Method for cultivating tissue culture seedlings of picrasma quassioides

Abstract

The invention belongs to the technical field of picrasma quassioides cultivation, and provides a picrasma quassioides tissue culture seedling cultivation method. The culture method comprises the following steps: (1) culturing rooting tissue culture seedlings; (2) seedling hardening: performing staged seedling hardening on the rooting tissue culture seedlings; (3) transplanting: preparing a transplanting substrate; disinfecting a transplanting matrix, treating the rooting tissue culture seedlings, and transplanting the treated rooting tissue culture seedlings to the transplanting matrix. According to the cultivation method, the seedling hardening and transplanting stages of picrasma quassioides tissue culture seedling production are improved, and the seedling hardening survival rate and the transplanting survival rate of the picrasma quassioides tissue culture seedlings are further increased, so that the production efficiency and the overall survival rate are increased, and industrial development of the picrasma quassioides tissue culture seedlings is promoted.

Description

Technical Field

[0001] This invention belongs to the field of *Palatinate* cultivation technology, specifically, it relates to a method for cultivating *Palatinate* tissue culture seedlings. Background Technology

[0002] Picrasma quassioides (D. Don) Benn., a deciduous tree belonging to the genus Picrasma in the family Simaroubaceae, is used as a traditional Chinese medicine. Its dried branches and leaves contain active ingredients such as alkaloids, triterpenes, and bitter principles, possessing properties that clear heat and detoxify, and dispel dampness. It has significant application value in the field of traditional Chinese medicine production. With the standardized development of the traditional Chinese medicine industry, the market demand for high-quality Picrasma quassioides raw materials continues to grow. However, traditional wild Picrasma quassioides resources are facing depletion due to over-harvesting, making large-scale artificial cultivation a key way to meet market demand.

[0003] Tissue culture technology, as a core technology for rapid plant propagation, provides an effective solution for the mass production of high-quality *Mallotus quassula* seedlings. The Hubei Provincial Forestry Society's T / HBSF 017-2024 "Technical Regulations for the Production of *Mallotus quassula* Tissue Culture Seedlings," as the first domestic group standard specifically for *Mallotus quassula* tissue culture seedling production, clarifies the basic technical requirements for key stages such as explant collection, tissue culture, hardening-off, and transplanting. This lays the technical framework for the industrialization of *Mallotus quassula* tissue culture seedlings and standardizes industry production practices.

[0004] Patent CN116391618A discloses a method for rapid propagation of medicinal *Mallotus idaeus* through tissue culture. The method involves inoculating sterilized explants into a primary induction medium to induce initial bud formation. These initial buds are then inoculated into a secondary proliferation medium to obtain secondary bud clusters. Single buds are then cut and inoculated into a rooting medium to induce rooting. After cultivation, rooted seedlings are obtained, which are then hardened off and transplanted to produce ready-to-grow seedlings. This invention achieves highly efficient induction, proliferation, and rooting, with high bud induction rate, proliferation coefficient, and rooting rate, resulting in high-quality seedlings and a short cultivation cycle, thereby reducing production costs in the industrial-scale cultivation of *Mallotus idaeus* seedlings through tissue culture.

[0005] However, in actual large-scale production, the tissue culture stage is relatively easy to control, while the survival rate in the seedling hardening and transplanting stages is often more prone to problems.

[0006] The T / HBSF 017-2024 "Technical Regulations for the Production of Tissue Culture Seedlings of *Mallotus oxypetalum*" specifies the following hardening-off process: "Select complete plants with a height ≥ 5 cm and at least 3 cm in length, and ≥ 3 adventitious roots. Place them in an environment with 6000 Lux light and 26℃ for 6 days of hardening-off; then loosen the bottle cap for 2 days." However, the standard uses a one-time bottle cap removal method for hardening-off, which makes the tissue culture seedlings prone to wilting due to the sudden environmental change, significantly impacting the survival rate.

[0007] Furthermore, the transplanting procedure in T / HBSF 017-2024, "Technical Regulations for the Production of Tissue Culture Seedlings of *Mallotus quassula*", includes the following: "Preparation of transplanting substrate: Mix nutrient soil and perlite evenly at a volume ratio of 7:3. One to two days before transplanting, thoroughly disinfect the substrate by watering it with a 500-800 times dilution of 75% mancozeb or 70% thiophanate-methyl. Transplanting of tissue culture seedlings: Remove the tissue culture seedlings, wash the culture medium from the roots with clean water, and place them in a tray with a small amount of water for transplanting. When transplanting, first fill a container with 6-8 cm in diameter and 10-15 cm in height halfway with substrate, then place the tissue culture seedling in the container, ensuring the roots are spread out, and add substrate to 1 / 3 to 1 / 2 of the seedling's height; then gently press the substrate, water thoroughly, and cover with a transparent plastic film or plastic cup to retain moisture." The transplanting method described in the standard still has significant room for improvement in terms of survival rate and average plant height growth rate one month after transplanting. Summary of the Invention

[0008] To address the aforementioned shortcomings in the existing technology, the present invention aims to provide a method for cultivating *Mallotus spp.* tissue culture seedlings. This method improves the hardening and transplanting stages of *Mallotus spp.* tissue culture seedling production, aiming to further increase the survival rate of hardening and transplanting seedlings, thereby improving production efficiency and overall survival rate, and promoting the industrialization of *Mallotus spp.* tissue culture seedlings.

