Rapid breeding method for weak-resistance tea seedlings

Abstract

The invention discloses a rapid breeding method for weak-resistance tea seedlings. The rapid breeding method comprises the following steps: S1, preparing a seedling raising shed, wherein the sunshade rate of the seedling raising shed is 60-80%; s2, hole tray preparation: selecting a light substrate as a seedling culture substrate and filling the seedling culture substrate into a hole tray; s3, preparation of cuttings: cutting tea seedling cuttings with 2 / 3-1.5 mature leaves and one full axillary bud; s4, cutting: inserting the cutting slips sheared in the S3 into the hole tray in the S2; s5, management after cuttage: watering thoroughly once after cuttage, spraying a rooting agent and a disinfectant, controlling the humidity of the substrate to be 60-70%, and controlling the air humidity to be 75-85%; s6, tea seedling outplanting. According to the method, the development completeness and quality of the tea seedlings are remarkably improved, the resistance of the tea seedlings is improved, and the method has a remarkable effect on improving growth and development in an adverse environment and large-scale breeding.

Description

Technical Field

[0001] The invention relates to the technical field of tea tree cultivation and breeding, and in particular to a method for rapid breeding of weakly resistant tea seedlings. Background Art

[0002] Tea trees are evergreen perennial shrubs of the genus Camellia in the family Theaceae. Because sexual reproduction cannot guarantee uniform seeding, tea cultivation primarily relies on asexual propagation, including internode cuttings and softwood cuttings. Excellent varieties such as Baojing Golden Tea No. 1 and Rucheng White Hair Tea have weak resistance and very low survival rates under traditional transplanting methods. Newly planted tea gardens are plagued by widespread problems such as low seedling survival rates, poor rooting efficiency, weak growth, and slow garden establishment. Furthermore, the frequent occurrence of extreme weather conditions such as high temperatures and droughts in recent years has led to high initial production and management costs for tea gardens, significantly impacting the breeding of tea varieties and the upgrading of the industry.

[0003] Seed plants are highly variable and generally unsuitable for mechanized harvesting and processing. Therefore, the most efficient and economical way to produce large numbers of homogeneous clones of tea plants is through asexual propagation. Researchers commonly use a portion of the plant's vegetative organs for asexual propagation to improve rooting efficiency. However, the lack of rapid propagation methods has hindered the widespread adoption of many superior varieties.

[0004] Chinese patent publication number CN102138408B discloses a method for tea seedling cultivation using light-substrate plug cuttings, which includes the following steps: collecting cuttings, preparing seedling cultivation facilities, preparing a seedling cultivation substrate, preparing a seedling cultivation plug, preparing cuttings, cutting, and post-cutting management. The method uses several soilless raw materials selected from peat, perlite, and decomposed straw to prepare a substrate; the cuttings collected in step (1) are cut into half-leaf-one-bud or one-third-leaf-one-bud cuttings 3-5 cm in length; for the first 15-20 days after cutting, the substrate must be fully moistened, with the relative humidity controlled at 85-95%, and mist irrigation is performed three times daily, once in the morning, noon, and evening, for 20-30 minutes each time; when watering manually, water thoroughly once in the morning and evening; after 10 days, mist irrigation is performed twice daily, once in the morning and evening, for 20-30 minutes each time, and when watering manually, water thoroughly once daily. The shading rate is 80%-90%. Compared with the present invention, this solution leads to increased mortality of cuttings during the cultivation of weakly resistant tea varieties under high temperatures. The present invention improves and enhances the adaptability of weakly resistant tea cuttings to high temperatures by addressing issues such as the lack of film covering the plug trays, spraying pesticides, and regulating temperature and humidity under high temperatures during the breeding process. Summary of the Invention

[0005] The present invention aims to provide a method for rapid propagation of weakly resistant tea seedlings, which can significantly improve the developmental integrity and quality of tea seedlings, enhance the resistance of tea seedlings, and has a significant effect on improving growth and development in adverse environments and large-scale propagation, thereby increasing the survival rate of weakly resistant tea seedlings.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for rapid propagation of weakly resistant tea seedlings comprises the following steps:

[0008] S1. Preparation of seedling shed: The shading rate of the seedling shed is 60-80%;

[0009] S2. Preparation of plug tray: Select light substrate as the seedling medium and put it into the plug tray;

[0010] S3. Cutting preparation: Cut tea seedling cuttings with 1 / 4-3 / 4 mature leaves and 1 full axillary bud;

[0011] S4, cuttings: insert the cuttings cut in S3 into the plug tray in S2;

[0012] S5. Management after cutting: After cutting, water the substrate to make it fully moist, spray rooting agent and disinfectant, control the substrate humidity at 60% to 70%, and the air humidity at 75 to 85%;

[0013] S6. Tea seedlings are transplanted.

[0014] Preferably, the shading rate of the seedling shed is 70-75%.

[0015] Preferably, the air humidity is controlled at 75-80%.

[0016] Preferably, it is best to cut the mature leaves to 1 / 2 to reduce the contact area between the leaves of the cuttings and maximize photosynthesis, which is suitable for large-scale cutting propagation.

[0017] Preferably, the best shading rate is 80%, which is suitable for large-scale tea seedling breeding in high temperature or extremely high temperature weather in summer.

[0018] Reduces light quantum but does not hinder photosynthesis.

[0019] Compared with the Chinese patent with the publication number CN102138408B, the substrate of this patent retains more water at high temperature, and the corresponding cutting survival rate is higher.

[0020] The invention improves the microenvironment of cuttings in a high temperature environment and establishes a steady state of substrate-cuttings-microclimate environment balance to achieve the ability to promote the growth and development of weakly resistant tea seedlings, so that the cuttings can be planted earlier and the garden can be established faster.

