Tissue culture rapid propagation and in-vitro induction method for screening banana fusarium wilt-resistant germplasm

Abstract

The invention discloses a tissue culture rapid propagation and in-vitro induction method for banana fusarium wilt resistance germplasm screening, and belongs to the technical field of plant biology. The method comprises the steps of banana tissue culture seedling propagation, white spherical bud group induction and rapid propagation, in-vitro toxin screening, resistant bud group regeneration, induced differentiation, seedling hardening, transplanting and inoculation and the like. According to the method disclosed by the invention, a large number of adventitious buds are screened in the technical combination of radiation mutagenesis, multi-round toxin screening and the like after induction of white spherical bud clusters, so that the probability of obtaining somatic variation strains can be greatly improved, the used culture medium is simple in formula, the tissue culture process is simple, the anti-Foc4 banana germplasm can be effectively cultured, and a key technical support is provided for prevention and treatment of banana wilt; later-stage unified planting management is facilitated, and industrial production can be achieved.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, and relates to plant tissue culture and plant disease resistance screening techniques. In particular, it relates to a technical method for selecting and breeding banana wilt-resistant germplasm through rapid propagation via tissue culture combined with in vitro induction screening. Background Technology

[0002] Banana wilt is a devastating soil-borne disease caused by Fusarium oxysporum, often referred to as the "cancer of bananas." The tropical race 4 is particularly damaging to widely cultivated triploid banana varieties, causing up to 100% yield loss once it invades the plantation. Since most cultivated banana varieties are seedless triploids, abnormal meiosis leads to sterility, making seed production virtually impossible. Traditional hybridization breeding methods struggle to introduce disease-resistant genes into cultivated varieties, have long breeding cycles, and are limited by the asexual reproduction characteristics of bananas.

[0003] The development of plant tissue culture technology has provided a new approach for breeding disease-resistant bananas. On the one hand, tissue culture allows for large-scale cloning and propagation of virus-free seedlings; on the other hand, somatic cell variations and mutagenesis generated during tissue culture can provide additional sources of genetic variation for disease resistance. Through in vitro screening, the toxins of pathogens or culture filtrate can be directly used during the tissue culture stage to apply selective pressure, eliminating susceptible materials and retaining resistant materials, thereby significantly improving the efficiency of disease-resistant germplasm screening. Referring to existing technologies CN111990255A and CN105941158A, both of which involve pre-treating large quantities of material for in vitro screening, avoiding the high costs and risks of large-scale field trials, this can be considered a direction for the development of breeding bananas resistant to Fusarium wilt. Summary of the Invention

[0004] Given the problems of unstable resistance in regenerated plants and high false positive rate in screening systems in existing tissue culture techniques, the breeding efficiency of banana germplasm resistant to Fusarium wilt is severely restricted.

[0005] This invention establishes a chlorophyll-free white spherical bud cluster culture system, combines radiation treatment and graded screening, and adds a combination of cell homeostasis regulators to the induction culture regeneration medium to retain the required resistant materials, thereby significantly improving the screening efficiency of disease-resistant germplasm.

[0006] A method for rapid propagation and in vitro induction of banana germplasm resistant to Fusarium wilt, characterized by the following steps:

[0007] Step 1) Banana tissue culture propagation;

[0008] 1.1) Select banana suckers or shoot tip meristems as explants;

[0009] 1.2) Disinfect with a mixture of mercuric chloride aqueous solution and sodium thiosulfate solution;

[0010] 1.3) Inoculate the shoot tip tissue into M1 solid medium containing 6-benzylaminopurine and indoleacetic acid.

[0011] Step 2) Induction and rapid propagation of white spherical bud clusters;

[0012] 2.1) Divide the adventitious buds into single buds and transfer them to M2 solid medium containing 6-benzylaminopurine;

[0013] 2.2) Irradiate the white bud clusters;

[0014] 2.3) Transfer to M3 and M4 medium containing thiadiazole phenylurea and paclobutrazol to induce small bud clusters with green leaves.

[0015] Step 3) Screening for bud mutations resistant to Fusarium wilt in vitro;

[0016] 3.1) Prepare Foc4 culture filtrate;

[0017] 3.2) After sampling the bud clusters obtained in step 2.3), inoculate them into M5 medium;

[0018] 3.3) Perform graded screening, and reduce the proportion of culture filtrate from 20 vol% to 10 vol%; add fusaric acid in the fourth round.

