Efficient preservation method of special germplasm of pinus massoniana based on resistance to pinewood nematode disease

Abstract

This invention relates to the field of germplasm resource preservation, and particularly to a method for the efficient preservation of Masson pine specific germplasm based on pine wilt disease resistance grading. This invention involves preliminary grading of fast-growing, high-yielding Masson pine stands for pine wilt disease resistance through field surveys. Then, different compound agents are used to cultivate robust seedlings. After nematode inoculation, the seedlings are further precisely graded based on a 30% incidence rate, obtaining susceptible and resistant germplasm. The preservation of these two types of germplasm is then optimized using tissue culture protocols, primarily through: optimizing the substrate for nursery preservation, and optimizing the tissue culture formula and cycle for in vitro preservation and rejuvenation culture. This precisely achieves the preservation of different types of Masson pine specific germplasm. Through optimization, the research group has derived different preservation schemes for resistant and susceptible germplasm, achieving precise grading and efficient preservation of fast-growing, high-yielding, disease-resistant, and susceptible Masson pine specific germplasm.

Description

[Technical Field]

[0001] This invention relates to the field of germplasm resource preservation, and in particular to a method for efficient preservation of Masson pine specific germplasm based on pine wilt disease resistance grading. [Background Technology]

[0002] Masson pine (Pinus massoniana Lamb.) is a major afforestation tree species in southern my country, characterized by its wide distribution, large stock volume, high economic value, and strong adaptability. Masson pine is planted in 17 provinces (regions) across my country, covering a forest area of ​​12.035 million hectares. 2 Masson pine accounts for 7.7% of the national arbor forest area, ranking first in total forest area and sixth in timber volume among all arbor tree species in China. It grows rapidly, providing abundant timber and resin within a short management period, making it a pillar industry for many timber, forestry, and paper industries. Furthermore, its resin, needles, and pollen are used in traditional medicine, its bark can be used to extract tannin, and its roots can be used to cultivate Poria cocos, making it a potential driving force for the development of emerging forestry industries. Masson pine possesses extremely strong vitality and wide ecological adaptability, making it a pioneering tree species for afforestation of barren mountains and the construction of ecological forests. It plays a crucial role in my country's ecological security and economic development.

[0003] Significant progress and achievements have been made in research areas such as geographical variation and germplasm selection of Masson pine, seed orchard construction, and targeted cultivation of pulpwood and building materials. Modern forestry emphasizes superior varieties and cultivation methods. Breeding superior Masson pine varieties to increase timber yield and quality per unit area is of great significance for addressing my country's forest resource scarcity, alleviating timber supply and demand imbalances, ensuring ecological security, and increasing the benefits of the forestry industry. Since the 1980s, numerous Chinese scholars have conducted extensive research on the collection and screening methods of Masson pine germplasm resources, and have bred a number of fast-growing and high-yielding superior varieties. However, compared to agricultural land, forest soils are relatively infertile, environmental conditions are harsh, and the requirements for tree resistance are high. Affected by pine wilt disease, the development of the Masson pine industry is currently severely hampered, and there is an urgent need for a batch of fast-growing, high-yielding, high-quality, and highly resistant Masson pine germplasm.

[0004] Forest germplasm resources are the original materials for the breeding and utilization of superior varieties. The quantity and quality of forest germplasm resources directly affect the efficiency of innovative utilization and the sustainable development of modern seed industry. The efficient preservation of forest germplasm resources, especially fast-growing, high-yielding, and highly resistant specific germplasm, is an essential part of current Masson pine breeding work. However, current Masson pine germplasm preservation still relies on the traditional method of nursery preservation, resulting in severe resource loss and extremely low preservation efficiency, which completely fails to meet the current demand for Masson pine specific germplasm. Therefore, this application, relying on tissue culture technology, establishes an efficient preservation method for Masson pine specific germplasm based on pine wilt disease resistance grading through steps such as germplasm screening, nursery preservation, in vitro preservation, and rejuvenation culture. [Summary of the Invention]

[0005] In view of the above, it is necessary to optimize the preservation of Masson pine germplasm to meet the growing social demand for Masson pine-specific germplasm. This invention improves tissue culture technology and establishes an efficient preservation method for Masson pine-specific germplasm based on pine wilt disease resistance grading through steps such as germplasm screening, nursery preservation, in vitro preservation, and rejuvenation culture.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for efficient preservation of Masson pine specific germplasm based on pine wilt disease resistance grading, the method being as follows:

[0008] (1) Conduct an investigation on Masson pine, select healthy and dominant branches of fast-growing and high-yielding trees with normal growth in disease-endemic areas as disease-resistant materials, and select seeds of fast-growing and high-yielding Masson pine families that are obviously susceptible to disease as disease-susceptible materials.

