Pine tissue culture medium, preparation method of the medium and application method thereof

Abstract

This invention discloses a rapid propagation culture medium for superior clonal lines of Pinus simonii, its preparation method, and its application method. Belonging to the field of forest tree tissue culture, the bud induction medium is based on DCR medium and contains a slow-release cytokinin prodrug (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one and naphthaleneacetic acid. The prodrug hydrolyzes in the aqueous phase to controllably release 6-benzylaminopurine and ferulic acid, ensuring that the instantaneous concentration of free cytokinin is lower than the concentration corresponding to its cumulative release, thereby decoupling the trade-off between bud induction rate and vitrification rate. The culture medium may also include a starting medium containing polyvinylpyrrolidone and ascorbic acid, a bud elongation subculture medium containing activated carbon, a rooting induction medium containing dual-load slow-release auxin, and a hardening substrate composed of pine needle humus, perlite, and vermiculite.

Description

Technical Field

[0001] This invention belongs to the field of forest tree tissue culture and plant biotechnology, specifically relating to a rapid propagation culture medium for superior clonal lines of Pinus sylvestris, the preparation method of the culture medium, and its application method. Background Technology

[0002] Simao pine ( Pinus kesiya var.langbianensis Pinus simaoense is an important native timber and resin-producing tree species in southwestern my country, widely distributed in Yunnan, Sichuan, Guizhou, and other provinces. It boasts advantages such as rapid growth, high yield, excellent wood quality, high resin extraction rate, and strong adaptability, making it one of the main fast-growing timber species in Yunnan Province. However, natural forests of Pinus simaoense have long faced the threat of pine wilt disease, a disease common to all pine species, and the pressure of unreasonable logging. Superior individual resources are becoming increasingly scarce, making the need for large-scale, genetically consistent preservation and propagation of its superior genotypes increasingly urgent.

[0003] Current methods for breeding superior varieties of *Pinus simonii* mainly rely on seed propagation and grafting. Chinese invention patent application CN1545844A, entitled "A Method for Afforestation by Mixing *Pinus simonii* Seeds with Crop Seeds," discloses a method for artificial afforestation using direct seeding or aerial seeding of *Pinus simonii* seeds. This method is essentially seed propagation, resulting in genetic differentiation in offspring, making it difficult to stably maintain the superior traits of the parent tree. Furthermore, its afforestation effect in tropical regions is not ideal, making it difficult to guarantee the establishment of mature forests and timber production. While grafting can maintain the genetic characteristics of asexual lines, its large-scale promotion is difficult due to limitations in rootstock resources and graft compatibility. Therefore, tissue culture rapid propagation technology, which can maintain the genetic consistency of superior asexual lines and achieve large-scale propagation, has become a key technological path for the breeding of superior *Pinus simonii* varieties.

[0004] Tissue culture rapid propagation can multiply a single superior genotype under in vitro aseptic conditions, and the resulting regenerated plants are genetically identical to the parent plant. It is an important technical approach for preserving and utilizing superior clones of forest trees. For coniferous species such as Pinus sylvestris, which have long generation cycles and low efficiency in both conventional breeding and asexual reproduction, establishing a stable and efficient tissue culture rapid propagation system is particularly significant for accelerating the promotion of superior varieties and the preservation of germplasm resources.

[0005] However, tissue culture of pine plants has long faced inherent technical bottlenecks such as severe polyphenol oxidation and browning, difficulty in rooting, and high vitrification rates, and *Pinus simaoensis* is no exception. Among existing pine tissue culture techniques, Chinese invention patent application CN202111086522.4, entitled "A Method for Rapid Propagation of *Pinus huangshanensis* by Tissue Culture," discloses a scheme using *Pinus huangshanensis* seeds as explants, employing an elongation medium based on DCR medium with constant addition of 6-benzylaminopurine and naphthaleneacetic acid, and a 1 / 2 DCR rooting medium with constant addition of indolebutyric acid for rapid propagation. This scheme uses seeds as explants, which are seed-grown materials, making it impossible to maintain the genetic consistency of specific superior clones. Furthermore, the constant addition of cytokinins in a free form fails to solve the vitrification problem associated with high cytokinin concentrations. Chinese invention patent application CN1915002A, entitled "Method for Inducing Embryogenic Callus in Larix and its Dedicated Culture Medium," discloses a scheme for inducing embryogenic callus in Larix spp. using DCR medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. This scheme targets the Larix spp., follows the somatic embryogenesis pathway, and aims to induce callus formation, which differs from the direct rapid propagation of superior clones of Pinus simonii via organogenesis. Furthermore, existing tissue culture reports on Pinus simonii mostly involve regeneration of shoot tips from young seedlings in MS medium supplemented with kinetin. This approach also uses young seedling material, avoids the phase transition problem of mature material, and cannot achieve genetically consistent propagation of superior adult clones.

[0006] Specifically, in pine tissue culture, phenolic substances released after explant inoculation are oxidized into quinones by polyphenol oxidase and phenylalanine ammonia-lyase, and further polymerize, leading to browning of the culture medium and explants, and in severe cases, explant death. During the induction and subculturing of shoot clusters, while higher cytokinin levels are beneficial for adventitious shoot formation, they can induce vitrification of seedlings, manifesting as transparent or water-soaked stems and leaves, low lignification, impaired stomatal function, and decreased mechanical strength. Vitrified seedlings are difficult to root and transplant successfully. Current techniques often alleviate browning by adding antioxidants or activated carbon, and vitrification is often addressed by increasing the amount of gelling agent, changing the type of cytokinin, or applying ethylene inhibitors. However, these methods do not address the underlying cause of vitrification—the interrelation between cumulative cytokinin dose and instantaneous concentration—nor do they comprehensively address the inhibition of phenolic oxidation during the rooting stage and the vitrification during the induction stage. Therefore, it is difficult to fundamentally reduce the vitrification rate and overcome rooting difficulties while ensuring the shoot cluster induction rate.

[0007] The study revealed that the core contradiction hindering the rapid propagation of superior clones of *Pinus kesiya* via tissue culture lies in the fact that the cytokinin concentration required to increase the induction rate of shoot clusters simultaneously drives vitrification of seedlings and phenolic oxidation browning of explants. The conventional method of adding cytokinin in a constant free form binds the cumulative effective dose of cytokinin to its instantaneous free concentration, making it difficult to suppress the instantaneous concentration leading to vitrification while maintaining the induction rate. Simultaneously, the accumulation of phenolic oxides at the base of pine seedlings during the rooting stage inhibits adventitious root formation, further exacerbating rooting difficulties. The aforementioned comparative studies failed to recognize this core contradiction, nor did they provide technical means to maintain the cumulative dose of cytokinin while suppressing its instantaneous free concentration, nor did they provide methods to simultaneously alleviate the inhibition of phenolic oxidation during the rooting stage. Therefore, there is an urgent need to develop a tissue culture medium and its application method system specifically for superior clones of *Pinus kesiya* that can simultaneously achieve high shoot cluster induction rates and low vitrification rates, while overcoming browning and rooting difficulties. Summary of the Invention

[0008] To address the problems existing in the prior art, the present invention aims to provide a rapid propagation culture medium for superior clonal lines of Pinus keseng, a method for preparing the culture medium, and a method for applying the medium, so as to resolve the contradiction between the induction rate of clustered shoots and the vitrification rate, and overcome the difficulties of explant browning and rooting in the tissue culture process of superior clonal lines of Pinus keseng, thereby achieving large-scale propagation while maintaining the genetic consistency of the clonal lines.

