Cytoplasmic male sterile plant of genus lactuca having improved low temperature growth ability

Abstract

To alleviate a reduction in the growth ability at low temperatures observed in previous CMS plants of the genus Lactuca and to provide a CMS plant of the genus Lactuca having low temperature growth ability.SOLUTION: The present invention relates to a cytoplasmic male sterile plant of the genus Lactuca having a low temperature growth ability comparable to that of a plant of the genus Lactuca with a normal cytoplasm, or a progeny thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a cytoplasmic male sterile Lactuca plant having improved low-temperature growth ability. [Background technology]

[0002] There are two types of plant varieties: fixed varieties and first generation hybrid (hereinafter referred to as "F1") varieties, and F1 varieties are widespread for major crops. F1 varieties have great advantages such as vigorous growth, fast growth, and high yield due to heterosis. Furthermore, F1 varieties are expected to have improved environmental adaptability, such as resistance to pests and diseases and cold and heat resistance, due to their vigorous growth. In addition, because the genotype of the F1 variety is heterozygous but identical, the phenotype is extremely uniform. This increases the marketability of the product. Furthermore, since the parents of the F1 variety can accumulate useful traits controlled by dominant genes, rapid breeding is possible.

[0003] Because of these advantages, F1 varieties have come to dominate the cultivars of major crops.

[0004] When producing F1 varieties, the parents are generally self-bred (inbred) lines, and the seed parents and pollen parents are selected from combinations that have the greatest effect on hybrid vigor.

[0005] Seed parents need to be emasculated to prevent self-fertilization, but manual emasculation requires an extremely large amount of labor. If a cytoplasmic male sterility (CMS) line, which is genetically male sterile, is used as a seed parent, manual emasculation becomes unnecessary, and F1 seeds can be produced economically and in large quantities. Commercial production systems for F1 seed production using CMS have been established for sunflower, sugar beet, potato, wheat, carrot, onion, leek, cabbage, broccoli, radish, and Chinese cabbage.

[0006] There were no closely related plant species in the genus Lactuca that had CMS. Therefore, the world's first CMS Lactuca plant was developed by using the CMS of sunflower, which is very distantly related, as a material and introducing CMS into the Lactuca plant using asymmetric cell fusion technology (Patent Document 1). This conventional CMS Lactuca plant has been deposited internationally. Conventional CMS Lactuca plants have very high male sterility stability, making manual emasculation unnecessary and enabling efficient F1 seed production.

[0007] In F1 breeding using CMS, it is important that the cytoplasm that expresses male sterility has as little effect as possible on traits other than male sterility.

[0008] For example, in corn, F1 varieties were developed by introducing T-type male sterile cytoplasm, but in 1970, the T race of the Southern leaf spot fungus appeared, and the T-type male sterile cytoplasm was dealt a major blow because it was specifically susceptible to this pathogen. For this reason, the use of T-type male sterile cytoplasm was immediately discontinued, and it was necessary to revert to the conventional artificial emasculation method (Non-Patent Document 1).

[0009] CMS in petunia has been known for a long time, and the causative gene S-pcf has been used as a research material in many cases. However, F1 varieties using this CMS are rarely used nowadays because they cause delayed flowering and halted flower bud development (Non-Patent Document 1).

[0010] The Ogura CMS is the most commonly used CMS for radish. However, due to the presence of a fertility restorer gene within the species, there has been a problem in that the introduction of CMS is difficult depending on the breeding line. Later, a new NWB-CMS with a different CMS factor was developed for radish, and the introduction rate of CMS improved (Patent Document 2). Furthermore, a new CMS was developed for radish that produces a small amount of sterile pollen and improves the efficiency of attracting pollinating insects (Patent Document 3). These new radish CMS were developed by successive backcrossing using wild radish species as material.

[0011] As in these examples, even if a CMS has been discovered a long time ago, there are cases in which the CMS is accompanied by poor traits or causes fertility restoration, making it difficult or limited to utilize the CMS.

[0012] However, because the utility value of CMS is still very high, when it is difficult to use an existing CMS, new CMS materials are explored and CMS with improved shortcomings are developed, as in the examples above.

[0013] The growth ability after germination at an optimum temperature condition (20°C) of the conventional CMS Lactuca plant (strain name "50125-3-V1") described in the aforementioned Patent Document 1 was equivalent to that of Lactuca plants having normal cytoplasm. However, although it was not clear at the time of the description of Patent Document 1, subsequent research has revealed that conventional CMS Lactuca plants can have reduced low-temperature growth ability. Since the quality of growth ability after germination greatly affects the quality of the subsequent seedlings, it is a very important agricultural trait. Therefore, if a CMS lettuce with improved low-temperature growth ability could be developed, its range of application would be further expanded and its industrial usefulness could be greatly increased.

[0014] The conventional CMS Lactuca plant described in Patent Document 1 has very excellent characteristics, except for the fact that its low-temperature growth ability may be reduced. Therefore, it was thought that using this CMS Lactuca plant as a material to improve the problematic low-temperature growth ability would be a shortcut to development. However, no effective methods for improving the poor characteristics of CMS had been reported, even for all plants, so it was thought that developing CMS lettuce with improved low-temperature growth ability would be extremely difficult. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] International Publication WO2007 / 049730 Brochure [Patent Document 2] Patent No. 3964368 [Patent Document 3] Patent No. 5089764 [Non-patent literature]

[0016] [Non-Patent Document 1] Cytoplasmic Male Sterility and Breeding Techniques, 1985, CMC Publishing Co., Ltd. [Non-Patent Document 2] C. RAMBAUD, A. BELLAMY, A. DUBREUCQ, J.-C. BOURQUIN, and J. VASSEUR (1997) Plant breeding Volume 116, Issue 5 p481-486“Molecular analysis of the fourth progeny of plants derived from cytoplasmic male sterile chicory cybrid” Summary of the Invention [Problem to be solved by the invention]

[0017] In view of the above-mentioned problem of the reduced low-temperature growth ability of conventional CMS Lactuca plants, the present invention aims to provide a CMS Lactuca plant having low-temperature growth ability, and to provide a method for producing F1 seeds of the Lactuca plant having low-temperature growth ability by utilizing the CMS Lactuca plant. [Means for solving the problem]

[0018] As a result of intensive research, the present inventors have now found that by performing asymmetric cell fusion (hereinafter sometimes referred to as "asymmetric back fusion") using a conventional CMS Lactuca plant as a cytoplasm donor parent and a Lactuca plant having normal cytoplasm as a cytoplasm recipient parent, it is possible to improve the cytoplasm, particularly the mitochondrial genome, produce a CMS Lactuca plant having low temperature growth properties, and use the CMS Lactuca plant to efficiently obtain F1 seeds of the Lactuca plant having low temperature growth properties. The present invention is based on these findings.

[0019] That is, according to the present invention, the following inventions are provided.

[0020] <1> A cytoplasmic male sterile Lactuca plant, or its progeny, which has low-temperature growth ability equivalent to that of a Lactuca plant having normal cytoplasm.

[0021] <2> The above-mentioned strain has a mitochondrial genome containing DNA derived from the mitochondrial genome of a plant of the genus Helianthus. <1> A cytoplasmically male sterile Lactuca plant, or its progeny.

[0022] <3> The above-mentioned Lactuca plant is obtained by performing asymmetric cell fusion multiple times using a Lactuca plant having normal cytoplasm as a cytoplasmic recipient parent. <1> or <2> A cytoplasmically male sterile Lactuca plant, or its progeny.

[0023] <4> The method is carried out by asymmetric cell fusion using an existing cytoplasmic male sterile Lactuca plant as a cytoplasm donor parent and a Lactuca plant having normal cytoplasm as a cytoplasm recipient parent. <1> ~ <3> any cytoplasmic male sterile Lactuca plant, or its progeny.

[0024] <5> A cytoplasmic male sterile Lactuca plant or its progeny, which has a mitochondrial genome containing DNA derived from the mitochondrial genome of a Helianthus plant, obtained by multiple asymmetric cell fusions using a Lactuca plant having normal cytoplasm as a cytoplasmic recipient parent; or A cytoplasmic male sterile Lactuca plant, or its progeny, obtained by asymmetric cell fusion using an existing cytoplasmic male sterile Lactuca plant as a cytoplasm donor parent and a Lactuca plant having normal cytoplasm as a cytoplasm recipient parent.