[0009] (2) Obtain tender seedlings from the disease-resistant or susceptible materials in step (1) through sowing or in vitro tissue culture. During cultivation, spray the seedlings of the disease-resistant or susceptible materials with compound agent to adjust the height-to-diameter ratio of the plants until the seedlings are cultivated to a height of ≥30cm, a ground diameter of ≥4mm, a height-to-diameter ratio of 65-90, and the seedlings have lignified below 10-15cm from the ground.

[0010] (3) Inoculate the seedlings cultivated in step (2) with nematodes and conduct grading verification of disease resistance indoors to obtain disease-resistant or disease-susceptible germplasm;

[0011] (4) Prune the tops of the disease-resistant or disease-susceptible germplasm from step (3) and apply the corresponding seedling substrate for nursery preservation to obtain disease-resistant or disease-susceptible plants.

[0012] Using disease-resistant or susceptible plants that have been preserved in the nursery for less than 2 years as the source of propagation material, collect the newly sprouted shoots of the current year as explants, and carry out sterile in vitro culture through the stem-bud culture method. After the seedlings have been subcultured a certain number of times, the subcultured buds are in vitro preserved. After each in vitro preservation for a certain period of time, they are rejuvenated and then transferred to the corresponding in vitro preservation medium for preservation.

[0013] Furthermore, the grading method for disease-resistant and disease-susceptible germplasm in step (3) is as follows: Select robust Masson pine seedlings that have been transplanted to the nursery for 8-12 months and have a high degree of lignification, inoculate them with nematodes at a rate of 4000 nematodes per seedling. Place the inoculated seedlings under conditions of 30-35℃, 60%-70% humidity, and 8000lx-12000lx light intensity, observe and measure the disease-susceptibility mortality rate of Masson pine. When the disease-susceptibility mortality rate is ≤30%, it is graded as disease-resistant germplasm; when the disease-susceptibility mortality rate is >30%, it is graded as disease-susceptible germplasm.

[0014] Furthermore, the compound agent of the disease-resistant material in step (2) is composed of 25-50 mg / L paclobutrazol, 0.05-0.15 g / L sodium dihydrogen phosphate, 0.2-0.3 g / L potassium nitrate, and 1-3 mg / L boric acid.

[0015] Furthermore, in step (4), the seedling substrate for disease-resistant germplasm consists of 3 parts peat moss, 3-4 parts perlite, 1-2 parts yellow clay, 1-2 parts coconut coir, and 2-3 kg / m³ of soil. 3 Composition of superphosphate.

[0016] Furthermore, the in vitro preservation culture medium for disease-resistant plants in step (5) consists of: 600 mg / L KNO3. -1 NH4NO3 150mg·L -1 CaCl2·2H2O 130mg·L -1 MgSO4·7H2O 125mg·L -1 Mg(NO3)2·6H2O 235mg·L -1 ;Ca(NO3)2·4H2O 110mg·L -1 KH2PO4 105 mg·L -1 MnSO4·H2O 6mg·L -1 ZnSO4·7H2O 5mg·L -1 CuSO4·5H2O 0.1 mg·L -1 H3BO3 3 mg·L -1 Na₂MoO₄·2H₂O 0.25 mg·L⁻¹ -1 CoCl2·6H2O 0.025 mg·L -1 Vitamin B1 1 mg·L -1 Vitamin B6 0.5 mg·L -1 ; Nicotinic acid 0.5 mg·L -1 Glycine 2 mg·L -1 Inositol 100 mg / L -1 6-BA 0.2 mg·L -1NAA 0.05 mg·L -1 Sodium thiosulfate 25 mg·L -1 .

[0017] Furthermore, the compounding agent for the disease-causing material in step (2) consists of 0-25 mg / L paclobutrazol, 0-0.15 g / L sodium dihydrogen phosphate, 0.1-0.3 g / L potassium nitrate, and 3-5 mg / L boric acid.

[0018] Furthermore, in step (4), the seedling substrate for the susceptible germplasm consists of 2 parts peat moss, 1-2 parts perlite, 1-2 parts yellow clay, 3-4 parts coconut coir, and 2-3 kg / m³ of soil. 3 Composition of superphosphate.