[0021] According to the embodiments of the present invention, the present invention can be further optimized. The following is the technical solution formed after optimization:

[0022] S3 tea seedlings include one or more of Baojing Golden Tea No. 1, Baojing Golden Tea No. 2, Baojing Golden Tea No. 6, Baojing Golden Tea No. 7, Baojing Golden Tea No. 8, Baojing Golden Tea No. 18, Baojing Golden Tea No. 168, and Rucheng White Hair Tea.

[0023] Excellent varieties such as Baojing Golden Tea No. 1 and Rucheng White Tea have weak resistance and their survival rate is very low under ordinary traditional transplanting methods. The breeding method of this application is suitable for excellent varieties with weak resistance such as Baojing Golden Tea No. 1 and Rucheng White Tea, and can significantly improve the developmental integrity and quality of tea seedlings and improve the resistance of tea seedlings.

[0024] In one preferred embodiment, when the air temperature in the seedling shed in S5 exceeds 30°C and the humidity is lower than 45% RH, the temperature inside the membrane is controlled to be higher than 33°C and the humidity is lower than 60% RH, and the soil temperature is higher than 35°C and the humidity is lower than 70% RH. In order to maintain the steady state of the microclimate environment, a greenhouse temperature and humidity control instrument is used. Compared with other patents, the present invention is not limited to any temperature attribute and significantly promotes the survival rate, rooting rate and germination rate of cuttings in a high temperature environment.

[0025] In one preferred embodiment, watering is performed once every 3 to 4 days.

[0026] In one preferred embodiment, when the air temperature is 30-33°C and the humidity is above 50% RH, the temperature inside the film is kept below 33°C and the humidity is above 85% RH, while the soil temperature is kept between 32-35°C and the humidity is above 80% RH. Using fans and intelligent spraying within the greenhouse to control the microclimate, this method can significantly improve the survival rate of cuttings in high-temperature summer environments. Compared with the Chinese patent application number CN102138408B, the present invention has a higher survival temperature and is more adaptable to weakly resistant tea seedlings, making it suitable for high-temperature summer cuttings.

[0027] In one preferred embodiment, watering is performed once every 6 to 7 days.

[0028] In one preferred embodiment, the light matrix in S2 includes the following components by volume: 6 to 12 parts of peat formula soil, 2 to 4 parts of perlite, and 2 to 4 parts of vermiculite.

[0029] Preferably, the light matrix is ​​in a volume ratio of peat soil: perlite: vermiculite = 3:1:1. 1 part = 1m 3 .

[0030] In one preferred embodiment, in S5, after the leaves are dried, a rooting agent and a disinfectant are sprayed.

[0031] Preferably, a seedbed is built in the seedling shed with a width of 0.8 to 1.6 m, a ditch width of 25 to 35 cm, a ditch depth of 10 to 20 cm, and a length generally not exceeding 15 m. The substrate is sprinkled with water and mixed evenly, then loaded into the plug tray, and then the plug tray is placed on the seedbed.

[0032] Preferably, the mother branches can be cut into spikelets 10 to 15 days after being toppled in batches according to standards.

[0033] Preferably, cutting of cuttings should be done to avoid periods of high temperature and strong light exposure, and shade and moisturizing measures should be taken during transportation and storage.

[0034] Preferably, the cuttings are 2 to 4 cm long.

[0035] Preferably, a 10-15 meter seedbed, a 32-64 hole tray, after cutting the leaves, plant 1-3 plants per hole, water each tray with 5-7L of water, water 200 trays with 15-20L / bag of rooting agent and disinfectant, and cover with a layer of white film every 8-12 meters.

[0036] Preferably, one pack of mancozeb, one bucket of water (16L), can cover: 200 trays of 32-hole trays;

[0037] Benzbromothiocarb: one package, one bucket of water (16L), can cover: 200 trays of 32-hole trays;

[0038] Chlorfenapyr: 15ml, one bucket of water (16L), can cover: 200 trays of 32-hole trays;

[0039] Methylotrophic Bacillus: One package, one bucket of water (16L), can cover: 200 trays of 32-hole trays;

[0040] Amino acid fertilizer: one bag, one bucket of water (16L), can cover: 200 trays of 32-hole trays.

[0041] Preferably, 1 to 2 cuttings are inserted into each hole in S4, and the cutting depth is such that the bottom of the cutting is located in the middle of the hole tray, the petiole cannot be close to the substrate, and the direction of the cutting leaves remains consistent.

[0042] Preferably, in S5, water is replenished once every 7 to 10 days;

[0043] Preferably, the monitoring in S5 is completed by an air temperature and humidity meter and a soil temperature and humidity meter.

[0044] Pest and disease control should be carried out 1-2 times every 15-25 days. In winter, when temperatures remain below 10°C, the frequency of pest and disease control can be reduced. After the root system has formed, 0.1-0.3% urea can be sprayed in conjunction with pest and disease control. Root formation generally occurs in September of the same year.

[0045] Preferably, the tea seedlings transplanted from plug trays in June or July can be transplanted in batches in early November of the same year. The transplant rate of the first batch can reach more than 55%, and the transplant standard is that the height of the tea seedlings is ≥8cm.

[0046] Ideally, after transplanting, the seedlings are shipped in plastic baskets measuring 60 cm in length, 40 cm in width, and 20 cm in height. Seedlings that don't meet the standards for transplanting can continue to be cultivated and transplanted in batches in mid-to-late February or mid-to-late March of the following year. This significantly shortens the seedling raising process.

