Method for producing bananas with enhanced resistance to fusarium oxysporum

Abstract

Methods for producing bananas with enhanced resistance to Fusarium oxysporum are provided. In particular, methods for producing bananas that stably exhibit resistance to Fusarium oxysporum even in the absence of allylalanine and probenazole are provided. The methods can be used to provide banana seedlings that have increased resistance to the race 4 mutant strain of Fusarium oxysporum f. sp. cubense and are agriculturally and horticulturally useful. Bananas that undergo a process of dedifferentiation and redifferentiation in the presence of allylalanine or probenazole acquire resistance to Fusarium oxysporum.

Description

Technical Field

[0001] This invention relates generally to the field of banana improvement and breeding, and more particularly to a method for conferring resistance to Fusarium oxysporum f. sp. cubense in bananas, which is useful in agriculture and horticulture. Background Technology

[0002] Bananas are cultivated worldwide, primarily in tropical regions, and have the highest yield among fruits, generating trillions of yen in economic impact. However, because cultivated varieties do not produce seeds, they are grown as clones through suckers and tissue culture, making them susceptible to disease and climate change. Indeed, in the early 20th century, the Gros Michel banana, then a major cultivated variety, suffered devastating damage from what is known as Panama disease, caused by the filamentous fungus *Fusarium oxysporum* race 1 Cuban specialization. In recent years, bananas worldwide are reportedly facing extinction due to what is known as neo-Panama disease (Fusarium wilt) caused by a variant of *Fusarium oxysporum* race 4 (TR4), a variant of the filamentous fungus *Fusarium oxysporum* race 1 Cuban specialization. Because bananas do not produce seeds, hybridization breeding cannot be applied to bananas. Therefore, research has been conducted on introducing disease-resistant genes through gene recombination (Patent Document 1).

[0003] However, there are serious concerns about potential legal and regulatory issues in producing and importing countries when using genetically modified organisms (GMOs), and a high possibility that the technology may not be accepted by end users. Currently, producing countries spray large quantities of pesticides, but concerns are growing about health hazards to workers due to pesticide exposure and pesticide residues, and all producing countries are grappling with these issues.

[0004] In the process of elucidating plant disease defense mechanisms, the inventors discovered that rice (belonging to the Poaceae family) and tobacco and tomato (belonging to the Solanaceae family) acquire resistance to rice blast and Botrytis cinerea, respectively, through a dedifferentiation / redifferentiation process in the presence of probenazole. Surprisingly, the resulting rice, tobacco, and tomato plants continued to exhibit resistance even in the absence of probenazole, and furthermore, they acquired genetically stable resistance that was maintained even after multiple sexual generations (Patent Document 2).

[0005] On the other hand, allylbenzylthiazide is used to control rice blast, but it is considered ineffective against highly saprophytic Fusarium species such as Fusarium head blight pathogens (Non-Patent Literature 1). Arie found that foliar spraying of validamycin A effectively controls soil-borne Fusarium diseases, such as tomato wilt and banana Panama disease, by inducing systemically acquired resistance (Non-Patent Literature 2). Furthermore, Ishikawa et al. reported that foliar spraying of validamycin A or validoxylamine A controls Fusarium wilt in tomatoes (Non-Patent Literature 3).

[0006] Therefore, the inventors attempted to induce dedifferentiation and redifferentiation in bananas in the presence of jinggangmycin A, thereby conferring resistance to *Fusarium oxysporum* Cuban specific form in the absence of jinggangmycin A. Surprisingly, the inventors found that bananas undergoing dedifferentiation and redifferentiation in the presence of jinggangmycin A did not acquire resistance to *Fusarium oxysporum* Cuban specific form, while bananas undergoing dedifferentiation and redifferentiation in the presence of allylbenzylthiazide or isothiazine acquired resistance to *Fusarium oxysporum* Cuban specific form, thus completing this invention.