[0009] To achieve the above objectives, the solution adopted by the present invention is as follows: A method for cultivating *Melia azedarach* tissue culture seedlings includes: (1) Cultivation of rooted tissue culture seedlings: pre-treatment, primary culture, proliferation culture, and rooting culture are performed on *Melia azedarach* explants in sequence to obtain robust rooted tissue culture seedlings; (2) Hardening off: complete plants with a height ≥5 cm and more than 3 cm long adventitious roots ≥3 are selected. On days 1-3: the rooted tissue culture seedlings and culture bottles are moved to the hardening off shed. One to two small holes are opened in the bottle cap. The temperature of the hardening off shed is set to 23-25℃, the humidity is set to 70%-80%, and the light intensity is set to 1500-2000 lx; ​​on days 4-6: the bottle cap is opened halfway, and the temperature of the hardening off shed is kept at 23-25℃, the humidity is set to 65%-75%, and the light intensity is set to 1500-2000 lx. 2000~2500 lx; ​​on days 7-10: completely open the bottle cap, set the temperature of the hardening shed to 22~24℃, the humidity to 60%~70%, and the light intensity to 2500~3000 lx; ​​(3) Transplanting: Transplanting substrate preparation: by weight, the transplanting substrate includes 4-6 parts of nutrient soil, 2-4 parts of perlite and 1-3 parts of vermiculite; disinfect the transplanting substrate, treat the rooted tissue culture seedlings, and transplant the treated rooted tissue culture seedlings into the transplanting substrate.

[0010] Furthermore, in a preferred embodiment of the present invention, in step (2), simultaneously, starting from the 4th day, a 0.1% potassium dihydrogen phosphate solution is sprayed onto the leaves of the rooted tissue culture seedlings every 2 days, with each spray amount sufficient to moisten the leaves but not drip water.

[0011] In this application, spraying potassium dihydrogen phosphate using the above method can provide phosphorus and potassium nutrition to tissue culture seedlings, enhance leaf photosynthesis, promote robust seedling growth, improve stress resistance, and lay a good foundation for subsequent transplanting.

[0012] Furthermore, in a preferred embodiment of the present invention, in step (3), the transplanting substrate further includes, by weight, 0.1-0.3 parts of well-rotted sheep manure.

[0013] Furthermore, in a preferred embodiment of the present invention, in step (3), the transplanting substrate further includes 0.04-0.06 parts of slow-release fertilizer by weight.

[0014] Furthermore, in a preferred embodiment of the present invention, in step (3), the raw materials of the slow-release fertilizer include, by weight, 100-110 parts of urea-formaldehyde, 32-36 parts of superphosphate, 24-28 parts of potassium sulfate, 10-12 parts of potassium chloride, 8-13 parts of urea, and 4-7 parts of zinc sulfate.

[0015] Furthermore, in a preferred embodiment of the present invention, the components by weight are: 105 parts urea-formaldehyde, 33 parts superphosphate, 26 parts potassium sulfate, 11 parts potassium chloride, 12 parts urea, and 6 parts zinc sulfate.

[0016] Furthermore, in a preferred embodiment of the present invention, in step (3), the preparation of the transplanting substrate includes: premixing slow-release fertilizer with perlite and vermiculite for 3 minutes, and then adding nutrient soil and well-rotted sheep manure and mixing.

[0017] Furthermore, in a preferred embodiment of the present invention, step (3) of disinfecting the transplanting substrate further includes: 12 days before transplanting: the transplanting substrate is piled into a trapezoidal pile with a height of 1.0~1.2m and a width of 1.5~2.0m, and a transparent plastic film is covered on the surface of the trapezoidal pile. The substrate is then disinfected using solar energy for 10 days; 2 days before transplanting: the solar-disinfected transplanting substrate is spread out, cooled to room temperature, and then thoroughly watered with a 500-fold dilution of 75% mancozeb.

[0018] In this application, the above-mentioned solar disinfection can effectively kill pathogens, insect eggs and weed seeds in the transplanting substrate. Then, chemical disinfection is used to ensure thorough disinfection of the substrate and reduce the risk of disease occurrence after transplanting.

[0019] Furthermore, in a preferred embodiment of the present invention, step (3) of treating the rooted tissue culture seedlings includes: taking out the rooted tissue culture seedlings, washing away the culture medium from the roots with clean water, immersing the roots of the rooted tissue culture seedlings in a 500-fold naphthaleneacetic acid solution for 10-15 minutes, taking them out, drying the moisture on the surface of the roots, and then transplanting them.

[0020] In this application, the above treatment method can promote root growth, improve root vitality, increase the number of new root sprouts, and improve the transplant survival rate.

[0021] Further, in a preferred embodiment of the present invention, in step (3), transplanting includes: first filling a container with a diameter of 6cm-8cm and a height of 10cm-15cm halfway with transplanting substrate, then placing the rooted tissue culture seedling in, keeping the roots spread out, and adding transplanting substrate to 1 / 3 to 1 / 2 of the seedling height; then gently pressing the transplanting substrate, watering thoroughly, covering the surface of the transplanting substrate with a layer of rice husks 1-2cm thick, and then covering it with a transparent plastic film or plastic cup to keep it moist.

[0022] In this application, the above-mentioned treatment method can reduce the evaporation of water from the transplanting substrate, keep the transplanting substrate moist, inhibit weed growth, regulate substrate temperature, and create a good environment for root growth.

[0023] The beneficial effects of the method for cultivating *Palatinate* tissue culture seedlings provided by this invention are: (1) The method for cultivating *Paspalum notatum* tissue culture seedlings provided by this invention adopts a three-step dynamic gradient hardening method during the hardening stage. By gradually expanding the bottle cap opening and controlling the temperature, humidity and light in stages, the transition of seedlings from a sterile and humid environment to the external environment during the hardening process is smoother, thereby effectively avoiding problems such as leaf wilting and dehydration. Experimental verification shows that the survival rate of hardened seedlings can be increased from about 90% in the standard method to more than 98.8%, laying a solid foundation for subsequent transplanting.