[0025] <6> The cytoplasmic male sterile Lactuca plant is derived from lettuce (Lactuca sativa L.) or an interspecific hybrid plant of the Lactuca plant. <1> ~ <5> any cytoplasmic male sterile Lactuca plant, or its progeny.

[0026] <7> A cytoplasmically male sterile Lactuca plant having a mitochondrial genome of a plant identified by accession number FERM BP-22373, or a progeny thereof. <8> A cytoplasmically male sterile Lactuca plant identified by accession number FERM BP-22373, or its progeny.

[0027] <9> The above <1> ~ <8> A part of any cytoplasmic male sterile Lactuca plant or its progeny. <10> The above <1> ~ <8> The present invention relates to a cytoplasmic male sterile Lactuca plant or a progeny thereof.

[0028] <11> The above <1> ~ <8> A cytoplasmic male sterile plant of the genus Lactuca, or a progeny thereof, <9> or a part of the plant body of said <10> The mitochondrial genome contained in the seeds of

[0029] <12> The plant obtained by asymmetric cell fusion or its progeny is a cytoplasmically male sterile Lactuca plant, which is used as a cytoplasmic donor parent; and A method for asymmetric backfusion in plants, comprising carrying out asymmetric cell fusion one or more times using a Lactuca plant having normal cytoplasm as a cytoplasmic recipient parent, one of the plants used in the first asymmetric cell fusion.

[0030] <13> The above <12> A method for improving the mitochondrial genome in the cytoplasm of a Lactuca plant by the method described above.

[0031] <14> A method for producing a cytoplasmically male sterile Lactuca plant, or a progeny thereof, which has low-temperature growth ability equivalent to that of a Lactuca plant having normal cytoplasm, comprising performing asymmetric cell fusion using an existing cytoplasmically male sterile Lactuca plant as a cytoplasm donor parent and a Lactuca plant having normal cytoplasm as a cytoplasm recipient parent. <15> The cytoplasmic male sterile Lactuca plant is derived from lettuce (Lactuca sativa L.) or an interspecific hybrid plant of the Lactuca plant. <14> How to.

[0032] <16> The above <1> ~ <8> A method for producing first generation hybrid seeds comprising: using any one of the cytoplasmically male sterile Lactuca plants or its progeny as a seed parent, crossing said plant with a Lactuca plant which is crossable with said plant as a pollen parent, and harvesting first generation hybrid seeds from the seed parent after crossing. <17> The pollen parent is lettuce (Lactuca sativa L.) or an interspecific hybrid plant of the genus Lactuca. <16> How to.

[0033] <18> The above <16> or <17> A first generation hybrid seed produced by the method of (a) or a first generation hybrid plant grown from said seed, its progeny, or a part of said plant.

[0034] <19> The above <1> ~ <8> A method for producing a Lactuca plant having a useful trait and expressing cytoplasmic male sterility, comprising successively backcrossing a Lactuca plant having a useful trait with any one of the cytoplasmic male sterile Lactuca plants described above, or a progeny thereof, to effect cytoplasmic substitution. <20> The Lactuca plant having useful characteristics is derived from lettuce (Lactuca sativa L.), <19> How to. Effect of the Invention

[0035] According to the present invention, it is possible to provide an improved CMS Lactuca plant having improved low-temperature growth ability. By using the improved CMS Lactuca plant according to the present invention, it is possible to efficiently produce F1 seeds of Lactuca plants having low-temperature growth ability.

[0036] The asymmetric back-fusion method of the present invention also allows for the modification of the cytoplasm, and in particular the mitochondrial genome. [Brief description of the drawings]

[0037] [Figure 1] The figure shows the results of a germination test 17 days after sowing for "Thermi" (A) with normal cytoplasm and "Thermi" (B) with conventional CMS cytoplasm, in a greenhouse environment with a night temperature of 0°C and a day temperature of 20°C. [Diagram 2] The figure shows the results of a germination test 17 days after sowing of "V lettuce" (A) with normal cytoplasm and "V lettuce" (B) with conventional CMS cytoplasm in a greenhouse environment with a night temperature of 0°C and a day temperature of 20°C. [Diagram 3]The figure shows the results of a germination test 16 days after sowing for "V lettuce" with normal cytoplasm (A) and "V lettuce" with conventional CMS cytoplasm (B), when the artificial weather chamber was set to the optimum temperature conditions for growth (night temperature 20°C, day temperature 20°C, 12 hours of light). [Figure 4] The figure shows the results of a germination test 17 days after sowing for "V lettuce" with normal cytoplasm (A) and "V lettuce" with conventional CMS cytoplasm (B), when the artificial weather chamber was set to low temperature conditions (night temperature 5°C, day temperature 20°C, 12-hour light period). [Diagram 5] The figure shows the results of a germination test 17 days after sowing of "V lettuce" (A) with normal cytoplasm and "V lettuce" (B) with improved CMS cytoplasm in a greenhouse environment with a night temperature of 0°C and a day temperature of 20°C. [Figure 6] The figure shows the results of a germination test 17 days after sowing for "M8-039" (A) with normal cytoplasm and "M8-039" (B) with improved CMS cytoplasm in a greenhouse environment with a night temperature of 0°C and a day temperature of 20°C. [Figure 7] The figure shows the results of a germination test 17 days after sowing for "Steady" (A) with normal cytoplasm and "Steady" (B) with improved CMS cytoplasm in a greenhouse environment with a night temperature of 0°C and a day temperature of 20°C. [Figure 8] The figure shows the results of a germination test 17 days after sowing for "Steady" with normal cytoplasm (A) and "Steady" with improved CMS cytoplasm (B) in a low-temperature environment in an artificial climate chamber (night temperature 5°C, day temperature 16°C, 12-hour light period). [Figure 9] The figure shows the BC7 generation of "Steady" (A) and "BF2MS1S" (B), which have normal cytoplasm, planted in a field (Azumino City, Nagano Prefecture) and grown to the heading stage. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] The present invention will be described in detail below.

[0039] Cytoplasmic male sterile Lactuca plants and their progeny with improved low-temperature growth ability The present invention relates to a cytoplasmic male sterile Lactuca plant or its progeny, which has improved low-temperature growth ability compared to conventional plants. As described above, this can be expressed as a cytoplasmic male sterile Lactuca plant or its progeny, which has low-temperature growth ability equivalent to that of a Lactuca plant having normal cytoplasm.

[0040] In this specification, "low temperature growth" refers to the growth of seedlings under low temperature conditions. Here, low temperature refers to a state lower than the temperature conditions that are suitable for the natural growth of Lactuca plants, and includes not only the case where the average temperature is low during the day, but also the case where the temperature is low only during a part of the day (for example, at night). Specifically, low temperature includes, for example, a case where the daytime temperature is 20°C, and the nighttime temperature is lower than the daytime temperature, for example, the nighttime temperature is 5 to 0°C. Low-temperature growth ability can be quantitatively evaluated by cultivating the target plants under the same low-temperature conditions, measuring and comparing the weights of the above-ground parts of the seedlings, and comparing them with the results of cultivation under appropriate temperature conditions.

[0041] In the present invention, the term "normal cytoplasm" is typically used to mean normal and not sterile, as opposed to the cytoplasm of a plant that exhibits male sterility, i.e., male sterile cytoplasm.

[0042] Furthermore, "equivalent" in the phrase "low-temperature growth ability equivalent to that of a Lactuca plant having normal cytoplasm" refers to a case where, when the low-temperature growth ability is measured based on the weight of the above-ground part of the seedling, the measured value of the target plant is within a range that can vary within 25% (preferably within 20%, more preferably within 15%, and even more preferably within 10%) compared to the value of a Lactuca plant having normal cytoplasm. Therefore, for example, when the measured value of the target plant is 90% of the value of the normal plant with respect to the above-ground part weight of the seedling of a "Lactuca plant having normal cytoplasm", the above-mentioned variation corresponds to 10%. "Equivalent" does not exclude cases where the low-temperature growth ability exceeds that of a "Lactuca plant having normal cytoplasm".