[0019] Furthermore, the in vitro preservation culture medium for the infected plants in step (5) consists of: 800 mg / L KNO3. -1 NH4NO3 200mg·L -1 CaCl2·2H2O 150mg·L -1 MgSO4·7H2O 125mg·L -1 Mg(NO3)2·6H2O 235mg·L -1 Ca(NO3)2·4H2O 165mg·L -1 KH2PO4 340 mg·L -1 MnSO4·H2O 6mg·L -1 ZnSO4·7H2O 5mg·L -1 CuSO4·5H2O 0.1 mg·L -1 H3BO3 3 mg·L -1 Na₂MoO₄·2H₂O 0.25 mg·L⁻¹ -1 CoCl2·6H2O 0.025 mg·L -1 Vitamin B1 1 mg·L -1 Vitamin B6 0.5 mg·L -1 ; Nicotinic acid 0.5 mg·L -1 Glycine 2 mg·L -1 Inositol 100 mg / L -1 6-BA 0.2 mg·L -1 NAA 0.05 mg·L -1 Sodium thiosulfate 30 mg·L -1 .

[0020] Furthermore, the seedlings of the disease-resistant germplasm are subcultured 4 to 8 times before being preserved in vitro, and the seedlings of the disease-susceptible germplasm are subcultured 8 to 12 times before being preserved in vitro.

[0021] Furthermore, the seedlings of the disease-resistant and disease-susceptible germplasms need to undergo rejuvenation culture after being stored in vitro for 2 years.

[0022] The present invention has the following beneficial effects:

[0023] This invention targets resistance to pine wilt disease. It involves preliminary resistance grading of Masson pine germplasm from epidemic areas to obtain resistant / susceptible materials. Then, using an optimized compound agent, the corresponding seedlings are vigorously cultivated. After nematode inoculation, the seedlings are graded based on a 30% incidence rate to determine whether they are resistant or susceptible. These two types of germplasm are then preserved. The overall preservation process is optimized in conjunction with tissue culture protocols, comprising: optimized substrate for nursery preservation, optimized tissue culture formula and cycle for in vitro preservation, and rejuvenation culture. This precisely achieves the preservation of different types of specific Masson pine germplasm with varying disease resistance, characterized by rapid growth and high yield. Through a series of technical parameter optimizations, the research team has derived different preservation schemes for resistant and susceptible germplasm, achieving precise grading and efficient preservation of specific Masson pine germplasm. [Attached Image Description]

[0024] Figure 1 Comparison of seedling growth under different compound agents for disease-resistant germplasm.

[0025] Figure 2 This is a curve showing the effect of disease-resistant germplasm matrix components.

[0026] Figure 3 The figure shows the effect of the number of subcultures on the in vitro preservation rate of germplasm.

[0027] Figure 4 A graph showing the effect of storage time on rejuvenation.

Detailed Implementation Methods

[0028] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0029] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0030] Example 1

[0031] When pine wilt disease occurs, Masson pine stands appear to be burned, exhibiting varying degrees of scorching and death. Based on the typical characteristics of pine wilt disease, we first investigated the susceptibility and mortality of Masson pine stands under natural growing conditions in the epidemic area, initially obtaining resistant and susceptible materials. However, in the natural environment, tree death is affected not only by disease but also by other factors, especially environmental stresses such as high temperature / drought and poor physiological activity of the trees themselves. Using only the survey results as the final resistance classification conclusion may lead to inaccurate or even erroneous classifications. Analyzing disease resistance under simulated nematode disease conditions, using seedlings as inoculation materials and employing artificial nematode inoculation, is currently the mainstream method for screening disease-resistant Masson pine germplasm. Therefore, we need to conduct precise disease resistance grading of Masson pine germplasm through indoor artificial inoculation of nematodes, while ensuring consistent environmental conditions and seedling growth traits. When conducting this precise disease resistance grading, we referenced the inoculation and identification scheme of the application filed on the same day, "A Precise Identification Method for Resistance to Pine Wilt Disease in Seedling Masson Pine," to grade and identify Masson pine. The specific scheme is as follows:

[0032] Seedlings of *Pinus massoniana* families were obtained through sowing or in vitro tissue culture, and then transplanted to a nursery for 8-12 months to obtain robust seedlings with high lignification. The seedlings were then inoculated with nematodes at a rate of 4000 nematodes per plant, and cultured under conditions of 30-35℃, 60%-70% humidity, and 8000-12000 lx light intensity. The disease mortality rate was observed and measured. A mortality rate ≤20% was considered resistant; 20% < mortality rate ≤30% was considered moderately resistant; 30% < mortality rate ≤40% was considered moderately susceptible; 40% < mortality rate ≤60% was considered susceptible; and a mortality rate >60% was considered highly susceptible. Specific grading is shown in Table 1.

[0033] Table 1. Comprehensive Evaluation Criteria for Disease Resistance of Masson Pine Seedlings

[0034]

[0035] The disease mortality frequency (DF) is calculated as follows: Disease mortality frequency (DF) = (Number of disease-infected and dead plants ÷ Total number of plants surveyed) × 100%.