[0024] (2) The method for cultivating *Paspalum notatum* tissue culture seedlings provided by this invention uses a substrate composed of nutrient soil, perlite, vermiculite, well-rotted sheep manure, and slow-release fertilizer. Vermiculite has good water retention, air permeability, and fertilizer retention. When mixed with nutrient soil and perlite, it further improves the physical properties of the substrate, enhances its water and fertilizer retention capacity, increases air permeability, and reduces root rot. Well-rotted sheep manure improves the substrate's aggregate structure and increases organic matter content; slow-release fertilizer continuously provides nutrients to the seedlings, preventing slow growth caused by nutrient deficiency in the early stages after transplanting.

[0025] The components of the above-mentioned transplanting substrate work synergistically. Through experiments, it has been verified that the improved transplanting substrate can increase the seedling survival rate from about 95% in the standard method to more than 99%. Furthermore, the improved transplanting substrate can increase the average plant height growth rate of seedlings within one month after transplanting by more than 26%, which greatly improves the seedling survival rate and growth rate. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0027] The following is a detailed description of a method for cultivating tissue culture seedlings of *Palatinate* provided by an embodiment of the present invention. It should be noted that for any aspects of the cultivation method not mentioned in this application, please refer to the relevant content of T / HBSF 017-2024 "Technical Regulations for the Production of *Palatinate* Tissue Culture Seedlings".

[0028] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0029] Example 1 This embodiment provides a method for cultivating tissue culture seedlings of *Melia azedarach*, including: (1) Cultivation of rooted tissue culture seedlings: The explants of *Mallotus spp.* were pretreated, primary cultured, proliferated, and rooted in sequence to obtain robust rooted tissue culture seedlings; (2) Hardening off seedlings: Select complete plants with a height of ≥5 cm and ≥3 adventitious roots of ≥3 cm in length. On days 1-3: move the rooted tissue culture seedlings along with the culture bottles to the hardening off shed. Open two small holes in the cap of the culture bottle. Set the temperature of the hardening off shed to 23℃, the humidity to 80%, and the light intensity to 1500 lx. On days 4-6: open the cap of the culture bottle halfway. Keep the temperature of the hardening off shed at 23℃, the humidity at 65%, and the light intensity at 2500 lx. On days 7-10: completely remove the cap of the culture bottle. Set the temperature of the hardening off shed to 22℃, the humidity at 70%, and the light intensity at 2500 lx. (3) Transplanting: Transplanting substrate preparation: By weight, the transplanting substrate includes 5 parts nutrient soil, 3 parts perlite and 2 parts vermiculite; disinfect the transplanting substrate, treat the rooted tissue culture seedlings, and transplant the treated rooted tissue culture seedlings into the transplanting substrate.

[0030] Example 2 This embodiment provides a method for cultivating tissue culture seedlings of *Melia azedarach*, including: (1) Cultivation of rooted tissue culture seedlings: The explants of *Mallotus spp.* were pretreated, primary cultured, proliferated, and rooted in sequence to obtain robust rooted tissue culture seedlings; (2) Hardening off seedlings: Select complete plants with a height of ≥5 cm and ≥3 adventitious roots of ≥3 cm in length. On days 1-3: move the rooted tissue culture seedlings along with the culture bottles to the hardening off shed. Open a small hole in the cap of the culture bottle. Set the temperature of the hardening off shed to 25℃, the humidity to 70%, and the light intensity to 2000 lx. On days 4-6: open the cap of the culture bottle halfway. Keep the temperature of the hardening off shed at 25℃, the humidity at 75%, and the light intensity at 2000 lx. On days 7-10: completely remove the cap of the culture bottle. Set the temperature of the hardening off shed to 24℃, the humidity at 60%, and the light intensity at 3000 lx. (3) Transplanting: Transplanting substrate preparation: By weight, the transplanting substrate includes 5 parts nutrient soil, 3 parts perlite and 2 parts vermiculite; disinfect the transplanting substrate, treat the rooted tissue culture seedlings, and transplant the treated rooted tissue culture seedlings into the transplanting substrate.

[0031] Example 3 This embodiment provides a method for cultivating tissue culture seedlings of *Melia azedarach*, including: (1) Cultivation of rooted tissue culture seedlings: The explants of *Mallotus spp.* were pretreated, primary cultured, proliferated, and rooted in sequence to obtain robust rooted tissue culture seedlings; (2) Hardening off seedlings: Select complete plants with a height of ≥5 cm and ≥3 adventitious roots of ≥3 cm in length. On days 1-3: move the rooted tissue culture seedlings along with the culture bottles to the hardening off shed. Open two small holes in the cap of the culture bottle. Set the temperature of the hardening off shed to 24℃, the humidity to 75%, and the light intensity to 1800 lx. On days 4-6: open the cap of the culture bottle halfway. Keep the temperature of the hardening off shed at 24℃, the humidity at 70%, and the light intensity at 2300 lx. On days 7-10: completely remove the cap of the culture bottle. Set the temperature of the hardening off shed to 23℃, the humidity at 65%, and the light intensity at 2800 lx. (3) Transplanting: Transplanting substrate preparation: By weight, the transplanting substrate includes 5 parts nutrient soil, 3 parts perlite and 2 parts vermiculite; disinfect the transplanting substrate, treat the rooted tissue culture seedlings, and transplant the treated rooted tissue culture seedlings into the transplanting substrate.

