A method for preserving and rapidly breeding tea germplasm resources by double-layer substrate and stereoscopic mode

Abstract

The present application relates to the field of plant germplasm resource preservation and propagation, and particularly relates to a tea tree germplasm resource preservation and rapid propagation method with double-layer substrate and a three-dimensional mode, comprising the following steps: laying double-layer substrate; hormone treatment overnight before mother plant cutting; selecting and cutting cuttings; synchronous treatment of rooting, browning prevention and nutrition increase; sand layer cutting; three-dimensional culture; watering before film covering; film covering; pre-rooting management and post-rooting management. The present application significantly reduces the land area and cost of tea tree resource preservation and propagation, and the land area of resource preservation can be reduced to 1 / 500, and the land area of resource propagation can be reduced to 1 / 10; the rooting speed of tea tree cuttings is significantly improved, and the rooting rate can reach 95% within 13 days; the operation cost is reduced; the survival rate of transplanting is significantly higher than that of the traditional method, and can reach 99%; the resource preservation and resource propagation mode can be switched according to needs, that is, the resources can be preserved and the resources can be rapidly expanded at any time.

Description

Technical Field

[0001] This invention relates to the field of plant germplasm resource preservation and propagation, specifically to a method for the preservation and rapid propagation of tea germplasm resources using a two-layer matrix and a three-dimensional model. Background Technology

[0002] Tea contains abundant pharmacologically active substances, such as theanine, catechins, anthocyanins, and caffeine, and has increasingly become a preferred health beverage, with the tea industry gradually becoming a pillar industry in mountainous regions. However, in recent years, factors such as over-harvesting, pests and diseases, natural disasters, and the decline and degradation of natural populations have severely damaged tea germplasm resources. Therefore, how to improve the preservation and propagation speed of tea germplasm resources is an urgent technical problem that needs to be solved.

[0003] Short-cut propagation of tea trees is currently the most common method of tea tree propagation. Rooting of tea tree cuttings is mainly due to the action of plant hormones and the directional flow of nutrients within the plant, with the primary purpose of promoting root development and propagation. Researchers separate roots, stems, and other vegetative organs from the mother plant and allow them to develop into complete and independent plants under suitable conditions. Using this method of propagation allows for the inheritance of superior varieties and has now become an important method for the propagation of excellent tea plants.

[0004] The rooting of tea tree cuttings is mainly affected by factors such as the cutting substrate, the timing of cutting, the temperature, exogenous hormones, and the tea variety. Although the efficiency of short-cut tea tree propagation has been continuously improved over the years, traditional methods of tea tree cultivation still have shortcomings. Currently, tea tree propagation by cuttings mainly involves field cuttings and seedling trays. While field cuttings are simple to operate, they are easily affected by external conditions, seedling production is seasonal, and root development is generally delayed and uneven due to soil temperature. Furthermore, field cuttings are difficult to move due to temperature changes and transplanting requirements. While seedling tray propagation can compensate for the shortcomings of field cuttings, it, like field cuttings, takes up considerable space, cannot fully utilize vertical space, and has higher production costs. In addition, existing tea tree propagation techniques have drawbacks such as slow rooting speed and underdeveloped root system. Rooting takes 2-3 months, resulting in an excessively long seedling cultivation cycle, low nursery rate, low seedling survival rate, and high seedling cost, which seriously hinders the efficiency of propagating superior tea tree germplasm resources.

[0005] Existing technologies for the preservation of tea germplasm resources all employ field preservation, which has disadvantages such as requiring a large area, being susceptible to pests and diseases, and being affected by natural disasters such as low and high temperatures, as well as high management costs.

[0006] In conclusion, current methods for preserving and propagating tea germplasm resources cannot meet production needs, and technological innovation is urgently needed. Summary of the Invention

[0007] The purpose of this invention is to provide a method for the preservation and propagation of tea germplasm resources using a two-layer matrix and a three-dimensional model, which shortens the rooting time of tea trees and reduces the cost of preserving and propagating tea germplasm resources.