[0019] Step 4) Regeneration culture of resistant bud clusters and plant recovery;

[0020] 4.1) Transfer the surviving bud clusters selected in step 3.3) to M6 medium for culture to induce their differentiation and regeneration;

[0021] 4.2) Transplant the roots obtained in step 4.1) into a substrate of perlite and vermiculite, and wait for the plant to grow steadily.

[0022] Step 5) Field planting and resistance assessment of regenerated plants;

[0023] 5.1) The Foc4 culture filtrate described in step 3.1) of root irrigation inoculation;

[0024] 5.2) Continue to observe for 280-300 days.

[0025] Step 1) describes banana tissue culture propagation, which includes the following: M1 medium is based on MS medium and also includes the following components: 6-benzylaminopurine, indoleacetic acid, sucrose, and agar.

[0026] The M2 culture medium mentioned in step 2) is an MS-based culture medium that also includes the following components: 6-benzylaminopurine, sucrose, and agar.

[0027] The M3 and M4 culture media mentioned in step 2) are MS-based culture media and also include the following components: thiadiazole phenylurea and paclobutrazol.

[0028] The M5 culture medium mentioned in step 3) is based on MS culture medium and also includes the following components: 6-benzylaminopurine and sucrose.

[0029] The resistant bud cluster regeneration culture described in step 4) uses M6 liquid culture medium with the following conditions: 26-28℃, 2000-3500lx light, and 12-20h / d.

[0030] As described above, step 4) involves the use of M6 liquid medium for the regeneration culture of resistant bud clusters, indolebutyric acid, 1-naphthaleneacetic acid, activated charcoal, and cell homeostasis regulators.

[0031] The cell homeostasis regulator contained in the M6 ​​liquid culture medium involved in step 4) is at least one of ectoin, oligogalacturonic acid, N-acetylcysteine, nitrosoglutathione, and polyglutamic acid.

[0032] The conditions for hardening off seedlings in step 5) are: humidity 80-85%, temperature 26-28℃, light intensity 600-800 lx, and hardening off period 7-21 days.

[0033] The beneficial effects of this invention are:

[0034] 1. Compared with the prior art, the present invention obtains uniform materials through white spherical bud cluster induction technology, and obtains resistant mutants through multiple rounds of in vitro toxin screening using γ-ray mutagenesis technology, thereby improving the rapid and targeted screening of banana wilt resistance materials.

[0035] 2. This invention uses tissue culture to induce white, spherical bud clusters without chlorophyll as selection material, which improves the uniformity of tissues and the frequency of resistance variations during the screening process.

[0036] 3. Introducing gamma radiation to induce mutagenesis in adventitious buds significantly increased the frequency of somatic cell mutations, which helped to generate disease-resistant mutants.

[0037] 4. Through multiple rounds of in vitro toxin screening at different concentrations, and by alternating the use of Fusarium wilt culture filtrate and key toxins such as fusarium oxysporum, sensitive buds are gradually eliminated, greatly improving the accuracy of disease-resistant bud screening.

[0038] 5. Through the above combination of technologies, the present invention can obtain plants with proven resistance to Fusarium wilt from a large number of banana tissue culture seedlings in a short period of time. The results of the disease resistance identification show that the resistance is significantly improved, which is an effective way to solve the current problem of banana Fusarium wilt control. Attached Figure Description

[0039] Figure 1 To induce white bud clusters, specifically, step 2.1) in Example 1 yields white bud clusters without leaf primitives.

[0040] Figure 2 (a) is an image of induced bud clusters with green leaves, specifically in the middle. Figure 2 (b) Enlarged view of the cluster of small buds with green leaves.

[0041] Figure 2 (b) is a diagram of induced bud clusters with green leaves, specifically the bud clusters with green leaves in step 2.3) of Example 1; some buds show no leaf differentiation.