[0032] Example 4 This embodiment provides a method for cultivating tissue culture seedlings of *Melia azedarach*, including: (1) Cultivation of rooted tissue culture seedlings: The explants of *Mallotus spp.* were pretreated, primary cultured, proliferated, and rooted in sequence to obtain robust rooted tissue culture seedlings; (2) Hardening off seedlings: Select complete plants with a height of ≥5 cm and ≥3 adventitious roots of ≥3 cm in length. On days 1-3: move the rooted tissue culture seedlings along with the culture bottles to the hardening off shed. Open two small holes in the cap of the culture bottle. Set the temperature of the hardening off shed to 24℃, the humidity to 75%, and the light intensity to 1800 lx. On days 4-6: open the cap of the culture bottle halfway. Keep the temperature of the hardening off shed at 24℃, the humidity at 70%, and the light intensity at 2300 lx. On days 7-10: completely remove the cap of the culture bottle. Set the temperature of the hardening off shed to 23℃, the humidity at 65%, and the light intensity at 2800 lx. (3) Transplanting: Transplanting substrate preparation: By weight, the transplanting substrate includes 4 parts nutrient soil, 4 parts perlite and 1 part vermiculite; disinfect the transplanting substrate, treat the rooted tissue culture seedlings, and transplant the treated rooted tissue culture seedlings into the transplanting substrate.

[0033] Example 5 This embodiment provides a method for cultivating tissue culture seedlings of *Melia azedarach*, including: (1) Cultivation of rooted tissue culture seedlings: The explants of *Mallotus spp.* were pretreated, primary cultured, proliferated, and rooted in sequence to obtain robust rooted tissue culture seedlings; (2) Hardening off seedlings: Select complete plants with a height of ≥5 cm and ≥3 adventitious roots of ≥3 cm in length. On days 1-3: move the rooted tissue culture seedlings along with the culture bottles to the hardening off shed. Open two small holes in the cap of the culture bottle. Set the temperature of the hardening off shed to 24℃, the humidity to 75%, and the light intensity to 1800 lx. On days 4-6: open the cap of the culture bottle halfway. Keep the temperature of the hardening off shed at 24℃, the humidity at 70%, and the light intensity at 2300 lx. On days 7-10: completely remove the cap of the culture bottle. Set the temperature of the hardening off shed to 23℃, the humidity at 65%, and the light intensity at 2800 lx. (3) Transplanting: Transplanting substrate preparation: By weight, the transplanting substrate includes 6 parts nutrient soil, 2 parts perlite and 3 parts vermiculite; disinfect the transplanting substrate, treat the rooted tissue culture seedlings, and transplant the treated rooted tissue culture seedlings into the transplanting substrate.

[0034] Example 6 This embodiment provides a method for cultivating tissue culture seedlings of *Melia azedarach*, including: (1) Cultivation of rooted tissue culture seedlings: The explants of *Mallotus spp.* were pretreated, primary cultured, proliferated, and rooted in sequence to obtain robust rooted tissue culture seedlings; (2) Hardening off seedlings: Select complete plants with a height of ≥5 cm and ≥3 adventitious roots of ≥3 cm in length. On days 1-3: move the rooted tissue culture seedlings along with the culture bottles to the hardening off shed. Open two small holes in the cap of the culture bottle. Set the temperature of the hardening off shed to 24℃, the humidity to 75%, and the light intensity to 1800 lx. On days 4-6: open the cap of the culture bottle halfway. Keep the temperature of the hardening off shed at 24℃, the humidity at 70%, and the light intensity at 2300 lx. On days 7-10: completely remove the cap of the culture bottle. Set the temperature of the hardening off shed to 23℃, the humidity at 65%, and the light intensity at 2800 lx. Meanwhile, starting from the 4th day, spray the leaves of the rooted tissue culture seedlings with a 0.1% potassium dihydrogen phosphate solution every 2 days. Each time, spray until the leaves are moist but not dripping. (3) Transplanting: Transplanting substrate preparation: By weight, the transplanting substrate includes 5 parts nutrient soil, 3 parts perlite and 2 parts vermiculite; disinfect the transplanting substrate, treat the rooted tissue culture seedlings, and transplant the treated rooted tissue culture seedlings into the transplanting substrate.

[0035] Example 7 This embodiment provides a method for cultivating tissue culture seedlings of *Melia azedarach*, including: (1) Cultivation of rooted tissue culture seedlings: The explants of *Mallotus spp.* were pretreated, primary cultured, proliferated, and rooted in sequence to obtain robust rooted tissue culture seedlings; (2) Hardening off seedlings: Select complete plants with a height of ≥5 cm and ≥3 adventitious roots of ≥3 cm in length. On days 1-3: move the rooted tissue culture seedlings along with the culture bottles to the hardening off shed. Open two small holes in the cap of the culture bottle. Set the temperature of the hardening off shed to 24℃, the humidity to 75%, and the light intensity to 1800 lx. On days 4-6: open the cap of the culture bottle halfway. Keep the temperature of the hardening off shed at 24℃, the humidity at 70%, and the light intensity at 2300 lx. On days 7-10: completely remove the cap of the culture bottle. Set the temperature of the hardening off shed to 23℃, the humidity at 65%, and the light intensity at 2800 lx. (3) Transplanting: Transplanting substrate preparation: By weight, the transplanting substrate includes 5 parts nutrient soil, 3 parts perlite, 2 parts vermiculite and 0.1 parts well-rotted sheep manure; disinfect the transplanting substrate, treat the rooted tissue culture seedlings, and transplant the treated rooted tissue culture seedlings into the transplanting substrate.