[0008] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0009] A method for the preservation and rapid propagation of tea germplasm resources using a two-layer matrix and three-dimensional model includes the following steps:

[0010] (1) Laying a double layer of substrate: Mix peat moss and vermiculite in a volume ratio of 1:2-3, water until wet but not dripping, then spread it into a flower pot that is 25cm wide × 55cm long × 18cm high, compact it to a thickness of 4-5cm, and then lay a 2-4cm thick layer of river sand on top.

[0011] (2) Hormone treatment overnight before cuttings of mother plant: Avoid rainy days. Before 24 hours of preservation and propagation, spray the leaves with 2-3 mg / L sodium naphthaleneacetate until they are wet but not dripping.

[0012] (3) Selecting cuttings: Select healthy, disease-free, and superior mother plant branches, cut the branches obliquely into 2-4cm long cuttings, leave 1 leaf, and cut off half of the front end of the leaf; propagation can be carried out at any time, the best time is when the terminal bud of the new shoot just enters the dormant period;

[0013] (4) Treatment of cuttings to promote root growth, prevent browning and increase nutrition: Align the cut ends of the cuttings and place them in the treatment solution. The treatment solution consists of NAA at a concentration of 5-7 mg / L, sucrose at 1%-3%, polyvinylpyrrolidone at 5%-10%, and MS salt at 2.0-2.4 g / L. The soaking height is 2 / 3 of the length of the cuttings. After soaking for 1-2 hours, take them out and let them dry.

[0014] (5) Sand layer cuttings: Insert the dried cuttings obliquely into the river sand layer in the substrate along the cut direction, with the leaves facing forward. The insertion depth is 2 / 3 of the length of the cutting. 35-45 cuttings are planted per pot.

[0015] (6) Three-dimensional cultivation: Place the flower pots with the tea cuttings on a multi-layer cultivation rack. The flower pots on the same layer can be placed together. The height of each layer of the cultivation rack is 30-50cm, and 3-7 layers can be placed. The cultivation rack can be placed indoors or in a corridor.

[0016] (7) Watering before covering: Water after cuttings are planted. Water with a fine stream of water or spray with a sprayer until the leaves are evenly and thoroughly wet.

[0017] (8) Covering with film: After wetting the leaves, cover the top edge of the flowerpot with a thin film. After the roots have grown, remove the film.

[0018] (9) Management before rooting:

[0019] (c) Watering: Do not water before removing the film. If the river sand turns white, spray water until the river sand is thoroughly wet.

[0020] (d) Temperature: The temperature should be controlled at 25-28℃;

[0021] (10) Post-rooting management;

[0022] A: Management of germplasm resource preservation

[0023] 1) Watering: Water once a week to keep the substrate moist;

[0024] 2) Hormone treatment: Spray the leaves with 0.2-0.3 mg / L sodium abscisate once a week, just wet them;

[0025] 3) Light: Supplement with 100-300 lux of light, 8-12 hours per day;

[0026] 4) Temperature: The temperature should be controlled at 10-15℃;

[0027] B: Management of germplasm resource breeding

[0028] v. Watering: Water once a week to keep the substrate moist;

[0029] vi. Hormone treatment: Spray the leaves with 0.015-0.020 mg / L brassinolide once a week, just wet them; vii. Light: Supplement with 100-300 lux of light, 8-12 hours a day.

[0030] viii. Temperature: The temperature should be controlled between 25-28℃;

[0031] ix. Fertilization: After the cuttings sprout, apply 2-4g of compound fertilizer to each pot, grind it into powder, dissolve it in water, and apply it to the substrate.

[0032] Preservation and propagation can be carried out at any time, with the best time being when the terminal buds of new shoots have just entered dormancy. Preservation and propagation can be carried out indoors or in a corridor.

[0033] The main innovations of this invention are:

[0034] (1) This invention is the first to use double-layer substrate culture. The double-layer substrate is one of the key factors for the success of this invention. It solves the problem of the contradiction between the survival rate of cuttings, the rooting rate of cuttings, the rooting speed and the growth rate of tea seedlings in nutrient pot seedling cultivation. It achieves the goal of having high cutting survival rate, rooting rate and rooting speed, and rapid growth of tea seedlings in the later stage.