[0043] In this specification, the term "progeny" includes not only progeny using a maintenance line of the cytoplasmic male sterile Lactuca plant, but also hybrids obtained by crossing the cytoplasmic male sterile Lactuca plant of the present invention with a Lactuca plant that can be crossed with the plant. Therefore, the term "progeny" also includes, for example, those obtained by crossing the cytoplasmic male sterile Lactuca plant of the present invention as a seed parent (female parent) with a Lactuca plant that can be crossed with the plant as a pollen parent (male parent). In addition, the term "progeny" also includes, for example, plants obtained by cell fusion between the cytoplasmic male sterile Lactuca plant of the present invention and a plant that can be crossed with the Lactuca plant, and inter-species hybrid plants.

[0044] Here, the "Lactuca plant" is preferably Lactuca sativa L., L. serriola, L. aculeate, L. scarioloides, L. azerbaijanica, L. georgica, L. dregeana, L. altaica, L. saligna, L. virosa, L. tatarica, L. indica, or L. debilis, or an interspecific hybrid plant thereof, and in particular, L. sativa L., which is a cultivated species of the Lactuca plant, or an interspecific hybrid plant of the Lactuca plant is preferred, and L. sativa L. is more preferred.

[0045] According to a preferred embodiment of the present invention, the cytoplasmic male sterile Lactuca plant of the present invention or its progeny has DNA derived from the mitochondrial genome of a Helianthus plant in its mitochondrial genome. Here, the "Helianthus plant" is preferably a sunflower cultivar Helianthus annuus L., or a cytoplasmic replacement line of H. annuus L. having cytoplasm derived from H. petiolaris, H. argophyllus, H. debilis, H. decapetalus, H. giganteus, H. rigidus, H. salicifolius, H. anomalus, H. bolanderi, H. exilis, H. maximiliani, H. neglectus, H. praecox, or H. tuberosus.

[0046] As used herein, asymmetric cell fusion refers to a method in which one of the nuclear genomes of isolated protoplasts used in cell fusion is inactivated before fusion and then cell fusion is performed using the inactivated nuclear genome. In this asymmetric cell fusion, a cell whose nuclear genome is inactivated during fusion and whose cytoplasm is donated to the fused cell by cell fusion is called a cytoplasm donor parent. A cell whose nuclear genome is not inactivated during fusion and that receives the cytoplasm from the cytoplasm donor parent is called a cytoplasm recipient parent.

[0047] Asymmetric back fusion (or asymmetric back cell fusion) refers to performing one or more (preferably one) additional asymmetric cell fusions using a plant obtained by asymmetric cell fusion or its progeny as a cytoplasm donor parent and one of the plants used in the first asymmetric cell fusion as a cytoplasm recipient parent. That is, in asymmetric back fusion, asymmetric cell fusion is performed two or more times, including the first time.

[0048] When obtaining the cytoplasmic male sterile Lactuca plant of the present invention, it is preferable to use a Lactuca plant having normal cytoplasm as a cytoplasmic recipient parent in asymmetric cell fusion. Therefore, according to a preferred embodiment of the present invention, the cytoplasmic male sterile Lactuca plant of the present invention can be obtained by performing asymmetric cell fusion multiple times, i.e., two or more times, using a Lactuca plant having normal cytoplasm as a cytoplasmic recipient parent. Alternatively, according to another preferred embodiment of the present invention, the cytoplasmic male sterile Lactuca plant of the present invention can be obtained by performing asymmetric cell fusion using an existing cytoplasmic male sterile Lactuca plant as a cytoplasmic donor parent and a Lactuca plant having normal cytoplasm as a cytoplasmic recipient parent. Here, the existing cytoplasmic male sterile Lactuca plant means a cytoplasmic male sterile Lactuca plant before being improved in the present invention, i.e., a conventional cytoplasmic male sterile Lactuca plant. In the present invention, the existing cytoplasmic male sterile Lactuca plant preferably means one which has room for improvement in low-temperature growth ability, i.e., one whose low-temperature growth ability is reduced compared to that of a Lactuca plant having normal cytoplasm.

[0049] Therefore, according to another aspect of the present invention, the cytoplasmic male sterile Lactuca plant according to the present invention, or a progeny thereof, is a cytoplasmic male sterile Lactuca plant having DNA derived from the mitochondrial genome of a Helianthus plant in its mitochondrial genome, or a progeny thereof, obtained by multiple asymmetric cell fusions using a Lactuca plant having normal cytoplasm as a cytoplasmic recipient parent; or It is obtained by asymmetric cell fusion using an existing cytoplasmic male sterile Lactuca plant as a cytoplasm donor parent and a Lactuca plant having normal cytoplasm as a cytoplasm recipient parent.

[0050] According to a more preferred embodiment of the present invention, the cytoplasmically male sterile Lactuca plant according to the present invention, or a progeny thereof, is a Lactuca plant having a mitochondrial genome of a plant identified by accession number FERM BP-22373 (described below), or a progeny thereof, and more preferably, is a Lactuca plant identified by accession number FERM BP-22373, or a progeny thereof.

[0051] According to another aspect of the present invention, there is provided a method for asymmetric back fusion in plants. This asymmetric back fusion method includes performing asymmetric cell fusion once or multiple times using a plant obtained by asymmetric cell fusion or its progeny and one of the plants used in the first asymmetric cell fusion. Here, preferably, the plant obtained by asymmetric cell fusion or its progeny is a cytoplasmic male sterile Lactuca plant, which is used as a cytoplasm donor parent, and one of the plants used in the first asymmetric cell fusion is a Lactuca plant with normal cytoplasm, which is used as a cytoplasm recipient parent, to perform asymmetric cell fusion.

[0052] According to yet another aspect of the present invention, there is provided a method for improving the mitochondrial genome in the cytoplasm of a plant by the asymmetric backfusion method described above.

[0053] As used herein, a "part of a plant" of a cytoplasmic hybrid Lactuca plant or its progeny includes one or more cells or cytoplasm from one or more cells of the plant, and specifically means organs or tissues such as flowers, leaves, stems, roots, etc., or cells (including protoplasts prepared from cells) or cytoplasm from these organs or tissues, or an aggregate of said cells or cytoplasm.

[0054] Methods for producing cytoplasmic male sterile Lactuca plants with improved low-temperature growth ability The improved cytoplasmic male sterile Lactuca plant according to the present invention can be produced, for example, by the following procedure. (1) Preparation of protoplasts (i) Isolation of plant protoplasts with normal cytoplasm (ii) Isolation of protoplasts from cytoplasmic male sterile plants (2) Protoplast fusion treatment (3) Cultivation of fused hybrid cells (4) Regeneration of plants from callus (5) Selection of cytoplasmic hybrid plants (6) Selection of superior lines (7) Continuous backcrossing of superior lines (8) Use of cytoplasmic male sterile plants and production of F1 seeds

[0055] In this specification, the term "production method" can be rephrased as "creation method." In other words, the terms "creation" and "production" are used herein with the same meaning.

[0056] More specifically, each of these steps is as follows.

[0057] (1) Preparation of protoplasts (i) Isolation of protoplasts from Lactuca plants with normal cytoplasm In the present invention, the Lactuca plant used as the cytoplasmic recipient is preferably Lactuca sativa L., L. serriola, L. aculeate, L. scarioloides, L. azerbaijanica, L. georgica, L. dregeana, L. altaica, L. saligna, L. virosa, L. tatarica, L. indica, or L. debilis, or an interspecific hybrid thereof, and among these, L. sativa L., a cultivated species of the Lactuca plant, is preferred.

[0058] As the cell tissue used to obtain protoplasts, it is preferable to use mesophyll tissue, which has high yield and division activity, but other tissues such as hypocotyls, stems, and calluses may also be used as materials.

[0059] The method for isolating protoplasts may be a 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 provides specific examples of procedures, but the present invention is not necessarily limited to these.

[0060] First, cell tissue of a Lactuca plant is cut into pieces and treated with an enzyme solution for isolating protoplasts to isolate protoplasts. This solution is an inorganic salt buffer solution mainly containing a cell wall decomposing enzyme and an osmotic pressure regulator. The cell wall decomposing enzyme is not particularly limited as long as it can be used to decompose the cell wall of a plant, and examples thereof include cellulase, hemicellulase, and pectinase. In the present invention, a combination of cellulase YC and macerozyme R-10 is preferred. As the osmotic pressure regulator, general sugar alcohols such as mannitol, sorbitol, and glucose can be used, and mannitol is preferred, and mannitol at a concentration of 0.3M to 0.7M is particularly preferred. Furthermore, it is desirable to add an inorganic salt to the enzyme solution in order to stabilize the membrane of the protoplast, and it is preferable to add, for example, CPW salt (Cocking and Peberdy, 1974) having the composition shown in Table 1 below. The enzyme treatment is preferably performed by leaving the solution at 25 to 30° C. for 8 to 20 hours.