[0042] Figure 3 The diagram shows the bud regeneration culture, specifically the small green plantlets obtained in step 4.1) of Example 1, which have grown roots. Detailed Implementation

[0043] A method for rapid propagation and in vitro induction of banana germplasm resistant to Fusarium wilt, characterized by the following steps:

[0044] Step 1) Banana tissue culture propagation;

[0045] 1.1) Select banana suckers or shoot tip meristems as explants;

[0046] 1.2) Disinfect with a mixture of mercuric chloride aqueous solution and sodium thiosulfate solution;

[0047] 1.3) Inoculate shoot tip tissues into M1 solid medium containing 6-benzylaminopurine and indoleacetic acid;

[0048] Step 2) Induction and rapid propagation of white spherical bud clusters;

[0049] 2.1) Divide the adventitious buds into single buds and transfer them to M2 solid medium containing 6-benzylaminopurine;

[0050] 2.2) Irradiate the white bud clusters;

[0051] 2.3) Transfer to M3 and M4 media containing thiadiazole phenylurea and paclobutrazol to induce small bud clusters with green leaves;

[0052] Step 3) Screening for bud mutations resistant to Fusarium wilt in vitro;

[0053] 3.1) Prepare Foc4 culture filtrate;

[0054] 3.2) After sampling the bud clusters obtained in step 2.3), inoculate them into M5 medium;

[0055] 3.3) Perform graded screening, decreasing the proportion of culture filtrate from 20 vol% to 10 vol%; add fusaric acid in the fourth round;

[0056] Step 4) Regeneration culture of resistant bud clusters and plant recovery;

[0057] 4.1) Transfer the surviving bud clusters selected in step 3.3) to M6 medium for culture to induce their differentiation and regeneration;

[0058] 4.2) Transplant the roots obtained in step 4.1) into a substrate of perlite and vermiculite, and wait for the plant to grow steadily;

[0059] Step 5) Field planting and resistance assessment of regenerated plants;

[0060] 5.1) The Foc4 culture filtrate described in step 3.1) of root irrigation inoculation;

[0061] 5.2) Continue to observe for 280-300 days.

[0062] The parameters and sources of some substances in the examples are as follows:

[0063] Banana: Variety: Brazilian banana tissue culture seedling, susceptible to Brazilian banana; Institute of Biotechnology, Guangxi Academy of Agricultural Sciences.

[0064] Fusarium oxysporum Cuban Specialized Type: Model: B215761; Source: Ningbo Mingzhou Biotechnology Co., Ltd.

[0065] Fusarium oxysine: CAS No.: 536-69-6, solid standard purity ≥98%.

[0066] MS medium: Duchefa MS medium; model: M0222-10L; source: Shanghai Yihui Biotechnology Co., Ltd.

[0067] PDA liquid culture medium: Model: 021050; Source: Guangdong Huankai Biotechnology Co., Ltd.

[0068] Ectocin: CAS No.: 96702-03-3.

[0069] N-acetylcysteine: CAS No.: 616-91-1.

[0070] Nitrosylglutathione: CAS No.: 57564-91-7.

[0071] Polyglutamic acid: agricultural grade; model: ft66; source: Shandong Fengtai Biotechnology Co., Ltd.

[0072] Example 1

[0073] The method for rapid propagation and in vitro induction of banana germplasm resistant to Fusarium wilt includes the following steps:

[0074] Step 1) Banana tissue culture propagation

[0075] 1.1) Material selection and pretreatment: Select meristematic tissues such as suckers and shoot tips from bananas as explants, rinse with running water for 20 minutes, and then dry.

[0076] 1.2) Disinfection treatment: First, soak in 75wt% ethanol for 45s in a sterile table, then soak in 0.1wt% mercuric chloride aqueous solution for 5min, then soak in 0.1wt% sodium thiosulfate solution for 30s, and finally rinse with sterile distilled water 5 times, 60s each time.

[0077] 1.3) Sampling: Using a sterile scalpel, extract the stem tip tissue containing meristems according to the standard of 10×2×2mm and fresh weight of 30mg;

[0078] 1.4) Primary culture: The above-mentioned shoot tip tissue was inoculated into M1 solid medium; after 30 days of culture, the shoot tip tissue swelled and formed adventitious buds. After 20 days of continued culture, the adventitious buds grew to 3 cm and were robust; the culture conditions were: 26℃, light intensity 2000 lx, 16 h / d.