[0036] Example 8 This embodiment provides a method for cultivating tissue culture seedlings of *Melia azedarach*, including: (1) Cultivation of rooted tissue culture seedlings: The explants of *Mallotus spp.* were pretreated, primary cultured, proliferated, and rooted in sequence to obtain robust rooted tissue culture seedlings; (2) Hardening off seedlings: Select complete plants with a height of ≥5 cm and ≥3 adventitious roots of ≥3 cm in length. On days 1-3: move the rooted tissue culture seedlings along with the culture bottles to the hardening off shed. Open two small holes in the cap of the culture bottle. Set the temperature of the hardening off shed to 24℃, the humidity to 75%, and the light intensity to 1800 lx. On days 4-6: open the cap of the culture bottle halfway. Keep the temperature of the hardening off shed at 24℃, the humidity at 70%, and the light intensity at 2300 lx. On days 7-10: completely remove the cap of the culture bottle. Set the temperature of the hardening off shed to 23℃, the humidity at 65%, and the light intensity at 2800 lx. (3) Transplanting: Transplanting substrate preparation: By weight, the transplanting substrate includes 5 parts nutrient soil, 3 parts perlite, 2 parts vermiculite and 0.3 parts well-rotted sheep manure; disinfect the transplanting substrate, treat the rooted tissue culture seedlings, and transplant the treated rooted tissue culture seedlings into the transplanting substrate.

[0037] Example 9 This embodiment provides a method for cultivating tissue culture seedlings of *Melia azedarach*, including: (1) Cultivation of rooted tissue culture seedlings: The explants of *Mallotus spp.* were pretreated, primary cultured, proliferated, and rooted in sequence to obtain robust rooted tissue culture seedlings; (2) Hardening off seedlings: Select complete plants with a height of ≥5 cm and ≥3 adventitious roots of ≥3 cm in length. On days 1-3: move the rooted tissue culture seedlings along with the culture bottles to the hardening off shed. Open two small holes in the cap of the culture bottle. Set the temperature of the hardening off shed to 24℃, the humidity to 75%, and the light intensity to 1800 lx. On days 4-6: open the cap of the culture bottle halfway. Keep the temperature of the hardening off shed at 24℃, the humidity at 70%, and the light intensity at 2300 lx. On days 7-10: completely remove the cap of the culture bottle. Set the temperature of the hardening off shed to 23℃, the humidity at 65%, and the light intensity at 2800 lx. (3) Transplanting: Transplanting substrate preparation: By weight, the transplanting substrate includes 5 parts nutrient soil, 3 parts perlite, 2 parts vermiculite and 0.2 parts well-rotted sheep manure; disinfect the transplanting substrate, treat the rooted tissue culture seedlings, and transplant the treated rooted tissue culture seedlings into the transplanting substrate.

[0038] Example 10 This embodiment provides a method for cultivating tissue culture seedlings of *Melia azedarach*, including: (1) Cultivation of rooted tissue culture seedlings: The explants of *Mallotus spp.* were pretreated, primary cultured, proliferated, and rooted in sequence to obtain robust rooted tissue culture seedlings; (2) Hardening off seedlings: Select complete plants with a height of ≥5 cm and ≥3 adventitious roots of ≥3 cm in length. On days 1-3: move the rooted tissue culture seedlings along with the culture bottles to the hardening off shed. Open two small holes in the cap of the culture bottle. Set the temperature of the hardening off shed to 24℃, the humidity to 75%, and the light intensity to 1800 lx. On days 4-6: open the cap of the culture bottle halfway. Keep the temperature of the hardening off shed at 24℃, the humidity at 70%, and the light intensity at 2300 lx. On days 7-10: completely remove the cap of the culture bottle. Set the temperature of the hardening off shed to 23℃, the humidity at 65%, and the light intensity at 2800 lx. (3) Transplanting: Transplanting substrate preparation: By weight, the transplanting substrate includes: 5 parts nutrient soil, 3 parts perlite, 2 parts vermiculite, 0.1 parts decomposed sheep manure and 0.04 parts slow-release fertilizer; The raw materials of the slow-release fertilizer by weight include: 105 parts urea-formaldehyde, 33 parts superphosphate, 26 parts potassium sulfate, 11 parts potassium chloride, 12 parts urea and 6 parts zinc sulfate; Disinfect the transplanting substrate, treat the rooted tissue culture seedlings, and transplant the treated rooted tissue culture seedlings into the transplanting substrate.

[0039] Example 11 This embodiment provides a method for cultivating tissue culture seedlings of *Melia azedarach*, including: (1) Cultivation of rooted tissue culture seedlings: The explants of *Mallotus spp.* were pretreated, primary cultured, proliferated, and rooted in sequence to obtain robust rooted tissue culture seedlings; (2) Hardening off seedlings: Select complete plants with a height of ≥5 cm and ≥3 adventitious roots of ≥3 cm in length. On days 1-3: move the rooted tissue culture seedlings along with the culture bottles to the hardening off shed. Open two small holes in the cap of the culture bottle. Set the temperature of the hardening off shed to 24℃, the humidity to 75%, and the light intensity to 1800 lx. On days 4-6: open the cap of the culture bottle halfway. Keep the temperature of the hardening off shed at 24℃, the humidity at 70%, and the light intensity at 2300 lx. On days 7-10: completely remove the cap of the culture bottle. Set the temperature of the hardening off shed to 23℃, the humidity at 65%, and the light intensity at 2800 lx. (3) Transplanting: Transplanting substrate preparation: By weight, the transplanting substrate includes: 5 parts nutrient soil, 3 parts perlite, 2 parts vermiculite, 0.1 parts decomposed sheep manure and 0.06 parts slow-release fertilizer; The raw materials of the slow-release fertilizer by weight include: 105 parts urea-formaldehyde, 33 parts superphosphate, 26 parts potassium sulfate, 11 parts potassium chloride, 12 parts urea and 6 parts zinc sulfate; Disinfect the transplanting substrate, treat the rooted tissue culture seedlings, and transplant the treated rooted tissue culture seedlings into the transplanting substrate.