[0007] Citation List

[0008] Patent documents

[0009] Patent Document 1: International Publication No. WO 2005 / 028651

[0010] Patent Document 2: International Publication No. WO 2020 / 218279

[0011] Non-patent literature

[0012] Non-patent document 1: Takumi Nishiuchi and Makoto Kimura, "Toward the development of safe and effective fungicides for controlling Fusarium headblight," Mycotoxins, 2021, Vol. 71, No. 1, pp. 13-19

[0013] Non-patent document 2: Arie T. Fusarium diseases of cultivated plants, control, diagnosis, and molecular and genetic studies. J Pestic Sci. 2019 Jul 25; 44(4):275-281.

[0014] Non-patent literature 3: Ishikawa R, Shirouzu K, Nakashita H, Lee HY, Motoyama T, Yamaguchi I, Teraoka T, Arie T. Foliar spray of validamycin or validoxylamine a controls tomato fusarium wilt. Phytopathology. 2005 Oct; 95(10):1209-16. Summary of the Invention

[0015] The problem the invention aims to solve

[0016] The problem to be solved by the present invention is to provide a method for producing bananas with enhanced resistance to *Fusarium oxysporum* var. *cubiclesii*. In particular, the present invention relates to a method for producing bananas that stably exhibit resistance to *Fusarium oxysporum* var. *cubiclesii* even in the absence of allylbenzylthiazol and isothiazine.

[0017] Solution for solving the problem

[0018] The present invention has the following structure.

[0019] [Implementation Plan 1]

[0020] A method for producing banana plants with enhanced resistance to Fusarium oxysporum Cuban variant, the method comprising:

[0021] (1) Banana explants containing shoot apical meristem were cultured under dedifferentiation induction conditions to form callus;

[0022] (2) Culture callus tissue under redifferentiation induction conditions to form shoots;

[0023] (A) Treatment of callus with a resistance inducer selected from the group consisting of allylbenzylthiazol (3-prop-2-enoxy-1,2-benzothiazolium-1,1-dioxide) and isothiazine (3,4-dichloro-2'-cyano-1,2-thiazol-5-formylaniline), wherein, compared with the control without resistance inducer treatment, the resistance inducer treatment did not reduce the callus survival rate to below 90%; and

[0024] (3) Cultivate shoots to obtain banana plants with enhanced resistance to Fusarium oxysporum Cuban variant;

[0025] Steps (1), (2), and (3) are performed in the order of (1), (2), and (3), and step (A) is performed in either or both of steps (1) and (2); and

[0026] In step (1) or step (2), no selection of resistant callus tissue based on antagonistic inducer treatment is performed.

[0027] [Implementation Plan 2]

[0028] A method for producing banana nursery plants, comprising:

[0029] As described in Implementation Scheme 1, steps (1), (2), (A) and (3) of the method; and

[0030] (4) The banana plants obtained in step (3) are propagated vegetatively to obtain banana seedlings;

[0031] Steps (1), (2), (3) and (4) are performed in the order of (1), (2), (3) and (4).

[0032] [Implementation Plan 3]

[0033] As described in embodiment 1 or 2, wherein the culture under dedifferentiation induction conditions is carried out on or in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), 6-benzylaminopurine (6-BA) or trans-zeatin (t-zeatin).

[0034] [Implementation Plan 4]

[0035] The method of any one of embodiments 1 to 3, wherein the culture under redifferentiation induction conditions is carried out on or in a medium containing kinetin, IAA, 6-BA, t-zeatin or 1-naphthaleneacetic acid (NAA).

[0036] [Implementation Plan 5]

[0037] The method of any one of embodiments 1 to 4, wherein the banana plant is a Cavendish cultivar.

[0038] [Implementation Plan 6]

[0039] The method of any one of embodiments 1 to 5, wherein Fusarium oxysporum Cuban specialization is Fusarium oxysporum Cuban specialization tropical race 4.

[0040] [Implementation Plan 7]

[0041] The method as described in any one of embodiments 1 to 6 does not include the step of gene recombination or the step of expressing a gene from a foreign vector.

[0042] [Implementation Plan 8]

[0043] Banana plants obtained by the method described in any one of embodiments 1 to 7.

[0044] [Implementation Plan 9]

[0045] Banana seedlings obtained by the method described in any one of embodiments 1 to 7. Attached Figure Description

[0046] Figure 1 This is a schematic diagram illustrating a method of inoculating pathogens into the roots during hydroponics. Figure 1 In diagram A, the part enclosed by the dashed line is a schematic diagram of the tuber and root. Figure 1 B is a schematic diagram of the tubers and roots immersed in the spore suspension. Figure 1 C is a schematic diagram showing the plant transplanted into the culture soil after inoculation.

