Breeding method for improving mutagenesis efficiency of stem tips of woody plants

Abstract

The invention belongs to the technical field of plant breeding, and discloses a breeding method for improving the mutagenesis efficiency of stem tips of woody plants. The CRISPR-Cas9 ribonucleoprotein complex and plant hormones 6-benzyladenine, gibberellin and jasmonic acid are co-encapsulated and then accurately delivered to stem tip meristem of woody ornamental plants such as acer truncatum, pteroceltis tatarinowii, crape myrtle and tassel, and the gene mutation obtaining rate of the stem tip meristem is effectively increased; the intelligent environment control system is used for dynamically optimizing illumination, temperature, humidity and oxidative stress, activating a plant endogenous DNA repair pathway and further enhancing homologous recombination efficiency, meanwhile, through collaborative optimization of induction hormone signals and environmental stress, the stress resistance is improved, the leaf color, the plant type and the growth vigor can be accurately regulated and controlled, and the yield of the plant is increased. According to the method, efficient, accurate and stress-resistant linked woody plant stem tip mutation breeding is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of plant breeding, and in particular relates to a breeding method for improving the mutagenesis efficiency of woody plant stem tips. Background Art

[0002] With the increasing importance of fruit trees and woody plants in food security, ecological restoration, and economic development, breeding efficiency and cultivar stress resistance have become key bottlenecks in research and production. Traditional shoot apex mutagenesis techniques primarily rely on single-agent mutagens such as ethyl methanesulfonate or physical mutagens such as gamma rays and ultraviolet light. While these techniques can produce genetic variation, they are often associated with low mutagenesis efficiency, random mutation patterns, and cytotoxicity, making precise and efficient genetic modification in shoot apex tissue difficult. In recent years, CRISPR-Cas9 gene editing technology has achieved breakthroughs in model plants, but delivery efficiency and stable expression in the shoot apex of woody plants remain limited, making it difficult to meet the demands of rapid breeding. Furthermore, plant growth regulators such as 6-benzyladenine and gibberellins, which promote cell division and proliferation, struggle to synergize with mutagenesis or editing tools, resulting in low regeneration rates after mutagenesis. Furthermore, existing breeding methods pay insufficient attention to the mutant plants' resistance to pests and diseases, drought, and salinity stresses. Consequently, new varieties often perform poorly in complex environments, limiting the widespread application of mutagenesis techniques. To address the above issues, there is an urgent need for an innovative breeding technology that integrates precise delivery, efficiency improvement, and stress resistance to accelerate the screening and cultivation of excellent woody plants and meet the sustainable development needs of modern agriculture and forestry. Summary of the Invention

[0003] In order to address the deficiencies mentioned in the above-mentioned background technology, the purpose of the present invention is to provide a breeding method for improving the efficiency of stem apex mutagenesis in woody plants. This method encapsulates chemical mutagens, gene editing tools and plant hormones in cationic liposome carriers, and precisely delivers them to the stem apex of woody plants through microinjection or foliar spraying, and induces mutagenesis in plants by controlling the external growth environment; combining gene regulation with hormone treatment to simultaneously enhance disease and insect resistance, drought resistance, salt and alkali resistance and other stress resistance, thereby realizing efficient, precise and stable stem apex mutagenesis breeding.

[0004] The purpose of the present invention can be achieved through the following technical solutions:

[0005] A breeding method for improving the efficiency of stem tip mutagenesis in woody plants comprises the following steps:

[0006] S1. Co-encapsulation of gene editing tools, chemical mutagens, and plant hormones using nanocarriers;

[0007] S2. The mixture is subjected to ultrasonic treatment and high-pressure homogenization to achieve the encapsulation of the active ingredient by the nanocarrier;

[0008] S3. quantitatively applying the nanocarrier solution to the stem tip of the woody plant;

[0009] S4. Inducing mutagenesis in the shoot apex using chemical mutagens and gene editing tools, and regulating cell division and proliferation in the shoot apex of woody plants through plant hormones;

[0010] S5. Regulate the stress resistance of woody plants during the mutagenesis process to improve their resistance to pests and diseases, drought, and salinity;

[0011] S6. Through the intelligent control system, the environmental factors during the mutagenesis process are dynamically adjusted to ensure the best mutagenesis effect.