[0079] The M1 solid medium is based on Duchefa MS medium and also includes the following components: 4.0 mg / L 6-benzylaminopurine, 0.5 mg / L indoleacetic acid, 3 g / L sucrose, and 5 g / L agar; pH adjusted to 5.8;

[0080] Step 2) Induction and rapid propagation of white spherical bud clusters

[0081] 2.1) Subculture: The adventitious buds obtained in step 1.4) were divided into single buds, retaining 2 nodes; under aseptic conditions, they were transferred to M2 solid medium for subculture for 42 days; the single buds proliferated to form clustered buds, and then subcultured to obtain leafless, primitive white bud clusters; culture conditions: 27℃, light intensity 1800 lx, light duration 16 h / d, 28 days per cycle;

[0082] The M2 solid medium is based on Duchefa MS medium and also includes the following components: 2.0 mg / L 6-benzylaminopurine, 3 g / L sucrose, and 5 g / L agar;

[0083] 2.2) Irradiation treatment: Under sterile conditions, the white bud clusters without leaf prototypic bases obtained in step 2.1) were subjected to 60Co-γ irradiation with an intensity of 40 Gy, a dose rate of 0.8 Gy / min, a target distance of 50 cm, and a temperature of 24 °C.

[0084] 2.3) Induced bud clusters: After 24 hours of radiation treatment, the buds were transferred to M3 solid medium. After 28 days of culture, the buds gradually differentiated into small bud clusters with green leaves. Some buds showed no leaf differentiation due to hormone effects and contained multiple meristematic tissues. Culture conditions: 27℃, light intensity 2000 lx, light duration 16 h / d.

[0085] The M3 solid medium is based on Duchefa MS medium and also includes the following components: 0.2 mg / L thiadiazole phenylurea and 0.2 mg / L paclobutrazol;

[0086] 2.4) Sampling and culture: The small bud clusters with green leaves obtained in step 2.3) were transferred to M4 solid medium and cultured for 28 days; the culture was repeated 3 times; white spherical bud clusters were obtained without leaf differentiation; culture conditions: 27℃, light intensity 2000 lx, light duration 16h / d;

[0087] The M4 solid medium is based on Duchefa MS medium and also includes the following components: 1 mg / L thiadiazole phenylurea and 1.5 mg / L paclobutrazol;

[0088] Step 3) In vitro toxin screening for bud mutations resistant to Fusarium wilt

[0089] 3.1) Preparation of Fusarium oxysporum culture filtrate (Foc4): Fusarium oxysporum Cuban-specific strain was inoculated into PDA medium and cultured on a shaker at 28℃ and 180 r / min for 7 days. The pathogenic bacterial block in the liquid medium was removed and inoculated into freshly prepared PDB medium. The culture was then cultured on a shaker at 28℃ and 180 r / min for 7 days to obtain the bacterial suspension. Subsequently, under aseptic conditions, 50 mL of the bacterial suspension was centrifuged at 8000 r / min to obtain the supernatant. The supernatant was resuspended in 50 mL of water. This process was repeated once to obtain the Foc4 culture filtrate, which was stored at 4℃ for later use.

[0090] 3.2) Sampling: First, cut the white spherical bud clusters obtained in step 2.4) into tissue blocks of moderate thickness (0.5 × 0.5 mm) using a sterile blade as the culture media to be screened;

[0091] 3.3) Grading and screening: The culture media from step 3.2) above were inoculated into the induction medium and then graded for culture and screening; culture conditions: temperature 27℃, light intensity 2000lx, light duration 16h / d, 30d per round;

[0092] First round: The induction medium was prepared by mixing 80 vol% M5 liquid medium and 20 vol% Foc4 culture filtrate evenly; wherein, the M5 liquid medium was based on Duchefa MS medium and also included the following components: 4 mg / L 6-benzylaminopurine, 30 g / L sucrose, pH 5.8;

[0093] The second round: The induction medium was obtained by mixing 85 vol% M5 liquid medium and 15 vol% Foc4 culture filtrate evenly; wherein, the M5 liquid medium was based on Duchefa MS medium and also included the following components: 4 mg / L 6-benzylaminopurine, 30 g / L sucrose, pH 5.8;

[0094] The third round: The induction medium was obtained by mixing 90 vol% M5 liquid medium and 10 vol% Foc4 culture filtrate evenly; among which, the M5 medium was based on Duchefa MS medium and also included the following components: 4 mg / L 6-benzylaminopurine, 30 g / L sucrose, pH 5.8;

