.

[0053] Method for creating novel cytoplasmically sterile petunia plants The novel cytoplasmically male-sterile Petunia plants according to the present invention can be produced, for example, by following the procedure below. (1) Preparation of protoplasts (2) Asymmetric cell fusion (3) Culture of fusion hybrid cells (4) Selection of cytoplasmic hybrid plants having cytoplasmic male sterility (5) Regeneration of plant tissue from callus (6) Acquisition of future generations and selection of superior bloodlines

[0054] In this specification, "manufacturing method" can also be referred to as "production method." In other words, the terms "production" and "manufacturing" used herein are used interchangeably.

[0055] These steps are described in more detail as follows:

[0056] (1) Preparation of protoplasts (i) Isolation of protoplasts from Petunia plants Petunia species that can be used for protoplast preparation include P. hybrida, P. axillaris, P. integrifolia, P. alpicola, P. altiplana, P. bajeensis, P. bonjardinensis, P. exserta, P. guarapuavensis, P. helianthemoides, P. humifusa, P. inflata, P. interior, P. ledifolia, P. littoralis, P. mantiqueirensis, P. occidentalis, P. patagonica, P. pubescens, P. reitzii, P. riograndensis, P. saxicola, P. scheideana, P. variabilis, and P. villadiana, but P. hybrida, a cultivated variety of petunia, is preferred.

[0057] For obtaining protoplasts, it is preferable to use mesophyll tissue, which has high yield and high mitotic activity. However, other tissues such as hypocotyls, stems, and callus may also be used as materials.

[0058] The method for isolating protoplasts may be any commonly used method known in the art (e.g., the method described in Matsumoto, E., Plant cell reports, 1991, vol. 9(10), etc.), and is not particularly limited. The following is a specific example of the procedure, but the present invention is not necessarily limited to it.

[0059] First, the cell tissue of a plant of the genus Petunia is finely chopped, and protoplasts are isolated by enzymatic treatment using an enzyme solution for protoplast isolation. This solution is mainly an inorganic salt buffer containing cell wall-degrading enzymes and osmotic regulators. The cell wall-degrading enzyme is not particularly limited as long as it can be used to degrade plant cell walls, but examples include cellulase, hemicellulase, and pectinase. In this invention, a combination of cellulase YC and macerozyme R-10 is preferred.

[0060] As an osmotic pressure regulator, common sugar alcohols such as mannitol, sorbitol, and glucose can be used, with mannitol being preferred, and mannitol at a concentration of 0.3 M to 0.7 M being particularly preferred. Furthermore, it is desirable to add an inorganic salt to the enzyme solution to stabilize the protoplast membrane, and for example, it is preferable to add CPW salt (Cocking and Peberdy, 1974) with the composition shown in Table 1 below. The enzyme treatment is preferably carried out by standing treatment at 25 to 30°C for 8 to 20 hours.

[0061] [Table 1]

[0062] Protoplasts isolated by enzymatic treatment are filtered through a nylon mesh with a pore size of 30-100 μm, and the protoplasts are collected by centrifugation to remove the enzyme solution. Next, the protoplasts are suspended in a washing solution and washed. As the washing solution, a commonly used CPW salt solution with sugar alcohols added as an osmotic pressure adjuster can be used.

[0063] Next, it is desirable to inactivate the protoplasts of Petunia plants to prevent them from dividing on their own. Inactivation can be performed by suspending the protoplasts in a CPW salt solution containing an iodine compound such as iodoacetic acid or iodoacetamide. In this invention, it is preferable to suspend the protoplasts in a CPW salt solution adjusted to a concentration of 5 mM to 30 mM and treat for 5 to 20 minutes.

[0064] Next, it is preferable to repeat the washing operation with CPW salt solution 1 to 3 times using a centrifuge. Since the protoplast suspension contains conduits and cell fragments, it is preferable to further purify the protoplasts by density gradient centrifugation or the like.

[0065] Reagents used for purification include sugars and synthetic colloids, but in this invention, the use of sucrose solution is preferred, and the use of a 15% to 20% sucrose solution is particularly preferred. After purification of the protoplasts, the cell density is measured using a hemocytometer, and the volume of the solution is adjusted with CPW salt solution to achieve a cell density suitable for cell fusion. The cell density of the protoplasts is 1 × 10⁻⁶. 5 ~1 × 10 7 A cell / ml ratio is preferred, and the use of a CPW salt solution is preferred for adjusting the volume.

[0066] (ii) Isolation of protoplasts from plants of the genus Nicotiana The cytoplasmic donor parent of the present invention can be a plant of the genus Nicotiana. Among Nicotiana plants, N. suaveolens, N. debneyi, N. acuminata, and N. longiflora are preferred, and N. suaveolens is particularly preferred as there are examples of producing cytoplasmic male-sterile lines of tobacco (N. tabacum).

[0067] Protoplasts from plants of the genus Nicotiana can be isolated, for example, by following the same method as the isolation of protoplasts from plants of the genus Petunia described above.