[0012] Further preferably, the nanocarrier in step S1 is a cationic liposome prepared from phosphatidylcholine, cholesterol and distearoylphosphatidylethanolamine in a molar ratio of 5:3:2.

[0013] Further preferably, the plant hormone used in step S1 is one or more of cytokinin, gibberellin, abscisic acid, and jasmonic acid.

[0014] Further preferably, the environmental conditions during the mutagenesis process are dynamically adjusted by an intelligent control system, and the ambient temperature during the mutagenesis process is controlled to 22-25°C, the humidity to 60%-80%, and the photoperiod to 12 hours of light and 12 hours of darkness to ensure the best mutagenesis effect.

[0015] Further preferably, in step S3, the nanocarriers are applied to the stem tips of woody plants by microinjection or foliar spraying, with an application volume of 2 to 50 μL per plant.

[0016] Further preferably, the chemical mutagen in step S4 is ethyl methanesulfonate or sodium azide with a concentration of 1 to 10 mM, the gene editing tool is the CRISPR-Cas9 system, and the Cas9 protein concentration is 10 to 50 μg / mL.

[0017] Further preferably, the breeding method for improving the efficiency of stem apex mutagenesis in woody plants also includes the following steps: inoculating the stem apex tissue induced in step S4 onto a solid culture medium containing MS medium, 0.8% agar, and 30 g / L sucrose, culturing for 2 to 4 weeks, and then performing molecular marker detection to screen and obtain target mutant plants.

[0018] Further preferably, the woody plant is any one or more of Acer truncatum, Pterocarpus tansy, Lagerstroemia indica, and Tassel.

[0019] Beneficial effects of the present invention:

[0020] The present invention uses cationic liposomes to encapsulate chemical mutagens, gene editing tools and plant hormones, which can be precisely targeted and delivered to the shoot apical meristem, achieving efficient cellular uptake and targeted release. Compared with traditional Agrobacterium or gene gun vectors, this solution has no exogenous DNA residue, significantly reducing the risk of genetic modification. At the same time, the action of the mutagen is limited to the target site, reducing toxic side effects on non-target tissues; the synergistic effect of cationic liposomes and plant hormones not only improves the efficiency of gene editing and mutagenesis, but also activates division and proliferation signals in shoot apical cells, greatly improving tissue regeneration rate and survival rate; combined with CRISPR-Cas9-mediated precise site-specific modification during the mutagenesis process, the plant's adaptability to multiple adversities such as disease and pest resistance, drought resistance, and salt and alkali resistance is effectively enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention will be further described below with reference to the accompanying drawings.

[0022] Figure 1 The figure is a histogram of the gene mutation rate of the mutation breeding method of Examples 1-3 and Comparative Examples 1-2;

[0023] Figure 2 It is a double column graph of tissue regeneration rate and survival rate of Examples 1-3 and Comparative Examples 1-2;

[0024] Figure 3 It is a daily dynamic line graph of disease-resistant healthy plants in Examples 1-3 and Comparative Examples 1-2. DETAILED DESCRIPTION

[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0026] Example 1

[0027] (1) Materials and reagents

[0028] Chemical mutagen: ethyl methanesulfonate was diluted to 5 mM with deionized water;

[0029] CRISPR-Cas9 solution: Dilute Cas9 protein and gRNA to 30 μg / mL and 40 μg / mL, respectively, and dissolve a total of 1.0 mL in PBS buffer (pH 7.4).

[0030] Plant hormones: 6-benzyladenine, gibberellin, abscisic acid, and jasmonic acid were prepared into 2.0 mg / L, 0.8 mg / L, 0.3 mg / L, and 75 μM solutions, respectively;

[0031] Plant hormone mixed solution: Mix the solution in a ratio of 1:1:1:1;

[0032] Nanocarriers: Phosphatidylcholine, cholesterol, and distearoylphosphatidylethanolamine were dissolved in chloroform:methanol (2:1) at a molar ratio of 5:3:2. The solvent was removed on a rotary evaporator at 50°C to form a thin film. The film was then degassed in a vacuum for 2 h. PBS buffer (pH 7.4) was added and resuspended to obtain a crude dispersion. The solution was sonicated at 20°C and 200 W for 10 min, followed by high-pressure homogenization at 700 bar three times. After cooling, the suspension was filtered to obtain a cationic liposome suspension.