[0036] According to this grading standard, we selected clonal lines with a seedling disease mortality rate of ≤30% as disease-resistant germplasm, and clonal lines with a seedling disease mortality rate of >30% as disease-susceptible germplasm.

[0037] Seedling quality is the prerequisite and foundation for ensuring the effectiveness of artificial nematode inoculation. Essentially, seedlings with excessively high (too old) or low (too young) lignification are unsuitable for nematode inoculation, thus affecting subsequent germplasm resistance grading and preservation. Therefore, to successfully implement nematode inoculation, seedlings need to be rejuvenated to obtain ideal inoculated seedlings. During the rejuvenation cultivation of seedlings, we found that although the same compound agent was used, the nutritional requirements of the compound agent for materials with different disease resistances were completely different (see Tables 3 and 7 for details). Through further cultivation and tissue culture methods, we also found that specific cultivation and tissue culture programs still differ. Therefore, our research group believes that two different efficient preservation methods can be established for two different germplasms.

[0038] Example 2

[0039] This embodiment describes a method for efficient preservation of disease-resistant Masson pine germplasm. The specific method is as follows:

[0040] 1. Germplasm screening

[0041] In areas affected by pine wilt disease, healthy and dominant trees with normal growth were selected from superior stands of fast-growing and high-yielding Masson pine as research subjects. Branches were collected and grafted back to the nursery. New shoots from grafted seedlings were used as explants and propagated into tissue culture seedlings through stem-bud in vitro culture technology.

[0042] When tissue culture seedlings were transferred from rooting bottles to the nursery, a self-made compound agent consisting of the growth regulator paclobutrazol and mineral nutrients sodium dihydrogen phosphate, potassium nitrate, and boric acid was applied as a foliar spray. This adjusted the plant height-to-diameter ratio, resulting in robust seedlings with high lignification levels within an 8-month seedling period. An orthogonal experimental design was used, with four levels for each component, totaling 16 treatments, with five replicates per treatment and 70 seedlings per replicate. The treatment was applied weekly, with 30 mL applied to each plant each time, to promote seedling growth. The results of this seedling growth experiment are shown in Tables 2-3.

[0043] When the seedlings are cultivated to a height of ≥30cm, ground diameter of ≥4mm, height-to-diameter ratio of 65-90, and the seedlings are lignified below 10-15cm from the ground, refer to the scheme of Example 1, inoculate them with pine nematodes for grading and verification, and select seedlings with a disease mortality rate of ≤30% as disease-resistant germplasm for the next step of cultivation.

[0044] Table 2 Factor Levels of Compound Agents

[0045]

[0046] Table 3. Differences in seedling growth performance under different compound treatments of disease-resistant germplasm.

[0047]

[0048]

[0049] Note: Different lowercase letters in the table indicate that the differences between different treatments reached a significant level (P<0.05), and the same applies below.

[0050] Table 3 shows that different compound agents resulted in different seedling growth performances. Among the 16 compound agent treatments tested, treatments 8 and 10 produced the most robust seedlings with the highest degree of lignification, and a height-to-diameter ratio ranging from 65.4 to 89.2. The growth of seedlings under different compound agent treatments is shown in the table below. Figure 1 As shown in the figure: A is the test group treated with compound agent 1, B is the test group treated with compound agent 8, C is the test group treated with compound agent 10, and D is the test group treated with compound agent 16. Figure 1 As can be seen, the growth of seedlings in treatments 16 and 10 was significantly lower than that in the control group, while treatment 8 showed the best growth. This indicates that more compound agent is not necessarily better. In our experiment, the best growth was achieved by foliar spraying of seedlings with a compound agent consisting of 25-50 mg / L paclobutrazol, 0.05-0.15 g / L sodium dihydrogen phosphate, 0.2-0.3 g / L potassium nitrate, and 1-3 mg / L boric acid. This combination significantly improved growth and could directly produce robust seedlings with a high degree of lignification within an 8-month seedling period.

[0051] 2. Nursery site preservation

[0052] In a well-lit, well-ventilated, and level nursery, select disease-resistant seedlings and prune them until they reach a height of approximately 40cm and a crown width of 35-40cm. Then, transplant them into non-woven planting bags with a diameter of 60cm and a height of 65cm, filled with a certain component of lightweight substrate. In the first year after transplanting, prune the tops to control the tree height to around 50cm. In the second year, the height should not exceed 100cm, and the height should increase by 50cm each year until the final tree height is controlled at around 1.5m.

[0053] The optimization of the seedling substrate composition was carried out using an orthogonal experimental method, with peat moss, perlite, yellow clay, coconut coir, and superphosphate as factors, each factor having 4 levels, for a total of 16 treatments. Each treatment was replicated 5 times, with 40 seedlings per replicate, and the survival rate was calculated three years after transplanting.