[0009] To achieve the above objectives, a first aspect of the present invention provides a rapid propagation culture medium for superior clonal lines of *Pinus kesiao*, comprising a shoot induction medium, wherein the shoot induction medium is based on DCR medium and contains (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one and naphthaleneacetic acid as a prodrug for the sustained release of cytokinin; wherein the (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one is a compound obtained by ferulic acid amidation of the N9 position of the purine ring of 6-benzylaminopurine, and its molecular formula is C0. 22 H 19N5O3, with a relative molecular mass of 401.43, can release 6-benzylaminopurine and ferulic acid through slow hydrolysis in an aqueous culture medium. Since the N9 position of the purine ring is the site where cytokinin is inactivated and stored in plants via N9-glycosylation, masking this site with a hydrolyzable ferulic acid bond can lower the instantaneous concentration of free 6-benzylaminopurine in the culture medium below the concentration corresponding to its cumulative release. This maintains the cumulative dose required for shoot induction while suppressing the instantaneous peak value for vitrification. Simultaneously, the released ferulic acid, as a phenolic acid antioxidant, can inhibit the activity of polyphenol oxidase in explants and scavenge reactive oxygen species, alleviating oxidative stress-induced vitrification and browning.

[0010] The culture medium described in this invention may further include a starter culture medium, a shoot elongation subculture medium, a rooting induction medium, and a hardening substrate, forming a functional culture medium system covering the entire process from explant start-up, shoot cluster induction, shoot elongation subculture, rooting induction to hardening and transplanting. The initiation medium is based on a modified DCR medium with reduced inorganic salt ion concentration and contains polyvinylpyrrolidone and ascorbic acid to adsorb and inhibit polyphenol oxidation products, overcoming the severe browning and death of Pinus sylvestris explants after inoculation. The shoot elongation subculture medium is based on DCR medium and contains activated carbon to adsorb metabolic products accumulated during subculture. The rooting induction medium is based on 1 / 2 DCR medium and contains 4-(1H-indol-3-yl)butyric acid-(2R)-2-[(2R)-3,4-dihydroxy-5-oxonyl-2,5-dihydrofuran-2-yl]-2-hydroxyethyl ester as a dual-loaded slow-release auxin. The molecular formula of the 4-(1H-indol-3-yl)butyric acid-(2R)-2-[(2R)-3,4-dihydroxy-5-oxonyl-2,5-dihydrofuran-2-yl]-2-hydroxyethyl ester is C 18 H 19 NO7, with a relative molecular mass of 361.35, releases indolebutyric acid and ascorbic acid upon hydrolysis. The former provides a continuous rooting signal, while the latter inhibits the oxidation of phenols at the base of the seedling to relieve their inhibition of adventitious root development. The hardening substrate is composed of pine needle humus, perlite, and vermiculite, simulating the soil characteristics of the native seedbed of Pinus kesao-yima.

[0011] The second aspect of the present invention provides a method for preparing the above-mentioned culture medium, comprising the preparation of (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one and the formulation of the culture medium; wherein the (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one is prepared in three steps: ferulic acid phenol hydroxyacetylation protection, selective acylation of the purine ring at the N9 position of 6-benzylaminopurine, and basic alcohol deprotection. The third aspect of the present invention provides a method for applying the above-mentioned culture medium, namely, using semi-lignified young branches of mature mother trees of superior clonal Pinus kesiao as explants, sequentially undergoing initiation culture, induction of clustered buds, bud elongation subculture, root induction after dark culture pretreatment, and hardening-off transplanting, ultimately obtaining genetically consistent and robust tissue culture seedlings.

[0012] In the above-mentioned technical solution of the present invention, the first aspect uses a slow-release cytokinin prodrug as a characteristic component of the shoot induction medium, which differs from the existing technology of constantly adding free cytokinin; the second aspect's preparation method focuses on the selective acylation of the purine ring at the N9 position and the construction of a hydrolyzable ferulic acid bond, so that the obtained prodrug is slowly hydrolyzed in the aqueous phase at the desired rate; the third aspect's application method uses semi-lignified young branches of superior clones as explants and introduces dark culture pretreatment before rooting induction, combined with a rooting induction medium containing dual-load slow-release auxin, thereby overcoming the phase transition problem and rooting difficulty of adult materials while maintaining the genetic consistency of clones. The three aspects work together to form a complete tissue culture rapid propagation technology system for superior clones of Pinus kesiao.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: First, by replacing free cytokinin with the slow-release cytokinin prodrug (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one, the cumulative dose required for induction and the instantaneous concentration for vitrification are decoupled, significantly reducing the vitrification rate while maintaining a high shoot induction rate, thus overcoming the inherent trade-off between induction rate and vitrification rate in the prior art; Second, the ferulic acid released by the hydrolysis of the prodrug, and the dual-load growth during the rooting stage... The ascorbic acid released by the hydrolysis of the clonal molecule removes phenolic oxidation products at both the induction and rooting ends, and works synergistically with polyvinylpyrrolidone and ascorbic acid in the initiation medium to inhibit browning at multiple stages and relieve rooting inhibition. Thirdly, using semi-lignified young branches of mature mother trees of superior clones as explants and propagating them through the entire process system maintains the genetic consistency of clones and overcomes the phase transition problem of mature materials. Fourthly, this invention is suitable for various application scenarios such as the breeding of superior Pinaceae plants in forestry research institutes, the industrialized seedling production of superior clones in forestry production units, and the preservation of forest germplasm resources.

[0014] In practical implementation, the present invention can achieve a bud induction rate of about 88% on superior clones of Pinus sylvestris while controlling the vitrification rate at about 6%, with an average of about 5.2 buds per explant, a rooting rate of about 82%, and a transplant survival rate of about 90%. Moreover, the resulting regenerated plants maintain the genetic consistency of superior clones. All comprehensive indicators are significantly better than the conventional scheme of constant addition of free cytokinin. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the entire process of the five-functional culture medium for rapid propagation of superior clonal lines of Pinus simonii in this invention. Figure 2 The structural formula of (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one of the present invention and the schematic diagram of its hydrolysis in the aqueous phase of the culture medium to release 6-benzylaminopurine and ferulic acid; Figure 3 The structural formula of 4-(1H-indole-3-yl)butyric acid-(2R)-2-[(2R)-3,4-dihydroxy-5-oxoyl-2,5-dihydrofuran-2-yl]-2-hydroxyethyl ester of the present invention and a schematic diagram of the hydrolysis pathway for the release of indolebutyric acid and ascorbic acid. Figure 4 This is a bar chart comparing the induction rate and vitrification rate of shoot clusters in Example 1 and Comparative Example 1. Figure 5 The bar chart shows the comparison of rooting rate and transplant survival rate between each embodiment and each comparative example. Detailed Implementation

[0016] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, all reagents used in the following embodiments are commercially available analytical grade or plant tissue culture grade; the DCR medium refers to the coniferous tissue culture basal medium reported by Gupta and Durzan; the modified DCR medium and 1 / 2 DCR medium refer to the standard DCR medium with its inorganic salt ion concentration reduced to a specified ratio and halved, respectively, while the other components remain unchanged; the superior clonal adult mother trees of *Pinus kesiao* are mature plants with excellent phenotypes and stable growth, selected through progeny testing. The entire culture process of the present invention, except for dark culture pretreatment and those specifically noted, is carried out under conditions of temperature 23-27°C, light intensity 1500-2500 lx, and light duration 10-12 h / d.