[0033] (2) Experimental steps

[0034] 1.0 mL of each chemical mutagen, CRISPR-Cas9 solution, and hormone mixed solution were taken, mixed, and vortexed for 5 minutes. 1.0 mL of cationic liposome suspension was added, and the mixture was ultrasonicated at 20°C for 8 minutes (200W). The mixture was then treated three times with a 700 bar high-pressure homogenizer to obtain 4 mL of composite nanocarrier suspension.

[0035] The composite nanocarrier suspension was loaded into a microsyringe and 20 μL was injected into the stem apex of each Acer truncatum seedling, treating a total of 20 plants. Foliar spraying was then performed, with 30 μL applied to each plant. The treated plants were placed in an incubator set at 24°C, 70% relative humidity, and alternating 12-hour light and 12-hour dark cycles for 14 consecutive days. Additional spraying with 20 μL of the composite nanocarrier suspension was performed every three days. On the seventh day of mutagenesis, the root zone was infused with a nutrient solution containing 10 mL of 6-benzyladenine and gibberellin. On the tenth day of mutagenesis, 10 mL of jasmonic acid was sprayed onto the leaves. On the 12th day of mutagenesis, the root zone was infused with 10 mL of a 150 mM sodium chloride solution for 48 hours.

[0036] The intelligent incubator was automatically adjusted to the following settings: temperature 24°C ± 1°C, humidity 60%–80%, and light intensity 150 μmol·m -2 ·s -1 .

[0037] Fourteen days after mutagenesis, approximately 50 mg of shoot apical tissue per plant was excised and inoculated onto solid plates of MS medium (containing 0.8% agar, 30 g / L sucrose, and 2.0 mg / L BAP) and cultured for 21 days under the same conditions as above. Regenerated shoots were then tested for molecular markers by PCR to screen for target mutant Acer truncatum plants.

[0038] Example 2

[0039] (1) Materials and reagents

[0040] Chemical mutagen: ethyl methanesulfonate was diluted to 5 mM with deionized water;

[0041] CRISPR-Cas9 solution: Dilute Cas9 protein and gRNA to 30 μg / mL and 40 μg / mL, respectively, and dissolve a total of 1.0 mL in PBS buffer (pH 7.4).

[0042] Plant hormones: 6-benzyladenine, gibberellin, abscisic acid, and jasmonic acid were prepared into 2.0 mg / L, 0.8 mg / L, 0.3 mg / L, and 100 μM solutions, respectively;

[0043] Plant hormone mixed solution: Mix the solution in a ratio of 1:1:1:1;

[0044] Nanocarriers: Phosphatidylcholine, cholesterol, and distearoylphosphatidylethanolamine were dissolved in chloroform:methanol (2:1) at a molar ratio of 5:3:2. The solvent was removed on a rotary evaporator at 50°C to form a thin film. The film was then degassed in a vacuum for 2 h. PBS buffer (pH 7.4) was added and resuspended to obtain a crude dispersion. The solution was sonicated at 20°C and 200 W for 10 min, followed by high-pressure homogenization at 700 bar three times. After cooling, the suspension was filtered to obtain a cationic liposome suspension.

[0045] (2) Experimental steps

[0046] The breeding operation steps for improving the efficiency of stem tip mutagenesis in woody plants are the same as those in Example 1.

[0047] Example 3

[0048] (1) Materials and reagents

[0049] Chemical mutagen: ethyl methanesulfonate was diluted to 5 mM with deionized water;

[0050] CRISPR-Cas9 solution: Dilute Cas9 protein and gRNA to 30 μg / mL and 40 μg / mL, respectively, and dissolve a total of 1.0 mL in PBS buffer (pH 7.4).

[0051] Plant hormones: 6-benzyladenine, gibberellin, abscisic acid, and jasmonic acid were prepared into 2.0 mg / L, 0.8 mg / L, 0.3 mg / L, and 75 μM solutions, respectively;

[0052] Plant hormone mixed solution: Mix the solution in a ratio of 1:1:1:1;

[0053] Nanocarriers: Phosphatidylcholine, cholesterol, and distearoylphosphatidylethanolamine were dissolved in chloroform:methanol (2:1) at a molar ratio of 5:3:2. The solvent was removed on a rotary evaporator at 50°C to form a thin film. The film was then degassed in a vacuum for 2 h. PBS buffer (pH 7.4) was added and resuspended to obtain a crude dispersion. The solution was sonicated at 20°C and 200 W for 10 min, followed by high-pressure homogenization at 700 bar three times. After cooling, the suspension was filtered to obtain a cationic liposome suspension.