[0054] Table 4 Matrix Factor Levels

[0055]

[0056] Note: The units for superphosphate in the table are expressed per m³. 3 Mass (kg) added to the matrix.

[0057] Table 5. Analysis of extremely poor nursery preservation effects of disease-resistant germplasm under different substrate treatments.

[0058]

[0059] The range analysis results in Table 5 show that peat moss and superphosphate had the greatest impact on seedling survival rate, with R values ​​ranging from 18.3 to 25.4, while perlite, yellow clay, and coconut coir had relatively smaller effects, with R values ​​ranging from 5.8 to 8.7. The experimental results indicate that, compared to substrate properties such as aeration and moisture retention, substrate nutrient supply is the key to improving seedling survival rate.

[0060] This application Figure 2 The matrix effect curve reflects the different effects of each matrix component. Peat moss and superphosphate showed the strongest effects, with moderate amounts of peat moss and higher amounts of superphosphate being beneficial for nursery preservation. Analysis of the differences in seedling survival rates among the 16 treatments in Table 4 shows that different treatments resulted in different nursery preservation effects, with treatments 11 and 12 showing the best preservation results, achieving survival rates as high as 96.5%–99.5%. Therefore, it is believed that a matrix composition of 3 parts peat moss, 3–4 parts perlite, 1–2 parts yellow clay, 1–2 parts coconut coir, and 2–3 kg / m³ is optimal. 3 Superphosphate is used as a substrate to achieve the best germplasm nursery preservation results.

[0061] 3. In vitro preservation

[0062] Disease-resistant plants less than two years old, preserved in the nursery, were used as propagation material. Newly sprouted shoots were collected as explants and cultured aseptically in vitro using a stem-bud culture method. After a certain number of subcultures, the subcultured shoots were transferred to an in vitro preservation medium. Culture was conducted at a temperature of 20±0.5℃, a light intensity of 2000–3000 lx, and a light duration of 14 h, with a subculture cycle of 28 days.

[0063] like Figure 3 It can be seen that the number of subcultures is closely related to the survival rate of seedlings preserved in vitro. Seedlings with 0–36 subcultures were used as experimental materials for in vitro preservation. After one year of preservation, the survival rate was statistically analyzed, revealing that the preservation effect varied depending on the number of subcultures. Among them, seedlings with 4–8 subcultures showed the best preservation effect, with a survival rate exceeding 94%.

[0064] The in vitro preservation culture medium consisted of: KNO3 600 mg·L⁻¹ -1 NH4NO3 150 mg·L -1 ;CaCl2·2H2O 130mg·L -1 MgSO4·7H2O 125mg·L -1 Mg(NO3)2·6H2O 235mg·L -1 ;Ca(NO3)2·4H2O 110mg·L-1 KH2PO4 105 mg·L -1 MnSO4·H2O 6mg·L -1 ZnSO4·7H2O 5mg·L -1 CuSO4·5H2O 0.1 mg·L -1 H3BO3 3 mg·L -1 Na₂MoO₄·2H₂O 0.25 mg·L⁻¹ -1 CoCl2·6H2O 0.025mg·L -1 Vitamin B1 1.0 mg / L -1 Vitamin B6 0.5 mg·L -1 ; Nicotinic acid 0.5 mg·L -1 Glycine 2.0 mg·L -1 Inositol 100 mg / L -1 6-BA 0.2 mg·L -1 NAA 0.05 mg·L -1 Sodium thiosulfate 25.0 mg·L -1 .

[0065] 4. Rejuvenation culture: Refer to the invention patent: "A culture method for the proliferation and rejuvenation of tissue culture subculture buds of Masson pine".

[0066] (ZL201810891292.0).

[0067] Example 3

[0068] This example demonstrates the preservation of diseased Masson pine germplasm, and the specific method is as follows:

[0069] 1. Germplasm screening

[0070] Based on the results of the epidemic investigation, the study focused on fast-growing, high-yielding but disease-susceptible Masson pine families. Seedlings of these Masson pine families were obtained through sowing and seedling cultivation or in vitro tissue culture.

[0071] When seedlings were transplanted to the nursery, a self-made compound agent consisting of the growth regulator paclobutrazol and mineral nutrients sodium dihydrogen phosphate, potassium nitrate, and boric acid was applied as a foliar spray. This adjusted the plant height-to-diameter ratio, cultivating robust seedlings with high lignification levels within an 8-12 month seedling period. An orthogonal experimental design was used, with four levels for each component, resulting in 16 treatments in total. Each treatment had five replicates, with 70 seedlings per replicate. Spraying was performed weekly, with 30 mL applied to each plant each time, to promote seedling growth. The experimental results for promoting seedling growth are shown in Tables 6-7.