[0017] Preparation Example 1: Preparation of (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one (sustained-release cytokinin prodrug). Figure 2As shown, this preparation example synthesizes the target compound in three steps. Step 1, phenolic hydroxyl acetylation protection: 13.04 g (67.13 mmol) of ferulic acid was dissolved in pyridine, and 13.71 g (134.27 mmol, the molar ratio of ferulic acid to acetic anhydride was 1:2) was added dropwise at 0°C. After the addition was complete, the temperature was raised to 25°C and stirred for 3 h under nitrogen protection. The reaction only acylated the phenolic hydroxyl group without affecting the carboxyl group. After post-treatment, (2E)-3-(4-acetoxy-3-methoxyphenyl)prop-2-enoic acid was obtained, with the molecular formula C 12 H 12 O5, yield approximately 92%. Step 2, N9-position selective acylation: The above (2E)-3-(4-acetoxy-3-methoxyphenyl)prop-2-enoic acid was activated with oxaloyl chloride to 4-O-acetylferuloyl chloride; 11.59 g (51.47 mmol) of 6-benzylaminopurine was placed in anhydrous N,N-dimethylformamide and deprotonated with sodium hydride (1.5 equivalents) at 0°C to generate an N9-position anion of the purine ring. After 30 min, the above acyl chloride (the molar ratio of 6-benzylaminopurine to (2E)-3-(4-acetoxy-3-methoxyphenyl)prop-2-enoic acid was added dropwise, and the reaction was carried out at 0~25°C under nitrogen protection for 6~12 h. Since the purine anion is an amphiphile competing for the N7 and N9 positions, and the N6-benzyl group of 6-benzylaminopurine spatially shields the adjacent N7 position, acylation yields a predominantly N9-position product and a minor N7-position product. Silica gel column chromatography was used to separate the pure N9-position isomer, namely 4-[(1E)-3-[6-(benzylamino)purine-9-yl]-3-oxoylideneprop-1-enyl]-2-methoxyphenyl ester, with the molecular formula C2. 24 H 21 N5O4, yield of this step approximately 55%. Step 3, alkaline alcohol deprotection: The above intermediate was dissolved in methanol, a catalytic amount of potassium carbonate was added, and the mixture was stirred at 0-25°C for 1-2 h under thin-layer chromatography monitoring. Utilizing the much faster hydrolysis rate of aryl acetate than that of the N9-position amide, the acetyl group was selectively removed while retaining the N9-position ferulic acid amide, which is crucial as a prodrug, to obtain 10.0 g of the target compound (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one, yield of this step approximately 88%, and the overall yield of the three steps approximately 44.5%. The molecular formula of the obtained compound is C 22 H 19 N5O3, with a relative molecular mass of 401.43, is given by electrospray mass spectrometry as [M+H] with an m / z of 402.2. +The 1H NMR spectrum (solvent: deuterated dimethyl sulfoxide) shows singlets at chemical shifts 8.6 and 8.4 for the hydrogens at positions 2 and 8 of the purine ring, two doublets at 7.5 and 6.7 for the feruloyl trans double bond, multiplets around 7.3 for the benzylbenzene ring, a singlet at 4.7 for the methylene group connected to N6, and a singlet at 3.8 for the methoxy group, consistent with the target structure.

[0018] In this preparation example, the (2E)-3-(4-acetoxy-3-methoxyphenyl)prop-2-enoic acid obtained in step one was confirmed by thin-layer chromatography to have complete acylation of the phenolic hydroxyl group and retention of the carboxyl group. In step two, strict anhydrous operation is required when generating the N9-position anion of the purine ring with sodium hydride. After the addition is completed, the ratio of the N9-position product to the N7-position product is monitored by thin-layer chromatography. The spatial shielding of the N7-position by the N6-benzyl group makes the ratio of the N9-position product to the N7-position product approximately 2:1. The two can be separated by gradient elution with dichloromethane and methanol on a silica gel column. In step three, the amount of potassium carbonate used for deacetylation should be catalytic, and the reaction time and temperature should be strictly controlled. Excessive alkali or prolonged reaction will cause partial hydrolysis of the N9-position ferulic acid, which is the key prodrug, thus reducing the yield. The resulting (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one is an off-white to pale yellow solid with a purity of not less than 98% as determined by high performance liquid chromatography. It should be noted that in this preparation example, the activation system of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole can also be used instead of the acyl chloride activation system to complete the acylation in step two, as both can form a ferulic acid bond at the N9 position of the purine ring.

[0019] To verify the sustained-release properties of (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one, it was added to shoot induction medium at a concentration of 0.2 mg / L and samples were taken at different culture times. The concentration of free 6-benzylaminopurine in the medium was determined by high performance liquid chromatography. The results showed that the concentration of free 6-benzylaminopurine gradually increased with the extension of culture time and was always maintained at a level lower than the concentration corresponding to its complete hydrolysis. This confirmed the design of the prodrug to slowly release cytokinin at the required rate in the aqueous phase of the culture medium, thereby suppressing the instantaneous free concentration.

[0020] Preparation Example 2: Preparation of 4-(1H-indol-3-yl)butyric acid-(2R)-2-[(2R)-3,4-dihydroxy-5-oxoylide-2,5-dihydrofuran-2-yl]-2-hydroxyethyl ester (dual-loaded sustained-release auxin). Figure 3As shown, this preparation example utilizes a one-step 6-position selective esterification catalyzed by lipase. 6.50 g of L-ascorbic acid and 7.50 g of indolebutyric acid (molar ratio approximately 1:1) were dissolved in tert-butanol. Candida antarcticis lipase B (Novozym 435) was added as a catalyst, and water was removed using molecular sieves. The reaction was stirred at 45–55°C for 24–48 h. This reaction exhibits high selectivity for the 6-position primary hydroxyl group of L-ascorbic acid, while retaining the 2,3-position enediol and the 5-position hydroxyl group, thus avoiding the destruction of the enediol structure by the concentrated sulfuric acid route. After the reaction, the lipase was removed by filtration, and the product was purified by column chromatography to obtain 10.0 g of the target compound, 4-(1H-indole-3-yl)butyric acid-(2R)-2-[(2R)-3,4-dihydroxy-5-oxoylide-2,5-dihydrofuran-2-yl]-2-hydroxyethyl ester, with a yield of approximately 75%. The molecular formula of the obtained compound is C 18 H 19 NO7, with a relative molecular mass of 361.35, is given by electrospray mass spectrometry as [M+H] with an m / z of 362.1. + Peak; it releases indolebutyric acid (C10) through slow hydrolysis in the aqueous phase of the culture medium. 12 H 13 NO2) and L-ascorbic acid (C6H8O6) have significantly improved antioxidant stability after acylation at the 6-position. Therefore, compared with free ascorbic acid, they can continuously exert antioxidant and root-inhibition effects during the rooting cycle.