[0068] It is preferable to use protoplasts from isolated Nicotiana plants in which the nuclei have been inactivated by radiation treatment. Examples of radiation used for this treatment include X-rays, gamma rays, and ultraviolet rays, but the type of radiation is not particularly limited as long as it can destroy the nuclei. The irradiation dose should preferably be as low as possible while still being able to destroy the nuclei. For example, in the case of soft X-ray irradiation in this invention, an irradiation dose of 100 Gy to 900 Gy is preferred.

[0069] (2) Protoplast fusion process Next, the two types of protoplasts obtained above are mixed and cell fusion is performed. Examples of fusion methods include conventional methods, such as the known electrofusion method (Planta, 151, 26-32, 1981), the PEG (polyethylene glycol) method (Planta, 120, 215-227, 1974), and the dextran method (Jap. J. Genet., 50, 235, 1975), but are not particularly limited. In this invention, the PEG method is preferably used.

[0070] (3) Culture of fusion hybrid cells The cells obtained by the fusion treatment are preferably cultured in a medium suitable for culturing protoplasts derived from plants of the genus Petunia. The method for culturing protoplasts of plants of the genus Petunia is not particularly limited, as long as it is based on the method for culturing protoplasts of petunias and modified as appropriate, but in this invention, it is preferable to use MS medium with a 1 / 2 concentration (Murashige, T. & Skoog, Physiol.Plant., 15, 473-497, 1962) with NH4NO3 reduced to 200 mg / l as the basic medium, and to add plant growth regulators, various additives, etc. as appropriate.

[0071] (4) Selection of cytoplasmic hybrid plants having cytoplasmic male sterility The fused cells are cultured until cell division begins and callus is visible, at which point the callus is transferred to a callus growth medium. Conventional callus growth media can be used, and although there are differences in response depending on the genotype of the plant material and the state of the callus, MS medium containing, for example, 0.1-3.0 mg / l naphthaleneacetic acid (NAA) and 0.1-3.0 mg / l thidiazurone (TDZ) is preferable.

[0072] For the selection of cytoplasmic hybrid individuals, it is preferable to extract DNA from the callus and efficiently select them using a marker that can specifically amplify the mitochondrial DNA of Nicotiana plants by PCR.

[0073] (5) Regeneration of plant tissue from callus Callus containing mitochondrial DNA from selected Nicotiana plants is transplanted into a redifferentiation medium and redifferentiated. Conventional redifferentiation media can be used, and although the reaction may vary depending on the genotype and callus state of the plant material, MS medium containing, for example, 0.1-1.0 mg / l of NAA and 0.1-1.0 mg / l of TDZ is preferable. The regenerated shoots are transplanted into MS medium supplemented with 3% sucrose and 0.8% agar to induce rooting and regenerate the plant. The regenerated plants are then acclimatized and grown in a greenhouse.

[0074] (6) Acquisition of future generations and selection of superior bloodlines The resulting cytoplasmic hybrid plants may be tetraploid or higher due to the fusion of protoplasts from two or more Petunia species. Since higher polyploids have poorer efficiency in acquiring offspring and require time and effort to return to diploidy, it is preferable to use a flow cytometer to test for ploidy and use only diploid individuals. The resulting cytoplasmic hybrid plants are grown in a greenhouse or similar environment to induce flowering, and individuals expressing male sterility are selected. These are then crossed with Petunia species having normal cytoplasm as the pollen parent, and cytoplasmic male sterility is confirmed in the offspring.

[0075] When different cell species fuse through cell fusion, their mitochondria repeatedly fuse and divide, resulting in various recombinations between the different mitochondrial genomes. In plants, hundreds to tens of thousands of mitochondria exist in a single cell, and each undergoes recombination, so cytoplasmic hybrid plants become heteroplasmic immediately after cell fusion. To resolve the heteroplasmic state and evaluate the stability of cytoplasmic male sterility and other traits, it is desirable to perform seven or more consecutive backcrosses.

[0076] Method for improving novel cytoplasmic male-sterile petunia plants If the resulting cytoplasmic male-sterile line exhibits undesirable traits, these traits can be improved through asymmetric backcell fusion. Asymmetric backcell fusion is a method of improving the mitochondrial genome by performing asymmetric cell fusion using a plant obtained through asymmetric cell fusion or its progeny as the cytoplasmic donor parent, while using a plant with normal cytoplasm as the cytoplasmic receptor parent.

[0077] Generally, the genes responsible for cytoplasmic male sterility are located in the mitochondrial genome. The novel petunia cytoplasmic male-sterile line obtained by the first asymmetric cell fusion is thought to possess not only the mitochondrial DNA of N. suaveolens that induces male sterility, but also the mitochondrial DNA of N. suaveolens that causes undesirable traits not involved in male sterility.