[0061] [Table 1]

[0062] The protoplasts isolated by enzyme treatment are filtered through a nylon mesh with a pore size of 30 to 100 μm, centrifuged to collect the protoplasts, and the enzyme solution is removed. The protoplasts are then suspended in a washing solution to wash them. The washing solution can be a commonly used CPW salt solution to which sugar alcohols have been added as an osmotic pressure regulator.

[0063] Next, it is desirable to carry out an inactivation treatment to prevent the protoplasts of Lactuca plants from dividing independently. The inactivation treatment can be carried out by suspending the protoplasts in a CPW salt solution in which an iodine compound such as iodoacetic acid or iodoacetamide is dissolved. In the present invention, it is preferable to suspend the protoplasts in a CPW solution in which the concentration of iodoacetamide has been adjusted to 5 mM to 30 mM, and carry out the treatment for 5 to 20 minutes.

[0064] Next, it is preferable to repeat the washing operation with CPW salt solution 1 to 3 times using centrifugation. Since fragments of vessels and cells are also mixed into the protoplast suspension, it is preferable to further purify the protoplasts by density gradient centrifugation or the like. Reagents used for purification include sugars and synthetic colloids, but in the present invention, the use of sucrose solution is preferable, and the use of 15% to 20% sucrose solution is particularly preferable. After purification of the protoplasts, the cell density is measured using a hemocytometer, and the liquid volume is adjusted with CPW salt solution to obtain a cell density suitable for cell fusion. The cell density of the protoplasts is 1 x 10 5 ~1×10 7 Cells / ml is preferred, and CPW salt solution is preferably used to adjust the volume.

[0065] (ii) Conventional CMS Lactuca protoplast isolation The CMS Lactuca plant used as the cytoplasm donor parent (donor) is not particularly limited as long as it has cytoplasmic male sterility, but is preferably a Lactuca plant with stable CMS. Specifically, for example, it is preferable to use the conventional CMS Lactuca plant described in Patent Document 1 (Accession No. FERM BP-10647) as the cytoplasm donor parent.

[0066] Conventional CMS Isolation of protoplasts from plants of the genus Lactuca can be carried out, for example, according to a method similar to that for isolating protoplasts from plants of the genus Lactuca described above.

[0067] It is preferable to use isolated protoplasts of conventional CMS Lactuca plants in which nuclear genes have been inactivated by radiation treatment. Examples of radiation to be irradiated for radiation treatment include X-rays, gamma rays, ultraviolet rays, etc., but are not particularly limited as long as they can inactivate nuclear genes. The irradiation dose is preferably as low as possible within the range that can inactivate nuclear genes. For example, in the case of irradiation with soft X-rays in the present invention, the irradiation dose is preferably 100 Gy to 900 Gy.

[0068] (2) Protoplast fusion Next, the two types of protoplasts obtained above are mixed together to carry out cell fusion. The fusion method includes, but is not limited to, conventional methods such as the well-known electric fusion method (Planta, 151, 26-32, 1981), PEG (polyethylene glycol) method (Planta, 120, 215-227, 1974), dextran method (Jap. J. Genet., 50, 235, 1975), etc. In the present invention, the PEG method is preferably used.

[0069] (3) Cultivation of fused 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 Lactuca. There are various known methods for culturing protoplasts derived from plants of the genus Lactuca, and although there is no limitation thereto, in the present invention, it is preferable to use a modified method of the method of Patent Document 1.

[0070] In Example 1 of Patent Document 1, asymmetric cell fusion is performed by combining protoplasts of the genus Helianthus and protoplasts of a plant of the genus Lactuca. On the other hand, in the present invention, asymmetric cell fusion is performed by combining protoplasts of plants of the genus Lactuca, and therefore the fused cells can be more suitably cultured by considering the optimum type and concentration of a plant growth regulator. Specifically, the optimal conditions for culturing fused cells can be achieved by appropriately combining the plant growth regulators NAA (α-Naphthaleneacetic Acid), 4-CPPU (N-(2-Chloro-4-pyridyl)-N'-phenylurea), and TDZ (Thidiazuron).

[0071] (4) Regeneration of plants from callus The fused cells are cultured, and when cell division begins and callus can be visually confirmed, the callus is transferred to a regeneration medium and allowed to regenerate. Conventional regeneration media can be used, and although the response varies depending on the Lactuca plant and the state of the callus, it is preferable to use, for example, MS medium containing 0.3 to 1.0 mg / l of 4-CPPU. The regenerated shoot is transplanted to a rooting medium, such as 1 / 2 concentration MS medium, and rooted to regenerate the plant. The regenerated plant is acclimatized and grown in a greenhouse.

[0072] (5) Selection of cytoplasmic hybrid plants DNA is extracted from the leaves of the plant body regenerated by the above procedure, as well as the conventional CMS Lactuca plant and the Lactuca plant with normal cytoplasm used as materials. In order to efficiently select cytoplasmic hybrid plants, it is preferable to detect the mitochondrial DNA sequence that can distinguish the conventional CMS Lactuca plant from the Lactuca plant with normal cytoplasm by PCR using a marker that can specifically amplify the mitochondrial DNA sequence.

[0073] The marker that can be used here is not particularly limited as long as it can specifically amplify only the mitochondrial DNA sequence derived from the conventional CMS Lactuca plant. In the present invention, as described above, it is preferable to use the conventional CMS Lactuca plant (Accession No. FERM BP-10647) described in Patent Document 1 as a cytoplasm donor parent, and in this case, the atp6 gene derived from sunflower can be detected by PCR using a marker that can specifically amplify the atp6 gene.

[0074] Cytoplasmic hybrid plants can be efficiently selected by performing PCR analysis using a marker that can specifically amplify the above-mentioned mitochondrial DNA sequence. In addition, the selection of cytoplasmic hybrid plants can be carried out, if necessary, by appropriately referring to the method of Patent Document 1.

[0075] The above PCR method is preferably carried out at the callus stage, but may also be carried out at other stages, such as on acclimatized plant bodies. It is also preferable to perform a test for ploidy by measuring the DNA content with flow cytometry or observing chromosomes to select diploids and eliminate higher polyploids. Since recombination of the mitochondrial genome by asymmetric cell fusion occurs frequently and randomly, it is preferable to produce more than 300 diploid cytoplasmic hybrid plants.

[0076] (6) Selection of superior lines The resulting cytoplasmic hybrid plants are cultivated and allowed to flower, and lines that have the male sterility trait but no morphological abnormalities in other organs are selected. The selected male sterile individuals are crossed with pollen from Lactuca plants with normal cytoplasm, and the progeny seeds are harvested. Furthermore, the selection of superior lines can be carried out, if necessary, by appropriately referring to the method of Patent Document 1.

[0077] Next, to select a line with excellent low-temperature growth ability, seeds are sown under low-temperature conditions, preferably with a night temperature of 0-5°C and a day temperature of 15-20°C. A Lactuca plant with normal cytoplasm is used as a control to compare its low-temperature growth ability with that of the CMS line produced, and individuals showing equal or better low-temperature growth ability are selected. If no individual showing the same or better low-temperature growth ability is obtained, it is desirable to select an individual with relatively high low-temperature growth ability and use it again as a cytoplasm donor parent for asymmetric cell fusion. If an individual showing the same or better low-temperature growth ability as a Lactuca plant with normal cytoplasm is obtained, proceed to the next step of successive backcrossing.

[0078] (7) Continuous backcrossing of superior lines Cytoplasmic hybrid plants obtained by cell fusion are in a heteroplasmic state, so even backcrossing progeny often have variations in their characteristics. Therefore, the progeny of cytoplasmic hybrids may have different properties at the cellular level, and it is desirable to select superior CMS lines for each individual. The aforementioned Non-Patent Document 2 suggests that cytoplasm requires continuous backcrossing for five or more generations before it becomes homoplasmic. Therefore, in order to produce a CMS line with low-temperature growth ability, it is desirable to repeat selection and backcrossing using low-temperature growth ability as an indicator for five or more generations until the low-temperature growth ability becomes stable.