[0035] (2) This invention is the first to adopt a three-dimensional culture mode, which solves the problem of large area required for germplasm resource preservation and propagation.

[0036] (3) This invention is the first to adopt a combination of mother plant and cutting. The combination of mother plant treatment and pruning treatment is also a key factor in the success of this invention. The two treatments work together to solve the problem of tea tree cutting difficulties.

[0037] (4) This invention is the first to add PVP to the cutting treatment solution, which solves the problem of browning of young cuttings and improves the rooting rate; MS and sucrose are added for the first time to increase the nutrition of cuttings and improve the rooting speed of cuttings; the optimal formula of each component of the treatment solution is determined, which further improves the rooting speed of cuttings.

[0038] (5) This invention has for the first time determined the optimal temperature for tea seedling dormancy and growth, as well as the optimal hormone and optimal concentration, realizing the artificial switching of tea seedling dormancy and dormancy release, realizing low-cost indoor preservation of tea germplasm resources, and also realizing rapid propagation of tea seedlings.

[0039] (6) This invention changes the traditional method of leaving whole leaves for tea tree cuttings. Instead, it removes some leaves, which promotes rooting of the cuttings and reduces the space occupied by a single cutting, thus improving space utilization efficiency.

[0040] (7) Through the above-mentioned combination of technologies and parameter optimization, the present invention solves the problem of seasonal and site limitations in the preservation and propagation of tea germplasm resources, and realizes that tea germplasm resources can be preserved or propagated at any time.

[0041] In summary, this invention significantly reduces the land area and cost required for tea tree resource preservation and propagation. The land area required for resource preservation can be reduced to 1 / 500, and the land area required for resource propagation can be reduced to 1 / 10. It significantly improves the rooting speed of tea tree cuttings, with a rooting rate of 95% within 13 days, compared to nearly 3 months using traditional methods. It eliminates the need for weeding, pest control, and other management, reducing operating costs. The transplant survival rate is significantly higher than traditional methods, reaching 99%. It allows for switching between resource preservation and propagation modes as needed, enabling both resource preservation and rapid propagation at any time. Attached Figure Description

[0042] Figure 1 Three-dimensional preservation of tea germplasm resources (left) and breeding model (right);

[0043] Figure 2 Rooting culture of tea cuttings (left) and rooting status on the 10th day (right);

[0044] Figure 3 This shows the growth of the tea seedlings on day 10.

[0045] Figure 4 For the later propagation of tea tree germplasm resources (left) and the preservation of tea tree germplasm resources (right). Detailed Implementation

[0046] The present invention will be further described below with reference to embodiments, but the present invention is not limited to any one of these embodiments or similar examples.