[0095] The fourth round: The induction medium was prepared by mixing 90 vol% M5 liquid medium and 10 vol% Foc4 culture filtrate evenly; wherein, the M5 liquid medium was based on Duchefa MS medium and also included the following components: 4 mg / L 6-benzylaminopurine, 30 g / L sucrose, 5 mg / L fusaric acid, pH 5.8;

[0096] After four rounds of screening, stable and surviving resistant bud clusters were obtained after the culture was completed;

[0097] Step 4) Regeneration culture of resistant bud clusters and plant recovery

[0098] 4.1) Regeneration culture: After screening in step three, the surviving bud clusters were transferred to M6 liquid medium to induce their differentiation and regeneration, forming small green plants and growing roots;

[0099] The M6 ​​liquid culture medium is based on Duchefa MS medium and also includes the following components: 1 mg / L indolebutyric acid, 0.8 mg / L 1-naphthaleneacetic acid, and 50 mg / L activated carbon; wherein, the culture conditions are: 27℃, 3000 lx light, 28 days;

[0100] 4.2) Hardening off and transplanting: After washing the culture medium off the roots, transplant them into a substrate with a perlite to vermiculite mass ratio of 1:1; under the conditions of 85% humidity, 27℃ temperature and 650lx light, observe for 14 days. After the new roots and leaves of the plants are growing stably and the plant height is 30cm, they can be judged as qualified seedlings.

[0101] Step 5) Transplanting and field planting of regenerated plants

[0102] 5.1) Transplanting and Inoculation: After hardening off in step four, transplant the seedlings to the seedbed. 30 days later, perform root irrigation inoculation on each seedling using the Foc4 culture filtrate described in step 3), with a concentration of 1×10⁻⁶. 7 CFU / mL, inoculation volume 100mL / plant, soil moisture 80%;

[0103] 5.2) Preliminary screening of potted plants: Small plants that do not show tuber browning symptoms after inoculation and infection are candidate strains that show resistance to Fusarium wilt;

[0104] (4) Management after transplanting into the disease nursery: Do not use chemical agents to prevent disease. Maintain a temperature of 27℃ and a humidity of 85%. Observe the wilting and vascular bundle browning symptoms for 300 days after inoculation.

[0105] Example 2

[0106] The methods for screening banana germplasm resistant to Fusarium wilt through tissue culture and in vitro induction are basically the same as in Example 1, with the only difference being:

[0107] Step 4) involves M6 liquid medium based on Duchefa MS medium, which also includes the following components: 1 mg / L indolebutyric acid, 0.8 mg / L 1-naphthaleneacetic acid, 50 mg / L activated charcoal, and 0.5 mg / L ectoine.

[0108] Example 3

[0109] The methods for screening banana germplasm resistant to Fusarium wilt through tissue culture and in vitro induction are basically the same as in Example 1, with the only difference being:

[0110] Step 4) involves M6 liquid medium based on Duchefa MS medium, which also includes the following components: 1 mg / L indolebutyric acid, 0.8 mg / L 1-naphthaleneacetic acid, 50 mg / L activated carbon; 0.5 mg / L oligogalacturonic acid.

[0111] Example 4

[0112] The methods for screening banana germplasm resistant to Fusarium wilt through tissue culture and in vitro induction are basically the same as in Example 1, with the only difference being:

[0113] Step 4) involves M6 liquid medium based on Duchefa MS medium, which also includes the following components: 1 mg / L indolebutyric acid, 0.8 mg / L 1-naphthaleneacetic acid, 50 mg / L activated charcoal; 0.5 mg / L N-acetylcysteine.

[0114] Example 5

[0115] The methods for screening banana germplasm resistant to Fusarium wilt through tissue culture and in vitro induction are basically the same as in Example 1, with the only difference being:

[0116] Step 4) involves M6 liquid medium based on Duchefa MS medium, which also includes the following components: 1 mg / L indolebutyric acid, 0.8 mg / L 1-naphthaleneacetic acid, 50 mg / L activated carbon; 0.5 mg / L nitrosoglutathione.

[0117] Example 6

[0118] The methods for screening banana germplasm resistant to Fusarium wilt through tissue culture and in vitro induction are basically the same as in Example 1, with the only difference being:

[0119] Step 4) involves M6 liquid medium, which is based on Duchefa MS medium and also includes the following components: 1 mg / L indolebutyric acid, 0.8 mg / L 1-naphthaleneacetic acid, 50 mg / L activated carbon, and 0.5 mg / L polyglutamic acid.