[0078] By performing an asymmetric back-cell fusion, or a second asymmetric cell fusion, using a novel cytoplasmic male-sterile petunia line obtained through the first asymmetric cell fusion as the cytoplasmic donor parent and a petunia plant with normal cytoplasm as the cytoplasmic recipient parent, it is possible to induce a new mitochondrial genome recombination. At this time, by using the novel cytoplasmic male-sterile petunia line obtained through the first asymmetric cell fusion, in which the entire mitochondrial genome is closer to the petunia type, it becomes easier to obtain a mitochondrial genome that is closer to the petunia type, and the selection of superior cytoplasmic male-sterile lines becomes more efficient.

[0079] The resulting cytoplasmic hybrid plants, like those from the first asymmetric cell fusion, become heteroplasmic immediately after fusion. Therefore, it is necessary to select superior cytoplasmic male-sterile lines by performing successive backcrosses. By repeating asymmetric backcrossing two or more times, it is also possible to further improve the mitochondrial genome. [Examples]

[0080] The present invention will be specifically described by the following embodiments, but the present invention is not limited to these embodiments.

[0081] Example 1: Method for creating a novel cytoplasmic male-sterile petunia line (1) Preparation of protoplasts (i) Isolation of protoplasts from Petunia plants with normal cytoplasm As a petunia plant with normal cytoplasm, we used 'Pt1', a Grandiflora variety with reddish-purple flowers. Sterilized seeds of 'Pt1' were placed on MS medium supplemented with 3% sucrose and 0.8% agar, and grown at 20°C under 16 hours of light for approximately one month. Approximately 1 g of unfolded true leaves were collected, finely chopped into strips about 2 mm wide, and then immersed in 10 ml of CPW salt solution containing 0.3% cellulase YC, 0.3% macerozyme R-10, and 0.5 M mannitol, and left to stand at 25°C for 16 hours.

[0082] The enzyme solution containing leaf tissue was filtered through a 59 μm nylon mesh to remove cell residue. The resulting protoplast suspension was transferred to a centrifuge tube and centrifuged at 800 rpm for 5 minutes. The supernatant was removed, and the resulting protoplasts were suspended in 5 ml of CPW salt solution containing 15 mM iodoacetamide and incubated at 4°C for 15 minutes. After incubation, the iodoacetamide-treated protoplast suspension was centrifuged at 800 rpm for 5 minutes, and the supernatant was removed. The protoplasts were washed by adding 10 ml of CPW salt solution to the protoplast suspension, centrifuging at 800 rpm for 5 minutes, and removing the supernatant, repeating this process three times.

[0083] The washed protoplast suspension was centrifuged at 800 rpm for 5 minutes, the supernatant was removed, and 2 ml of CPW salt solution was added to suspend the protoplasts. 5 ml of CPW salt solution with 20% sucrose was added to a new centrifuge tube, and the protoplast suspension was placed on top. Centrifuged at 800 rpm for 5 minutes. Cell residue settled at the bottom of the centrifuge tube, and the purified protoplasts floated to the upper layer of CPW salt solution, which were then transferred to a new centrifuge tube using a Pasteur pipette. A small amount of the suspension was taken, the cell density of the protoplasts was determined using a hemocytometer, and CPW solution was added to obtain a cell density of 1 × 10⁶. 6 Prepared at 1 / ml.

[0084] (ii) Isolation of protoplasts from plants of the genus Nicotiana As a Nicotiana species, N. suaveolens was used, which has been used to create cytoplasmically sterile tobacco plants. Tobacco seeds N. suaveolens were provided by the Tobacco Leaf Research Institute of Japan Tobacco Inc. Sterilized N. suaveolens seeds were placed on MS medium supplemented with 3% sucrose and 0.8% agar and grown at 20°C under 16 hours of illumination for approximately one month. Approximately 1 g of developed true leaves were harvested, finely chopped to approximately 2 mm in size, and then immersed in 10 ml of CPW salt solution containing 0.3% cellulase YC, 0.3% macerozyme R-10, and mannitol, and left to stand at 25°C for 16 hours.

[0085] The enzyme solution containing leaf tissue was filtered through a 59 μm nylon mesh to remove cell residue. The protoplasts were transferred to a plastic petri dish using a Pasteur pipette and irradiated with 900 Gy of soft X-rays.

[0086] The obtained protoplast suspension was transferred to a centrifuge tube and centrifuged at 800 rpm for 5 minutes. The supernatant was removed, and 2 ml of CPW salt solution was added to suspend the protoplasts. 5 ml of CPW salt solution with 20% sucrose was added to a new centrifuge tube, and the protoplast suspension was placed on top. Centrifuged at 800 rpm for 5 minutes. Cell residue settled at the bottom of the centrifuge tube, and the purified protoplasts floated to the upper layer of CPW salt solution. These were then transferred to a new centrifuge tube using a Pasteur pipette. A small amount of the suspension was taken, and the cell density of the protoplasts was determined using a hemocytometer. CPW salt solution was added to obtain a cell density of 1 × 10⁶. 6 Prepared at 1 / ml.