[0021] In this preparation example, using methyl or vinyl ester of indolebutyric acid as the acyl donor can further improve the esterification conversion rate. The lipase used in the reaction can be reused after filtration and recovery. The obtained 4-(1H-indole-3-yl)butyric acid-(2R)-2-[(2R)-3,4-dihydroxy-5-oxonyl-2,5-dihydrofuran-2-yl]-2-hydroxyethyl ester is a light yellow solid with a purity of not less than 97% as determined by high performance liquid chromatography. The ester bond is slowly hydrolyzed at a suitable rate in the aqueous phase of a neutral to slightly acidic culture medium, thereby continuously supplying indolebutyric acid and ascorbic acid during the rooting cycle of approximately 40 days. Compared to the situation where free indolebutyric acid dissolves once and free ascorbic acid is oxidized and inactivated within a few days, this ester can maintain a stable rooting signal and antioxidant environment throughout the entire rooting cycle. This is the material basis for its ability to relieve phenolic oxidation inhibition at the rooting end and improve the rooting rate and transplant survival rate.

[0022] Example 1 (Optimal conditions for intermediate values ​​of various hormones, complete process). This example provides a set of tissue culture rapid propagation media for superior clonal Pinus sylvestris and uses it to complete the entire propagation process. The media includes five functional media: starter medium, shoot cluster induction medium, shoot elongation subculture medium, rooting induction medium, and hardening substrate. The initiation medium was based on a modified DCR medium with an inorganic salt ion concentration of 65% of the standard DCR medium, containing 25 g / L sucrose, 7 g / L agar, 3 g / L polyvinylpyrrolidone, and 120 mg / L ascorbic acid, with a pH of 5.8. The shoot induction medium was based on DCR medium, containing 25 g / L sucrose, 7 g / L agar, 0.2 mg / L (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one obtained in Preparation Example 1, and 0.1 mg / L naphthaleneacetic acid, with a pH of 5.8. The shoot elongation subculture medium was based on DCR medium, containing 25 g / L sucrose, 7 g / L agar, and 1.0 g / L activated carbon, with a pH of 5.8. The rooting induction medium was based on half of the DCR medium with half the inorganic salt ion concentration, containing 25 g / L sucrose, 7 g / L agar, 0.2 mg / L (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one, and 0.1 mg / L naphthaleneacetic acid, with a pH of 5.8. The concentration of 4-(1H-indol-3-yl)butyric acid-(2R)-2-[(2R)-3,4-dihydroxy-5-oxoylide-2,5-dihydrofuran-2-yl]-2-hydroxyethyl ester obtained in Preparation Example 2 was 1.0 mg / L, and the pH was 5.8; the hardening substrate was composed of pine needle humus, perlite and vermiculite in a volume ratio of 4:1.5:1.5.

[0023] The general rules for preparing the above-mentioned culture media are as follows: Prepare inorganic salt stock solutions, organic stock solutions, and iron salt stock solutions of DCR or modified DCR according to the formula of each culture medium. Measure the required amounts according to the proportions, add sucrose and bring to a final volume, then add agar and heat to dissolve. Adjust the pH to 5.6-5.9 with 0.1 mol / L hydrochloric acid or 0.1 mol / L sodium hydroxide. After dispensing, autoclave at 121°C and 0.1 MPa for 20 min. (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one, naphthaleneacetic acid, 4-(1H-indol-3-yl)butyric acid-(2R)-2-[(2R)-3,4-dihydroxy-5-oxonyl-2,5-dihydrofuran-2-yl]-2-hydroxyethyl ester, and ascorbic acid are all prepared as stock solutions beforehand and sterilized at 0.22... Sterilization is achieved by filtration through a μm microporous membrane. The culture medium is added aseptically after being sterilized and cooled to approximately 50°C to avoid damage to its structure during autoclaving. Polyvinylpyrrolidone and activated carbon can be autoclaved together with the culture medium. The seedling hardening substrate is autoclaved at 121°C for 30 minutes and moderately rehydrated before use.

[0024] The collection and pretreatment of explants used in this embodiment are as follows: During the spring or autumn growing season of mature mother trees of superior clonal Pinus yunnanensis, healthy, disease-free, and semi-lignified young shoots of the current year were selected. After collection in the early morning, they were immediately placed in water containing a small amount of ascorbic acid to maintain moisture and brought back to the site. The needles were removed, and the young shoots were cut into 1.5–2.0 cm long stem segments with axillary buds. They were first soaked in a neutral detergent solution and rinsed with running water for 30 minutes, then subjected to aseptic disinfection. Semi-lignified young shoots have a higher regeneration capacity than fully lignified old branches, and a lower contamination and browning rate than non-lignified shoots, making them a suitable explant that balances regeneration capacity and initiation success rate.

[0025] The propagation method using the above-mentioned culture medium is as follows. First, take the current year's semi-lignified tender branches of mature mother trees of superior clonal Pinus simonii, cut them into stem segments with axillary buds, rinse them with running water, then treat them sequentially with 75% ethanol (v / v) for 30 s and 1.5% sodium hypochlorite (w / w) for 9 min, rinse them 5 times with sterile water, and then inoculate them into the starter culture medium. After culturing for 14 days, due to the synergistic adsorption and reduction of phenolic oxidation products released by the explants by polyvinylpyrrolidone and ascorbic acid in the starter culture medium, the browning rate of the explants is low and the proportion of surviving healthy explants is high. Secondly, healthy explants after induction were transferred to shoot clustering induction medium and cultured for 30 days to induce shoot clustering. During this period, (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one was slowly hydrolyzed to maintain a low and stable concentration of free 6-benzylaminopurine. It was also formulated with naphthaleneacetic acid in an appropriate ratio to regulate the balance of cytokinin and auxin, thereby achieving a high shoot clustering induction rate while inhibiting vitrification. Thirdly, the shoot clusters were cut and transferred to shoot elongation subculture medium for subculture. Activated carbon adsorbed the metabolic products accumulated during subculture, extending the subculture cycle and maintaining robust seedlings. Subsequently, when the seedlings elongated to 2-4 cm, they were pretreated by dark culture at 23-27°C for 7 days, and then transferred to rooting induction medium to induce rooting. During this period, 4-(1H-indole-3-yl)butyric acid-(2R)-2-[(2R)-3,4-dihydroxy-5-oxoylide-2,5-dihydrofuran-2-yl]-2-hydroxyethyl ester continuously released indolebutyric acid to provide rooting signals and released ascorbic acid to inhibit phenolic oxidation at the base of the seedlings, thus relieving rooting inhibition. Finally, the rooted seedlings were transplanted to a hardening substrate to complete acclimatization. Figure 1 The diagram illustrates the entire process from explant initiation to hardening and transplanting. Testing in this embodiment showed an explant browning rate of 12%, a shoot induction rate of 88%, an average of 5.2 shoots per explant, a vitrification rate of 6%, a rooting rate of 82%, and a transplant survival rate of 90%.