[0120] Example 7

[0121] The methods for screening banana germplasm resistant to Fusarium wilt through tissue culture and in vitro induction are basically the same as in Example 1, with the only difference being:

[0122] Step 4) involves M6 liquid medium based on Duchefa MS medium, which also includes the following components: 1 mg / L indolebutyric acid, 0.8 mg / L 1-naphthaleneacetic acid, 50 mg / L activated carbon; 0.25 mg / L ectoine, and 0.25 mg / L polyglutamic acid.

[0123] Example 8

[0124] The methods for screening banana germplasm resistant to Fusarium wilt through tissue culture and in vitro induction are basically the same as in Example 1, with the only difference being:

[0125] Step 4) involves M6 liquid medium based on Duchefa MS medium, which also includes the following components: 1 mg / L indolebutyric acid, 0.8 mg / L 1-naphthaleneacetic acid, 50 mg / L activated carbon; 0.25 mg / L ectoine, and 0.5 mg / L N-acetylcysteine.

[0126] Test Example 1

[0127] Disease investigation

[0128] Field symptom observation was conducted according to the "NY-T4235-2022 Technical Specification for the Prevention and Control of Banana Fusarium Wilt": After the field planting in Examples 1-8, 45 plants were randomly selected. From these, leaves from 15 plants, stems from 15 plants, and stem bases from 15 plants were randomly selected for observation. Data were recorded and calculated, and the average results were taken. The results are shown in Table 1. The formula is as follows:

[0129] Yellowing rate (%) = (Number of yellowed leaves / Total number of leaves surveyed) × 100%;

[0130] Fusarium wilt incidence rate (%) = (number of infected plants / total number of plants surveyed) × 100%;

[0131] Basal rot incidence (%) = (Number of plants with basal rot / Total number of plants) × 100%

[0132] Table 1. Results of field survey on banana wilt disease

[0133] Yellow leaf rate / % wilting rate / % Base rot incidence rate / % Example 1 85.64 4.27 6.58 Example 2 79.78 3.85 5.63 Example 3 81.02 4.06 6.15 Example 4 80.56 3.95 5.92 Example 5 71.93 4.13 5.84 Example 6 60.49 3.74 5.39 Example 7 54.51 3.12 4.87 Example 8 71.14 3.87 5.70

[0134] Compared to the conventional addition of indolebutyric acid, 1-naphthaleneacetic acid, and activated carbon in Example 1 to promote root development, enhance water absorption capacity, prevent browning, and reduce wilting rate, Examples 2-8, during the post-screening recovery and early field hardening stages, showed a reduction in possible symptoms such as yellowing and wilting after the addition of cell homeostasis regulators. This indicates that the cell homeostasis regulators effectively alleviated leaf yellowing caused by pathogens.

[0135] Example 2: Added ectoine can alleviate water loss and secondary oxidative stress, delay vascular bundle browning, reduce yellowing and wilting, and prevent secondary necrosis in basal tissues. Example 3: Added oligogalacturonic acid, as a cell wall-derived oligosaccharide signal, activates pathways related to cell wall integrity sensing and regeneration, deposits callosity and lignin, and physically isolates pathogens. Example 4: Added N-acetylcysteine ​​can scavenge excess reactive oxygen species and inhibit polyphenol oxidation and browning cross-linking; in the early hardening-field stage, a lower oxidative load is beneficial to the vascular function of the root and stem base and chloroplast stability. Example 5: Added nitrosoglutathione can promote adventitious root development and differentiation of protox or phloem; in the early stage of field planting, it improves water absorption and transport capacity and inhibits disease progression, corresponding to reduced damage to basal tissues, thus decreasing the incidence of basal rot. In Example 6, polyglutamic acid was added. Polyglutamic acid is a highly hydrophilic biopolymer with water retention and weak coordination ability; however, it lacks the inhibitory effect on ethylene signal.

[0136] In a further embodiment 7, ectoine and polyglutamate work synergistically to build homeostasis both inside and outside the cell. Ectoine maintains cell membrane stability, inhibits ethylene synthase, blocks the Foc4 toxin-induced ethylene signaling pathway, and reduces leaf abscission and wilting; polyglutamate activates the phenylpropanone metabolic pathway, preventing cell structural breakage due to dehydration. The two work synergistically to form a weak interaction, promoting the accumulation of ectoine around the cell periphery, increasing its intracellular transport efficiency, and thus enhancing the scavenging effect of reactive oxygen species.