[0047] Optimally, seedlings grown in plug trays are transplanted to nutrient pots after six months and then transplanted to the field after one year. This seedling-based approach saves costs and maximizes profitability. This approach can be used directly in conjunction with the tea season, with fertilization during the process ensuring a healthy supply of nutrients for the seedlings' growth. This allows for large-scale tea seedling cultivation, eliminating the need for traditional methods such as clearing nursery beds and digging subsoil.

[0048] The effects of substrate ratios on tea seedling growth vary significantly. Tea plug propagation using light substrates such as peat, vermiculite, and perlite, in specific proportions, is suitable. Light substrate plug propagation can significantly increase the propagation speed and quality of improved clones, offering significant advantages over other asexual propagation techniques. Combined with appropriate cutting management techniques, it can significantly improve root system and seedling quality after transplantation. By enhancing specific metabolic response pathways, it can also enhance the resistance and adaptability of less resistant tea seedlings.

[0049] At present, there are no reports on the research on the application of light substrate formula combined with cutting nursery management technology to breed high-quality but weak-resistant excellent varieties such as Baojing Golden Tea No. 1 and Rucheng White Hair Tea, and the related research on large-scale reproduction to enhance resistance, rooting survival rate, and special metabolic response pathways of tea seedlings.

[0050] Therefore, the light substrate formula and management method provided by the present invention can significantly improve the callus rate, rooting rate, germination rate, survival rate, callus quality, stem quality, leaf quality and the completeness and quality of root development of tea seedlings, improve the resistance of tea seedlings, and have a significant effect on improving growth and development in adverse environments and large-scale breeding.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] (1) Under normal conditions, using light substrate plug trays and controlling the shading rate, temperature and humidity for tea seedling cuttings can significantly improve the rooting rate, germination rate, survival rate and callus formation rate, and improve the rooting efficiency and survival ability of weakly resistant tea seedling cuttings. It has a significant effect on improving the growth condition and development ability of tea seedling cuttings under general or adverse conditions.

[0053] (2) The present invention makes the tea seedling cuttings grow very well, the cuttings grow vigorously, and the root buds are fully developed and strong, overcoming the problems of poor growth and incomplete root development under traditional cutting technology.

[0054] (3) The cutting operation method and materials used in the present invention are relatively simple and convenient, which solves the shortcomings of traditional technology that requires relatively complex and harsh conditions to ensure rooting rate and survival rate.

[0055] (4) The nursery management method of the present invention can make the cuttings take root more quickly than the traditional technology, solving the problem that the weakly resistant tea seedlings have difficulty taking root under the traditional technology management. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is the technical roadmap for this application.

[0057] Figure 2 This is the sampling process and testing diagram for this application.

[0058] Figure 3 For climate microenvironment data monitoring and optimization.

[0059] Figure 4 This is a comparison of the agronomic properties of two soil matrices under high temperature.

[0060] Figure 5 It is the phenotypic changes and anatomical structure differences of cutting seedlings.

[0061] Figure 6 Comparison of paraffin sections of leaves and roots of cutting seedlings at the third and fifth stages.

[0062] Figure 7 The changes in physiological and biochemical components of leaves, stems, callus tissues, buds and roots of cutting seedlings at different stages.

[0063] Figure 8 This is the change of malondialdehyde (MDA) in the root system (callus), stem, leaf and bud of cutting seedlings at different stages.

[0064] Figure 9 Significance comparison of enzyme activities in leaves and buds of cutting seedlings at different stages.

[0065] Figure 10 This is a significant comparison of enzyme activities in cutting seedling stems at various stages.

[0066] Figure 11 This is a significant comparison of enzyme activities between cutting seedling roots and calli at various stages.

[0067] Figure 12 Principal component analysis (A) and Pearson correlation analysis (B) of transcriptome data.

[0068] Figure 13The bar graph and scatter plot of differential gene expression of samples.

[0069] Figure 14 GO enrichment analysis of differentially expressed genes in cutting seedling development.

[0070] Figure 15 KEGG enrichment scatter plot of differentially expressed genes.

[0071] Figure 16 OPLS-DA analysis of amino acids in roots and leaves.

[0072] Figure 17 OPLS-DA analysis of plant hormones in callus and roots.

[0073] Figure 18 Transcriptome sequencing diagrams of roots (see Figure a) and leaves (see Figure b).

[0074] Figure 19 Component analysis of each sample (see Figure a), targeted plant hormone analysis of roots (see Figure b), callus tissue (see Figure c), and heat map of the correlation between targeted plant hormones and amino acids (see Figure d).

[0075] Figure 20 Differential gene expression of samples.

[0076] Figure 21 The third stage effect comparison diagram of Example 1 and Comparative Example 2 is shown.

[0077] Figure 22 These are three groups of control experiments in Example 1 and Comparative Example 3.

[0078] Figure 23 The survival rate of Baojing Golden Tea (weakly resistant variety) under high temperature environment is compared between Example 1 (Figure b) and the comparative patent (Figure a).

[0079] Figure 24 This is a graph showing the growth of resistant tea varieties in Example 1 and Comparative Example 3 under high temperature conditions.

[0080] Figure 25 This is a graph showing the growth of the resistant tea varieties in Example 1 and Comparative Example 4. DETAILED DESCRIPTION

[0081] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments. It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention may be combined with each other.