[0087] (2) Protoplast fusion process A suspension of protoplasts from petunia plants with normal cytoplasm treated with iodoseacetamide and a suspension of protoplasts from Nicotiana plants irradiated with soft X-rays were mixed in a 1:3 ratio, and 2 ml of the mixture was dropped into the center of the bottom of a 9 cm petri dish. After standing for 30 minutes, 3 ml of 500 g / l PEG solution (polyethylene glycol #6000 (nacalai tesque Inc.), 1,500 mg / l CaCl2·2H2O, 100 mg / l KH2PO4, pH 5.5) was dropped around the protoplast mixture.

[0088] After 1 minute, 3.5 ml of CPW salt solution was added dropwise around the protoplast mixture. After another 2 minutes, another 3.5 ml of CPW salt solution was added dropwise around the protoplast mixture. After 5 minutes, the added liquid was gently aspirated and removed from the edge of the petri dish, and 20 ml of CPW salt solution was added from the edge of the petri dish. This washing procedure with CPW salt solution was repeated three times at 5-minute intervals.

[0089] (3) Culture of fusion hybrid cells After removing the washing solution, 10 ml of MS medium (pH 5.8) containing 0.5 M mannitol, 150 mg / l casamino acid, 100 mg / l L-glutamine, 0.1 mg / l NAA, 0.1 mg / l 2,4-D (2,4-dichlorophenoxyacetic acid), 0.1 mg / l TDZ, and 1% sucrose, with NH4NO3 reduced to 200 mg / l, was added, and the cells were incubated at 25°C in the dark.

[0090] Seven days after the start of culture, 5 ml of MS medium (pH 5.8) containing 150 mg / l casamino acid, 100 mg / l L-glutamine, 0.1 mg / l NAA, 0.1 mg / l 2,4-D, 0.1 mg / l BA, and 1% sucrose, with NH4NO3 reduced to 200 mg / l, was added to lower the mannitol concentration and the culture was continued.

[0091] Ten days after the start of culture, the cells attached to the bottom of the petri dish were gently scraped off with the tip of tweezers, and 7.5 ml of a solution containing 0.2 M mannitol, 4% sucrose, and 0.6% gellan gum was added and mixed to form a semi-solid gel medium, which was then used to continue the culture.

[0092] Approximately one month after the start of culture, the callus became visible to the naked eye, so the callus was transferred to callus growth medium (MS medium containing 1 mg / l NAA, 1 mg / l TDZ, 3.0% sucrose, and 0.8% agar, pH 5.8).

[0093] (4) Selection of cytoplasmic hybrid plants having cytoplasmic male sterility To select cytoplasmic hybrid plants, we designed molecular markers that can detect DNA specific to N. suaveolens using PCR. Since the mitochondrial genome sequence information for N. suaveolens is not publicly available, we used the nucleotide sequence information (GenBank registration number BA000042) of N. tabacum, another species in the same genus, to design primers specific to the nad3 gene (Table 2).

[0094] When the callus had grown to a size of 5 mm or more, a portion of the callus was sampled and DNA was extracted. Using the extracted whole-genome DNA as a template, PCR was performed using primer No. 1. The PCR was performed by repeating the following cycle 35 times: denaturation at 94°C for 1 minute, annealing at 60°C for 2 minutes, and extension reaction at 72°C for 2 minutes. PCR products were subjected to electrophoresis on a 1.8% agarose gel, immersed in ethidium bromide solution, and photographed under UV irradiation. Individuals with a band of the expected size (456 bp) were selected.

[0095] [Table 2]

[0096] (5) Regeneration of plant tissue from callus When the callus reached a size of approximately 1 cm, it was divided into pieces of approximately 2 mm in size and transplanted into a redifferentiation medium (MS medium containing 0.1 mg / l NAA, 1.0 mg / l TDZ, 3.0% sucrose, and 0.8% agar, pH 5.8).

[0097] Approximately one month after transplantation to the redifferentiation medium, the callus began to differentiate into shoots. The differentiated shoots were transplanted into MS medium (pH 5.8) containing 3.0% sucrose and 0.8% agar, which induced rooting. Cytoplasmic hybrid plants were transplanted into 72-well cell trays for acclimatization. Flow cytometry analysis of the cytoplasmic hybrid plants revealed diploid or tetraploid plants; no aneuploidy was observed.

[0098] (6) Acquisition of future generations and selection of superior bloodlines Cytoplasmic hybrid plants were transplanted into 9cm pots and continued to be grown, and male sterility was investigated after flowering. As a result, 12 male-sterile lines were obtained. Twelve lines expressing male sterility were used as seed parents (single-generation parents), and the multiflora petunia parent line "Pt2," which has normal cytoplasm and pink flower color, was used as the pollen parent (recurrent parent) in a series of backcrosses.