[0072] Once the seedlings have been cultivated to a height of ≥30cm, a ground diameter of ≥4mm, a height-to-diameter ratio of 65-90, and have lignified below 10-15cm from the ground, refer to the scheme in Example 1, inoculate with pine nematodes for grading and verification, and select seedlings with a disease mortality rate >30% as susceptible seedlings for the next stage of cultivation.

[0073] Table 6 Factor Levels of Compound Agents

[0074]

[0075] Table 7. Differences in seedling growth performance under different compound treatments of susceptible germplasm.

[0076]

[0077] Note: Different lowercase letters in the table indicate that the differences between different treatments reached a significant level (P<0.05), and the same applies below.

[0078] As shown in Table 7, the requirements of susceptible germplasm for compound agents are quite different from those of resistant germplasm, indicating that susceptible and highly resistant germplasm have different nutritional requirements for growth. Among the 16 compound agent treatments mentioned above, the seedlings treated with treatments 4 and 5 grew the most robustly and had the highest degree of lignification, which is different from the growth status of highly resistant germplasm under these treatments (Table 2). From the perspective of the composition of the compound agent, it is not the case that the more types or the greater the amount added, the stronger the seedlings will grow. For example, as the amount of paclobutrazol added is continuously increased, the seedlings will become shorter and shorter. This indicates that the application of foliar compound agents needs to be continuously explored and verified. In this application, the seedlings of susceptible germplasm were best after foliar spraying with a compound agent composed of 0-25 mg / L paclobutrazol, 0-0.15 g / L sodium dihydrogen phosphate, 0.1-0.3 g / L potassium nitrate, and 3-5 mg / L boric acid. This significantly improved the growth quality of the seedlings and could directly cultivate strong seedlings with a high degree of lignification within an 8-12 month seedling period.

[0079] 2. Nursery site preservation

[0080] In a well-lit, well-ventilated, and level nursery, select disease-resistant seedlings and prune them until they reach a height of approximately 40cm and a crown width of 35-40cm. Then, transplant them into non-woven planting bags with a diameter of 60cm and a height of 65cm, filled with a certain component of lightweight substrate. In the first year after transplanting, prune the tops to control the tree height to around 50cm. In the second year, the height should not exceed 100cm, and the height should increase by 50cm each year until the final tree height is controlled at around 1.5m.

[0081] The optimization of the seedling substrate composition was carried out using an orthogonal experimental method, with peat moss, perlite, yellow clay, coconut coir, and superphosphate as factors, each factor having 4 levels, for a total of 16 treatments. Each treatment was replicated 5 times, with 40 seedlings per replicate, and the survival rate was calculated three years after transplanting.

[0082] Table 8 Matrix Factor Levels

[0083]

[0084] Note: The units for superphosphate in the table are expressed per m³. 3 Mass (kg) added to the matrix.

[0085] Table 9. Analysis of extremely poor nursery preservation effects of disease-susceptible seedlings under different substrate treatments.

[0086]

[0087]

[0088] The range analysis results in Table 9 show that peat moss and perlite had the greatest impact on seedling survival rate, with R values ​​ranging from 9.3 to 29.2, while the effects of yellow clay, coconut coir, and superphosphate were relatively smaller, with R values ​​ranging from 3.2 to 5.4. The results indicate that for susceptible germplasm, the aeration and moisture retention of the substrate have a greater impact than nutrient supply. Combining the analysis of the differences in seedling survival rates among the 16 treatments in the table above, different treatments resulted in different preservation effects in the nursery. Treatments 5 and 6 showed the best preservation effect for susceptible germplasm, with survival rates as high as 94.2% to 96.4%. Therefore, it is believed that a mixture of 2 parts peat moss, 1-2 parts perlite, 1-2 parts yellow clay, 3-4 parts coconut coir, and 2-3 kg / m³ of substrate is suitable. 3 Superphosphate is used as a substrate to achieve the best germplasm nursery preservation results.

[0089] 3. In vitro preservation

[0090] Using diseased plants less than two years old preserved in the nursery as propagation material, newly emerging shoots of the current year were collected as explants and cultured aseptically in vitro using a stem-bud culture method. After a certain number of subcultures, the subcultured shoots were transferred to an in vitro preservation medium. Culture was carried out under conditions of 20±0.5℃, light intensity of 2000–3000 lx, and light duration of 14 h, with a subculture cycle of 28 days.

[0091] The number of subcultures is closely related to the survival rate of seedlings preserved in vitro. Figure 3 Seedlings with 0 to 36 subcultures were used as experimental materials for in vitro preservation. After one year of preservation, the survival rate was counted. It was found that the preservation effect varied with the number of subcultures, with seedlings with 8 to 12 subcultures showing the best preservation effect and a survival rate of over 90%.