[0082] The present invention provides a management method for a mother tea garden for budding, comprising the following steps: selecting a tea garden with 6-10 years of age and 100% cultivar purity under normal cultivation and management conditions as the mother tea garden for budding, producing only premium spring tea, pruning in mid-to-late April to remove slender, thin branches and clumps, and pruning to a depth sufficient to produce robust shoots. New shoots sprouting that year are then cultivated for cuttings. After pruning, fertilizer and water management is strengthened, increasing the ratio of phosphorus to potassium fertilizer to 3:1 in addition to conventional fertilization to produce shoots with strong branching ability. In summer, the light-shading ratio is strictly maintained at 60-80%, the soil and tea plantation air temperature at 26-30°C, the water and soil humidity at 70-80% relative humidity, and the air circulation within the tea plantation and fertilizer utilization rate are strictly maintained. During the growth of new shoots, special attention is paid to pest and disease control.

[0083] In the present invention, the weakly resistant tea tree varieties include any one or more of Baojing Golden Tea No. 1, Baojing Golden Tea No. 2, Baojing Golden Tea No. 6, Baojing Golden Tea No. 7, Baojing Golden Tea No. 8, Baojing Golden Tea No. 18, Baojing Golden Tea No. 168, and Rucheng White Hair Tea. These varieties are all extremely superior but show poor adaptability, slow growth, and weak stress resistance during transplanting or propagation.

[0084] The present invention provides a light matrix formula for rapid plug propagation, comprising the following components by volume: 6-12 parts of peat soil, 2-4 parts of perlite, and 2-4 parts of vermiculite, preferably in a volume ratio of peat soil: perlite: vermiculite = 3:1:1. 1 part = 1m 3 Preferably, 9:3:3 = 9 parts peat soil + 3 parts perlite + 3 parts vermiculite, mix well and fill into the hole tray.

[0085] The invention provides a rapid propagation cutting technology method. The cutting is generally carried out in June or July each year and mainly comprises the following steps.

[0086] (1) Preparation of the seedling shed.

[0087] Choose a plastic greenhouse or greenhouse to grow seedlings, cover the outside of the greenhouse or greenhouse with a shade net with a shading rate of 80%, and equip the greenhouse with irrigation facilities.

[0088] (2) Preparation of seedbed and plug tray.

[0089] Build a seedbed in the greenhouse. The bed should be 1.2 meters wide, with a 30-cm wide, 15-cm deep trench, and generally no longer than 15 meters. Use a 9:3:3 ratio by volume of peat soil, perlite, and vermiculite as the seedling medium. Use a 50-hole tray as the container for seedling cuttings. Mix the medium thoroughly with water and then place it in the tray. Place the tray on the seedbed.

[0090] (3) Preparation of cuttings.

[0091] Cuttings can be made 10-15 days after the mother branches are toppled in batches according to standards. Cuttings should be kept away from periods of high temperature and strong sunlight. Shade and moisture-retaining measures should be taken during transportation and storage. Cuttings should be handled promptly and planted. Cuttings should be approximately 3 cm long, with half a mature leaf and one plump axillary bud.

[0092] (4) Cuttings.

[0093] Insert the cut cuttings into the prepared hole tray, 1-2 cuttings per hole. The cutting depth should be such that the bottom of the cutting is in the middle of the hole tray. The petiole cannot be close to the substrate, and the direction of the cutting leaves should be consistent.

[0094] (5) Management after cutting.

[0095] After grafting, water thoroughly once. After the leaves have dried, spray with a rooting agent and disinfectant. Maintain substrate humidity at 60% to 70%, and air humidity at around 80%. Maintain air temperature at around 30°C (this patent allows temperatures up to 37°C), film temperature at 32°C, and soil temperature at 31°C. Generally, rehydrate every 7 to 10 days, and conduct pest and disease control every 20 days. In winter, when temperatures remain below 10°C, the frequency of pest control can be reduced. Roots will generally form in September of the same year, and after this, spray with 0.2% urea in conjunction with pest and disease control.

[0096] (6) Tea seedlings transplanting and transportation.

[0097] Tea seedlings transplanted from plug trays in June and July are generally ready for transplanting in batches in early November of the same year. The first batch can achieve a transplant rate of over 55%, with a transplant height of 8 cm or greater. After transplanting, seedlings are typically shipped in plastic baskets measuring 60 cm in length, 40 cm in width, and 20 cm in height. Seedlings that do not meet transplant standards can be further cultivated and transplanted in batches in mid-to-late February or mid-to-late March of the following year.

[0098] Preferably, the method comprises the following steps: preparing a temperature-controlled and humidity-controlled seedling shed with 70-80% sunshade, preparing a 10-15 meter seedling bed and a 32-64 hole tray, cutting the cuttings obtained from the mother garden by 1 / 4-3 / 4 of the leaves, pruning and transplanting 1-3 plants per hole, watering 5-10L, spraying pesticides and watering 180-220 trays per 18L, and covering 1 layer of white film every 10-15 meters. Preferably, the seedling shed has a sunshade efficiency of 80%, a 10 meter seedling bed, a 32-hole tray, cutting 1 / 2 of the leaves, transplanting 2 plants per hole, watering 7L per tray, pouring 200 trays of rooting agent and disinfectant per 18L / bag, and covering 1 layer of white film every 10 meters.

[0099] Mancozeb: One pack, one bucket of water (16L), can cover: 200 trays of 32-hole trays;

[0100] Benzbromothiocarb: one package, one bucket of water (16L), can cover: 200 trays of 32-hole trays;

[0101] Chlorfenapyr: 15ml, one bucket of water (16L), can cover: 200 trays of 32-hole trays;

[0102] Methylotrophic Bacillus: One package, one bucket of water (16L), can cover: 200 trays of 32-hole trays;

[0103] Amino acid fertilizer: one bag, one bucket of water (16L), can cover: 200 trays of 32-hole trays.