[0099] After performing seven consecutive backcrosses, we selected a new petunia cytoplasmic male-sterile line, "P4," which exhibited stable cytoplasmic male sterility, high female fertility, and normal morphology (Figure 1). The anthers of "P4" were completely degenerated and browned, and no pollen was produced at all. "P4" had undergone seven consecutive backcrosses with "Pt2," and it was thought that the mitochondrial genome was homoplasmyated and the cytoplasm was stable.

[0100] Next, in order to use "P4" as a seed parent for F1 variety development, 32 different fertile parent lines (inbred lines) were successively backcrossed to induce male sterility in the parent lines.

[0101] As a result, a decrease in seedling growth vigor was observed in the progeny of most parent lines through continuous backcrossing, revealing problems with practical application. A notable example of the decreased seedling growth vigor is shown in Figure 2.

[0102] Furthermore, as shown in Figure 2, it was confirmed that the growth of seedlings in the BC3 progeny of the new petunia cytoplasmic male sterile line "P4" through successive backcrosses was reduced compared to its repeating parent line.

[0103] In the seventh generation (BC7) of the continuous backcross of "Pt2," no decrease in seedling growth was observed, suggesting that the decrease in growth (nucleo-cytoplasmic incompatibility) is caused by a genotype in the nuclear genome of the parent breeding line. Thus, it is thought that the cytoplasmic male-sterile line "P4" exhibits reduced seedling growth in most of the progeny of the continuous backcross of the parent lines, and that improvement of the mitochondrial genome is necessary for its use in breeding.

[0104] Example 2: Method for improving the novel petunia cytoplasmic male sterility line "P4" To improve the mitochondrial genome of the novel cytoplasmic male-sterile petunia line "P4" obtained by the first asymmetric cell fusion, "P4" obtained by the first asymmetric cell fusion was used as the cytoplasmic donor parent, while "Pt3," a multiflora parent line of the Petunia genus with normal cytoplasm and red flower color, was used as the cytoplasmic recipient parent. Asymmetric cell fusion (asymmetric reversal cell fusion) was performed in the same manner as in Example 1.

[0105] Since "Pt3" is a line that exhibits reduced seedling growth when repeatedly backcrossed with "P4", it is possible to select the desired cytoplasmic male sterility line by comparing the seedling growth of lines obtained by asymmetric back-cell fusion.

[0106] The obtained callus was selected for cytoplasmic hybrids using a primer specific to the tobacco nad3 gene, as in Example 1. Cytoplasmic hybrid plants were transplanted into 9cm pots and continued to be grown, and male sterility was investigated after flowering. As a result, 14 cytoplasmic male-sterile lines were obtained.

[0107] Fourteen lines expressing male sterility were used as seed parents (single-generation parents), and a multiflora petunia plant "Pt3" (same as the cytoplasmic receptor parent) with normal cytoplasm and red flower color was used as the pollen parent (recurrent parent) in a series of backcrosses.

[0108] Seven consecutive backcrosses were performed to select a new petunia cytoplasmic male sterile line, "Q15," which exhibited stable cytoplasmic male sterility, high female fertility, and normal morphology (Figure 3).

[0109] Figure 4 shows the anther morphology of "Pt3" with normal cytoplasm, "pcf-CMS" which is a cytoplasmically male-sterile line obtained by sequential backcrossing "Pt3" (BC7), and "Q15".

[0110] "pcf-CMS" showed production of degenerated anthers and infertile pollen grains, but "Q15" had completely degenerated anthers and produced no pollen. The cytoplasmically sterile line "Q15" underwent seven backcrosses, and it was thought that the mitochondrial genome was homoplasmic and the cytoplasm was stable.

[0111] While "Q15" was confirmed to possess the tobacco nad3 gene at the callus stage obtained during asymmetric back-cell fusion, the tobacco nad3 gene disappeared in the BC7 generation after seven backcrosses. This was thought to be because, while confirming the expression of male sterility in each generation of the backcross, selection was repeatedly performed based on seedling growth, resulting in selection proceeding in a direction that eliminated mitochondrial genes derived from N. suaveolens, which are thought to be the cause of incompatibility between the nuclear and cytoplasmic genomes of petunias.

[0112] Therefore, because the tobacco nad3 gene, which was used as a cytoplasmic selection marker for "Q15," was lost, it was necessary to design a new molecular marker and analyze the cytoplasm in order to confirm that N. suaveolens mitochondrial DNA had been introduced into "Q15."

[0113] Since the nucleotide sequence information for the mitochondrial genome of N. suaveolens is not publicly available, we used the complete nucleotide sequence information (GenBank registration number BA000042) of N. tabacum, another species in the same genus, to design primers that specifically amplify 16 mitochondrial genes, which are shown as primers No. 2 to 17 in Table 3. Furthermore, based on the nucleotide sequence information of known petunia CMS causative genes (pcf), primers that specifically amplify the pcf gene were designed and are shown as primer No. 18 in Table 3.