[0079] Once a CMS line with stable low-temperature growth characteristics is obtained, that line is selected as an improved CMS line (i.e., a cytoplasmic male sterile Lactuca plant with improved low-temperature growth characteristics) for use in actual breeding.

[0080] (8) Utilization of cytoplasmic male sterile plants and production of F1 seeds When producing F1 seeds, self-fertilized (inbred) lines are generally used as the parent lines. The seed parent and the pollen parent are determined, and the parent lines must be crossed artificially. Therefore, to prevent the seed parent from self-fertilizing, emasculation is required, but in the case of Lactuca plants, artificial emasculation is difficult due to the structure of the flower, so it is preferable to use CMS.

[0081] Since the male sterility of the improved CMS line is inherited through cytoplasmic inheritance, any excellent Lactuca plant (i.e., a Lactuca plant having useful traits) can be easily made into a CMS plant by successive backcrossing using any excellent Lactuca plant as the pollen parent and the improved CMS line as the seed parent, thereby carrying out nuclear substitution. In other words, by using the improved CMS line deposited in the present invention, any Lactuca plant that can be crossed can be made into an excellent CMS plant without using the asymmetric backfusion technique that requires skilled techniques.

[0082] By successive backcrossing, any excellent CMS-treated Lactuca plant can be used as a seed parent and any pollen parent can be crossed by hand or insects to efficiently obtain F1 seeds of Lactuca plants with low-temperature growth properties.

[0083] By creating an F1 variety of lettuce using the above procedure, it will be possible to rapidly breed lettuce varieties that have low-temperature growth properties and excellent traits, and to create marketable lettuce varieties that excel in product uniformity and environmental adaptability. EXAMPLES

[0084] The present invention will be specifically described with reference to the following examples, but the present invention is not limited to these examples.

[0085] Example 1: Creation of improved CMS Lactuca plants (1) Preparation of protoplasts (i) Isolation of protoplasts from Lactuca plants with normal cytoplasm As a Lactuca plant having normal cytoplasm, lettuce cultivar "Steady" (available from Tsuruta Seed Co., Ltd.) was used. First, sterilized seeds were placed on MS medium supplemented with 10 g / l sucrose and 8 g / l agar, and cultured for one month at 20°C under 16-hour lighting. Approximately 1 g of expanded primary leaves was collected and cut into pieces of approximately 2 mm in size, then immersed in 10 ml of CPW salt solution containing 0.4% cellulase YC, 0.4% Macerozyme R-10, and mannitol, and allowed to stand at 25°C for 16 hours.

[0086] The enzyme solution containing the leaf tissue was filtered through a 92 μm nylon mesh to remove cell debris. The resulting protoplast suspension was transferred to a centrifuge tube and centrifuged at 800 rpm for 5 minutes. The protoplasts obtained by removing the supernatant were suspended in 5 ml of CPW salt solution containing 15 mM iodoacetamide and incubated at 25 °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 three times.

[0087] 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 containing 20% ​​sucrose was added to a new centrifuge tube, and the above protoplast suspension was layered on top of it, followed by centrifugation at 800 rpm for 5 minutes. The cell debris sank to the bottom of the centrifuge tube, and the purified protoplasts floated to the top of the CPW salt solution layer, which was then transferred to a new centrifuge tube with a Pasteur pipette. A small amount of the suspension was taken and the protoplast cell density was determined using a hemocytometer, and CPW solution was added to obtain a cell density of 1 × 10 6 The concentration was adjusted to 1 / ml.

[0088] (ii) Isolation of protoplasts from conventional CMS lettuce As the conventional CMS lettuce, the lineage name "50125-3-V1" (accession number FERM BP-10647) described in Patent Document 1 was used. It should be noted that "50125-3-V1" is a progeny obtained by crossing the CMS lettuce line "50125-3" with "V lettuce" (available from Kaneko Seed Co., Ltd.), and seeds of "50125-3-V1" have been internationally deposited with the National Institute of Advanced Industrial Science and Technology (6 Central, 1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture) on July 31, 2006 (identification mark attached by the depositor SSC-LET-06-001, accession number FERM BP-10647) (Patent Document 1).

[0089] First, sterilized seeds were placed on MS medium supplemented with 10 g / l sucrose and 8 g / l agar, and cultured for one month at 20°C under 16-hour lighting. Approximately 1 g of expanded true leaves were harvested and cut into pieces of approximately 2 mm in size, then immersed in 10 ml of CPW salt solution containing 0.4% cellulase YC, 0.4% Macerozyme R-10, and mannitol, and allowed to stand at 25°C for 16 hours.

[0090] The enzyme solution containing the leaf tissue was filtered through a 92-μm nylon mesh to remove cellular debris. The protoplasts were transferred to a plastic petri dish with a Pasteur pipette and irradiated with 900 Gy of soft X-rays.

[0091] The resulting 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 containing 20% ​​sucrose was added to a new centrifuge tube, and the above protoplast suspension was layered on top of it and centrifuged at 800 rpm for 5 minutes. The cell debris sank to the bottom of the centrifuge tube, and the purified protoplasts floated to the top of the CPW salt solution layer, which was then transferred to a new centrifuge tube with a Pasteur pipette. A small amount of the suspension was taken and the protoplast cell density was determined using a hemocytometer, and CPW salt solution was added to obtain a cell density of 1 × 10 6 The concentration was adjusted to 1 / ml.

[0092] (2) Protoplast fusion treatment A suspension of lettuce protoplasts treated with iodoacetamide and a suspension of conventional CMS lettuce protoplasts irradiated with soft X-rays were mixed in a ratio of 1:3, and 2 ml of the mixture was dropped onto the center of the bottom of a 9 cm petri dish. After leaving it to stand for 30 minutes, 3 ml of PEG solution (polyethylene glycol #6000 (Nacalai Tesque Inc.) 500 g / l, CaCl2·2H2O 1,500 mg / l, KH2PO4 100 mg / l, pH 5.5) was dropped onto the periphery of the protoplast mixture.

[0093] After 1 minute, 3.5 ml of CPW salt solution was dropped around the protoplast mixture. After another 2 minutes, 3.5 ml of CPW salt solution was dropped around the protoplast mixture. After 5 minutes, the dropped solution was gently sucked up and removed from the edge of the dish, and 20 ml of CPW salt solution was added from the edge of the dish. This washing procedure with CPW salt solution was repeated three times at 5-minute intervals.

[0094] (3) Cultivation of fused hybrid cells After removing the washing solution, 10 ml (pH 5.8) of 1 / 2 concentration MS medium (hereinafter referred to as lettuce protoplast culture medium) containing 2.7 g / l disodium succinate hexahydrate, 0.5 g / l casamino acids, 0.1 mg / l NAA, 1.0 mg / l 4-CPPU, 0.1 mg / l TDZ, and 0.3 M sucrose with NH4NO3 reduced to 200 mg / l was added, and the medium was cultured in the dark at 25°C.

[0095] Three days after the start of cultivation, 5 ml of 4x concentrated lettuce protoplast cultivation medium (0.3 M sucrose concentration) was mixed with 5 ml of 0.3 M sucrose solution (pH 5.8) containing 0.6% gellan gum to prepare a semi-solid gel medium, in which cultivation was continued.

[0096] Ten days after the start of culture, 10 ml of medium containing the fused hybrid cells was transferred together with the gel into 10 ml of lettuce protoplast culture medium with the sucrose concentration modified to 0.15 M. Twenty days after the start of culture, callus could be confirmed with the naked eye, and the cultured callus was transferred to lettuce protoplast culture medium (solid medium, pH 5.8) containing 0.2 M sucrose and 0.3% gellan gum.

[0097] (4) Regeneration of plants from callus Thirty days after the start of culture, when the calli had grown to a size of about 2 mm, they were transferred to MS medium (pH 5.8) containing 0.3 mg / l 4-CPPU, 1.0% sucrose, and 0.8% agar.

[0098] After 40 to 60 days of culture, shoots regenerated from the callus were rooted by transplanting them onto 1 / 2 MS medium (pH 5.8) containing 1.0% sucrose and 0.8% agar, and more than 300 cytoplasmic hybrid plants were regenerated.