[0104] The present invention provides a cutting nursery technical management method, comprising the following steps:

[0105] ① Application of pesticides: Apply pesticides and supplement rooting agents on the 15th to 20th day after cutting. Preferably, it is done on the 15th day. Callus tissue can be seen on the 23rd to 25th day, entering the callus period.

[0106] ② Temperature and humidity: including greenhouse air temperature and humidity, film temperature and humidity, and soil temperature and humidity; when the greenhouse air temperature exceeds 30°C and the humidity is lower than 45% RH, the film temperature is higher than 33°C and the humidity is lower than 60% RH, the soil temperature is higher than 35°C and the humidity is lower than 70% RH, water once every 4 days; when the air temperature is between 25 and 30°C and the humidity is higher than 50% RH, the film temperature is lower than 30°C and the humidity is higher than 85% RH, the soil temperature is between 26 and 29°C and the humidity is higher than 80% RH, water once every 7 days; monitoring is completed by air temperature and humidity meters and soil temperature and humidity meters.

[0107] ③ Transplantation in nutrient pots: Take out the seedlings from the hole tray with the substrate and transplant them into nutrient pots for cultivation. Transplant 2 plants per pot with a plant spacing of 2 to 3 cm. Choose a nutrient pot made of non-woven fabric with a diameter of about 9 cm and a height of about 10 cm. Use peat soil and organic fertilizer in a ratio of 10:1 for nutrient soil. Arrange the nutrient pots of the transplanted tea seedlings neatly and tightly in the seedbed, keeping the direction of the mother leaves of the cuttings consistent, and place them every 667m. 2 The nursery can accommodate 90,000 tea pots and cultivate approximately 180,000 tea seedlings. Large seedlings in different sizes can also be produced for the desired seedling specifications. These large seedlings are then transplanted to the mountain, and tea can be harvested six months after planting. After transplanting, tea seedlings in tea pots are cultivated according to conventional methods. Generally, by July or August, when the seedlings reach a height of over 40 cm, they are topped to promote aging of the branches. Seven to ten days later, the seedlings are pruned to a height of 12 to 15 cm. This pruning process yields an average of seven high-quality cuttings per tea plant.

[0108] Figure 5 The stomatal data analysis of the leaves of the medium-length cutting seedlings at stages ③, ④, and ⑤ is shown in Table 1.

[0109] Table 1 Stomatal data analysis of cutting seedling leaves at stages ③, ④, and ⑤

[0110]

[0111] In stages ③, ④, and ⑤, letters indicate significant differences between groups, P < 0.05.

[0112] Wherein T refers to Example 1, and CK refers to Comparative Example 1.

[0113] The stomatal opening of the experimental group was much larger than that of the control group under high temperature in summer, indicating that the matrix significantly improved the adaptability of weakly resistant cuttings and had a higher photosynthetic rate potential.

[0114] The data analysis of vein cross-section of cutting seedling leaves at stages ③ and ⑤ is shown in Table 2.

[0115] Table 2 Analysis of vein cross-section data of cutting seedling leaves at stages ③ and ⑤

[0116]

[0117] Note: ③④⑤ represent the third, fourth and fifth stages, and letters indicate significant differences between the groups, P < 0.05.

[0118] in

[0119] The stomatal opening and closing of the experimental group were larger than those of the control group, but the leaf vein vascular bundles were smaller than those of the control group. Considering the environment and survival efficiency, the large vascular bundles of the control group did not achieve an advantage in transport efficiency, indicating that the cuttings in the experimental group did not respond to stress in the matrix, while the control group had insufficient survival adaptability.

[0120] Comparative Example 1( Figure 1 CK group in

[0121] The difference between Comparative Example 1 and Example 1 is that light matrix cuttings are not used, but traditional soil cuttings are used.

[0122] Example 2

[0123] The difference between Example 2 and Example 1 is that the sun shading rate is 75%.

[0124] The experimental group can alleviate the adaptability of cuttings under high temperature stress.

[0125] Comparative example leaf (CK-Y), comparative example callus (CK-S), comparative example root (CK-G), comparative example stem (CK-J), comparative example bud (CK-YY), example leaf (TY), example callus (TS), example root (TG), example stem (TJ), example bud (T-YY).

[0126] Figure 2The first to fifth stages represent growth nodes ①-⑤. Boxes of the same color represent the same sampling or experimental level.

[0127] Figure 3 A. Fluctuations in light intensity inside and outside the greenhouse; B. Changes in soil temperature and humidity at the plug tray sampling holes; C1. Violin plot of humidity differences inside and outside the greenhouse; C2. Violin plot of temperature differences inside and outside the greenhouse; D. Heat map of pH changes in the plug tray soil matrix; E. Range of soil moisture variation at the plug tray sampling holes in the experimental group; F. Optimization of soil moisture parameters using response surface methodology; G. Optimization of parameter contour thresholds using response surface methodology. Determine the temperature and humidity ranges for air, inside the film, and soil.

[0128] Figure 4 A. Leaf weight and bud weight; B. Stem weight; C. Callus weight and root weight; D. Survival rate; E. Callus rate; F. Rooting rate; G. Germination rate; H. Side branch length of the fifth sampling (10 groups); I. Main root length of the fifth sampling (10 groups). * indicates a significant difference (P < 0.05), ** indicates P < 0.01, *** indicates P < 0.005, **** indicates P < 0.001. All samples were biologically replicated in three groups. This shows that under high temperature conditions, the cuttings of the embodiment are significantly better than those of the control example in all agronomic trait indicators.

[0129] Figure 5 A. Growth and development phenotypes of different substrates; B. Scanning electron micrographs of root development in different substrates; C. Scanning electron micrographs of leaf stomata in different substrates. ①-⑤ represent the first to fifth sampling stages; red boxes indicate special attention; CK represents the subsoil substrate cutting group; T represents the light substrate cutting group. Compared to the comparative example, the example shows better root vascular development, larger stomatal openings, and greater gas exchange capacity.