[0114] Furthermore, to identify the origin of the "Q15" chloroplast, we used the complete nucleotide sequence information of the N. tabacum chloroplast genome (GenBank registration number Z00044) to design primers that specifically amplify the spacer region between the atpA and atpH genes, which are shown as primer No. 19 in Table 3. The PCR products were digested with the restriction enzyme TaqI, and the origin of the chloroplasts was identified by detecting RFLP based on differences in restriction enzyme sites.

[0115] [Table 3]

[0116] Table 4 shows the results of cytoplasmic analysis of N. suaveolens, "Pt3", "P4", "Q15", and "pcf-CMS" strains using the newly designed primer sets shown in Table 3.

[0117] [Table 4]

[0118] The primers No. 2-17 designed in this study were confirmed to induce specific amplification in N. suaveolens. In normal cytoplasmic petunias, specific amplification did not occur with any primer except for No. 6, which was confirmed to induce petunia-specific amplification.

[0119] Neither "P4" nor "Q15" showed specific amplification of the N. suaveolens type in any of the 16 primer sets designed in this study. This suggests that the majority of the mitochondrial genomes of "P4" and "Q15" have already been recombined into the petunia type. "pcf-CMS" is thought to be derived from a wild species of the genus Petunia, and no specific amplification of the N. suaveolens type was observed.

[0120] Furthermore, specific amplification of the pcf gene was confirmed in "pcf-CMS" by PCR using primer No. 18, which amplifies the pcf gene. However, no specific amplification was observed in N. suaveolens, "P4", and "Q15", indicating that the pcf gene is absent in N. suaveolens, "P4", and "Q15", and that "P4" and "Q15" express cytoplasmic male sterility through a factor other than the pcf gene.

[0121] PCR-RFLP analysis of the chloroplast gene atpA using primer No. 19 showed that chloroplasts "P4" and "Q15" were of the petunia type. Since it is known that chloroplast recombination does not fundamentally occur during cell fusion, it was confirmed that "P4" and "Q15" possess chloroplasts derived from petunia.

[0122] Based on these results, it was confirmed that "P4" and "Q15" express CMS through a mechanism different from existing pcf genes, possess petunia-derived chloroplasts, and have major mitochondrial genes recombined into the petunia type.

[0123] In tobacco (N. tabacum), "sua-CMS," which involves introducing the cytoplasm of N. suaveolens into N. tabacum via cell fusion, is used, and the nucleotide sequence information (GenBank registration number KR071121) of the mitochondrial genome (recombinant mitochondrial genome of N. tabacum and N. suaveolens) of its CMS strain "ms zhongyan100" has been made public. Furthermore, the nucleotide sequence information (GenBank registration number KR780036) of the mitochondrial genome (N. tabacum) of "zhongyan100," a normal cytoplasmic cell with the same nuclear genome, has also been made public.

[0124] Therefore, by comparing the mitochondrial genomes of "ms zhongyan100" and "zhongyan 100" using BLAST (Basic Local Alignment Search Tool), we estimated regions specific to the mitochondrial genome of N. suaveolens and designed primers that specifically amplify N. suaveolens DNA (Table 5).

[0125] [Table 5]

[0126] Table 6 shows the results of cytoplasmic analysis of N. suaveolens, "Pt3", "P4", "Q15", and "pcf-CMS" strains using primer sets designed for regions presumed to be specific to N. suaveolens.

[0127] [Table 6]

[0128] The 22 primer sets designed in this study resulted in specific DNA amplification in N. suaveolens. In normal cytoplasmic petunias, specific amplification did not occur except with primer combinations No. 24 and 35, while petunia-specific amplification occurred with primer combinations No. 24 and 35.

[0129] "P4" showed amplification of N. suaveolens-specific DNA with five primer sets (primer Nos. 21, 27, 29, 30, and 34), while "Q15" showed amplification of N. suaveolens-specific DNA with two primer sets (primer Nos. 30 and 34). "pcf-CMS" is thought to be derived from a wild species of the genus Petunia, and, as with Table 4, no specific amplification of the N. suaveolens type was observed. Furthermore, "Q15" underwent asymmetric reversal cell fusion using "P4" as the material, and it was thought that the N. suaveolens region was being eliminated while retaining the DNA region involved in CMS, thus approaching the mitochondrial genome of petunia.

[0130] The new petunia cytoplasmic male-sterile line "Q15" was introduced into 51 diverse parent lines (BC1-BC4), but no restoration of fertility was observed. Furthermore, no fertility recovery was observed in 91 F1 test crosses using these parent lines.

[0131] Therefore, the male sterility of "Q15" was considered to be extremely stable. On the other hand, as described in the "Background Art" section of this specification, existing "pcf-CMS" were known to restore fertility through single dominant or multiple fertility restoration genes and through changes in the environment (Non-Patent Literature 3).