[0047] Figure 2 A schematic diagram illustrating the methods for evaluating disease resistance. Figure 2 A is a schematic diagram showing the position 4cm above the base of the banana. Figure 2 B is a schematic diagram showing the cutting of the tissue from the base of the banana up to 4cm above it at 1cm intervals. "1", "2", "3" and "4" respectively represent the positions 1cm, 2cm, 3cm and 4cm above the bottom. Figure 2 C is a schematic diagram showing a culture of fungi on PDA medium, in which tissues from positions of 1 cm, 2 cm, 3 cm and 4 cm are placed on the medium.

[0048] Figure 3 shows the phenotype of banana plants inoculated with the Cuban variant of Fusarium oxysporum. Figure 3A The control banana plant is shown, and the arrows indicate yellowed leaves. Figure 3B The banana plant of the present invention is shown.

[0049] Figure 4 A graph showing the results of assessing the presence or absence of pathogen infection in banana tissue. Figure 4 Figure A shows the results obtained by placing tissue from a control banana on PDA medium and culturing fungi. The arrows indicate hyphal growth from tissues at 1 cm and 2 cm locations. Figure 4 B shows the results obtained by placing banana tissue treated with PBZ by the method of the present invention on PDA medium and culturing fungi. Figure 4 C shows the results of PCR using Fusarium oxysporum Cuban-specific primers on tissues from control banana plants and banana plants of the present invention. Cont: control banana plant; PBZ: banana plant treated with PBZ according to the method of the present invention; 1cm: tissue at 1cm position; 2cm: tissue at 2cm position; Origin: positive control; M: marker.

[0050] Figure 5 The figure shows the results of verifying the results of the isolation of Fusarium oxysporum Cuban specialization race 4 (TR4) (the pathogen that causes new Panama disease) from a banana pseudostem sample preserved at Mie University. Figure 5 Figure A shows the growth of fungi when banana roots infected with TR4 strain are cultured on PDA medium. Figure 5 Figure B shows the growth of fungi when banana stems infected with TR4 strain are cultured on PDA medium. Figure 5 C shows the growth of fungi when banana roots infected with non-TR4 strains are cultured on PDA medium. Figure 5 D shows the growth of fungi when banana stems infected with non-TR4 strains are cultured on PDA medium. Figure 5 Figure E shows the results of PCR using TR4-specific primers on Cuban-specific strains of Fusarium oxysporum (Nob1 and Nob2) isolated from banana pseudostems. Nob1 and Nob2: TR4 strains isolated from banana pseudostems; FocTR4: TR4-specific primers; FO1 and FO2: Fusarium oxysporum (non-TR4 strains isolated from banana pseudostems in Okinawa); 1 kb and 100 bp: markers.

[0051] Figure 6 shows the phenotypes of banana plants inoculated with TR4 strain. Figure 6A shows uninoculated banana plants 19 days after inoculation. Figure 6B shows control banana plants 19 days after inoculation. Figure 6C shows PBZ-treated banana plants 19 days after inoculation. Figure 6D shows control banana plants 8 days after inoculation. Figure 6E shows VMA-treated banana plants 8 days after inoculation. Figure 6F shows ITN-treated banana plants 8 days after inoculation.

[0052] Figure 7A graph showing the SPAD values ​​of control banana plants and banana plants of the present invention. Figure 7 In A, "Cont" represents the control banana plant, "PBZ150" represents the PBZ-treated banana plant, "Mock" represents the uninoculated banana plant, "Leaf 3" represents the three-leaf stage, and "Leaf 4" represents the four-leaf stage. Figure 7 In B, "Cont" represents the control banana plant, and "VMA" represents the VMA-treated banana plant.