[0054] (2) Experimental steps

[0055] The ultrasonic treatment time was extended from 8 min to 12 min, and the high-pressure homogenization pressure was increased from 700 bar to 900 bar. The remaining breeding operation steps for improving the mutagenesis efficiency of the woody plant stem tip were the same as those in Example 1.

[0056] Comparative Example 1

[0057] (1) Materials and reagents

[0058] Chemical mutagen: ethyl methanesulfonate was diluted to 5 mM with deionized water;

[0059] CRISPR-Cas9 solution: Dilute Cas9 protein and gRNA to 30 μg / mL and 40 μg / mL, respectively, and dissolve a total of 1.0 mL in PBS buffer (pH 7.4).

[0060] Plant hormones: 6-benzyladenine, gibberellin, abscisic acid, and jasmonic acid were prepared into 2.0 mg / L, 0.8 mg / L, 0.3 mg / L, and 75 μM solutions, respectively;

[0061] Plant hormone mixed solution: Mix the solution in a ratio of 1:1:1:1;

[0062] (2) Experimental steps

[0063] 1.0 mL of each chemical mutagen, CRISPR-Cas9 solution, and hormone mixed solution were taken, mixed, and vortexed for 5 minutes. The mixed solution was placed in a microsyringe and 20 μL was injected into the stem tip of each Acer truncatum seedling. A total of 20 plants were treated, and 30 μL was sprayed on each plant using a foliar sprayer. The treated plants were placed in an incubator at 24°C, 70% relative humidity, and a 12-hour light and 12-hour dark cycle. The culture was continued for 14 days, and the above spraying treatment was repeated every 3 days. On the 7th day of mutagenesis, 10 mL of nutrient solution containing 6-benzyladenine and gibberellin was injected into the root zone. On the 10th day of mutagenesis, 10 mL of jasmonic acid was sprayed on the leaves. On the 12th day of mutagenesis, 10 mL of 150 mM sodium chloride solution was injected into the root zone for 48 hours. The intelligent incubator settings were automatically adjusted to: temperature 24°C ± 1°C, humidity 60%–80%, and light intensity 150 μmol·m-2 ·s -1 .

[0064] 14 days after mutagenesis, approximately 50 mg of shoot apical tissue per plant was cut and inoculated onto MS medium solid plates and cultured for 21 days under the same conditions as above; PCR molecular marker detection was performed on the regenerated shoots to screen for target mutant Acer truncatum plants.

[0065] Comparative Example 2

[0066] (1) Materials and reagents

[0067] Chemical mutagen: ethyl methanesulfonate was diluted to 5 mM with deionized water;

[0068] CRISPR-Cas9 solution: Dilute Cas9 protein and gRNA to 30 μg / mL and 40 μg / mL, respectively, and dissolve a total of 1.0 mL in PBS buffer (pH 7.4).

[0069] Plant hormones: Prepare 6-benzyladenine and gibberellin into 2.0 mg / L and 0.8 mg / L solutions, respectively;

[0070] Plant hormone mixed solution: Prepare a hormone mixed solution in a ratio of 1:1;

[0071] The preparation of nanocarriers was the same as in Example 1.

[0072] (2) Experimental steps

[0073] 1.0 mL of each chemical mutagen, CRISPR-Cas9 solution, and hormone mixed solution were taken, mixed, and vortexed for 5 minutes. 1.0 mL of cationic liposome suspension was added, and the mixture was ultrasonicated at 20°C for 8 minutes (200W). The mixture was then treated three times with a 700 bar high-pressure homogenizer to obtain 4 mL of composite nanocarrier suspension.