[0132] The reason for the unstable cytoplasmic male sterility of the existing "pcf-CMS" is thought to be that it originates from interspecific hybrids within the Petunia genus, and multiple fertility-restoring genes have emerged within the Petunia genus during the course of evolution. On the other hand, the reason for the stable cytoplasmic male sterility of the novel cytoplasmic male-sterile petunia "Q15" is thought to be that it uses cytoplasm derived from the Tobacco genus, and therefore fertility-restoring genes are not present in the distantly related Petunia genus.

[0133] Example 3: Evaluation of seedling growth of a novel petunia cytoplasmic male-sterile line "Q15" To confirm the usefulness of the novel petunia cytoplasmic male sterility line "Q15" produced by Example 2, a comparative growth test was conducted on seedlings of "Pt3" with normal cytoplasm and cytoplasmic replacement lines of "Q15" and "pcf-CMS" "Pt3".

[0134] The comparative study of seedling growth used "Q15," a novel CMS line created by seven consecutive backcrosses (BC7) with "Pt3" as the control (which has normal cells), and "pcf-CMS," an existing CMS line. Since each CMS line is BC7, its nuclear genome is replaced with that of "Pt3," resulting in identical nuclear genomes, making it possible to compare differences in cytoplasm.

[0135] Seeds from each strain were sown in 128-cell seed trays and cultivated in an artificial climate chamber set to a daytime temperature of 22°C, a nighttime temperature of 15°C, and 16 hours of lighting. To quantitatively evaluate the growth of seedlings, 30 days after sowing, the above-ground parts of seedlings from each strain were cut at the base, and the weight per plant was measured. The results are shown in Table 7. Figure 5 also shows photographs illustrating the differences in growth characteristics of each strain at that time.

[0136] [Table 7]

[0137] As shown in the column for test plot A in Table 7, the "pcf-CMS" line showed low growth, with a relative above-ground weight of "39" compared to "Pt3". In contrast, the new CMS line "Q15" showed extremely high growth potential, with a relative above-ground weight of "137" compared to "Pt3".

[0138] Next, the same test was conducted under the conditions of a glass greenhouse. The glass greenhouse (located in Kakegawa City, Shizuoka Prefecture) was set to a daytime temperature of 22°C and a nighttime temperature of 15°C. On April 1, 2020, seeds of each strain were sown in 128-cell seed trays and cultivated. To quantitatively evaluate the growth of seedlings, 30 days after sowing, the above-ground parts of seedlings from each strain were cut at the base, and the weight per plant was measured. The results are also shown in Table 7.

[0139] As shown in the column for test plot B in Table 7, the "pcf-CMS" line showed low growth, with a relative above-ground weight of "47" compared to "Pt3". In contrast, the new CMS line "Q15" showed high growth potential, with a relative above-ground weight of "119" compared to "Pt3".

[0140] Based on these results, while the existing "pcf-CMS" line showed reduced growth during the seedling stage in both artificial climate chamber and greenhouse environments, the new petunia cytoplasmic male sterile line "Q15" showed superior growth compared to "Pt3," which has normal cytoplasm, in both environments, confirming its usefulness.

[0141] Seeds of the new petunia cytoplasmic male sterile line "Q15" were internationally deposited (originally deposited) on August 28, 2020, at the National Institute of Technology and Evaluation (NITE) Patent Organism Depository Center (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) (depositor's identification marking: SSC-PET-20-001, accession number: FERM BP-22398).

[0142] Example 4: Introduction of a novel cytoplasmic male sterility in petunia into intergeneric hybrid plants. To introduce the novel cytoplasmic male sterility of petunias, created using N. suaveolens in this invention, into fertile intergeneric hybrid plants of the petunia family (petunia × calibrachoa), cytoplasmic replacement was performed by successive backcrosses using the novel cytoplasmic male-sterile petunia line as the seed parent (single parent) and seven lines of fertile intergeneric hybrid plants of the petunia family as the pollen parents (recurrent parents).

[0143] Intergeneric hybrid plants in which the cytoplasm was replaced by that of a new cytoplasmic male-sterile line through repeated backcrossing showed degenerated anthers and no pollen grains were formed at all, as shown in Figure 6. All 112 individuals from 7 intergeneric hybrid plant lines tested were male-sterile, confirming that the cytoplasmic male sterility of the new cytoplasmic male-sterility line is stably expressed even in intergeneric hybrid plants.

[0144] Therefore, it was confirmed that the cytoplasm of the novel CMS line derived from N. suaveolens according to the present invention can be introduced into closely related intergeneric hybrid plants and expresses cytoplasmic male sterility.

Claims

1. A cytoplasmic male-sterile petunia plant having DNA derived from the mitochondrial genome of Nicotiana suaveolens in its mitochondrial genome, or a hybrid plant with the cytoplasmic male-sterile petunia plant, or their progeny (wherein the hybrid plant and the progeny have DNA derived from the mitochondrial genome of Nicotiana suaveolens in their mitochondrial genome).