[0053] Figure 8 To illustrate the measured lesion area (mm²) of control banana plants and banana plants of the present invention. 2 The results are shown in the figure. Mock: Uninoculated banana plants; PBZ150: PBZ-treated banana plants; Cont: Control banana plants. Detailed Implementation

[0054] [definition]

[0055] Banana: As used herein, the term “banana” refers to bananas belonging to the genus Musa in the family Musaceae, particularly including Cavendish cultivars, and preferably Cavendish cultivars.

[0056] Shoot apical meristem: As used herein, the term "shoot apical meristem" refers to the undifferentiated group of cells located at the apex of the banana stem.

[0057] Banana explant containing shoot apical meristem: As used herein, the term "banana explant containing shoot apical meristem" can have any shape and size, as long as it contains shoot apical meristem capable of forming callus. In one aspect, the banana explant containing shoot apical meristem is an explant comprising the boundary region between the tuber and the pseudostem, and preferably an explant comprising a tuber portion with a thickness and width of 0.5 to 2 cm, 0.8 to 1.5 cm, or about 1 cm, and a pseudostem portion with a thickness and width of 1 to 5 cm, 2 to 4 cm, or about 3 cm. In one aspect, the volume of the banana explant containing shoot apical meristem is 0.13 cm³. 3 Up to 27cm 3 1cm 3 Up to 27cm 3 3.4cm 3 Up to 27cm 3 1cm 3 Up to 15.6cm 3 3.4cm 3 Up to 15.6cm 3 or 3.4 to 8cm 3 .

[0058] Vegetative propagation: As used herein, the term “vegetative propagation” refers to propagation through organs other than seeds. Vegetative propagation includes propagation through propagules such as tubers, rhizomes, bulbs, and bulbs, as well as artificial propagation such as cuttings, grafting, root cuttings, leaf cuttings, and tissue culture (see Takeshi Nishio et al., “Shokubutsu Ikushugaku (Plant Breeding)”, 4th ed., 2013, Bun-eido Shuppan).

[0059] Dedifferentiation induction conditions: As used herein, “culture under dedifferentiation induction conditions” means culture in the presence of a dedifferentiation inducer such as 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), 6-benzylaminopurine (6-BA) or trans-zeatin (t-zeatin), and more specifically means culture on or in a medium containing 2,4-D, IAA, 6-BA or trans-zeatin. The concentration of the dedifferentiation inducer can be suitably determined by those skilled in the art, and is typically from 0.01 mg / L to 2 mg / L in terms of the final concentration, for example, 0.00125 to 10.24 mg / L, 0.0025 to 5.12 mg / L, 0.005 to 2.56 mg / L, 0.01 to 1.28 mg / L, 0.02 to 0.64 mg / L, 0.04 to 0.32 mg / L, or 0.08 to 0.16 mg / L. In one aspect of the invention, the period of "culturing under dedifferentiation induction conditions" is 1 to 60 days, 2 to 59 days, 3 to 58 days, 4 to 57 days, 5 to 56 days, 6 to 55 days, 7 to 54 days, 8 to 53 days, 9 to 52 days, 10 to 51 days, 11 to 50 days, 12 to 49 days, 13 to 48 days, 14 to 47 days, 15 to 46 days, 16 to 45 days, 17 to 44 days, 18 to 43 days, 19 to 42 days, 20 to 41 days, 21 to 40 days, 22 to 39 days, 23 to 38 days, 24 to 37 days, 25 to 36 days, 26 to 35 days, 27 to 34 days, 28 to 33 days, 29 to 32 days, and so on. 1 day, or 30 to 31 days, or optionally, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days. 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, or 60 days.