[0074] The composite nanocarrier suspension was loaded into a microsyringe, and 20 μL was injected into the stem tip of each Acer truncatum seedling. The foliar sprayer was then used for spraying, with 30 μL applied to each plant. The treated plants were placed in an incubator set at 24°C, 70% relative humidity, 12 h light and 12 h dark cycle, and cultured continuously for 14 days. 20 μL of composite nanocarrier suspension was sprayed every 3 days. On the 7th day of mutagenesis, 10 mL of nutrient solution containing 6-benzyladenine and gibberellin was injected into the root zone. The intelligent incubator was automatically adjusted to the following settings: temperature 24°C ± 1°C, humidity 60%–80%, and light intensity 150 μmol·m -2 ·s -1 .

[0075] 14 days after mutagenesis, approximately 50 mg of shoot apical tissue per plant was cut and inoculated onto MS medium solid plates and cultured for 21 days under the same conditions as above; the regenerated shoots were tested for molecular markers by PCR, and mutant Acer truncatum plants were screened after 21 days.

[0076] Performance testing

[0077] 1. Mutagenesis efficiency detection

[0078] The stem tip tissue (50 mg / plant) grown for 14 days after mutagenesis in Examples 1-3 and Comparative Examples 1-2 was taken respectively, and genomic DNA was extracted using a DNA extraction kit according to the instructions and dissolved in 50 μL TE buffer. The extracted genome was amplified by PCR, and the amplified product was digested with T7E1. The digested product was loaded onto a 1.5% agarose gel and electrophoresed at 120 V for 30 min. After ultraviolet imaging, the grayscale values ​​of the uncut band (A) and the two cut bands (B and C) were measured using ImageJ software, and the mutation rate of each sample was calculated according to the following formula:

[0079]

[0080] The results are shown in Table 1 below.

[0081] Table 1 Gene mutation rate

[0082]

[0083] As can be seen from Table 1, based on the use of cationic liposomes, the optimization of different parameters has a significant effect on the efficiency of stem tip mutagenesis. Example 1 achieved a gene mutation rate of 45% under the conditions of 8 minutes of ultrasound, 700 bar high-pressure homogenization process and 75 μM jasmonic acid, which is 20% higher than that of Comparative Example 1. In Example 2, in which the jasmonic acid concentration was increased to 100 μM, the mutation rate increased to 50%, indicating that the optimization of the dosage of anti-stress hormones helps to activate cell division and gene editing signals. By extending the ultrasound to 12 minutes and raising the homogenization pressure to 900 bar in Example 3, the encapsulation efficiency and release performance of the cationic liposomes were improved, and the mutation rate jumped to 60%, far exceeding the 30% of Comparative Example 2. In summary, the dual optimization of the physical preparation parameters of the cationic liposomes and the concentration of anti-stress hormones can synergistically improve the delivery and gene editing efficiency of the CRISPR-Cas9 system in the stem tips of woody plants, thereby greatly enhancing the success rate of mutagenesis.

[0084] 2. Tissue regeneration rate and survival rate

[0085] The stem tip tissues of each mutagenesis scheme were cultured for 14 days, and 50 tissue blocks with a diameter of 5 mm and a weight of about 50 mg were cut and inoculated into MS solid medium. The culture medium was kept at a temperature of 24 °C, a relative humidity of 70%, and an illumination of 150 μmol·m -2 ·s-1 The culture was continuously cultured for 21 days in an incubator with alternating light and darkness for 12 hours. If the culture medium dried up during this period, it could be aseptically covered and supplemented with a small amount of sterile water. After 21 days, the total number of initial regenerated buds on all tissue blocks was counted, and the regeneration rate and survival rate of the seedlings were calculated. The results are shown in Table 2 below.

[0086] Table 2 Tissue regeneration rate and survival rate

[0087] sample Number of regenerated buds Regeneration rate (%) Number of surviving seedlings Survival rate (%) Example 1 30 60 24 80 Example 2 33 66 28 84.8 Example 3 38 76 35 92.1 Comparative Example 1 15 30 8 53.3 Comparative Example 2 23 46 15 65.2