[0026] Throughout the entire process, it was observed that after explants were inoculated onto the starting medium, only slight browning was observed at the cut site, which stabilized within 2-3 days. No significant browning spread was observed in the medium, and no large-scale browning or death was observed. After being transferred to the shoot induction medium, green buds appeared at the axils and cut site after approximately 10 days, forming clusters of shoots by day 30. The buds were bright green with well-developed leaf needles, and no obvious transparent or water-soaked vitrified seedlings were observed. After being transferred to the shoot elongation subculture medium, the seedlings elongated rapidly and the stems became robust. The adsorption effect of activated carbon extended the subculture period to approximately 35 days without causing premature senescence. After the seedlings reached 2-4 cm in length and underwent a 7-day dark culture pretreatment, they were transferred to the rooting induction medium. Adventitious roots were induced from the base of the seedlings after approximately 30 days. The roots were white, numerous, and showed no significant browning or necrosis at the base. After transplanting to the hardening substrate, growth resumed within approximately 7 days, and new leaves emerged normally. At time d, the surviving plants grew uniformly, and their phenotypic traits matched those of the superior clonal mother trees, indicating that this invention maintains the genetic consistency of the clonal lines while achieving efficient propagation. The entire process, from explant inoculation to seedling hardening, takes approximately 120-140 days.

[0027] Example 2 (Concentration ratio of each hormone at the lower end). The only difference between this example and Example 1 is that the concentration of (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one in the shoot induction medium is 0.05 mg / L at the lower end, and naphthaleneacetic acid is 0.05 mg / L. The concentration of polyvinylpyrrolidone in the starting medium is 1 g / L, ascorbic acid is 50 mg / L, and the concentration of 4-(1H-indol-3-yl)butyric acid-(2R)-2-[(2R)-3,4-dihydroxy-5-oxonyl-2,5-dihydrofuran-2-yl]-2-hydroxyethyl ester in the rooting induction medium is 0.5 mg / L. The composition of the other media and the operation steps are the same as in Example 1. The results showed that in this embodiment, the explant browning rate was 12%, the shoot induction rate was 72%, the average number of shoots per explant was 3.5, the vitrification rate was 4%, the rooting rate was 78%, and the transplant survival rate was 86%. It can be seen that at the lower end concentration, the vitrification rate further decreased, but due to the low dose of cytokinin cumulatively released from the prodrug, the shoot induction rate and the number of shoots decreased accordingly.

[0028] The bud clusters obtained in this embodiment are robust and almost without vitrification. However, the number of bud clusters formed by a single explant is reduced compared to Example 1. This reflects that when the amount of sustained-release prodrug added is low, the cumulative release of free cytokinin in the culture medium is insufficient to fully stimulate the dense generation of buds. Therefore, the lower end concentration is more suitable for clones that are extremely sensitive to vitrification and can appropriately sacrifice the proliferation multiple in exchange for the quality of the seedlings.

[0029] Example 3 (Concentration ratio of each hormone at the upper end). The only difference between this example and Example 1 is that the concentration of (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one in the shoot induction medium is 0.5 mg / L at the upper end and 0.2 mg / L of naphthaleneacetic acid. The concentration of polyvinylpyrrolidone in the starting medium is 5 g / L and ascorbic acid is 200 mg / L. The concentration of 4-(1H-indol-3-yl)butyric acid-(2R)-2-[(2R)-3,4-dihydroxy-5-oxonyl-2,5-dihydrofuran-2-yl]-2-hydroxyethyl ester in the rooting induction medium is 2 mg / L. The composition of the other media and the operation steps are the same as in Example 1. The results showed that in this example, the explant browning rate was 12%, the shoot induction rate was 85%, the average number of shoots per explant was 5.0, the vitrification rate was 9%, the rooting rate was 80%, and the transplant survival rate was 88%. It is evident that at the upper limit concentration, the shoot induction rate was higher, but the instantaneous concentration of free 6-benzylaminopurine increased with the cumulative release, and the vitrification rate also increased slightly accordingly. Based on Examples 1 to 3, it can be seen that (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one can achieve a balance between high induction and low vitrification within the range of 0.05~0.5 mg / L, with the intermediate value of 0.2 mg / L being optimal.

[0030] The number of clustered shoots obtained in this embodiment is similar to that in Example 1, but a small number of seedling leaf needles are observed to be slightly vitrified in a semi-transparent state. This indicates that the instantaneous concentration of free cytokinin at the upper end of the concentration is close to the vitrification threshold. Therefore, the upper end concentration is more suitable for clones that require a higher proliferation rate and can be rejuvenated through subculture.

[0031] The basis for setting the numerical ranges of this invention is further explained below. For the concentration of (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one in the shoot induction medium, the cumulative release of cytokinin is low at the lower end of 0.05 mg / L, and the shoot induction rate is about 72%; at the middle value of 0.2 mg / L, the induction rate and vitrification rate reach the best balance, with an induction rate of about 88% and a vitrification rate of only about 6%; at the upper end of 0.5 mg / L, the induction rate is still high, but the vitrification rate rises to about 9%. Therefore, the concentration is limited to 0.05~0.5 mg / L, with 0.2 mg / L being preferred. For naphthaleneacetic acid (NAA) at 0.05–0.2 mg / L, it is combined with slow-release 6-benzylaminopurine to regulate the ratio of cytokinin to auxin. A low ratio results in insufficient differentiation of shoot clusters, while a high ratio easily induces excessive proliferation of basal callus tissue; therefore, 0.1 mg / L is preferred. For the inorganic salt ion concentration of the starting medium at 50%–80%, coniferous explants are sensitive to high inorganic salt ion concentrations; excessively high concentrations are detrimental to initiation, while excessively low concentrations result in insufficient nutrients; 65% is preferred. For polyvinylpyrrolidone (PVP) at 1–5 g / L and ascorbic acid (Ascorbic acid) at 50–200 mg / L in the starting medium, excessively low concentrations result in insufficient resistance to browning, while excessively high concentrations increase costs without further benefit; therefore, 3 g / L and 120 mg / L are preferred, respectively. For the rooting induction medium, the concentration of 4-(1H-indol-3-yl)butyric acid-(2R)-2-[(2R)-3,4-dihydroxy-5-oxonyl-2,5-dihydrofuran-2-yl]-2-hydroxyethyl ester is 0.5~2 mg / L, with 1.0 mg / L being preferred. For the volume ratio of pine needle humus, perlite and vermiculite in the hardening substrate, 4:1.5:1.5 is preferred, where the pine needle humus simulates the original seedbed of Pinus kesiao and provides an organic substrate with mycorrhizal affinity, while perlite and vermiculite ensure the aeration and water retention of the substrate.

[0032] Comparative Example 1 (using free 6-benzylaminopurine instead of the sustained-release prodrug). The only difference between this comparative example and Example 1 is that 0.2 mg / L of free 6-benzylaminopurine was used instead of (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one in the shoot induction medium. All other components and procedures were identical to Example 1. This comparative example omits the essential technical feature of the sustained-release prodrug in the independent claims to verify its necessity in resolving the trade-off between induction rate and vitrification rate. The results showed that the browning rate of the explants in this comparative example was 13%, the shoot induction rate was 86%, the average number of shoots per explant was 5.0, the vitrification rate was 38%, the rooting rate was 55%, and the transplant survival rate was 50%. It can be seen that at a similar shoot induction rate as in Example 1, the vitrification rate is as high as 38% because the instantaneous concentration of free 6-benzylaminopurine cannot be reduced. This leads to difficulties in rooting vitrified seedlings and a sharp drop in transplant survival rate. This demonstrates the irreplaceable nature of the characteristics of the sustained-release prodrug from both the underlying mechanism (instantaneous concentration leading to vitrification) and the molecular level (slow-release prodrug).