[0060] Redifferentiation induction conditions: As used herein, “culture under redifferentiation induction conditions” means culture in the presence of a redifferentiation inducer such as kinetin, IAA, 6-BA, t-zeatin, or 1-naphthaleneacetic acid (NAA), and more specifically, culture on or in a medium containing kinetin, etc. The concentration of the redifferentiation inducer can be suitably determined by those skilled in the art, and is typically from 0.01 mg / L to 2 mg / L, for example, 0.00125 to 10.24 mg / L, 0.0025 to 5.12 mg / L, 0.005 to 2.56 mg / L, 0.01 to 1.28 mg / L, 0.02 to 0.64 mg / L, 0.04 to 0.32 mg / L, or 0.08 to 0.16 mg / L. In one aspect of the invention, the period of "culturing under redifferentiation-inducing conditions" is 1 to 60 days, 2 to 59 days, 3 to 58 days, 4 to 57 days, 5 to 56 days, 6 to 55 days, 7 to 54 days, 8 to 53 days, 9 to 52 days, 10 to 51 days, 11 to 50 days, 12 to 49 days, 13 to 48 days, 14 to 47 days, 15 to 46 days, 16 to 45 days, 17 to 44 days, 18 to 43 days, 19 to 42 days, 20 to 41 days, 21 to 40 days, 22 to 39 days, 23 to 38 days, 24 to 37 days, 25 to 36 days, 26 to 35 days, 27 to 34 days, 28 to 33 days, 29 to 32 days, and so on. 1 day, or 30 to 31 days, or optionally, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days. 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, or 60 days.

[0061] Callus: As used herein, the term "callus" generally refers to a mass of undifferentiated plant cells cultured on a solid medium or similar medium. Those skilled in the art will understand that callus can exist not only on dedifferentiation induction medium (callus induction medium) but also on redifferentiation medium (e.g., immediately after being placed on redifferentiation medium), as long as it is in a dedifferentiated state.

[0062] Selection: As used herein, the term “selection” for callus tissue means, for example, subjecting callus tissue to low-temperature treatment and selecting callus tissue that is resistant to low-temperature treatment, subjecting callus tissue to low-temperature treatment for a certain period of time and selecting callus tissue that grows at room temperature, culturing callus tissue in a selective medium containing a drug and selecting callus tissue that exhibits resistance to the drug, etc.

[0063] Resistance: As used in this article, the phrase "enhanced resistance to Fusarium oxysporum cuboid" means that resistance to Fusarium oxysporum cuboid is enhanced. In this case, it does not mean that the plant is completely immune to Fusarium oxysporum cuboid infection.

[0064] Example

[0065] [Example 1] Conferring disease resistance on banana plants

[0066] I. Experimental Materials and Methods

[0067] (A) Banana tissue culture

[0068] (1) Trimming and sterilization of suckers

[0069] 1. Separate suckers, approximately 40 to 100 cm in height and free from BBTV (Banana bunchy topvirus) symptoms, from the main stem. Transplant the collected suckers into pots filled with plant growing soil (a mixture of potting soil, river sand, and Kanuma soil (fine granules) in a 1:1:1 ratio) and cultivate them in a greenhouse until used in experiments.

[0070] 2. Remove the tuber from the pot and wash it under running tap water to remove any attached soil. Shorten the pseudostem, leaving about 30cm, and remove the rest of the upper part.

[0071] 3. Trim the cut tubers by removing the surface with residual roots and soil, thus preserving the growth point located at the junction between the root and the bud, and then immerse them in a container with a 1 / 2 dilution (2.5%) of household bleach (5.25% NaOCl) for 40 minutes.

[0072] 4. Remove the browned tissue from the pseudostem and tuber, and then immerse the adventitious roots again in a container with a 1 / 2 dilution (2.5%) of household bleach for 30 minutes.

[0073] 5. Remove the browned tissue again and immerse the adventitious roots in a container with a 1 / 2 dilution (2.5%) of household bleach (5.25% NaOCl) for 10 minutes.

[0074] 6. The following processes are performed in a clean bench.

[0075] 7. Transfer the roots to a clean cutting dish and remove the browned tissue. Cut off the tuber, including about 1 cm of the stem apical meristem and about 3 cm of the pseudostem, in chunks. Sterilize the surface with 70% ethanol and 2.5% sodium hypochlorite solution, and then wash with sterile water.

[0076] (2) Callus induction and resistance inducing treatment in bananas

[0077] 8. Transfer the shoot tip to a new cutting dish and cut it longitudinally into four equal parts through the center. Transplant each quarter portion into the callus and shoot induction medium (CIM & SIM medium) in the growing box.

[0078] 9. Seal the growing box containing the culture medium with medical tape and incubate it in the dark at 25°C for 7 days.