[0047] Example 1

[0048] Taoyuan Daye is a distinctive tea germplasm resource of Hunan Province. Its leaves are rich in nutrients and have strong fermentation capabilities. Black tea made from this resource possesses excellent qualities such as a glossy black color, a bright red liquor, and a rich, aromatic flavor. However, this resource has poor reproductive capacity and a very low seed set rate, thus facing the risk of extinction. Therefore, the effective preservation and propagation of Taoyuan Daye is urgently needed. This case study demonstrates the preservation of Taoyuan Daye tea, such as... Figure 1 As shown, first, peat moss and vermiculite are exposed to strong sunlight for 2 days to kill insects and harmful bacteria. Then, 2L of peat moss and 6L of vermiculite are mixed and watered until moist but not dripping. This mixture is then spread at the bottom of two rectangular flowerpots, compacted to a thickness of about 5cm, and then topped with a layer of river sand about 3cm thick. A 100mL solution of 3mg / L NAA is prepared and sprayed onto the leaves of the *Prunus persica* large-leaf mother plant. 24 hours later, healthy, disease-free branches are selected from the NAA-sprayed *Prunus persica* large-leaf mother plant, and the branches are obliquely cut to a length of about 3cm. Cuttings were prepared, each with one leaf, and the tip of the leaf was cut off in half, resulting in 90 cuttings in total. A 10mL solution of 5mg / L NAA + 3% sucrose + 5% PVP + 2.0g / L MS saline was prepared. The lower cut ends of the cuttings were immersed in this solution for 1 hour, then removed and air-dried. The cuttings were then inserted obliquely into the river sand layer of the substrate, with the leaves facing forward, to a depth of 2 / 3 of the cutting length, with 45 cuttings per pot. The cuttings were then placed side-by-side on a 5-layer cultivation rack, sprayed with water until the leaves were evenly moistened, covered with a thin film, and incubated naturally at room temperature indoors. The rooting and cultivation of the tea cuttings were as follows. Figure 2 As shown on the left, the tea cuttings have rooted on the 10th day. Figure 2 As shown on the right. On day 15, 89 cuttings survived, achieving a 100% rooting rate. Afterward, the film was removed, and 20 mL of 0.2 mg / L ABA was sprayed once. The cuttings were then naturally cultured indoors at 13℃. Two days later, the maximum length of the new shoots was <3 cm. Subsequently, 20 mL of 0.015 mg / L BR was sprayed once, and the cuttings were moved to 28℃ with 300 lux light for 9 hours daily. 3 g of compound fertilizer (ground into powder, dissolved in water) was applied to each pot and drenched in the substrate. Watering was done once a week. The growth of the tea seedlings on day 10 is as follows. Figure 3 As shown, the average length of new shoots was 13.6 cm after 60 days, and the survival rate after transplanting to the field was 100%. The propagation of tea germplasm resources is as follows... Figure 4 As shown on the left, the preservation of tea germplasm resources is as follows: Figure 4 As shown on the right.

[0049] Example 2

[0050] Golden Tea is the most popular premium green tea in Hunan in recent years, and tea seedlings are in high demand. Therefore, this example demonstrates the propagation of the Baojing Golden Tea No. 1 variety. First, peat moss and vermiculite were exposed to strong sunlight for two days to kill insects and harmful bacteria. Then, 40L of peat moss and 120L of vermiculite were mixed, watered until moist but not dripping, and then spread at the bottom of 36 rectangular flowerpots, compacting them to a thickness of about 5cm. A layer of river sand about 3cm thick was then laid on top. 1L of... A 2.5 mg / L NAA aqueous solution was sprayed onto the leaves of the Baojing Golden Tea No. 1 mother plant. 24 hours later, healthy, disease-free, green-stemmed branches were selected from the NAA-sprayed Baojing Golden Tea No. 1 mother plants. These branches were obliquely cut into cuttings approximately 3 cm long, retaining one leaf and removing half of the leaf tip, resulting in 1440 cuttings. A 500 mL solution of 6 mg / L NAA + 1% sucrose + 10% PVP + 2.2 g / L saline was prepared. The lower cut ends of the cuttings were immersed in this solution for 1 hour, then removed and dried. The cuttings were then obliquely inserted into the river sand layer of the substrate, with the leaves facing forward, to a depth of 2 / 3 of the cutting length. Each pot was used for propagation. Forty cuttings were planted, totaling 36 pots. The pots were placed on cultivation racks, with 5 layers on each rack and 7 pots on each layer. The leaves were sprayed with water until evenly moistened, then covered with a thin film and cultivated indoors at room temperature. On the 11th day, 1440 cuttings survived, and 50 cuttings were randomly selected for inspection, with a rooting rate of 98%. After 15 days, the film was removed, and 0.015 mg / L of BR was sprayed once, just enough to moisten the leaves. The cultivation temperature was adjusted to 26℃, and 100 lux of light was provided for 10 hours daily. 3g of compound fertilizer was applied to each pot, ground into powder, dissolved in water, and applied to the substrate. Watering was done once a week. After 40 days, the average length of the new shoots was 15.3 cm. After 60 days, the cuttings were transplanted to the field with a 100% survival rate.