[0099] Cytoplasmic hybrid plants were acclimatized by transplanting them onto vermiculite and then grown in a greenhouse.

[0100] (5) Selection of cytoplasmic hybrid plants with mitochondrial genes specific to conventional CMS lettuce To detect DNA specific to conventional CMS lettuce by PCR, primers specific to the atp6 gene were designed based on the known base sequence information (Gene Bank accession number X82387.1) (Table 2).

[0101] PCR was performed using the extracted total genomic DNA as a template and a combination of primers atp6-F and atp6-R. PCR consisted of 35 cycles of thermal denaturation at 94°C for 1 minute, annealing at 60°C for 2 minutes, and extension reaction at 72°C for 2 minutes. The PCR products were electrophoresed on a 1.8% agarose gel, immersed in an ethidium bromide solution, and then photographed under UV irradiation to confirm the presence or absence of an amplified product of the expected size (209 bp). Individuals in which amplification was confirmed were selected.

[0102] In this way, we obtained the CMS line "BF2MS1S" with improved low-temperature growth ability.

[0103] [Table 2]

[0104] Example 2: Detailed development process of improved CMS lines The CMS line "BF2MS1S" produced in Example 1 was crossed with lettuce (variety "Steady") having normal cytoplasm to obtain 156 seeds of BC1 (first generation backcross progeny). 156 seeds of the obtained BC1 seeds of one line were sown in a 288-hole tray and raised in a greenhouse set at a night temperature of 0°C and a day temperature of 20°C. The seedlings of BC1 generally showed high low-temperature growth ability, but germination was somewhat variable. 24 lines with excellent low-temperature growth ability were selected from the germinated individuals of the 156 BC1 seeds, and backcrossed with Steady to obtain BC2 seeds.

[0105] The 20 seeds of each of the 24 BC2 lines were sown in 200-well trays and raised in a greenhouse under the same conditions. The BC2 seedlings showed high low-temperature growth ability overall. Germination was somewhat variable, but was improved over the BC1 seedlings. From the 480 germinated BC2 seeds, 38 lines with excellent low-temperature growth ability were selected, and steady backcrossing was performed to obtain BC3 seeds.

[0106] The BC3 seeds obtained were sown in 200-well trays of 20 seeds each from 38 lines, and raised in a greenhouse under the same conditions. The BC3 seedlings showed high low-temperature growth ability overall, with little variation in germination. 49 lines with excellent low-temperature growth ability were selected from the 760 germinated BC3 seeds, and steady backcrossing was performed to obtain BC4 seeds.

[0107] Twenty seeds from each of the 49 BC4 lines were sown in 200-well trays and grown in a greenhouse under the same conditions. The BC4 seedlings showed high low-temperature growth ability overall, with little variation in germination. In other words, it is believed that as generations progressed, the cytoplasm became homoplasmic, and the differences in low-temperature growth ability between individuals became smaller. Four lines with excellent low-temperature growth ability were selected from the 980 germinated BC4 seeds, and steady backcrossing was performed to obtain BC5 seeds.

[0108] The BC5 seeds were sown in 288-well trays of 96 seeds each from the four lines, and raised in a greenhouse under the same conditions. The BC5 seedlings showed high low-temperature growth and little variation in germination. Five lines with excellent low-temperature growth were selected from the 384 germinated BC5 seeds, and steady backcrossing was performed to obtain BC6 seeds.

[0109] The obtained BC6 seeds, 96 seeds each of the five lines, were sown in 288-well trays and raised in a greenhouse under the same conditions. The seedlings of BC6 showed high low-temperature growth ability, and no variation in germination was observed. Since the cytoplasm was considered to have been homoplasmic, it was determined that there was no need to select multiple lines thereafter. From the 480 germinated BC6 seeds, one line with excellent low-temperature growth ability was selected, and steady backcrossing was performed to obtain BC7 seeds.

[0110] The BC7 seeds (96 seeds) were sown in a 288-well tray and grown in a greenhouse under the same conditions. The BC7 seedlings showed high low-temperature growth and no variation in germination was observed. Steady backcrossing was performed on one line with excellent low-temperature growth ability from the 96 germinated BC7 seeds, and BC8 seeds were obtained.

[0111] The 96 seeds of one lineage of BC8 obtained were sown in a 288-well tray and raised in a greenhouse under the same conditions. The seedlings of BC8 had high low-temperature growth potential and showed no variation in germination, suggesting that the cytoplasm had become homoplasmic and was stable. Steady backcrossing was performed on one lineage with excellent low-temperature growth potential from among the 96 germinated BC8 seeds, and BC9 seeds were obtained.

[0112] In addition, BC10 seeds of the progeny "BF2MS1S" lineage obtained by crossing "BF2MS1S" cultivated from seeds of BC9 with "Steady" have been internationally deposited (original deposit) at the National Institute of Technology and Evaluation, Patent Organism Depositary Center (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) on February 1, 2019 (identification marking added by the depositor: SSC-LET-19-001, deposit number: FERM BP-22373).

[0113] Example 3: Confirmation of the characteristics of improved CMS lettuce by germination test In order to confirm the usefulness of the improved CMS line produced in Example 1, a germination test was carried out on various lettuces having normal cytoplasm and CMS cytoplasm. Since lettuce may have differences in seed quality and germination ability depending on the seed harvesting environment, all the lines used in this example were harvested under the same environment. In addition, since lettuce seeds may go dormant, all germination tests in this example were performed after refrigerating the cell trays after sowing in a refrigerator at 4°C for 2 days to ensure that dormancy was broken.

[0114] First, the low-temperature growth ability of the backcross progeny between the line "1216-2-T1" (accession number FERM BP-10421), which is a conventional CMS lettuce described in Patent Document 1, and the line "50125-3-V1" was confirmed.

[0115] Here, "1216-2-T1" is a progeny obtained by crossing the CMS line "1216-2" with "Thermy" (Sakata Seed Corporation), and seeds of "1216-2-T1" have been internationally deposited with the National Institute of Advanced Industrial Science and Technology (6 Central, 1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture) on September 29, 2005 (identification mark SSC-LET-05-001 attached by the depositor, accession number FERM BP-10421) (Patent Document 1).

[0116] 64 seeds of each of "Thermi" with normal cytoplasm and "1216-2-T1", a CMS lettuce described in Patent Document 1, "V lettuce" with normal cytoplasm and "50125-3-V1", a CMS lettuce described in Patent Document 1, were sown in a 128-well cell tray. The sown cell trays were kept in a greenhouse (Kakegawa City, Shizuoka Prefecture) with a night temperature set at 0°C and a day temperature set at 20°C.

[0117] Figure 1 shows the results of the germination test on the 17th day after sowing for "Thermi" and "1216-2-T1". Compared to "Thermi," "1216-2-T1" showed very poor uniformity in germination and was visually confirmed to have inferior low-temperature growth ability.

[0118] For quantitative evaluation, the above-ground parts of seedlings of each line were cut off at ground level and the weights per plant were compared. The results are shown in Table 3.

[0119] As shown in No. 1 and No. 2 in Table 3, the weight of the above-ground parts of "1216-2-T1" per plant was 25% of the weight of the above-ground parts of "Thermi" per plant. Since the nuclear genome of “1216-2-T1” is identical to that of “Thermi,” the decrease in growth rate of “1216-2-T1” due to low temperatures was thought to be due to the influence of the CMS cytoplasm.

[0120] Figure 2 shows the results of the germination test on the 17th day after sowing of "V lettuce" and "50125-3-V1." Compared to "V lettuce," "50125-3-V1" showed less uniform germination and was visually confirmed to have inferior low-temperature growth ability.

[0121] For quantitative evaluation, the above-ground parts of seedlings of each line were cut off at ground level and the weights per plant were compared. The results are shown in Table 3.

[0122] As shown in No. 3 and No. 4 in Table 3, the weight of the above-ground parts of “50125-3-V1” per plant was 55% of the weight of the above-ground parts of “V lettuce” per plant. Because the nuclear genome of "50125-3-V1" is identical to that of "V lettuce," the reduced growth rate of "50125-3-V1" due to low temperatures was previously thought to be due to the influence of the CMS cytoplasm.

[0123] As described above, germination tests conducted in a greenhouse under low temperature conditions confirmed that conventional CMS lettuce had reduced growth ability at low temperatures.