[0079] 10. On day 7 of incubation in the dark, transfer the growing box to an incubator set at 25°C, with a photosynthetic photon flux (PPF) of approximately 60 μE / m²s¹ and a photoperiod of 16 h, and continue culturing for another 3 to 5 weeks until the callus reaches a size of approximately 2 cm. During the incubation period, transplant the callus to fresh culture medium every two weeks.

[0080] 11. Transplant the growing callus to fresh CIM&SIM medium, then to CIM&SIM medium containing resistance inducers, and culture under light.

[0081] 12. Callus tissue sections cultured on a medium containing a resistance inducer for 7 days were transplanted to fresh medium.

[0082] (3) Banana redifferentiation process

[0083] 13. When the shoot tip emerging from the leaf axis reaches a height of approximately 2 cm, the shoot tip is considered ready for subculture. Subculture is then performed again when resistance inducer treatment is repeated or when an increase in the number of plants is required.

[0084] 14. For subculture, when the shoot height exceeds 2cm, the tip of the growing shoot is divided longitudinally into four equal parts.

[0085] 15. Subculture should be repeated no more than five times. When the number of subculture cycles is high, abnormal variations such as dwarfing and elongation are more likely to occur in banana plants.

[0086] 16. Cultivate the buds for 3 to 4 weeks.

[0087] 17. Cut each shoot from the shoot cluster and transfer it to rooting induction medium. For best results, the rooting induction medium (RIM medium) should be used within one week of preparation.

[0088] 18. Roots form within 3 to 4 weeks.

[0089] 19. When the plant has 3 to 4 leaves and the roots are firmly established, open the lid of the planting box and cover it with plastic wrap. Gradually remove the plastic wrap to acclimate the plant.

[0090] 20. Finally, remove the plastic wrap to complete the acclimatization process.

[0091] 21. Once you have confirmed that the plant is absorbing water through its roots, fill the plastic cup with potting soil and transplant the plant into it.

[0092] (4) Composition and adjustment methods of various culture media

[0093] (i) Callus and shoot induction medium (CIM & SIM medium)

[0094] [Table 1]

[0095]

[0096] *Adjust the pH of the vitamin-containing Murashige & Skoog medium (M0222, Duchefa Biochemie BV) to 5.8 with 1N NaOH, add 2 g / L gellan gum, and then autoclave. In the treatment zone, add the resistance inducer, and aliquot approximately 50 mL of medium into each sterilized growing box.

[0097] (a) Method of adding resistance inducers

[0098] - Jinggangmycin A (VMA)

[0099] 1.07 mL of jinggangmycin solution 5 (Sumitomo Chemical Co., Ltd.; jinggangmycin A, 5%) was added to 1 L of autoclaved callus and shoot induction medium. However, when the concentration of commercially available jinggangmycin varies, the concentration of jinggangmycin A should be adjusted to 150 μM before addition.

[0100] -Activated ester (Acibenzolar-S-methyl, ASM)

[0101] The activated ester (Syngenta Japan KK; active ingredient activated ester, 50%) was ground into powder using a mortar and pestle, and 0.213 g of the powder was suspended in 5 mL of acetone. 1 mL of the activated ester suspension was added to 1 L of autoclaved callus and shoot induction medium. However, when the concentration of commercially available activated ester varies, the concentration was adjusted to 100 μM before addition.

[0102] -Isothiamethoxam (ITN)

[0103] 0.248 g of Routine FS (Bayer SeedGrowth; containing 18% of the active ingredient isothiazamide) was added to 1 L of autoclaved callus and shoot induction medium. However, when the concentration of isothiazamide in commercially available routines varies, the concentration of isothiazamide was adjusted to 150 μM before addition.

[0104] - Allylphenthiazide (PBZ)

[0105] Oryzemate (Meiji Seika Pharma Co., Ltd.; active ingredient allylphenthiazole [3-allyloxy-1,2-benzisothiazole 1,1-dioxide], 24%, mineral powder, etc., 76%) was ground into powder using a mortar and pestle, and 0.140 g of the powder was suspended in 10 mL of sterile water. However, when the concentration of allylphenthiazole in commercially available oryzemate varies, oryzemate was added to achieve an allylphenthiazole concentration of 150 μM. The oryzemate suspension was then added as a whole to 1 L of autoclaved callus and bud induction medium.