[0040] Example 12 This embodiment provides a method for cultivating tissue culture seedlings of *Melia azedarach*, including: (1) Cultivation of rooted tissue culture seedlings: The explants of *Mallotus spp.* were pretreated, primary cultured, proliferated, and rooted in sequence to obtain robust rooted tissue culture seedlings; (2) Hardening off seedlings: Select complete plants with a height of ≥5 cm and ≥3 adventitious roots of ≥3 cm in length. On days 1-3: move the rooted tissue culture seedlings along with the culture bottles to the hardening off shed. Open two small holes in the cap of the culture bottle. Set the temperature of the hardening off shed to 24℃, the humidity to 75%, and the light intensity to 1800 lx. On days 4-6: open the cap of the culture bottle halfway. Keep the temperature of the hardening off shed at 24℃, the humidity at 70%, and the light intensity at 2300 lx. On days 7-10: completely remove the cap of the culture bottle. Set the temperature of the hardening off shed to 23℃, the humidity at 65%, and the light intensity at 2800 lx. (3) Transplanting: Transplanting substrate preparation: By weight, the transplanting substrate includes: 5 parts nutrient soil, 3 parts perlite, 2 parts vermiculite, 0.1 parts decomposed sheep manure and 0.05 parts slow-release fertilizer; The raw materials of the slow-release fertilizer by weight include: 105 parts urea-formaldehyde, 33 parts superphosphate, 26 parts potassium sulfate, 11 parts potassium chloride, 12 parts urea and 6 parts zinc sulfate; Disinfect the transplanting substrate, treat the rooted tissue culture seedlings, and transplant the treated rooted tissue culture seedlings into the transplanting substrate.

[0041] Example 13 This embodiment provides a method for cultivating tissue culture seedlings of *Melia azedarach*, including: (1) Cultivation of rooted tissue culture seedlings: The explants of *Mallotus spp.* were pretreated, primary cultured, proliferated, and rooted in sequence to obtain robust rooted tissue culture seedlings; (2) Hardening off seedlings: Select complete plants with a height of ≥5 cm and ≥3 adventitious roots of ≥3 cm in length. On days 1-3: move the rooted tissue culture seedlings along with the culture bottles to the hardening off shed. Open two small holes in the cap of the culture bottle. Set the temperature of the hardening off shed to 24℃, the humidity to 75%, and the light intensity to 1800 lx. On days 4-6: open the cap of the culture bottle halfway. Keep the temperature of the hardening off shed at 24℃, the humidity at 70%, and the light intensity at 2300 lx. On days 7-10: completely remove the cap of the culture bottle. Set the temperature of the hardening off shed to 23℃, the humidity at 65%, and the light intensity at 2800 lx. (3) Transplanting: Transplanting substrate preparation: By weight, the transplanting substrate includes 5 parts nutrient soil, 3 parts perlite and 2 parts vermiculite; Disinfecting the transplanting substrate: 12 days before transplanting: pile the transplanting substrate into a trapezoidal pile 1.1m high and 1.8m wide, cover the surface of the trapezoidal pile with a layer of transparent plastic film, and disinfect it using solar energy for 10 days; 2 days before transplanting: spread out the solar-disinfected transplanting substrate, cool it to room temperature, and then water the transplanting substrate thoroughly with a 500-fold dilution of 75% mancozeb; Then treat the rooted tissue culture seedlings and transplant the treated rooted tissue culture seedlings into the transplanting substrate.

[0042] Example 14 This embodiment provides a method for cultivating tissue culture seedlings of *Melia azedarach*, including: (1) Cultivation of rooted tissue culture seedlings: The explants of *Mallotus spp.* were pretreated, primary cultured, proliferated, and rooted in sequence to obtain robust rooted tissue culture seedlings; (2) Hardening off seedlings: Select complete plants with a height of ≥5 cm and ≥3 adventitious roots of ≥3 cm in length. On days 1-3: move the rooted tissue culture seedlings along with the culture bottles to the hardening off shed. Open two small holes in the cap of the culture bottle. Set the temperature of the hardening off shed to 24℃, the humidity to 75%, and the light intensity to 1800 lx. On days 4-6: open the cap of the culture bottle halfway. Keep the temperature of the hardening off shed at 24℃, the humidity at 70%, and the light intensity at 2300 lx. On days 7-10: completely remove the cap of the culture bottle. Set the temperature of the hardening off shed to 23℃, the humidity at 65%, and the light intensity at 2800 lx. (3) Transplanting: Transplanting substrate preparation: By weight, the transplanting substrate includes 5 parts nutrient soil, 3 parts perlite and 2 parts vermiculite; After disinfecting the transplanting substrate, the treatment of the rooted tissue culture seedlings includes: taking out the rooted tissue culture seedlings, washing off the culture medium from the roots with clean water, immersing the roots of the rooted tissue culture seedlings in a 500-fold naphthaleneacetic acid solution for 12 minutes, taking them out and drying the moisture on the surface of the roots before transplanting.

[0043] Example 15 This embodiment provides a method for cultivating tissue culture seedlings of *Melia azedarach*, including: (1) Cultivation of rooted tissue culture seedlings: The explants of *Mallotus spp.* were pretreated, primary cultured, proliferated, and rooted in sequence to obtain robust rooted tissue culture seedlings; (2) Hardening off seedlings: Select complete plants with a height of ≥5 cm and ≥3 adventitious roots of ≥3 cm in length. On days 1-3: move the rooted tissue culture seedlings along with the culture bottles to the hardening off shed. Open two small holes in the cap of the culture bottle. Set the temperature of the hardening off shed to 24℃, the humidity to 75%, and the light intensity to 1800 lx. On days 4-6: open the cap of the culture bottle halfway. Keep the temperature of the hardening off shed at 24℃, the humidity at 70%, and the light intensity at 2300 lx. On days 7-10: completely remove the cap of the culture bottle. Set the temperature of the hardening off shed to 23℃, the humidity at 65%, and the light intensity at 2800 lx. (3) Transplanting: Transplanting substrate preparation: By weight, the transplanting substrate includes 5 parts nutrient soil, 3 parts perlite and 2 parts vermiculite; disinfect the transplanting substrate, treat the rooted tissue culture seedlings, and transplant the treated rooted tissue culture seedlings into the transplanting substrate; the transplanting process includes: first filling a container with a diameter of 7cm and a height of 12cm halfway with the transplanting substrate, then placing the rooted tissue culture seedlings in, keeping the roots spread out, and adding the transplanting substrate to 1 / 2 of the seedling height; then lightly pressing the transplanting substrate, watering thoroughly, covering the surface of the transplanting substrate with a 2cm thick layer of rice husks, and then covering it with a transparent plastic film or plastic cup to retain moisture.