[0137] Test Example 2

[0138] Resistance determination

[0139] The disease severity index (DI) of 30 randomly selected plants from those planted in the field after the completion of examples 1-8 was calculated using the International Society for Plant Pathology's disease severity index system. The results are shown in Table 2. A susceptible control group and a blank control group were also set up.

[0140] Disease control group: Thirty tissue culture seedlings from the same batch were selected and inoculated with Fusarium oxysporum Cuban-specific culture medium at a concentration of 1×10⁻⁶ using the root immersion method. 7 CFU / mL, and those inoculated were marked as the disease control group;

[0141] Blank control group: 30 healthy banana tissue culture seedlings were selected and cultivated using sterilized substrate. The daily management conditions were the same as in Examples 1-8, and the seedlings were not exposed to pathogenic spore suspension throughout the process.

[0142] Grading: Grade 0: No symptoms of disease, plant growth is normal (no yellowing or wilting of leaves, no browning at the base of the stem);

[0143] Grade 1: Mild disease, leaves with a DI value ≤25% show yellowing or slight wilting, and there is no obvious browning at the base of the stem;

[0144] Grade 2: Moderate disease, leaves with DI values ​​of 26-50% are yellowed or wilted, and a small amount of browning appears at the base of the stem (browning range ≤ 1 / 3 of the stem circumference);

[0145] Grade 3: Severe disease, leaves with a DI value of 51-75% are severely yellowed and wilted, and the base of the stem is obviously browned (browning range 1 / 3-2 / 3);

[0146] Grade 4: Extremely severe disease, with leaves withering or the entire plant dying if the DI value is ≥76%, and severe browning and rotting at the base of the stem (browning range ≥2 / 3).

[0147] Referring to the "Technical Specification for Identification of Banana Wilt Resistance" (GB / T 35339-2017), plants with a DI ≤ 20% were identified as candidate strains resistant to wilt.

[0148] Table 2 Results of Resistance Determination

[0149] DI / % Blank control group 0 Disease control group 95.15 Example 1 75.45 Example 2 28.72 Example 3 29.60 Example 4 30.00 Example 5 37.05 Example 6 24.85 Example 7 18.36 Example 8 21.05

[0150] The blank control group, having not been exposed to Foc4 throughout the process, showed no symptoms of disease, consistent with the resistance of healthy plants. The infected control group, inoculated only with the pathogen, generally exhibited extremely severe disease, confirming the strong pathogenicity of Foc4 to bananas.

[0151] Examples 1-8 all underwent the resistance screening process of this invention, and their DI values ​​were significantly lower than those of the susceptible control group, indicating that this screening system can effectively reduce the disease index of banana wilt. Among them, the DI values ​​of Examples 2-6 were all reduced, and the cell homeostasis regulators can significantly improve the resistance of the selected germplasm to wilt. In Example 3, oligogalacturonic acid may activate the cell wall integrity sensing and regeneration pathway, promote the deposition of callose and lignin in the vascular bundle cell wall, and physically isolate the pathogen hyphae invasion and toxin diffusion. In Example 4, N-acetylcysteine, as a glutathione precursor, replenishes the cell's antioxidant system reserves, efficiently scavenges reactive oxygen species and inhibits polyphenol oxidase activity, and reduces browning cross-linking at the stem base. In Example 5, nitrosoglutathione regulates the NO signaling pathway, promotes adventitious root formation and protist xylem differentiation, enhances the plant's water absorption and nutrient transport capacity, and alleviates wilting.

[0152] Furthermore, Example 7 demonstrates that the combination of ectoine and polyglutamic acid blocks disease development from both the signaling molecule and pathogen structure perspectives; ectoine directly inhibits ACS enzyme activity, reducing ethylene synthesis; and polyglutamic acid reduces the synthesis of salicylic acid in vascular bundle parenchyma cells caused by pathogen infection. The combination of these two substances promotes the formation of an ectoine enrichment zone around the cell periphery, significantly enhancing its intracellular transport efficiency. The synergistic effect of this combination in disease resistance is verified through the three major stages of the conventional disease process: toxin invasion, oxidative damage, and structural destruction.