[0106] -Thiamid (TDN)

[0107] 0.134 g of V-GET Flowable (Nihon Nohyaku Co., Ltd.; active ingredient thiamethoxam, 30%) was added to 1 L of autoclaved callus and shoot induction medium. However, when the concentration of thiamethoxam contained in commercially available V-GET Flowable varies, the concentration of thiamethoxam was adjusted to 150 μM before addition.

[0108] (ii) Rooting induction medium (RIM medium)

[0109] [Table 2]

[0110]

[0111] *Adjust the pH of the vitamin-containing Murashige & Skoog medium (M0222, Duchefa Biochemie BV) to 5.8 with 1N NaOH, add 7.5 g / L agar, and then autoclave. Dispense approximately 50 mL of the medium into each sterilized growing box.

[0112] (B) Pathogen inoculation and disease resistance evaluation methods

[0113] (1) Fusarium oxysporum Cuban-specific strain

[0114] As a preliminary experiment, *Fusarium oxysporum* var. *cubicans*, which has relatively weak pathogenicity to *Cavendish*, was used. Banana roots planted as described above were immersed in a spore suspension for 3 days for pathogen inoculation. Figure 1 (A and B).

[0115] (2) Fusarium oxysporum, Cuban-specific type TR4

[0116] The pathogen was inoculated using *Fusarium oxysporum* Cuban-specific variant TR4 (the causative fungus of new Panama disease) in the same manner as *Fusarium oxysporum* Cuban-specific variant. This fungus was isolated from banana pseudostem samples preserved at Mie University.

[0117] (C) Disease resistance evaluation

[0118] (1) Measurement of SPAD values, etc.

[0119] Inoculate the roots of the plants hydroponically, and transplant them into potting soil 3 days after inoculation and cultivate them until 8 to 19 days post-inoculation. Thereafter, measure the SPAD value* and observe the phenotype. Figure 1 (B and C). *SPAD value: The value measured using a chlorophyll meter that measures chlorophyll content. Because this measuring device was developed under the Soil and Plant Analyzer Development (SPAD) project, its output is called the SPAD value. It enables the quantification of leaf yellowing caused by pathogen infection, etc.

[0120] (2) Observation of fungal growth

[0121] The tissue from the banana root, which has been inoculated with the pathogen and incubated, up to 4 cm above the root, is cut at 1 cm intervals. Figure 2 (A and B), and these portions were incubated on potato dextrose agar (PDA) medium at 25°C for 3 days. Figure 2 (C). Observe fungal growth and sample the fungi for electrophoresis to confirm the presence of pathogen DNA.

[0122] II. Results and Discussion

[0123] (A) Production of regenerated plants

[0124] No regenerated plants were obtained when activated esters or thiamethoxam were added to the callus and shoot induction media. In contrast, regenerated plants were obtained when isothiazine, allylbenzylthiazide, or jinggangmycin A were added to the callus and shoot induction media. Therefore, the disease resistance of these regenerated plants was evaluated.

[0125] (B) Evaluation of resistance to Fusarium oxysporum cucumeriforme in banana plants produced by the method of the present invention.

[0126] Four weeks after inoculation, in control banana plants where no resistance inducer was added to the callus and shoot induction medium, the entire leaf turned yellow, and early symptoms of new Panama disease were observed. Figure 3A In contrast, no particular changes in leaf color or morphology were observed in the banana plants of this invention. Figure 3B ).

[0127] Mycelial growth was observed in the tissues of control banana plants. Figure 4 A), while no pathogens were detected in the tissue of the banana of the present invention ( ). Figure 4 (B).

[0128] Furthermore, as a result of PCR using pathogen-specific primers, a specific band of *Fusarium oxysporum* Cuban variant was detected in control banana plants, while no band was detected at all in the banana plants of this invention. Figure 4 (C).

[0129] (C) Evaluation of resistance to Fusarium oxysporum var. copaiform TR4 in banana plants produced by the method of the present invention

[0130] First, the *Fusarium oxysporum* strains (Nob1 and Nob2) isolated from banana pseudostem samples preserved at Mie University were confirmed to be tropical race 4 (TR4) (the pathogenic fungus of new Panama disease). Infection with TR4 strains ( Figure 5 A and C) or non-TR4 strains ( Figure 5 The roots and stems of banana plants (B and D) were cultured on PDA medium to allow fungal growth. PCR was performed on these fungi using TR4-specific primers, and TR4-specific bands were detected in strains Nob1 and Nob2, confirming these strains as TR4( Figure 5 (E).