[0044] Comparative Example 1 This comparative example provides a method for cultivating tissue culture seedlings of *Palatinate*, including: (1) Cultivation of rooted tissue culture seedlings: The explants of *Mallotus spp.* were pretreated, primary cultured, proliferated, and rooted in sequence to obtain robust rooted tissue culture seedlings; (2) Hardening off seedlings: Select complete plants with a height of ≥5 cm and a length of ≥3 adventitious roots, and move them to an environment with 6000 Lux light and 26℃ temperature for 6 days to harden off seedlings; then loosen the bottle cap for 2 days. (3) Transplanting: Transplanting substrate preparation: by weight, the transplanting substrate includes 7 parts nutrient soil and 3 parts perlite; disinfect the transplanting substrate, treat the rooted tissue culture seedlings, and transplant the treated rooted tissue culture seedlings into the transplanting substrate.

[0045] Comparative Example 2 This comparative example provides a method for cultivating tissue culture seedlings of *Palatinate*, including: (1) Cultivation of rooted tissue culture seedlings: The explants of *Mallotus spp.* were pretreated, primary cultured, proliferated, and rooted in sequence to obtain robust rooted tissue culture seedlings; (2) Hardening off seedlings: Select complete plants with a height of ≥5 cm and a length of ≥3 adventitious roots, and move them to an environment with 6000 Lux light and 26℃ temperature for 6 days to harden off seedlings; then loosen the bottle cap for 2 days. (3) Transplanting: Transplanting substrate preparation: By weight, the transplanting substrate includes 5 parts nutrient soil, 3 parts perlite and 2 parts vermiculite; disinfect the transplanting substrate, treat the rooted tissue culture seedlings, and transplant the treated rooted tissue culture seedlings into the transplanting substrate.

[0046] Comparative Example 3 This comparative example provides a method for cultivating tissue culture seedlings of *Palatinate*, including: (1) Cultivation of rooted tissue culture seedlings: The explants of *Mallotus spp.* were pretreated, primary cultured, proliferated, and rooted in sequence to obtain robust rooted tissue culture seedlings; (2) Hardening off seedlings: Select complete plants with a height of ≥5 cm and ≥3 adventitious roots of ≥3 cm in length. On days 1-3: move the rooted tissue culture seedlings along with the culture bottles to the hardening off shed. Open two small holes in the cap of the culture bottle. Set the temperature of the hardening off shed to 24℃, the humidity to 75%, and the light intensity to 1800 lx. On days 4-6: open the cap of the culture bottle halfway. Keep the temperature of the hardening off shed at 24℃, the humidity at 70%, and the light intensity at 2300 lx. On days 7-10: completely remove the cap of the culture bottle. Set the temperature of the hardening off shed to 23℃, the humidity at 65%, and the light intensity at 2800 lx. (3) Transplanting: Transplanting substrate preparation: by weight, the transplanting substrate includes 7 parts nutrient soil and 3 parts perlite; disinfect the transplanting substrate, treat the rooted tissue culture seedlings, and transplant the treated rooted tissue culture seedlings into the transplanting substrate.

[0047] Comparative Example 4 This comparative example provides a method for cultivating tissue culture seedlings of *Palatinate*, including: (1) Cultivation of rooted tissue culture seedlings: The explants of *Mallotus spp.* were pretreated, primary cultured, proliferated, and rooted in sequence to obtain robust rooted tissue culture seedlings; (2) Hardening off seedlings: Select complete plants with a height of ≥5 cm and ≥3 adventitious roots of ≥3 cm in length. On days 1-3: move the rooted tissue culture seedlings along with the culture bottles to the hardening off shed. Open 3 small holes in the cap of the culture bottle. Set the temperature of the hardening off shed to 20℃, the humidity to 85%, and the light intensity to 1200 lx. On days 4-6: open the cap of the culture bottle halfway. Keep the temperature of the hardening off shed at 20℃, the humidity at 60%, and the light intensity at 3000 lx. On days 7-10: completely remove the cap of the culture bottle. Set the temperature of the hardening off shed to 25℃, the humidity at 75%, and the light intensity at 3500 lx. (3) Transplanting: Transplanting substrate preparation: By weight, the transplanting substrate includes 5 parts nutrient soil, 3 parts perlite and 2 parts vermiculite; disinfect the transplanting substrate, treat the rooted tissue culture seedlings, and transplant the treated rooted tissue culture seedlings into the transplanting substrate.

[0048] Experimental Example 1 Tissue culture seedlings of *Paspalum notatum* with the same batch and consistent growth status were selected and cultured according to the standard of T / HBSF 017-2024 "Technical Specification for Production of *Paspalum notatum* Tissue Culture Seedlings" to obtain rooted tissue culture seedlings. The rooted tissue culture seedlings were divided into groups 1-15 of Examples and groups 1-4 of Comparative Examples, with 500 seedlings per group. Each group was hardened off and transplanted according to the cultivation methods provided in Examples 1-15 and Comparative Examples 1-4, respectively. The survival rate of hardened off, the survival rate of transplanted seedlings, and the average growth rate of plant height one month after transplanting were counted for each group. The results are shown in Table 1.