[0033] Comparative Example 2 (using a physical mixture of free 6-benzylaminopurine and free ferulic acid instead of the covalently coupled prodrug). This comparative example differs from Example 1 only in that the shoot induction medium contains a physical mixture of 0.11 mg / L free 6-benzylaminopurine and 0.10 mg / L free ferulic acid instead of an equimolar amount of (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one; all other components and procedures are identical to Example 1. The results showed that the browning rate of the explants in this comparative example was 12%, the shoot induction rate was 80%, the average number of shoots per explant was 4.6, the vitrification rate was 22%, the rooting rate was 70%, and the transplant survival rate was 74%. It is evident that although the glass transition rate of physical mixing decreased compared to Comparative Example 1 due to the antioxidant effect of ferulic acid, free ferulic acid was easily oxidized and rendered ineffective, and the instantaneous concentration of free 6-benzylaminopurine could not be continuously reduced. Its effect was significantly inferior to that of covalently coupled sustained-release prodrug, which confirms the necessity of the covalent coupling and controlled sustained-release design at the molecular level.

[0034] Comparative Example 3 (using free indolebutyric acid instead of dual-loaded slow-release auxin). The only difference between this comparative example and Example 1 is that 1.5 mg / L of free indolebutyric acid was used instead of 1.5 mg / L of 4-(1H-indole-3-yl)butyric acid-(2R)-2-[(2R)-3,4-dihydroxy-5-oxoylide-2,5-dihydrofuran-2-yl]-2-hydroxyethyl ester in the rooting induction medium. All other components and procedures were identical to Example 1. Due to the change only in the rooting process, the browning rate, induction rate, number of shoot clusters, and vitrification rate of this comparative example were essentially the same as in Example 1, at 12%, 88%, 5.2 shoots / explant, and 6%, respectively. However, the rooting rate was only 58%, and the transplant survival rate was only 62%. This indicates that in the absence of ascorbic acid leaving groups to continuously inhibit phenolic oxidation at the base of the seedling, the accumulation of phenolic oxides at the base inhibits adventitious root formation, thus confirming the necessity of the dual-load slow-release auxin feature from both the underlying mechanism and the molecular level at the rooting end.

[0035] Comparative Example 4 (using young seedling explants instead of mature clonal shoots). The only difference between this comparative example and Example 1 is that the shoot tips of young *Pinus kesiaoensis* seedlings were used instead of the semi-lignified clonal shoots of the superior clonal mother tree as explants. All other components and procedures were the same as in Example 1. Testing showed that the browning rate of the explants in this comparative example was 15%, the bud induction rate was 90%, the average number of buds per explant was 5.5, the vitrification rate was 7%, the rooting rate was 84%, and the transplant survival rate was 91%. It is evident that young seedlings have a slightly higher regeneration capacity due to their younger physiological age. However, the resulting regenerated plants originate from genetically differentiated offspring and cannot maintain the genetic consistency of a specific superior clonal line. This contradicts the purpose of this invention to preserve and propagate superior clonal lines, thus highlighting the necessity of using mature clonal shoots as explants from an application perspective.

[0036] Comparative Example 5 (starting medium without polyvinylpyrrolidone and ascorbic acid). This comparative example differs from Example 1 only in that the starting medium does not contain polyvinylpyrrolidone and ascorbic acid; all other components and procedures are the same as in Example 1. Testing revealed that the browning rate of explants in this comparative example was as high as 45%. Due to the large number of browning and death of explants after inoculation, the number of healthy explants capable of subsequent induction was significantly reduced, thus confirming the necessity of polyvinylpyrrolidone and ascorbic acid in adsorbing and inhibiting polyphenol oxidation products.

[0037] As can be seen from the comparison between Example 1 and Comparative Examples 1 to 5, the sustained-release cytokinin prodrug as defined in the independent claims of this invention, and the dual-load sustained-release auxin, anti-browning initiating component, and superior clonal mature shoot explants as defined in the dependent claims, respectively correspond to the decoupling of induction rate and vitrification rate at the induction end, the relief of phenolic oxidation inhibition at the rooting end, and the inhibition of browning at the initiating end and the maintenance of clonal genetic consistency. Removing any of these features would significantly degrade the technical effect of the corresponding step. This series of comparative examples corresponds one-to-one with each necessary technical feature, jointly verifying the indispensability of each technical feature of this invention from both the underlying mechanism and molecular design levels. It also shows that the technical effect of this invention is not a simple superposition of the effects of each component, but a synergistic result of the mutual promotion of the induction end cause and the rooting end effect.

[0038] Detection methods. The browning rate of explants was defined as the percentage of browned explants counted 14 days after inoculation out of the total number of inoculated explants; the shoot induction rate was the percentage of explants forming shoot clusters 30 days after induction out of the total number of healthy explants; the average number of shoot clusters was the average number of adventitious shoots formed by each successfully induced explant 30 days after induction; the vitrification rate was the percentage of seedlings exhibiting transparent, translucent, or water-soaked vitrification symptoms after subculturing elongation out of the total number of seedlings; the rooting rate was the percentage of seedlings forming adventitious roots 40 days after rooting induction out of the total number of inoculated seedlings; the transplant survival rate was the percentage of surviving plants transplanted to the hardening substrate 60 days after transplanting out of the total number of transplanted plants. Each treatment was repeated in triplicate, with at least 30 explants or seedlings inoculated in each replicate, and the results were averaged.

[0039] The test data of each treatment group were compared between groups by one-way ANOVA. The differences between Example 1 and Comparative Example 1 in vitrification rate, rooting rate and transplant survival rate, as well as the differences between Example 1 and Comparative Example 3 in rooting rate and transplant survival rate, were all significant. Figure 4 The induction rate and vitrification rate of shoot clusters in Example 1 and Comparative Example 1 were visually compared using a bar chart. Figure 5 The rooting rate and transplant survival rate of each embodiment and each comparative example are visually compared using bar charts. The two charts together reflect the technical effect of the present invention in significantly reducing the vitrification rate and thereby improving the rooting rate and transplant survival rate at the same induction level.

[0040] In addition, to address the issue of severe endophytic bacterial contamination in adult Pinus sylvestris explants, this invention employs a two-step disinfection process using ethanol and sodium hypochlorite in the surface disinfection stage, and maintains the health of the explants in the initiation culture medium using polyvinylpyrrolidone and ascorbic acid to indirectly reduce the contamination rate. Statistical analysis shows that the initiation contamination rate of explants can be controlled at a low level after the above treatment, thereby ensuring that a sufficient number of healthy explants are available for use in subsequent stages.

[0041] The detection data of each embodiment and comparative example are summarized in Table 1. To quantitatively characterize the nonlinear synergistic effect of the present invention, the effective propagation coefficient is defined as the product of the bud induction rate, (1 minus vitrification rate), rooting rate, and transplant survival rate. This coefficient comprehensively reflects the propagation efficiency of the entire chain from induction to seedling formation.

[0042]

[0043] Note: The data in Table 1 are the average of three replicates; due to the large number of browning and death of explants in the initial stage, there were too few healthy explants in the subsequent stages of Comparative Example 5, and the relevant indicators were not counted.