[0130] Figure 6 A. Paraffin-sectioned cross section of roots and leaves at the third stage; B. Paraffin-sectioned cross section of roots and leaves and longitudinal section of leaves at the fifth stage. This shows that the embodiment plays a positive role in the root system development and leaf vein development of tea tree cuttings, and can significantly affect the development ability of cuttings.

[0131] Figure 7 * indicates significant difference (P<0.05). This indicates that under high temperature environment, the physiological metabolite conditions of each part of the cuttings in the example are higher than those in the control example, showing stronger adaptability.

[0132] Figure 8 * indicates significant difference (P<0.05).

[0133] Malondialdehyde (MDA) is one of the main end products of lipid peroxidation. When cells are subjected to oxidative stress (such as free radical attack), unsaturated fatty acids on the cell membrane undergo peroxidation and decompose to produce MDA, a marker of oxidative damage. Its level directly reflects the degree of damage to the cell membrane system and the level of oxidative stress experienced by the organism.

[0134] The higher the MDA value, the more severe the oxidative damage to the plant and the more severely the cell membrane integrity is destroyed, which usually means that the plant is in a worse condition and has weaker resistance under the stress condition.

[0135] according to Figure 8 The results showed that the MDA content of the leaves of the comparative example 1 showed a fluctuating upward trend, while the MDA content of the leaves of the experimental group showed a trend of first increasing and then decreasing in the first to fifth stages. In the third stage, the MDA content of the leaves of the comparative example 1 was lower than that of the experimental group, but in the fourth and fifth stages, the MDA content of the leaves of the comparative example 1 was significantly higher than that of the experimental group, and in the fifth stage, the MDA content of the leaves of the comparative example 1 increased significantly, much higher than that of the experimental group, indicating that the comparative example 1 was under strong stress throughout the entire stage. The bud MDA value of the experimental group was also significantly different from that of the comparative example 1. The buds of the experimental group developed in the fourth stage, and the MDA decreased in the fourth and fifth stages, while the comparative example 1 did not develop buds throughout the entire experimental stage.

[0136] The MDA value of the stem showed an overall upward trend. From stage one to stage two, the stem MDA content of both the comparative example 1 and the experimental group decreased, but the rate of decrease in the experimental group was lower than that of the comparative example 1. From stage two to stage three, the stem MDA content of both the experimental group and the comparative example 1 increased simultaneously, with the rate of decrease in the experimental group higher than that of the comparative example 1. From stage three to stage five, the MDA content of the experimental group decreased slowly, while that of the comparative example 1 first increased and then decreased. Ultimately, the stem MDA content of the experimental group was higher than that of the comparative example 1, which may be related to substance transport.

[0137] In terms of callus tissue, the callus tissue of the comparative example 1 experienced a decline in the early stage, then rose rapidly and then fell again, while the callus tissue of the experimental group began to decline in the third stage. The root system of the comparative example 1 began to decline in the fourth stage. Except for the first stage, the callus tissue and root system of the comparative example 1 and the experimental group showed significant differences in other stages.

[0138] Figure 9* indicates significant differences (P < 0.05). PPO (polyphenol oxidase) can catalyze the oxidation of phenolic substances into quinones. Quinones are toxic to insects / pathogens and aggregate to form physical barriers (such as wound browning). When injured or stimulated by pathogens, PPO activity rises sharply, which is a biochemical marker of plant resistance to insects / diseases. POD (peroxidase) uses H2O2 to oxidize a variety of substrates (such as phenols, lignin precursors), decomposes H2O2 (cooperates with CAT), reduces the production of hydroxyl radicals (·OH), and participates in cell wall lignification (enhancing mechanical resistance), wound healing and disease resistance. Its activity increases significantly under adversity (pathogen infection, mechanical damage). High POD activity can enhance cell wall strength and limit the expansion of pathogens. It is a key indicator of disease resistance. SOD (superoxide dismutase), SOD is the first line of defense in the plant antioxidant defense system. It catalyzes superoxide radicals (·O2 - ) into hydrogen peroxide (H2O2) and oxygen (O2), which scavenge superoxide radicals, the most toxic of reactive oxygen species (ROS), preventing damage to proteins, DNA, and membrane lipids. The generated H2O2 can be further broken down by subsequent enzymes (such as POD and CAT) and can also serve as a signaling molecule to activate defense pathways. Higher SOD activity generally indicates a stronger plant's ability to scavenge ROS and greater resistance to stresses (such as drought, salt, and heavy metals). In stress resistance assessment, increased SOD and POD activity, a moderate PPO response, and reduced MDA content are positive signals of plant stress tolerance.

[0139] The PPO, POD, and SOD activities of the leaves from Comparative Example 1 were all higher than those from the experimental group. Regarding CAT activity, both the leaves from Comparative Example 1 and the experimental group showed an inflection point in the third stage: CAT activity in the leaves from Comparative Example 1 leveled off and then rose, while that in the leaves from the experimental group rose and then fell. The PPO value of the leaves from Comparative Example 1 first decreased, then increased, and then decreased again, while the PPO value of the leaves from the experimental group showed an overall downward trend. The POD value of the leaves from Comparative Example 1 showed an upward trend, while the POD value of the leaves from the experimental group showed a fluctuating equilibrium trend. The SOD activity of the leaves from Comparative Example 1 first increased, then decreased, and then slowly increased, while the SOD activity of the leaves from the experimental group first increased, then decreased, with less fluctuation.