[0088] As can be seen from Table 2, under the same culture conditions, different schemes have significant differences in the regeneration and survival ability of the shoot apex tissue. Example 1 achieved a regeneration rate of 60% and a survival rate of 80%, far exceeding Comparative Example 1, indicating that cationic liposomes combined with standard hormone formulas can effectively promote cell division and regeneration and improve seedling survival. In Example 2, in which the jasmonic acid concentration was increased from 75 μM to 100 μM, the regeneration rate increased to 66% and the survival rate increased to 84.8%, indicating that the optimization of the dose of anti-stress hormones helps to improve tissue regeneration efficiency. Example 3 enhances the encapsulation efficiency and stability of cationic liposomes by extending ultrasound and increasing the homogenization pressure, and the regeneration rate and survival rate reach 76% and 92.1% respectively, which are significantly better than all comparative examples, indicating that physical process optimization plays a key role in improving regeneration ability. Although Comparative Example 2 uses a carrier but no jasmonic acid, the regeneration rate and survival rate are lower than those of the embodiment group, further highlighting the importance of the linkage of anti-stress hormones in improving tissue regeneration and survival ability.

[0089] 3. Disease resistance testing

[0090] Leaf spot pathogens were revived in LB liquid medium and centrifuged at 8000×g for 10 minutes. The supernatant was discarded, and the pellet was resuspended in PBS and diluted to 1×10^6 CFU / mL. 10 μL of bacterial solution was inoculated onto the underside of each leaf or stem base of each seedling (20 plants per treatment). After inoculation, the plants were incubated in a high-humidity incubator (28°C, 90% relative humidity, 12h light / 12h dark) for 7 days to promote pathogen infection. Plant health was observed daily during this period, and plant health percentage was calculated on day 7. The results are shown in Table 3 below.

[0091] Table 3 Plant disease resistance results

[0092]

[0093] As can be seen from Table 3, all examples maintained a relatively high number of healthy plants on the 7th day, while the number of healthy plants in the control group dropped sharply. Among them, Example 1 still had 16 healthy plants on the 7th day, with a health rate of 80%, which was 40% higher than that of Control Example 1; Example 2 maintained 17 healthy plants on the 7th day, with a health rate of 85%, which was 5% higher than that of Example 1; Example 3 maintained 18 healthy plants until the 7th day, with a health rate of 90%, which was the highest level, reflecting the superimposed synergy of vector preparation and hormone linkage on disease resistance. On the 7th day, only 8 healthy plants remained in Control Example 1, with a health rate of 40%. Although there were 12 healthy plants and a health rate of 60% left in Control Example 2 on the 7th day, it was still far lower than that of each example, highlighting the key role of cationic liposomes themselves and anti-stress hormones in synergizing to improve the disease resistance of woody plants.

[0094] 4. Salt and alkali resistance test

[0095] Twenty seedlings of each of Examples 1-3 and Comparative Examples 1-2, grown for 14 days after mutagenesis, were taken and the root zone of each seedling was placed in 10 mL of 150 mM NaCl solution for incubation. The incubator conditions (24°C, 70% RH, 12 h light and 12 h dark alternation) were maintained for 7 consecutive days. After the treatment, the number of surviving seedlings with green leaves and no wilting was observed and recorded.

[0096] Relative electrolyte leakage rate: Three leaves of equal size (approximately 100 mg) were cut from each surviving seedling, placed in 10 mL of deionized water, and gently shaken at room temperature for 2 h. The initial conductivity C1 was measured. The same sample was placed in a 100°C water bath or autoclave and heated for 20 min to completely disrupt the cells. After cooling, the total conductivity C2 was measured. The relative permeability was calculated based on the relative electrolyte leakage rate (%) = (C1 ÷ C2) × 100. The results are shown in Table 4.

[0097] Table 4 Survival rate and relative permeability results

[0098]

[0099] As can be seen from Table 4, under the same salt stress conditions, the survival rate of seedlings and the integrity of cell membranes in the example group were significantly better than those in the control group. In Example 1, 15 strains still survived after 7 days of treatment with 150mM NaCl, with an average initial conductivity of only 87.5μS / cm, compared with a total conductivity of 350μS / cm and a relative leakage rate of 25%, indicating that most cell membranes remained intact; in Example 2, the survival rate was 80%, and the relative leakage rate was 22%, further reducing electrolyte leakage; in Example 3, the survival rate was as high as 85%, and the relative leakage rate was only 18%, showing optimal membrane stability and salt tolerance. In contrast, the survival rate of Control 1 was only 40%, and the relative leakage rate was as high as 50%, indicating that the cell membrane was severely damaged; in Control 2, the survival rate was 55%, and the leakage rate was 35%. Although there was carrier protection, there was a lack of hormone linkage, and the effect was still insufficient. In summary, it shows that cationic liposomes combined with anti-stress hormone treatment have a synergistic effect in improving the salt and alkali tolerance of woody plants.