[0044] Data Analysis. As shown in Table 1, the core effect of this invention is the decoupling of the inherent trade-off between shoot induction rate and vitrification rate. Comparing Example 1 and Comparative Example 1: the shoot induction rates of both are comparable, at 88% and 86% respectively, but the vitrification rate of Example 1 is only 6%, while that of Comparative Example 1 is as high as 38%. That is, at the same induction level, the vitrification rate is reduced to about one-sixth by using a sustained-release cytokinin prodrug. This comparison shows that the determining factor for vitrification is not the cumulative dose of cytokinin, but its instantaneous free concentration; free 6-benzylaminopurine cannot suppress the instantaneous concentration while maintaining the induction dose, while (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one decouples the cumulative dose and instantaneous concentration through sustained release, thereby breaking the trade-off between induction rate and vitrification rate. Further comparison of Example 1 and Comparative Example 2 shows that although the physical mixing of equimolar amounts of free 6-benzylaminopurine and free ferulic acid reduced the glass transition rate to 22%, it was still significantly higher than the 6% in Example 1. This indicates that ferulic acid must be covalently coupled with cytokinin and released in a controlled manner with the hydrolysis of the prodrug in order to continuously exert its antioxidant and stable release effects.

[0045] The aforementioned effects exhibit a significant non-linear amplification in the effective propagation coefficient. Calculations show that the effective propagation coefficient of Example 1 is 0.88 × (1 - 0.06) × 0.82 × 0.90, approximately equal to 0.61; while that of Comparative Example 1 is 0.86 × (1 - 0.38) × 0.55 × 0.50, approximately equal to 0.15. Although the bud induction rate of Example 1 differs from that of Comparative Example 1 by only about 2 percentage points, the effective propagation coefficient of Example 1 is approximately 4.1 times that of Comparative Example 1 because the vitrification rate, rooting rate, and transplant survival rate are progressively amplified through a product. This indicates that the benefits of the slow-release prodrug suppressing the vitrification rate at the induction stage are amplified progressively along the rooting and transplanting stages through a product, producing a non-linear synergistic effect far exceeding the linear superposition of individual indicators. Furthermore, in Comparative Example 3, with all indicators at the induction end consistent with Example 1, the rooting rate decreased from 82% to 58% and the transplant survival rate decreased from 90% to 62% simply because the rooting induction medium was changed from dual-loaded slow-release auxin to free indolebutyric acid. This confirms the independent contribution of dual-loaded slow-release auxin in relieving phenol oxidation inhibition at the rooting end. Although the numerical indicators of Comparative Example 4 were similar to or even slightly higher than those of Example 1, its regenerated plants lost the genetic consistency of the superior clone, deviating from the purpose of this invention. Comparative Example 5, due to the lack of anti-browning components at the initiation end, saw the browning rate increase from 12% to 45%. The above comparative examples correspond one-to-one with the necessary technical features at the induction end, rooting end, explant source, and initiation end, respectively, jointly confirming the completeness of the technical solution of this invention and the indispensability of each feature.

[0046] Furthermore, comparing Examples 1, 2, and 3, it can be seen that as the concentration of (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one increased from 0.05 mg / L to 0.2 mg / L and then to 0.5 mg / L, the shoot induction rate increased from 72% to 88% and then slightly decreased to 85%, while the vitrification rate increased from 4% to 6% and then to 9%. This shows a trend of initial increase followed by stabilization of induction and monotonic increase of vitrification rate with concentration. This trend is consistent with the mechanism that the cumulative release of the sustained-release prodrug increases synchronously with the instantaneous free concentration as the amount added increases. The three concentration points together define the concentration window that balances high induction and low vitrification, with the median value of 0.2 mg / L being optimal, which is consistent with the calculation results of the effective reproduction coefficient mentioned above.

[0047] The core mechanism of this invention can be summarized as the molecular-level decoupling of two sets of coupled contradictions. First, at the induction end of shoot clusters, the cumulative effective dose of cytokinin, which is conventionally added in free form, is bound to the instantaneous free concentration. Vitrification is mainly caused by oxidative stress and water metabolism disorders caused by excessively high instantaneous free concentration. The (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one of this invention, by mimicking the natural mechanism of controlled storage and release of cytokinin through N9-position glycosylation in plants, decouples the cumulative dose from the instantaneous concentration, so that the former is sufficient to maintain induction and the latter is below the vitrification threshold. Furthermore, the ferulic acid released simultaneously by hydrolysis clears reactive oxygen species at the induction end. Secondly, at the rooting end, the accumulation of phenolic oxides at the base of pine tissue culture seedlings inhibits adventitious root formation. The hydrolysis of 4-(1H-indole-3-yl)butyric acid-(2R)-2-[(2R)-3,4-dihydroxy-5-oxonyl-2,5-dihydrofuran-2-yl]-2-hydroxyethyl ester of this invention releases indolebutyric acid, providing a continuous rooting signal. Simultaneously, it transforms ascorbic acid, acting as a leaving group, from an inert carrier into an active antioxidant, thus inhibiting basal phenolic oxidation in situ and relieving rooting inhibition. The slow-release prodrug at the induction end is the cause, and the dual-load auxin at the rooting end is the effect. Their mechanisms are orthogonal and complementary, promoting growth. Combined with polyvinylpyrrolidone and ascorbic acid at the initiation end and activated carbon at the subculture end, this constitutes a multi-stage, synergistic, end-to-end antioxidant and hormone regulation system.

[0048] Furthermore, the beneficial effects of this invention exhibit non-linear amplification because rapid tissue culture propagation is a chain-like process sequentially linked from induction, elongation, rooting to transplanting. The health status of the seedlings in each stage directly determines the success rate of the next. Reducing the vitrification rate at the induction end not only minimizes the loss of vitrified seedlings but also improves the overall quality of seedlings entering the elongation and rooting stages, thus amplifying the rooting rate and transplant survival rate simultaneously through a multiplicative approach. Conversely, if the vitrification rate at the induction end is too high, even with a high rate of bud induction, the number of healthy tissue culture seedlings that ultimately survive will be progressively reduced. This is the fundamental reason why this invention uses a molecular-level slow-release method to decouple the induction rate and vitrification rate at the induction end, thereby achieving an overall propagation efficiency improvement far exceeding the improvement of any single indicator across the entire chain.

[0049] Based on the aforementioned technical effects, the culture medium, preparation method, and application method of this invention can be adapted to various application scenarios. For forestry research institutes, it can be used for the breeding and regeneration system research of superior clones of Pinaceae plants, especially Pinus yunnanensis; for forestry production units, it can be used for the factory-scale seedling production of superior clones, and the resulting genetically consistent and robust tissue culture seedlings can be used for the construction of superior seed orchards and large-scale afforestation; for forest germplasm resource preservation institutions, it can be used to preserve the germplasm resources of superior individuals of Pinus yunnanensis through in vitro culture. In practical applications, those skilled in the art can make adaptive fine adjustments to the hormone concentration and substrate ratio within the numerical ranges defined in this invention, based on the genotypic differences of specific clones, without departing from the technical concept of this invention.