[0140] The CAT, PPO, POD, and SOD activities in the shoots of the experimental group were all higher than those in Comparative Example 1. CAT and SOD in the shoots of the experimental group showed an upward trend from the fourth to fifth stages, while PPO and POD showed a downward trend from the fourth to fifth stages. The POD values ​​of the experimental group were significantly lower than those of Comparative Example 1 in the fourth and fifth stages, but the POD activity in the shoots of the experimental group was much higher than that of Comparative Example 1, indicating that POD activity is stronger in young tissues. This suggests that the experimental group has completed the source-sink transition, with older leaves gradually transitioning to functional leaves. With the exception of the first stage, enzyme activities in leaves and shoots showed significant differences between Comparative Example 1 and the experimental group.

[0141] Figure 10 * indicates significant difference (P<0.05)

[0142] In the stems of Comparative Example 1, CAT and SOD activities showed a fluctuating equilibrium, PPO activity showed a steady increase, and POD activity showed a trend of first increasing, then decreasing, and then increasing again. This may indicate that under the conditions of Comparative Example 1, CAT and SOD function while maintaining a certain equilibrium. The continued increase in PPO activity may be related to certain physiological processes or environmental factors, while the fluctuations in POD activity may be influenced by multiple factors. In the stems of the experimental group, CAT activity showed a trend of first decreasing, then increasing, PPO activity showed a trend of first increasing, then decreasing, and POD activity showed a fluctuating trend of slowly increasing. SOD activity, however, returned to its initial value after experiencing fluctuations in the fourth stage. This suggests that under the experimental conditions, changes in enzyme activity may be affected by the experimental treatments, and different treatments may lead to different trends in enzyme activity. Significant differences in enzyme activity were observed between Comparative Example 1 and the experimental group at all other stem stages, except for the first stage. This suggests that the effects of the experimental treatments on enzyme activity vary significantly across different stages, possibly related to factors such as the plant growth stage and the treatment method. Clear differences in enzyme activity between Comparative Example 1 and the experimental group may reflect changes in the plant's physiological state under different conditions. Further research on the physiological mechanisms and influencing factors behind these differences will help to gain a deeper understanding of plant growth and adaptability under different environmental conditions.

[0143] Figure 11 * indicates significant difference (P<0.05)

[0144] It can be observed that the activities of CAT and SOD in the roots and callus of Comparative Example 1 showed a downward trend, among which CAT showed an upward fluctuation in the fourth stage, PPO activity rose slowly, and POD activity showed an overall upward trend, with a peak in the third stage and a trough in the fourth stage. Except for the first stage, the enzyme activities of the roots and callus of Comparative Example 1 showed significant differences compared with those of the experimental group. This indicates that the metabolism and nutrient uptake capacity of the experimental group are significantly better than those of Comparative Example 1 in the root system and callus. In addition, the activities of POD and SOD in the roots and callus showed a relative increase and decrease between Comparative Example 1 and the experimental group. This occurred in the fourth to fifth stages and may be an important turning point for germination of the experimental group. It can be inferred that POD and SOD may have an impact on the growth of buds.

[0145] Figure 12The squared Pearson correlation coefficient (R²) between biological replicates was at least 0.8. CK: Control Example 1, subsoil substrate; T: Experimental group, light substrate; Y: leaf; S: callus; G: root; 1, 3, 5: cuttings at the first, third, and fifth growth and development stages; CKT: Samples at the first stage were identical, so the same group number was used.

[0146] Figure 12 The results of principal component analysis showed that the transcriptome results of the 10 groups of samples were highly reproducible and the same group could be clustered together. Figure 12 As shown in Figure B, the Pearson correlation coefficient within the 10 sample groups was as high as 0.98 or higher, indicating stable biological reproducibility within each group. The transcriptomes of callus and roots of the two substrate-based cuttings grown and developed under high temperature showed little difference in principal component 1 (PC1), but showed significant differences in principal component 2 (PC2). In particular, the callus and roots of the light-substrate-based cuttings at the fifth stage (T-5-S and T-5-G) accounted for a large proportion of PC2. Furthermore, the transcriptomes of leaves (CKT-Y, CK-5-Y, and T-5-Y) of the two substrate-based cuttings during development under high temperature showed significant differences in both PC1 and PC2.

[0147] Figure 13 This shows that the developmental differences between the comparative example (see groups ① to ⑨, and groups 11 to 13 in FIG. b ) and the embodiment (see group ⑩ in FIG. b ) under high temperature conditions are manifested as differences in gene expression.

[0148] Figure 14 GO enrichment analysis of differentially expressed genes in callus tissues at the third and fifth stages and root systems at the fifth stage between CK and T indicated that the stages where differentially expressed genes were generated between the embodiment and the comparative example were the third and fifth stages, which are the most critical stages for cutting growth and development under high temperature conditions.

[0149] Figure 15 The vertical axis represents the KEGG pathway. The horizontal axis represents the Rich factor. The Rich factor refers to the ratio of the number of differentially enriched genes (Sample number) to the number of annotated genes (Background number) in the pathway. The larger the Rich factor, the greater the degree of enrichment. The larger the point, the more differentially enriched genes there are in the pathway. P-value: significance test p-value, Corrected_P-value (qvalue): p-value after correction for multiple hypothesis testing. The smaller the qvalue, the redder the color of the point, and the more significant the enrichment. CK: Control Example 1, subsoil matrix; T: Experimental group, light matrix; Y: leaf; S: callus tissue; G: root system; 1, 3, 5: the first, third, and fifth growth and development stages of cutting seedlings.

[0150] Figure 16 The amino acid composition analysis diagrams (A, B), model evaluation diagrams (C, D) and OPLS-DA analysis diagrams (E, F) show that the amino acid composition and content in the examples are higher than those in the comparative examples, and the model is stable and highly reliable.