[0092] The in vitro preservation culture medium consisted of: 800 mg·L⁻¹ KNO₃. -1 NH4NO3 200 mg·L -1 ;CaCl2·2H2O 150mg·L -1 MgSO4·7H2O 125mg·L -1 Mg(NO3)2·6H2O 235mg·L -1 ;Ca(NO3)2·4H2O 165mg·L -1 KH2PO4 340 mg·L -1 MnSO4·H2O 6mg·L -1 ZnSO4·7H2O 5mg·L -1 CuSO4·5H2O 0.1 mg·L -1 H3BO3 3 mg·L -1 Na₂MoO₄·2H₂O 0.25 mg·L⁻¹ -1 CoCl2·6H2O 0.025mg·L -1 Vitamin B1 1 mg·L -1 Vitamin B6 0.5 mg·L -1 ; Nicotinic acid 0.5 mg·L -1 Glycine 2 mg·L -1 Inositol 100 mg / L -1 6-BA 0.2 mg·L -1 NAA 0.05 mg·L -1 Sodium thiosulfate 30 mg·L -1 .

[0093] 4. Rejuvenation Culture: Refer to the invention patent: "A Culture Method for Proliferation and Rejuvenation of Tissue Culture Subculture Buds of Pinus massoniana"

[0094] (ZL201810891292.0).

[0095] The applicant's extensive and long-term research revealed that, for both susceptible and resistant germplasm, storage time significantly impacts seedling viability. Rejuvenation culture is necessary after a period of storage to ensure the effectiveness of long-term in vitro seedling preservation. The proliferation coefficient reflects seedling viability and regeneration capacity; a higher proliferation coefficient indicates stronger seedling viability. Figure 4It was found that the longer the storage time, the lower the seedling activity. Specifically, when the storage time was 0.5–2 years, the seedling proliferation coefficient during rejuvenation culture showed no significant difference, reaching 6.9–8.4. However, when the storage time was 2.5 years, the proliferation coefficient decreased slightly to 6.1, and when the storage time was more than 3 years, the proliferation coefficient decreased significantly, only 1.1–3.5. Considering the actual needs of Masson pine tissue culture production, a seedling subculture proliferation coefficient of over 6.0 fully meets the production requirements. Therefore, seedling rejuvenation culture is necessary every 2 years of in vitro storage.

[0096] In summary, this invention, after preliminary grading of the resistance to pine wilt disease in fast-growing, high-yielding Masson pine stands through field surveys, combined with different compound agents to cultivate robust seedlings. After nematode inoculation, the seedlings were further precisely graded based on a 30% incidence rate to obtain disease-resistant and susceptible germplasm. Furthermore, the preservation of these two types of germplasm was optimized for individual plants. The specific preservation scheme included: optimizing the substrate for nursery preservation, optimizing the tissue culture formula and cycle for in vitro preservation and rejuvenation culture, thereby precisely achieving efficient preservation of disease-resistant and susceptible specific germplasm of fast-growing, high-yielding Masson pine with varying disease resistance.