[0131] Next, the phenotypes of the control banana plants and the banana plants of the present invention were observed. In the control banana plants, obvious yellowing and necrosis of the lower leaves were clearly observed (Figs. 6B and 6D), while no obvious disease symptoms were observed in the banana plants treated with allylbenzylthiazide (PBZ) using the method of the present invention (Fig. 6C) and in the banana plants treated with isothiazine (ITN) (Fig. 6F). In contrast, the banana plants treated with jinggangmycin A (VMA) showed lesions and did not acquire resistance (Fig. 6E).

[0132] Similarly, when measuring the SPAD values ​​of control banana plants and banana plants of the present invention, high SPAD values ​​were obtained in banana plants treated with PBZ and ITN using the method of the present invention, while low SPAD values ​​were obtained in control banana plants and banana plants treated with VMA. Figure 7 (A and B).

[0133] Furthermore, when the lesion area was measured in banana plants inoculated with TR4 strain, the lesion area in banana plants treated with PBZ using the method of the present invention was significantly smaller than that in control banana plants. Figure 8 (A and B).

[0134] Based on the SPAD values ​​and lesion area percentages obtained above, it is considered that banana plants produced by the method of the present invention have acquired resistance to Fusarium oxysporum Cuban-specific TR4.

[0135] Industrial availability

[0136] The method of this invention can be used to confer resistance to the new Panama disease on banana plants. In particular, the method of this invention can be used by research institutions or seed companies other than farmers or horticulturists to provide banana seedlings with enhanced resistance to the new Panama disease that are useful both agriculturally and horticulturally.

Claims

1. A method for producing banana plants with enhanced resistance to Fusarium oxysporum var. cucumeroides, the method comprising: (1) Banana explants containing shoot apical meristems were cultured under dedifferentiation induction conditions to form callus; (2) The callus was cultured under redifferentiation-inducing conditions to form shoots; (A) The callus was treated with a resistance inducer selected from the group consisting of allylbenzylthiazol (3-prop-2-enoxy-1,2-benzothiazolium 1,1-dioxide) and isothiazine (3,4-dichloro-2'-cyano-1,2-thiazol-5-formylaniline), wherein the resistance inducer treatment did not reduce the survival rate of the callus to below 90% compared with a control without treatment; and (3) Cultivate the aforementioned shoots to obtain banana plants with enhanced resistance to Fusarium oxysporum Cuban variant; Steps (1), (2), and (3) are performed in the order of (1), (2), and (3), and step (A) is performed in either or both of steps (1) and (2); and In step (1) or step (2), the selection of the callus tissue based on resistance to the resistance inducer treatment is not performed.

2. A method for producing banana plantlets, said method include: Steps (1), (2), (A) and (3) of the method according to claim 1; and (4) The banana plants obtained in step (3) are propagated vegetatively to obtain banana seedlings; Steps (1), (2), (3) and (4) are performed in the order of (1), (2), (3) and (4).

3. The method according to claim 1 or 2, wherein the culture under the dedifferentiation induction conditions is carried out on a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), 6-benzylaminopurine (6-BA) or trans-zeatin (t-zeatin) or in said medium.

4. The method according to claim 3, wherein the culture under the redifferentiation induction conditions is carried out on a medium containing kinetin, IAA, 6-BA, t-zeatin or 1-naphthylacetic acid (NAA) or in the medium.

5. The method according to claim 1 or 2, wherein the banana is a Cavendish cultivar.

6. The method according to claim 5, wherein the *Fusarium oxysporum* Cuban variant is *Fusarium oxysporum* Cuban variant tropical race 4.

7. The method according to claim 1 or 2, excluding the step of gene recombination or the step of expressing a gene from a foreign vector.

8. A banana plant obtained by the method according to claim 1.

9. A banana seedling obtained by the method according to claim 2.

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