[0151] Figure 17 The plant hormone component analysis diagrams (A, B, C), model evaluation diagrams (D, E, F) and OPLS-DA analysis diagrams (G, H, I) show that the plant hormone components and contents in the examples are higher than those in the comparative examples, and the model is stable and highly reliable.

[0152] Comparative Example 2

[0153] The difference between this comparative example and Example 1 is that no vermiculite was added to the light substrate. Compared with the addition of vermiculite, the growth and development of the cuttings in Comparative Example 2 was significantly weaker than that in Example 1, and the water retention of the substrate was weaker than that in Example 1. Figure 21 It is the third stage effect, wherein Figure a is the third stage effect of Example 1, and Figure b is the third stage effect of Comparative Example 2.

[0154]

[0155] Comparative Example 3

[0156] This comparative example includes three groups of experiments, which differ from Example 1 in that: ① the ratio of peat formula soil, perlite and vermiculite is 3:1:2; ② the ratio of old matrix soil, perlite and sand is 3:1:1; ③ the ratio of subsoil, perlite and vermiculite is 3:1:1. ① Increasing the ratio of vermiculite enhances the water retention and moisture retention capacity of the matrix; ② replacing the soil matrix with the old matrix (after storage for a period of time), the soil nutrient capacity is lost to a certain extent; ③ replacing the soil with traditional soil. Figure 22 As shown, compared with Example 1, the root system development of Comparative Example 3 is weaker than that of Example 1 due to excessive soil moisture, and the tea fruit development phenomenon occurs due to excessive humidity and nutrient problems. Compared with the comparative patent, the present invention is suitable for tea tree cuttings in high temperature environments and can improve the survival adaptability of weakly resistant tea seedlings. Figure 23 All of them are comparisons of the survival rate of Baojing Golden Tea (weakly resistant variety) under high temperature environment.

[0157]

[0158] like Figure 24 As shown, Example 1 can greatly enhance the adaptability of weakly resistant tea varieties (such as Baojing Golden Tea No. 1) in high temperature environments. This adaptability is achieved by alleviating the stress effects brought about by high temperature environments through measures such as light substrate formulation and tube culture, thereby placing the tea seedlings in a state suitable for reproductive growth and greatly improving their growth potential.

[0159] Comparative Example 4

[0160] The difference between this comparative example and Example 1 is that the temperature is in the range of 26°C to 28°C and the humidity is in the range of 60% to 65% RH (compared with the patent). Compared with Comparative Example 4, the survival rate of Example 1 is lower than that of Comparative Example 4. However, combined with Comparative Example 3, it can be concluded that Example 1 can improve the adaptability of weakly resistant tea seedlings in high temperature environments, which is superior to Comparative Example 4.

[0161]

[0162] like Figure 25 , indicating that comparative example 4 is more suitable for tea seedling cuttings propagation at room temperature or non-stress temperature, and example 1 is more suitable for large-scale tea seedling propagation under high temperature environment in summer. Example 1 is the best choice for propagating weakly resistant tea varieties in July in summer.

Claims

1. A method for rapid propagation of weakly resistant tea seedlings, characterized in that: The steps include: S1. Preparation of seedling shed: The shading rate of the seedling shed is 60-80%; S2. Preparation of plug tray: Select light substrate as the seedling medium and put it into the plug tray; S3. Cutting preparation: Cut tea seedling cuttings with 2 / 3-1.5 mature leaves and 1 full axillary bud; S4, cuttings: insert the cuttings cut in S3 into the plug tray in S2; S5. Management after cutting: After cutting, water the substrate to make it fully moist, spray rooting agent and disinfectant, control the substrate humidity at 60% to 70%, and the air humidity at 75 to 85%; S6. Tea seedlings are transplanted.

2. The method for rapid breeding of weakly resistant tea seedlings according to claim 1, wherein S3 tea seedlings include one or more of Baojing Golden Tea No. 1, Baojing Golden Tea No. 2, Baojing Golden Tea No. 6, Baojing Golden Tea No. 7, Baojing Golden Tea No. 8, Baojing Golden Tea No. 18, Baojing Golden Tea No. 168, and Rucheng White Hair Tea.

3. The method for rapid breeding of weakly resistant tea seedlings according to claim 1, wherein When the air temperature of the seedling shed in S5 exceeded 30°C and the humidity was lower than 45% RH, the temperature inside the film was controlled to be higher than 33°C and the humidity to be lower than 60% RH, and the soil temperature was higher than 35°C and the humidity to be lower than 70% RH.

4. The method for rapid breeding of weakly resistant tea seedlings according to claim 3, wherein: Water once every 3 to 4 days.

5. The method for rapid breeding of weakly resistant tea seedlings according to claim 1, wherein: When the air temperature is 25-30℃ and the humidity is higher than 50% RH, the temperature inside the film should be lower than 30℃ and the humidity should be higher than 85% RH. The soil temperature should be 26-29℃ and the humidity should be higher than 80% RH.

6. The method for rapid breeding of weakly resistant tea seedlings according to claim 5, wherein: Water once every 6 to 7 days.

7. The method for rapid propagation of weakly resistant tea seedlings according to any one of claims 1 to 6, characterized in that: The light matrix in S2 includes the following components by volume: 6 to 12 parts of peat formula soil, 2 to 4 parts of perlite, and 2 to 4 parts of vermiculite.

8. The method for rapid breeding of weakly resistant tea seedlings according to any one of claims 1 to 6, characterized in that: In S5, after the leaves are dried, spray them with rooting agent and disinfectant.

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