2. The cytoplasmically male-sterile Petunia plant, hybrid plant, or progeny according to claim 1, wherein the cytoplasmically male-sterile Petunia plant is derived from Petunia hybrida or an interspecific hybrid thereof.

3. The cytoplasmically male-sterile Petunia plant, hybrid plant, or progeny according to claim 1 or 2, wherein the cytoplasmically male-sterile Petunia plant is derived from one obtained by performing asymmetric cell fusion using Nicotiana suaveolens as the cytoplasmic donor parent.

4. A cytoplasmically sterile petunia plant, a hybrid plant, or a progeny according to any one of claims 1 to 3, wherein the hybrid plant with the cytoplasmically sterile petunia plant is derived from an intergeneric hybrid plant of a petunia plant and a calibrachoa plant.

5. A cytoplasmic male-sterile Petunia plant, hybrid plant, or progeny according to any one of claims 1 to 4, comprising a plant-derived mitochondrial genome identified by accession number FERM BP-22398.

6. A cytoplasmic male-sterile petunia plant, hybrid plant, or progeny according to any one of claims 1 to 5, wherein at least one mitochondrial DNA region identified by a mitochondrial genome marker using one or more primers selected from the group consisting of primer sets having the nucleotide sequences shown in SEQ ID NOs. 59 and 60, and primers included in primer sets having the nucleotide sequences shown in SEQ ID NOs. 67 and 68, is of the Nicotiana suaveolens type.

7. A cytoplasmic male-sterile petunia plant, hybrid plant, or progeny according to any one of claims 1 to 6, having the mitochondrial genome of a plant identified by accession number FERM BP-22398.

8. A cytoplasmically male-sterile Petunia plant, hybrid plant, or progeny according to any one of claims 1 to 6, as identified by accession number FERM BP-22398.

9. A cytoplasmically male-sterile Petunia plant, a hybrid plant, or a progeny obtained by asymmetric cell fusion using a cytoplasmically male-sterile Petunia plant or a hybrid plant with a cytoplasmically male-sterile Petunia plant having the mitochondrial genome of a plant identified by accession number FERM BP-22398 as the cytoplasmic donor parent, and a Petunia plant or a hybrid plant with a Petunia plant having normal cytoplasm as the cytoplasmic recipient parent.

10. A part of a plant body of a cytoplasmically male-sterile Petunia plant, hybrid plant, or progeny according to any one of claims 1 to 9.

11. Seeds of a cytoplasmically male-sterile Petunia plant, hybrid plant, or progeny according to any one of claims 1 to 9.

12. A cytoplasmic male-sterile Petunia plant, hybrid plant, or progeny described in any one of claims 1 to 9, a part of the plant body described in claim 10, or a seed described in claim 11, comprising a mitochondrial genome.

13. A method for producing a cytoplasmically male-sterile Petunia plant, a hybrid plant with the cytoplasmically male-sterile Petunia plant, or their offspring, comprising the step of performing asymmetric cell fusion using Nicotiana suaveolens as the cytoplasm-donor parent and a Petunia plant having normal cytoplasm or a hybrid plant with a Petunia plant as the cytoplasm-receiving parent.

14. A method for producing a cytoplasmically sterile petunia plant with an improved mitochondrial genome, a hybrid plant with a cytoplasmically sterile petunia plant, or a progeny thereof, comprising the step of performing asymmetric cell fusion using a cytoplasmic donor parent (wherein the hybrid plant and the progeny have DNA derived from the mitochondrial genome of Nicotiana suaveolens in their mitochondrial genomes), and a petunia plant having normal cytoplasm or a hybrid plant with a petunia plant as the cytoplasmic recipient parent.

15. The method for producing a petunia plant according to claim 13 or 14, wherein the cytoplasmically male-sterile petunia plant is derived from Petunia hybrida or an interspecific hybrid thereof.

16. The manufacturing method according to any one of claims 13 to 15, wherein the hybrid plant with the cytoplasmically male-sterile Petunia plant is derived from an intergeneric hybrid plant of the Petunia and Calibrachoa genera.

17. A method for producing first-generation hybrid seeds, comprising using a cytoplasmically male-sterile Petunia plant, hybrid plant, or progeny described in any one of claims 1 to 9 as the seed parent, crossing it with a Petunia plant capable of crossing with the said plant and an intergeneric hybrid derived therefrom as the pollen parent, and collecting first-generation hybrid seeds from the seed parent after the cross.

18. Hybrid first-generation seeds produced by the method described in claim 17, or hybrid first-generation plants grown from said seeds, their progeny, or parts of such plants.

19. A method for producing a cytoplasmic male-sterile Petunia plant and an intergeneric hybrid plant derived therefrom that exhibits cytoplasmic male sterility, comprising sequentially backcrossing a cytoplasmic male-sterile Petunia plant, a hybrid plant, or an offspring with any Petunia plant and an intergeneric hybrid plant derived therefrom, and performing cytoplasmic replacement.

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