Claims

1. A method for rapid propagation and in vitro induction of banana germplasm resistant to Fusarium wilt, characterized in that, Includes the following steps: Step 1) Banana tissue culture propagation; Step 2) Induction and rapid propagation of white spherical bud clusters; Step 3) Screening for bud mutations resistant to Fusarium wilt in vitro; Step 4) Regeneration culture of resistant bud clusters and plant recovery; Step 5) Field planting and resistance assessment of regenerated plants; In step 4), the resistant bud cluster regeneration culture uses M6 liquid medium, which is based on Murashige and Skoog medium and also includes the following components: indolebutyric acid, 1-naphthaleneacetic acid, activated carbon, and cell homeostasis regulators.

2. The method for rapid propagation and in vitro induction of banana germplasm resistant to Fusarium wilt according to claim 1, characterized in that, Step 1) describes banana tissue culture propagation, which includes: 1.1) Select banana suckers or shoot tip meristems as explants; 1.2) Disinfect with ethanol, mercuric chloride aqueous solution, or sodium thiosulfate solution; 1.3) Inoculate shoot tip tissues into M1 solid medium containing 6-benzylaminopurine and indoleacetic acid; The M1 medium is based on MS and also includes the following components: 6-benzylaminopurine, indoleacetic acid, sucrose, and agar.

3. The method for rapid propagation and in vitro induction of banana germplasm resistant to Fusarium wilt according to claim 1, characterized in that, Step 2) describes the induction of white spherical bud clusters, which includes: 2.1) Divide the adventitious buds into single buds and transfer them to M2 solid medium containing 6-benzylaminopurine; 2.2) Irradiate the white bud clusters; 2.3) Transfer to M3 and M4 media containing thiadiazole phenylurea and paclobutrazol to induce small bud clusters with green leaves; The M2 medium is based on MS and also includes the following components: 6-benzylaminopurine, sucrose, and agar. The M3 and M4 media are based on MS media and also include the following components: thiadiazole phenylurea and paclobutrazol.

4. The method for rapid propagation and in vitro induction of banana germplasm resistant to Fusarium wilt according to claim 1, characterized in that, Step 3) describes the in vitro toxin screening, which includes: 3.1) Prepare Foc4 culture filtrate; 3.2) After sampling the bud clusters obtained in step 2.3), inoculate them into M5 medium; 3.3) Graded screening was carried out, with the proportion of culture filtrate decreasing from 20 vol% to 10 vol%; in the fourth round, fusaric acid was added. The M5 medium is based on MS and also includes the following components: 6-benzylaminopurine and sucrose.

5. The method for rapid propagation and in vitro induction of banana germplasm resistant to Fusarium wilt according to claim 1, characterized in that, Step 4) describes the regeneration culture and plant recovery of resistant bud clusters, which includes: 4.1) Transfer the surviving bud clusters selected in step 3.3) to M6 medium for culture to induce their differentiation and regeneration; 4.2) Transplant the roots obtained in step 4.1) into a substrate of perlite and vermiculite, and wait for the plant to grow steadily.

6. The method for rapid propagation and in vitro induction of banana germplasm resistant to Fusarium wilt according to claim 1 or 5, characterized in that, The resistant bud cluster regeneration culture described in step 4) uses M6 liquid medium culture conditions: 26-28℃, 2000-3500lx light, 12-20h / d.

7. The method for rapid propagation and in vitro induction of banana germplasm resistant to Fusarium wilt according to claim 1 or 6, characterized in that, Step 4) involves a cell homeostasis regulator contained in the M6 ​​liquid culture medium, wherein the cell homeostasis regulator is at least one of ectoin, oligogalacturonic acid, N-acetylcysteine, nitrosoglutathione, and polyglutamic acid.

8. The method for rapid propagation and in vitro induction of banana germplasm resistant to Fusarium wilt according to claim 1, characterized in that, Step 5) describes the field resistance identification, which includes: 5.1) The Foc4 culture filtrate described in step 3.1) of root irrigation inoculation; 5.2) Continue to observe for 280-300 days.

9. The method for rapid propagation and in vitro induction of banana germplasm resistant to Fusarium wilt according to claim 1 or 5, characterized in that, The hardening conditions are: humidity 80-85%, temperature 26-28℃, light intensity 600-800 lx, and hardening period 7-21 days.

Images