[0124] However, since it was thought that tests in a greenhouse would be easily affected by the natural environment, a germination test was also carried out in an artificial environment using an artificial weather chamber, which is not affected by the natural environment.

[0125] First, a germination test was conducted using an artificial weather chamber under optimal temperature conditions for growth of "50125-3-V1," a conventional CMS lettuce described in Patent Document 1. The artificial weather chamber was set to a constant temperature of 20°C and 12 hours of light, which are optimal temperatures for lettuce germination and growth.

[0126] Figure 3 shows the results of a germination test 16 days after sowing of "V lettuce" and "50125-3-V1" when the artificial weather chamber was set to the optimum temperature conditions for growth. Under optimal temperature conditions, "50125-3-V1" showed growth that was visually comparable to "V lettuce."

[0127] For quantitative evaluation, the above-ground parts of seedlings of each line were cut off at ground level and the weights per plant were compared. The results are shown in Table 3.

[0128] As shown in No. 5 and No. 6 in Table 3, the weight of the above-ground parts of “50125-3-V1” per plant was 116% of the weight of the above-ground parts of “V lettuce” per plant, and under optimum temperature conditions, it showed growth potential slightly higher than that of “V lettuce” which has normal cytoplasm.

[0129] Next, a low-temperature condition was artificially created using an artificial weather chamber, and a germination test was conducted. The artificial weather chamber was set to a night temperature of 5°C, a day temperature of 20°C, and 12 hours of lighting. Because it was not possible to set the night temperature to 0°C due to the performance of the artificial weather chamber, the lower limit of 5°C was used.

[0130] Figure 4 shows the results of a germination test on the 17th day after sowing of "V lettuce" and "50125-3-V1" when the artificial weather chamber was set to low temperature conditions. Compared to "V lettuce," "50125-3-V1" showed less uniform germination and was visually confirmed to have inferior low-temperature growth ability.

[0131] For quantitative evaluation, the above-ground parts of seedlings of each line were cut off at ground level and the weights per plant were compared. The results are shown in Table 3.

[0132] As shown in No. 7 and No. 8 in Table 3, the weight of the above-ground parts of "50125-3-V1" per plant was 66% of the weight of the above-ground parts of "V lettuce" per plant. It was confirmed that the decrease in growth of "50125-3-V1" due to low temperatures could be reproduced even under the low temperature conditions created in the weather chamber. Furthermore, in the test using the weather chamber, it was revealed that a decrease in growth due to low temperatures was caused simply by the nighttime temperature being low, since the daytime temperature was 20°C, which is the optimum temperature for growth.

[0133] As described above, the CMS lettuce disclosed in Patent Document 1 showed higher growth than "V lettuce" with normal cytoplasm under optimal temperature conditions, but it was revealed that its growth decreased under low night temperatures. In other words, at the time of filing of Patent Document 1, the decrease in growth of conventional CMS Lactuca plants under low temperature conditions had not been noticed or made clear. Naturally, Patent Document 1 does not state or suggest this point at all.

[0134] Next, an improved CMS line "BF2MS4V" was obtained in the same manner as in Example 1, except that "V lettuce" was used as a Lactuca plant having normal cytoplasm. "BF2MS4V" was obtained by using "50125-3-V1" as the cytoplasmic donor parent and "V lettuce" as the cytoplasmic recipient parent in asymmetric cell fusion.

[0135] The improved CMS line "BF2MS4V" produced according to Example 1 was produced by asymmetric cell fusion using "50125-3-V1" as the cytoplasmic donor parent, and therefore has the same nuclear genome as "V lettuce."

[0136] Figure 5 shows the results of a germination test on the 17th day after sowing "V lettuce" and "BF2MS4V" in a greenhouse environment with a night temperature of 0°C and a day temperature of 20°C. Visually, "BF2MS4V" showed uniform germination and low-temperature growth properties equivalent to "V lettuce."

[0137] For quantitative evaluation, the above-ground parts of seedlings of each line were cut off at ground level and the weights per plant were compared. The results are shown in Table 3.

[0138] As shown in No. 3 and No. 9 in Table 3, the weight of the above-ground parts per plant of "BF2MS4V" was 90% of the weight of the above-ground parts per plant of "V lettuce." The weight of the above-ground parts per plant of "BF2MS4V" under low temperature conditions was about 10% lower than that of normal cytoplasm, but a comparison of No. 4 and No. 9 in Table 3 shows that the weight ratio of the above-ground parts per plant of "BF2MS4V" was improved to 165% of that of "50125-3-V1," the CMS lettuce of Patent Document 1. These results demonstrated that the CMS cytoplasm of CMS lettuce, which has poor traits such as reduced growth ability at low temperatures, can be improved by successive asymmetric cell fusion with lettuce having normal cytoplasm.

[0139] Next, an improved CMS line "BF2MS2M" was obtained in the same manner as in Example 1, except that a breeding line "M8-039" (Sakata Seed Corporation) was used as a Lactuca plant having normal cytoplasm. "BF2MS2M" was obtained by using "50125-3-V1" as the cytoplasmic donor parent and "M8-039" as the cytoplasmic recipient parent in asymmetric cell fusion.

[0140] FIG. 6 shows the results of a germination test on the breeding lines "M8-039" and "BF2MS2M" 17 days after sowing in a greenhouse environment with a night temperature of 0°C and a day temperature of 20°C.

[0141] As shown in the figure, "BF2MS2M" showed low-temperature growth properties visually equivalent to those of "M8-039."

[0142] For quantitative evaluation, the above-ground parts of seedlings of each line were cut off at ground level and the weights per plant were compared. The results are shown in Table 3.

[0143] As shown in No. 10 and No. 11 in Table 3, the weight of the above-ground parts per plant of "BF2MS2M" was 93% of the weight of the above-ground parts per plant of "M8-039". The difference in the weight of the above-ground parts per plant was only a 7% decrease compared to the case of normal cytoplasm, which was considered to be a significant improvement in the CMS cytoplasm compared to the 75% decrease in "1216-2-T1", the CMS lettuce in Patent Document 1, and the 45% decrease in "50125-3-V1".

[0144] Therefore, it was confirmed that cytoplasmic improvement by asymmetric back fusion is possible even when the nuclear genome is different.

[0145] Next, Figure 7 shows the results of a germination test on the 17th day after sowing "Steady" and "BF2MS1S" in a greenhouse environment with a night temperature of 0°C and a day temperature of 20°C. The development process of "BF2MS1S" was performed by asymmetric cell fusion using "50125-3-V1" as the cytoplasmic donor parent, as described in detail in Example 1, and "Steady" was used as the cytoplasmic recipient parent during the asymmetric cell fusion. Visual inspection revealed that the germination was more uniform than that of "Steady," and its low-temperature growth ability appeared to be equivalent.

[0146] For quantitative evaluation, the above-ground parts of seedlings of each line were cut off at ground level and the weights per plant were compared. The results are shown in Table 3. As shown in No. 12 and No. 13 in Table 3, the weight of the above-ground parts of "BF2MS1S" per plant was 101% of the weight of the above-ground parts of "Steady" per plant.

[0147] In addition, to confirm the cytoplasmic characteristics of "BF2MS1S" in an artificial environment, a germination test was conducted using an artificial weather chamber. The artificial weather chamber was set at a night temperature of 5°C, a day temperature of 20°C, and 12 hours of lighting. The night temperature was not set to 0°C, as in the greenhouse, because the lower limit of the temperature setting for the artificial weather chamber was 5°C.

[0148] Figure 8 shows the results of a germination test 17 days after sowing for "Steady" and "BF2MS1S" using an artificial weather chamber. Visual inspection revealed that the low-temperature growth characteristics of "Steady" and "BF2MS1S" were equivalent.

[0149] For quantitative evaluation, the above-ground parts of seedlings of each line were cut off at ground level and the weights per plant were compared.

[0150] The results are shown in Table 3. As shown in No. 14 and No. 15 in Table 3, the weight of the above-ground parts of "BF2MS1S" per plant was 106% of that of "Steady," and even under artificial conditions, "BF2MS1S" showed low-temperature growth ability equal to or greater than that of "Steady," which has normal cytoplasm. Since the nuclear genome of "BF2MS1S" is identical to that of "Steady," which has normal cytoplasm, it was confirmed that the cytoplasm of "BF2MS1S" has low-temperature growth ability equal to or greater than that of normal cytoplasm.