[0100] 5. Determination of antioxidant enzyme activity

[0101] 3 g of healthy leaves from Examples 1-3 and Comparative Examples 1-2, cultured 14 days after mutagenesis, were collected and placed on ice. 15 mL of pre-chilled 0.1 M potassium dihydrogen phosphate buffer (pH 7.8) was added and ground into a homogenate. The mixture was centrifuged at 12,000 × g for 15 min at 4°C. The supernatant was collected as the enzyme extract and placed on ice until further use. The protein concentration of the enzyme extract was determined using the Coomassie Brilliant Blue method with bovine serum albumin as the standard and converted to mg protein.

[0102] SOD activity determination: Take 2.8 mL of 50 mM potassium dihydrogen phosphate buffer, 0.1 mL of 0.1 mM EDTA, 0.1 mL of 13 mM toluidine blue, 0.05 mL of 2 mM ethylenediaminetetraacetic acid ferrous iron, 0.05 mL of 50 μM xanthogenin chloride and an appropriate amount of enzyme extract, place the mixture under a 15 W fluorescent lamp for 10 min, and measure the absorbance change at 450 nm. 1 U of SOD activity is defined as the amount of enzyme required to inhibit the NBT photoreduction rate by 50%.

[0103] CAT activity assay: 2.8 mL of 50 mM potassium dihydrogen phosphate buffer (pH 7.0), 0.1 mL of 15 mM H2O2, and an appropriate amount of enzyme extract were mixed and immediately recorded at 240 nm the linear rate of change of absorbance over time (0–1 min) ΔA240 / min. 1 UCAT activity was defined as the amount of enzyme required to decompose 1 μmol H2O2 per minute. The results are shown in Table 5.

[0104] Table 5 Antioxidant enzyme activity results

[0105]

[0106]

[0107] As can be seen from Table 5, the antioxidant enzyme activity of the example group far exceeds that of the comparative example group: in Example 1, under the action of cationic liposomes and hormones, the superoxide dismutase activity reached 150U / mg and the catalase activity reached 80U / mg, which were increased by 87.5% and 100% respectively compared with comparative example 1 (80U / mg, 40U / mg); in Example 2, the activity was further increased to 160U / mg and 90U / mg after increasing the dosage of jasmonic acid; in Example 3, after process optimization, the SOD and CAT activities were as high as 180U / mg and 100U / mg, which were increased by 125% and 150% compared with comparative example 1, showing the best antioxidant defense ability. The enzyme activity of comparative example 2 was only increased to 100U / mg and 60U / mg, which was far lower than that of the example group, highlighting the key role of anti-stress hormones in activating the plant antioxidant system. Overall, the optimization of the physical preparation parameters of cationic liposomes and the linkage with anti-stress hormones significantly enhanced the antioxidant defense level of breeding materials.

[0108] 6. Comprehensive growth and ornamental performance testing

[0109] (1) Flower color phenotype and pigment content: 100 mg of petals from each protocol at the flowering stage were collected and CIELab parameters were measured using a portable colorimeter to evaluate hue and saturation. The petals were extracted in 80% ethanol for 12 h and centrifuged at 12,000 × g. The absorbance at 530 nm was measured by a spectrophotometer, and the total anthocyanin content was calculated using the molar absorption coefficient.

[0110] (2) Plant morphology: Plant height and stem diameter at the third node were measured 30, 60, and 90 days after mutagenesis, and the total number of branches above the base of the main stem was recorded to evaluate the degree of branching.

[0111] (3) Growth potential and biomass: During the 0–30 d and 30–60 d periods, the dry weights W1 and W2 of the plants in each scheme were taken, and the relative growth rate was calculated according to RGR = (lnW2–lnW1) / (t2–t1). The leaf area was measured and the leaf area index was calculated. The results are shown in Table 6 below.