[0051] Example 3

[0052] Fuding Da Bai tea is the most widely cultivated tea variety in my country. This example demonstrates the propagation of the Fuding Da Bai tea variety. First, peat moss and vermiculite were exposed to strong sunlight for two days to eliminate insects and harmful bacteria. Then, 60L of peat moss and 180L of vermiculite were mixed, watered until moist but not dripping, and then spread at the bottom of 60 rectangular flowerpots, compacting them to a thickness of about 5cm. A layer of river sand about 3cm thick was then laid on top. 1L of... A 2 mg / L NAA aqueous solution was sprayed onto the leaves of Fuding Da Bai tea mother plants. 24 hours later, healthy, disease-free branches with green bark were selected from the NAA-sprayed mother plants. These branches were obliquely cut into 3 cm long cuttings, each retaining one leaf, with the tip of the leaf cut off in half. A total of 2100 cuttings were prepared. A 700 mL solution of 7 mg / L NAA + 1% sucrose + 7% PVP + 2.5 g / L saline was prepared. The lower cut ends of the cuttings were immersed in this solution for 1 hour. After soaking, the cuttings were removed, dried, and obliquely inserted into the river sand layer of the substrate, with the leaves facing forward, to a depth of 2 / 3 of the cutting length. Three cuttings were planted per pot. Five plants were planted, resulting in 60 pots. The pots were placed on cultivation racks, with five layers on each rack and seven pots on each layer. The leaves were sprayed with water until evenly moistened, then covered with a thin film and cultivated naturally at room temperature indoors. On the 13th day, 2097 plants survived, and 50 plants were randomly selected for inspection, resulting in a rooting rate of 99%. After 15 days, the film was removed, and 0.015 mg / L of BR was sprayed once, just enough to moisten the leaves. The cultivation temperature was adjusted to 25℃, and 100 lux of light was provided for 12 hours daily. 3g of compound fertilizer was applied to each pot, ground into powder, dissolved in water, and applied to the substrate. Watering was done once a week. After 50 days, the average length of the new shoots was 11.7 cm. After 60 days, the plants were transplanted to the field with a survival rate of 99%.

[0053] Example 4

[0054] Jianghua bitter tea belongs to the small arbor type with large leaves. It represents a transitional type in the evolution of tea tree germplasm resources from the original arbor type to the shrub type. Originating in the Nanling Mountains, it is one of the four major characteristic local tea tree groups in Hunan Province. It is an ancient and precious tea tree group variety, with the largest and most concentrated distribution in Jianghua County. Jianghua bitter tea has a high content of tea polyphenols and contains bitter theophylline, resulting in significant health benefits. It is suitable for making fermented teas such as red and black tea. The resulting black tea has a dark, oily appearance, a bright golden liquor, a fresh and full-bodied taste, a floral and honey-sweet aroma, a long-lasting aftertaste, and is highly resistant to multiple infusions, making it of excellent quality. This example demonstrates the propagation of Jianghua bitter tea. First, peat moss and vermiculite were exposed to strong sunlight for two days to eliminate insects and harmful bacteria. Then, 30L of peat moss and 60L of vermiculite were mixed thoroughly, watered until moist but not dripping, and then spread at the bottom of 30 rectangular flowerpots, compacting them to a thickness of approximately 4cm. A 3cm layer of river sand was then laid on top. A 500L... A 3 mg / L NAA aqueous solution was sprayed onto the leaves of the mother plants of Jianghua bitter tea. 24 hours later, healthy, disease-free branches with green bark were selected from the NAA-sprayed mother plants. These branches were obliquely cut into cuttings approximately 4 cm long, retaining one leaf and removing half of the leaf tip, resulting in 1200 cuttings. A 300 mL solution of 6 mg / L NAA + 1% sucrose + 7% PVP + 2.2 g / L saline solution was prepared. The lower cut ends of the cuttings were immersed in this solution for 2 hours, then removed and dried. The cuttings were then obliquely inserted into the river sand layer of the substrate, with the leaves facing forward, to a depth of approximately 3 cm. 40 cuttings were planted per pot. A total of 40 pots were prepared. The pots were placed on cultivation racks, with 5 layers on each rack and 7 pots on each layer. Water was sprayed until the leaves were evenly moistened, and a thin film was covered. The plants were then naturally cultivated indoors at room temperature. On the 10th day, 1193 plants survived, and 50 plants were randomly selected for inspection, resulting in a rooting rate of 99%. After 14 days, the film was removed, and 0.02 mg / L of BR was sprayed once, just enough to moisten the leaves. The cultivation temperature was adjusted to 27℃, and 300 lux of light was provided for 8 hours daily. 2.5g of compound fertilizer was applied to each pot, ground into powder, dissolved in water, and applied to the substrate. Watering was done once a week. After 50 days, the average length of the new shoots was 13.5 cm. After 50 days, the plants were transplanted to the field with a survival rate of 99%.