[0097] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for efficient preservation of specific germplasm of *Pinus massoniana* based on resistance grading for pine wilt disease, characterized in that, The method is as follows: (1) Conduct an investigation on Masson pine, select healthy and dominant branches of fast-growing and high-yielding trees with normal growth in disease-endemic areas as disease-resistant materials, and select seeds of fast-growing and high-yielding Masson pine families that are obviously susceptible to disease as disease-susceptible materials. (2) Obtain tender seedlings from the disease-resistant or susceptible materials in step (1) through sowing or in vitro tissue culture. During cultivation, spray the seedlings of the disease-resistant or susceptible materials with compound agent to adjust the height-to-diameter ratio of the plants until the seedlings are cultivated to a height of ≥30 cm, a ground diameter of ≥4 mm, a height-to-diameter ratio of 65~90, and the seedlings have lignified below 10~15 cm from the ground. (3) Inoculate the seedlings cultivated in step (2) with nematodes and conduct grading verification of disease resistance in the room to obtain disease-resistant or disease-susceptible germplasm; (4) Prune the tops of the disease-resistant or disease-susceptible germplasm from step (3) and apply the corresponding seedling substrate for nursery preservation to obtain disease-resistant or disease-susceptible plants. (5) Use the disease-resistant plants or susceptible plants that have been preserved in the nursery for 2 years or less in step (4) as the source of propagation material, collect the tender shoots that have emerged in the current year as explants, and carry out sterile in vitro culture through the stem and bud culture method. When the seedlings have been subcultured a certain number of times, the subcultured buds are in vitro preserved. After each in vitro preservation for a certain period of time, they are rejuvenated and then transferred to the corresponding in vitro preservation medium for preservation. The in vitro preservation culture medium for the disease-resistant plants in step (5) consists of: 600 mg / L KNO3. -1 NH4NO3 150 mg·L -1 CaCl2·2H2O 130 mg·L -1 MgSO4·7H2O 125 mg·L -1 Mg(NO3)2·6H2O 235 mg·L -1 ;Ca(NO3)2·4H2O 110 mg·L -1 KH2PO4 105 mg·L -1 MnSO4·H2O 6 mg·L -1 ZnSO4·7H2O 5mg·L -1 CuSO4·5H2O 0.1 mg·L -1 H3BO3 3 mg·L -1 Na₂MoO₄·2H₂O 0.25 mg·L⁻¹ -1 CoCl2·6H2O 0.025 mg·L -1 Vitamin B1 1 mg·L -1 Vitamin B6 0.5 mg / L -1 ; Nicotinic acid 0.5 mg·L -1 Glycine 2 mg·L -1 Inositol 100 mg·L -1 ; 6-BA 0.2 mg·L -1 NAA 0.05 mg·L -1 Sodium thiosulfate 25 mg·L -1 ; The in vitro preservation culture medium for the infected plants in step (5) consists of: 800 mg / L KNO3. -1 NH4NO3 200 mg·L -1 CaCl2·2H2O 150 mg·L -1 MgSO4·7H2O 125 mg·L -1 Mg(NO3)2·6H2O 235 mg·L -1 Ca(NO3)2·4H2O 165 mg·L -1 KH2PO4 340 mg·L -1 MnSO4·H2O 6 mg·L -1 ZnSO4·7H2O 5mg·L -1 CuSO4·5H2O 0.1 mg·L -1 H3BO3 3 mg·L -1 Na₂MoO₄·2H₂O 0.25 mg·L⁻¹ -1 CoCl2·6H2O 0.025 mg·L -1 Vitamin B1 1 mg·L -1 Vitamin B6 0.5 mg / L -1 ; Nicotinic acid 0.5 mg·L -1 Glycine 2 mg·L -1 Inositol 100 mg·L -1 ; 6-BA 0.2 mg·L -1 NAA 0.05 mg·L -1 Sodium thiosulfate 30 mg·L -1 .

2. The efficient preservation method according to claim 1, characterized in that, The grading method for disease-resistant and disease-susceptible germplasm in step (3) is as follows: Select robust Masson pine seedlings that have been transplanted to the nursery for 8-12 months and have a high degree of lignification. Inoculate them with nematodes at a rate of 4000 nematodes per seedling. Place the seedlings inoculated with nematodes under conditions of 30-35℃, 60%-70% humidity, and 8000 lx-12000 lx light intensity. Observe and measure the disease mortality rate of the seedlings. When the disease mortality rate is ≤30%, the seedlings are graded as disease-resistant germplasm. When the disease mortality rate is >30%, the seedlings are graded as disease-susceptible germplasm.

3. The efficient preservation method according to claim 1, characterized in that, The compound agent for the disease-resistant material in step (2) consists of 25~50 mg / L paclobutrazol, 0.05~0.15 g / L sodium dihydrogen phosphate, 0.2~0.3 g / L potassium nitrate, and 1~3 mg / L boric acid.

4. The efficient preservation method according to claim 1, characterized in that, In step (4), the seedling substrate for disease-resistant germplasm consists of 3 parts peat moss, 3-4 parts perlite, 1-2 parts yellow clay, 1-2 parts coconut coir, and 2-3 kg / m³ of soil. 3 Composition of superphosphate.

5. The efficient preservation method according to claim 1, characterized in that, The compound agent for the disease-causing material in step (2) consists of 0~25 mg / L paclobutrazol, 0~0.15 g / L sodium dihydrogen phosphate, 0.1~0.3 g / L potassium nitrate, and 3~5 mg / L boric acid.

6. The efficient preservation method according to claim 1, characterized in that, In step (4), the seedling substrate for susceptible germplasm consists of 2 parts peat moss, 1-2 parts perlite, 1-2 parts yellow clay, 3-4 parts coconut coir, and 2-3 kg / m³ of soil. 3 Composition of superphosphate.

7. The efficient preservation method according to claim 1, characterized in that, The seedlings of the disease-resistant germplasm were subcultured 4 to 8 times before being preserved in vitro, while the seedlings of the disease-susceptible germplasm were subcultured 8 to 12 times before being preserved in vitro.

8. The efficient preservation method according to claim 1, characterized in that, The seedlings of the disease-resistant and disease-susceptible germplasms need to undergo rejuvenation culture after two years of in vitro preservation.

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