[0151] As described in Example 2, "BF2MS1S" was subjected to successive backcrossings to develop BC7. BC7 of "BF2MS1S" was planted in a field (Azumino City, Nagano Prefecture) and the characteristics during the heading stage were investigated.

[0152] The results are shown in FIG. There was no morphological difference between "Steady" with normal cytoplasm, and "BF2MS1S" was confirmed to be an excellent CMS. Furthermore, no individuals with restored fertility were observed until the BC7 stage, confirming that the properties of CMS are extremely stable.

[0153] [Table 3]

[0154] Example 4 In order to analyze the cytoplasmic type of the improved CMS lettuce "BF2MS1S" produced in Example 1, 44 primer sets shown in Table 4 were designed based on the known nucleotide sequence information of the sunflower mitochondrial genome (Gene Bank accession number KF815390). When primers were capable of amplifying intergenic regions, markers were designed to preferentially target those sites, and markers in other regions were designed so that the physical distance between markers was 5,000 to 10,000 bp apart.

[0155] The test materials used were "V lettuce" having normal cytoplasm, the CMS sunflower "IB5", the CMS lettuces "1216-2-T1" and "50125-3-V1" described in Patent Document 1, and the improved CMS lettuce "BF2MS1S" produced according to the present invention.

[0156] Total genomic DNA was extracted from each test material and used as a template to perform PCR using the primer set shown in Table 4. PCR consisted of 35 cycles of thermal denaturation at 94°C for 1 minute, annealing at 60°C for 2 minutes, and extension reaction at 72°C for 2 minutes. When no polymorphism was detected between "V lettuce" with normal cytoplasm and CMS sunflower "IB5", the PCR products were treated with the restriction enzymes listed in Table 4 for PCR-RFLP analysis. The PCR products were electrophoresed on a 1.8% agarose gel, immersed in an ethidium bromide solution, and photographed under UV irradiation to investigate the presence or absence of PCR products and polymorphism.

[0157] The results of cytoplasmic analysis using PCR and PCR-RFLP are shown in Table 5. In Table 5, "Ha" indicates H. annuus type, "Ls" indicates L. sativa type, and "Hetero" indicates heterozygous for both species. "0" indicates no detection by the marker. Of the 44 primer sets, sunflower-type polymorphisms were detected in 24 sets (including heterozygotes) with "1216-2-T1," 10 sets with "50125-3-V1," and 3 sets with "BF2MS1S."

[0158] These results confirmed that the proportion of sunflower-derived mitochondrial genomic regions in CMS lettuce decreased each time asymmetric cell fusion was performed back-fusion, and that in "BF2MS1S," most of the mitochondrial genome had been recombined with the lettuce-derived mitochondrial genome.

[0159] As described above, the improvement in the low-temperature growth ability of "BF2MS1S" was achieved by backfusion through asymmetric cell fusion, which resulted in the replacement of most of the mitochondrial genome region with the lettuce type while retaining the sunflower-derived genomic region involved in CMS. This eliminated the incompatibility between the nuclear genome and the mitochondrial genome, and is believed to have improved low-temperature growth ability while maintaining the CMS trait.

[0160] However, the results in Table 5 show only one example of the analysis results of an individual expressing cytoplasmic male sterility, and it is not certain that CMS lettuce with improved low-temperature growth properties will necessarily exhibit only this type of mitochondrial genome structure.

[0161] [Table 4-1]

[0162] [Table 4-2]

[0163] [Table 4-3]

[0164] [Table 5]

[0165] (Explanation of symbols in the table) Ha: H. annuus type Ls: L. sativa type Hetero: Heterotype of both species 0: No detection for this marker

Claims

1. A cytoplasmic male sterile Lactuca plant or its progeny, which has low-temperature growth ability equivalent to that of a Lactuca plant having normal cytoplasm.

2. 2. The cytoplasmically male sterile Lactuca plant according to claim 1, or a progeny thereof, which has DNA derived from the mitochondrial genome of a Helianthus plant in its mitochondrial genome.

3. 3. The cytoplasmic male sterile Lactuca plant according to claim 1 or 2, or a progeny thereof, which is obtained by multiple rounds of asymmetric cell fusion using a Lactuca plant having normal cytoplasm as a cytoplasmic recipient parent.

4. The cytoplasmic male sterile Lactuca plant according to any one of claims 1 to 3, or a progeny thereof, obtained by asymmetric cell fusion using an existing cytoplasmic male sterile Lactuca plant as a cytoplasm donor parent and a Lactuca plant having normal cytoplasm as a cytoplasm recipient parent.

5. A cytoplasmic male sterile Lactuca plant having DNA derived from the mitochondrial genome of a Helianthus plant in its mitochondrial genome, or a progeny thereof, obtained by multiple asymmetric cell fusions using a Lactuca plant having normal cytoplasm as the cytoplasmic recipient parent, or A cytoplasmic male sterile Lactuca plant, or its progeny, obtained by asymmetric cell fusion using an existing cytoplasmic male sterile Lactuca plant as a cytoplasm donor parent and a Lactuca plant having normal cytoplasm as a cytoplasm recipient parent.

6. The cytoplasmic male sterile Lactuca plant according to any one of claims 1 to 5, wherein the cytoplasmic male sterile Lactuca plant is derived from lettuce (Lactuca sativa L.) or an interspecific hybrid plant of the Lactuca plant, or a progeny thereof.

7. A cytoplasmically male sterile Lactuca plant having the mitochondrial genome of the plant identified by accession number FERM BP-22373, or a progeny thereof.

8. A cytoplasmically male sterile Lactuca plant identified by accession number FERM BP-22373, or a progeny thereof.

9. A part of the cytoplasmic male sterile Lactuca plant or its progeny according to any one of claims 1 to 8.

10. A seed of the cytoplasmic male sterile Lactuca plant or a progeny thereof according to any one of claims 1 to 8.

11. A mitochondrial genome contained in a cytoplasmic male sterile Lactuca plant according to any one of claims 1 to 8, or a progeny thereof, a part of a plant body according to claim 9, or a seed according to claim 10.

12. The plant obtained by asymmetric cell fusion or its progeny is a cytoplasmically male sterile Lactuca plant, which is used as a cytoplasmic donor parent; and A method for asymmetric backfusion in plants, comprising carrying out asymmetric cell fusion one or more times using a Lactuca plant having normal cytoplasm as a cytoplasmic recipient parent, one of the plants used in the first asymmetric cell fusion.

13. A method for improving the mitochondrial genome in the cytoplasm of a plant of the genus Lactuca by the method of claim 12.

14. A method for producing a cytoplasmically male sterile Lactuca plant, or a progeny thereof, which has low-temperature growth ability equivalent to that of a Lactuca plant having normal cytoplasm, comprising performing asymmetric cell fusion using an existing cytoplasmically male sterile Lactuca plant as a cytoplasm donor parent and a Lactuca plant having normal cytoplasm as a cytoplasm recipient parent.

15. The method according to claim 14, wherein the cytoplasmic male sterile Lactuca plant is derived from lettuce (Lactuca sativa L.) or an interspecific hybrid plant of the Lactuca plant.

16. A method for producing a first generation hybrid seed, comprising: using a cytoplasmically male sterile Lactuca plant according to any one of claims 1 to 8, or a progeny thereof, as a seed parent; crossing said plant with a Lactuca plant which is capable of crossing with said plant as a pollen parent; and harvesting first generation hybrid seeds from the seed parent after crossing.

17. 17. The method of claim 16, wherein the pollen parent is lettuce (Lactuca sativa L.), or an interspecific hybrid plant of the Lactuca genus.

18. A first generation hybrid seed produced by the method according to claim 16 or 17, or a first generation hybrid plant grown from said seed, its progeny, or a part of said plant.

19. A method for producing a Lactuca plant having a useful trait and expressing cytoplasmic male sterility, comprising successively backcrossing a Lactuca plant having a useful trait with the cytoplasmic male sterile Lactuca plant according to any one of claims 1 to 8, or a progeny thereof, and carrying out cytoplasmic substitution.

20. The method according to claim 19, wherein the Lactuca plant having useful traits is derived from lettuce (Lactuca sativa L.).

Images