Claims

1. A method for the preservation and rapid propagation of tea germplasm resources using a two-layer matrix and three-dimensional model, characterized in that, Includes the following steps: Lay a double layer of substrate: Mix peat moss and vermiculite, water until moist but not dripping, then spread it in the flower pot, compact it to a thickness of 4-5cm, and then spread a 2-4cm thick layer of river sand on top. Hormone treatment overnight before mother plant cuttings: Avoid rainy days, spray the leaves with sodium naphthaleneacetate 24 hours before preservation and propagation, until wet but not dripping; Selecting cuttings: Select healthy, disease-free branches from the mother plant. Cut the branches at an angle to make cuttings 2-4 cm long, leaving one leaf and cutting off half of the leaf tip. Simultaneous treatment of cuttings to promote root growth, prevent browning, and increase nutrition: Align the lower cut ends of the cuttings, place them in the treatment solution, and immerse them to 1 / 2-2 / 3 of the length of the cuttings. After soaking for 1-2 hours, take them out and let them dry. Sand cuttings: Insert the dried cuttings obliquely into the river sand layer of the substrate along the cut direction, with the leaves facing forward. The insertion depth is 1 / 2 to 2 / 3 of the length of the cutting. 35-45 cuttings are planted per pot. Vertical cultivation: Place the flowerpots with tea cuttings on a multi-layer cultivation rack, with the flowerpots on the same layer placed close together; Watering before mulching: Water after cuttings are taken, until the leaves are evenly and thoroughly wet; Covering with film: After wetting the leaves, cover the pot with a thin film. Remove the film after the roots have grown. Pre-rooting management: Watering: Do not water before removing the film. If the river sand turns white, spray water until the river sand is thoroughly wet. Temperature: The temperature should be controlled between 25-28℃; Post-rooting management; A: Management of germplasm resource preservation Watering: Water weekly to keep the substrate moist; Hormone treatment: Spray the leaves with 0.2-0.3 mg / L sodium abscisate once a week, just wet them; Light: Supplement with 100-300 lux of light for 8-12 hours per day; Temperature: The temperature should be controlled at 10-15℃; B: Management of germplasm resource breeding Watering: Water weekly to keep the substrate moist; Hormone treatment: Spray the leaves with 0.015-0.020 mg / L brassinolide once a week, just wet them; Light: Supplement with 100-300 lux of light, for 8-12 hours per day; Temperature: The temperature should be controlled between 25-28℃; Fertilization: After the cuttings sprout, apply 2-4 g of compound fertilizer to each pot, dissolving the compound fertilizer in water and applying it to the substrate. Peat moss and vermiculite are mixed in a volume ratio of 1:2-3; In step (2), the concentration of sodium naphthaleneacetate is 2-3 mg / L; In step (4), the treatment solution consists of NAA at a concentration of 5-7 mg / L, sucrose at 1%-3%, polyvinylpyrrolidone at 5%-10%, and MS salt at 2.0-2.4 g / L. Watering during the later stages of rooting should be done 1-3 times per week.

2. The method for preserving and rapidly propagating tea germplasm resources using a double-layer matrix and three-dimensional model according to claim 1, characterized in that, The best time to preserve the propagation time is when the terminal buds of new shoots have just entered dormancy.

3. The method for preserving and rapidly propagating tea germplasm resources using a double-layer matrix and three-dimensional model according to claim 1, characterized in that, In step (6), the height of each layer of the multi-layer culture rack is 30-50 cm.

4. The method for preserving and rapidly propagating tea germplasm resources using a double-layer matrix and three-dimensional model according to claim 1, characterized in that, In step (6), the flowerpots with the tea cuttings can be placed in 3-7 layers.

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