[0049] Table 1

[0050] As shown in Table 1, the method for cultivating *Panicum esculentum* tissue culture seedlings provided in this application increases the hardening survival rate from approximately 90% to over 98.8% compared to the standard method, and the transplant survival rate from approximately 95% to over 99% compared to the standard method. Furthermore, it increases the average height growth rate of seedlings within one month after transplanting by over 26%. This demonstrates that the method for cultivating *Panicum esculentum* tissue culture seedlings provided in this application can improve the hardening and transplant survival rates, thereby increasing production efficiency and overall survival rate. This is of great significance for promoting the industrialization of *Panicum esculentum* tissue culture seedlings.

[0051] In summary, the method for cultivating *Panicum esculentum* tissue culture seedlings provided by this invention improves the hardening and transplanting stages of *Panicum esculentum* tissue culture seedling production, aiming to further increase the survival rate of hardening and transplanting seedlings, thereby improving production efficiency and overall survival rate, and promoting the industrialization of *Panicum esculentum* tissue culture seedlings.

[0052] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for cultivating tissue culture seedlings of *Palatinate*, characterized in that: include: (1) Cultivation of rooted tissue culture seedlings: The explants of *Mallotus spp.* were pretreated, primary cultured, proliferated, and rooted in sequence to obtain robust rooted tissue culture seedlings; (2) Hardening off seedlings: Select complete plants with a height of ≥5 cm and at least 3 adventitious roots of ≥3 cm in length. On days 1-3: the rooted tissue culture seedlings along with the culture bottles are moved to the hardening shed. One or two small holes are opened in the cap of the culture bottles. The temperature of the hardening shed is set to 23-25℃, the humidity is set to 70%-80%, and the light intensity is set to 1500-2000 lx. On days 4-6: Open the cap of the culture bottle halfway, maintain the temperature of the hardening shed at 23-25℃, the humidity at 65%-75%, and the light intensity at 2000-2500 lx; On days 7-10: completely open the caps of the culture bottles, set the temperature of the hardening shed to 22-24℃, the humidity to 60%-70%, and the light intensity to 2500-3000 lx; (3) Transplanting: Transplanting substrate preparation: By weight, the transplanting substrate comprises: 4-6 parts nutrient soil, 2-4 parts perlite and 1-3 parts vermiculite; The transplanting substrate is disinfected, the rooted tissue culture seedlings are treated, and the treated rooted tissue culture seedlings are transplanted into the transplanting substrate.

2. The method for cultivating *Palatinate* tissue culture seedlings according to claim 1, characterized in that: In step (2), starting from day 4, spray the leaves of the rooted tissue culture seedlings with a 0.1% potassium dihydrogen phosphate solution every 2 days. Each time, the amount of spray should be enough to moisten the leaves but not drip water.

3. The method for cultivating *Palatinate* tissue culture seedlings according to claim 1, characterized in that: In step (3), the transplanting substrate also includes 0.1-0.3 parts of well-rotted sheep manure by weight.

4. The method for cultivating *Palatinate* tissue culture seedlings according to claim 3, characterized in that: In step (3), the transplanting substrate also includes 0.04-0.06 parts of slow-release fertilizer by weight.

5. The method for cultivating *Palatinate* tissue culture seedlings according to claim 3, characterized in that: In step (3), the raw materials of the slow-release fertilizer include, by weight, 100-110 parts of urea-formaldehyde, 32-36 parts of superphosphate, 24-28 parts of potassium sulfate, 10-12 parts of potassium chloride, 8-13 parts of urea, and 4-7 parts of zinc sulfate.

6. The method for cultivating *Palatinate* tissue culture seedlings according to claim 5, characterized in that: By weight, the urea-formaldehyde is 105 parts, the superphosphate is 33 parts, the potassium sulfate is 26 parts, the potassium chloride is 11 parts, the urea is 12 parts, and the zinc sulfate is 6 parts.

7. The method for cultivating *Palatinate* tissue culture seedlings according to claim 1, characterized in that: In step (3), the preparation of the transplanting substrate includes: premixing the slow-release fertilizer with the perlite and the vermiculite for 3 minutes, and then adding the nutrient soil and the decomposed sheep manure and mixing.

8. The method for cultivating *Palatinate* tissue culture seedlings according to claim 1, characterized in that: In step (3), disinfecting the transplanting substrate further includes: 12 days before transplanting: piling the transplanting substrate into a trapezoidal pile with a height of 1.0~1.2m and a width of 1.5~2.0m, covering the surface of the trapezoidal pile with a layer of transparent plastic film, and using solar energy for disinfection for 10 days; 2 days before transplanting: spreading out the solar-disinfected transplanting substrate, cooling it to room temperature, and then thoroughly watering the transplanting substrate with a 500-fold dilution of 75% mancozeb.

9. The method for cultivating *Palatinate* tissue culture seedlings according to claim 1, characterized in that: In step (3), the treatment of the rooted tissue culture seedlings includes: taking out the rooted tissue culture seedlings, washing away the culture medium from the roots with clean water, immersing the roots of the rooted tissue culture seedlings in a 500-fold naphthaleneacetic acid solution for 10-15 minutes, taking them out, drying the moisture on the surface of the roots, and then transplanting them.

10. The method for cultivating *Palatinate* tissue culture seedlings according to claim 1, characterized in that: In step (3), transplanting includes: first filling a container with a diameter of 6cm-8cm and a height of 10cm-15cm halfway with the transplanting substrate, then placing the rooted tissue culture seedling in, keeping the roots spread out, and adding the transplanting substrate to 1 / 3 to 1 / 2 of the seedling height; then gently pressing the transplanting substrate, watering thoroughly, covering the surface of the transplanting substrate after transplanting with a layer of rice husks 1-2cm thick, and then covering it with a transparent plastic film or plastic cup to retain moisture.

Images