[0050] It should be further pointed out that there is a causal complementary relationship between the sustained-release cytokinin prodrug at the induction end and the dual-load sustained-release auxin at the rooting end of this invention: the induction end provides seedlings with normal lignification and healthy physiological state for the rooting stage by decoupling the induction rate and vitrification rate, which is the cause; the rooting end then efficiently transforms the above-mentioned healthy seedlings into transplantable rooted seedlings by relieving the inhibition of adventitious root formation by phenolic oxidation at the base of the seedling, which is the effect. The two act on different stages and address different coupling contradictions. Their mechanisms are orthogonal and cannot be substituted for each other, but they are connected and mutually promote each other throughout the entire process, jointly ensuring the efficient transformation from explant to seedling. This molecular-level sustained-release regulation design that runs through the induction end and the rooting end is the fundamental feature that distinguishes this invention from existing tissue culture technologies that only make local improvements at a single stage.

[0051] In summary, the technical contribution of this invention cannot be measured by the improvement of any single indicator, but should be evaluated by the effective propagation coefficient of the entire chain. Under the premise that the induction rate of clustered shoots is basically the same, this invention reduces the vitrification rate to about one-sixth at the induction end and amplifies it step by step along the rooting and transplanting stages, ultimately achieving an increase of about four times in the effective propagation coefficient. This nonlinear amplification is the core value brought about by the decoupling of the induction rate and vitrification rate of the slow-release cytokinin prodrug.

[0052] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements 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 rapid propagation medium for superior clonal lines of Pinus simonii, including a bud induction medium, characterized by: The shoot induction medium is based on DCR medium, which contains 20-30 g / L sucrose, 6-8 g / L agar, and a pH of 5.6-5.

9. The shoot induction medium contains a slow-release cytokinin prodrug (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one, which is a compound obtained by ferulic acid amidation of the N9 position of the purine ring of 6-benzylaminopurine. Its concentration in the shoot induction medium is 0.05-0.5 mg / L, and it is hydrolyzed in the aqueous phase of the shoot induction medium to release 6-benzylaminopurine and ferulic acid. The shoot induction medium also contains 0.05-0.2 mg / L naphthaleneacetic acid. mg / L.

2. The rapid propagation culture medium for superior clonal lines of Pinus simonii according to claim 1, characterized in that, The concentration of (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one in the shoot induction medium was 0.2 mg / L; the ferulic acid released by the hydrolysis of (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one inhibited the activity of explant polyphenol oxidase and scavenged reactive oxygen species.

3. The rapid propagation culture medium for superior clonal lines of Pinus simonii according to claim 1, characterized in that, The sustained-release cytokinin prodrug is a compound obtained by cycloazonamide reaction of cytokinin and phenolic acid. The cytokinin is selected from 6-benzylaminopurine, kinetin, and thiabendazole, and the phenolic acid is selected from any one or a combination of ferulic acid, caffeic acid, chlorogenic acid, and sinapic acid.

4. The rapid propagation culture medium for superior clonal lines of Pinus simonii according to claim 1, characterized in that, It also includes a starter culture medium, a shoot elongation subculture medium, a rooting induction medium, and a hardening substrate; the starter culture medium is based on a modified DCR medium with an inorganic salt ion concentration of 50%~80% of the standard DCR medium, and contains 1~5 g / L polyvinylpyrrolidone and 50~200 mg / L ascorbic acid; the shoot elongation subculture medium is based on the DCR medium and contains 0.5~2 g / L activated carbon; the rooting induction medium is based on half of the DCR medium with a halved inorganic salt ion concentration, and contains 0.5~2 g / L 4-(1H-indol-3-yl)butyric acid-(2R)-2-[(2R)-3,4-dihydroxy-5-oxoylide-2,5-dihydrofuran-2-yl]-2-hydroxyethyl ester. mg / L, the 4-(1H-indole-3-yl)butyric acid-(2R)-2-[(2R)-3,4-dihydroxy-5-oxonyl-2,5-dihydrofuran-2-yl]-2-hydroxyethyl ester is hydrolyzed to release indolebutyric acid and ascorbic acid; the seedling hardening substrate is composed of pine needle humus, perlite and vermiculite.

5. The rapid propagation culture medium for superior clonal lines of Pinus simonii according to claim 4, characterized in that, The concentration of the 4-(1H-indol-3-yl)butyric acid-(2R)-2-[(2R)-3,4-dihydroxy-5-oxoylide-2,5-dihydrofuran-2-yl]-2-hydroxyethyl ester in the rooting induction medium was 1.0 mg / L; the volume ratio of pine needle humus, perlite and vermiculite in the seedling hardening substrate was 3~5:1~2:1~2.

6. The method for preparing the tissue culture medium for rapid propagation of superior clonal lines of Pinus simonii as described in claim 1, characterized in that, The preparation steps of (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one and the culture medium preparation steps are included; the preparation of (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one includes: Step 1, ferulic acid is acetylated with acetic anhydride to obtain (2E)-3-(4-acetoxy-3-methoxyphenyl)prop-2-enoic acid; Step 2, 6-benzylaminopurine is deprotonated with a base and then reacted with (2E)-3-(4-acetoxy-3-methoxyphenyl)prop-2-enoic acid. The activated product is acylated at the N9 position of the purine ring to obtain acetic acid-4-[(1E)-3-[6-(benzylamino)purine-9-yl]-3-oxoylide-1-enyl]-2-methoxyphenyl ester; in step three, the acetyl group is removed by alkaline alcoholysis to obtain (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one; the culture medium preparation steps are as follows: (2E)-1-[6-(benzylamino)purine-9-yl]-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one and naphthaleneacetic acid are added to the basal culture medium and the volume is adjusted, the pH is adjusted, and sterilization is performed.

7. The preparation method according to claim 6, characterized in that, In step one, the molar ratio of ferulic acid to acetic anhydride is 1:2; in step two, the base is sodium hydride, the activation product is 4-O-acetylferuloyl chloride, and the molar ratio of 6-benzylaminopurine to (2E)-3-(4-acetoxy-3-methoxyphenyl)prop-2-enoic acid is 1:1.

2. The acylation is carried out in N,N-dimethylformamide at 0~25°C; in step three, the alkaline alcoholysis is performed with a methanol solution of potassium carbonate at 0~25°C.

8. The method of applying the tissue culture medium for rapid propagation of superior clonal Pinus simonii as described in claim 4, characterized in that, Includes the following steps: Step S1: Take semi-lignified tender branches from mature mother trees of superior clonal Pinus yunnanensis, disinfect them on the surface, and inoculate them into the initiation culture medium; Step S2: Transfer the healthy explants after initiation into the shoot induction culture medium to induce shoot clustering; Step S3: Cut the shoot clusters and transfer them into the shoot elongation subculture medium for subculture elongation; Step S4: After pretreatment by dark culture, transfer the elongated seedlings into the rooting induction culture medium to induce rooting; Step S5: Transplant the rooted seedlings into the hardening substrate to complete acclimatization.

9. The application method according to claim 8, characterized in that, The dark culture pretreatment in step S4 is to culture in the dark at 23~27°C for 5~10 days; in step S4, the seedlings that have grown to 2~4 cm are transferred into the rooting induction medium.

10. The application method according to claim 8, characterized in that, The surface disinfection in step S1 is to treat with 75% ethanol for 30 seconds and sodium hypochlorite for 8 to 10 minutes in sequence; the culture conditions in steps S2 to S3 are a temperature of 23 to 27°C, a light intensity of 1500 to 2500 lx, and a light exposure time of 10 to 12 h / d.

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