[0112] Table 6 Comprehensive growth and ornamental performance

[0113]

[0114]

[0115] As shown in Table 6, the Example group outperformed the Comparative Example group in all comprehensive growth and ornamental indicators. In terms of anthocyanin content, Examples 1–3 reached 5.2, 6.0, and 7.2 mg / g FW, respectively, significantly higher than the 3.0 mg / g FW of Comparative Example 1 and the 4.0 mg / g FW of Comparative Example 2. Plant height and stem diameter also showed significant increases: Example 3 had a plant height of 24, 48, and 72 cm at 30, 60, and 90 days, respectively, and a stem diameter of 10, 20, and 30 mm, respectively, all approximately 60% higher than Comparative Example 1. Examples 1 and 2 also showed a steady, gradual increase in the number of branches, increasing from 2–3 to 5–9, further demonstrating the effective improvement in lush foliage. In terms of growth potential, the RGR 0–30d of the embodiment group reached 0.036–0.055d-1, and the RGR 30–60d was 0.030–0.040d-1, both higher than the control group, indicating a significant improvement in relative growth rate; the LAI also increased from 0.7–0.8 of the control group to 1.0–1.5, showing a stronger photosynthetic light-harvesting ability.

[0116] Throughout this specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0117] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications fall within the scope of the invention as claimed.

Claims

1. A breeding method for improving the efficiency of stem tip mutagenesis in woody plants, characterized in that: The following steps are involved: S1. Co-encapsulation of gene editing tools, chemical mutagens, and plant hormones using nanocarriers; S2. The mixture is subjected to ultrasonic treatment and high-pressure homogenization to achieve the encapsulation of the active ingredient by the nanocarrier; S3. quantitatively applying the nanocarrier solution to the stem tip of the woody plant; S4. Inducing mutagenesis in the shoot apex using chemical mutagens and gene editing tools, and regulating cell division and proliferation in the shoot apex of woody plants through plant hormones; S5. Regulate the stress resistance of woody plants during the mutagenesis process to improve their resistance to pests and diseases, drought, and salinity; S6. Through the intelligent control system, the environmental factors during the mutagenesis process are dynamically adjusted to ensure the best mutagenesis effect.

2. The breeding method for improving the efficiency of woody plant stem tip mutagenesis according to claim 1, wherein: The nanocarrier in step S1 is a cationic liposome prepared from phosphatidylcholine, cholesterol and distearoylphosphatidylethanolamine in a molar ratio of 5:3:

2.

3. The breeding method for improving the efficiency of induced mutagenesis of woody plant stem tips according to claim 1, wherein: The plant hormones used in step S1 are one or more of cytokinin, gibberellin, abscisic acid, and jasmonic acid.

4. The breeding method for improving the efficiency of stem tip mutagenesis in woody plants according to claim 1, wherein: The environmental conditions during the mutagenesis process are dynamically adjusted by an intelligent control system, and the ambient temperature during the mutagenesis process is controlled at 22-25°C, the humidity is 60%-80%, and the photoperiod is 12 hours of light and 12 hours of darkness to ensure the best mutagenesis effect.

5. The breeding method for improving the efficiency of stem tip mutagenesis in woody plants according to claim 1, wherein: In step S3, the nanocarriers are applied to the stem tips of woody plants by microinjection or foliar spraying, with the application volume for each plant being 2 to 50 μL.

6. The breeding method for improving the efficiency of stem tip mutagenesis in woody plants according to claim 1, characterized in that: The chemical mutagen in step S4 is one or more of ethyl methanesulfonate, sodium azide, anhydrous lithium chloride and acridine orange, with a concentration of 1 to 10 mM. The gene editing tool is the CRISPR-Cas9 system, and the Cas9 protein concentration is 10 to 50 μg / mL.

7. The breeding method for improving the efficiency of stem tip mutagenesis in woody plants according to claim 1, wherein: The following steps are also included: The stem tip tissue induced by step S4 was inoculated on a solid culture medium containing MS medium, 0.8% agar, and 30 g / L sucrose, and cultured for 2 to 4 weeks, followed by molecular marker detection to screen for target mutant plants.

8. The breeding method for improving the efficiency of stem tip mutagenesis in woody plants according to claim 1, wherein: The woody plants are any one or more of Acer truncatum, Pterocarpus serrata, Lagerstroemia indica, and Tassel.

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