A method for inducing and culturing embryogenic callus from Pinus simonii

Abstract

This invention provides a method for inducing and culturing embryogenic callus from *Pinus kesiya*, relating to the field of biotechnology. The method includes: collecting male cones of *Pinus kesiya* and performing surface disinfection on the collected male cones; immersing the surface-disinfected male cones in a detoxification solution for detoxification; washing the detoxified male flower explants and drying them; removing the dried explants, separating the male gametophytes from the explants, and culturing them in the dark in a culture medium until callus is induced. This method uses male gametophytes of *Pinus kesiya* for explant cultivation. Anthers, as reproductive organs of plants, possess stable genetic information, which helps maintain the superior traits of *Pinus kesiya*. Using *Pinus kesiya* anthers as breeding material allows for early and stable segregation of offspring, improving the reliability and accuracy of selection. Furthermore, the callus induced by pollen can maintain the superior traits of the maternal parent, which is of great significance for subsequent propagation of superior plants.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for inducing and culturing embryogenic callus from Pinus simaoides. Background Technology

[0002] *Pinus simonii*, an evergreen tree belonging to the genus *Pinus* of the family Pinaceae, has become an important forest resource in my country due to its rapid growth, good wood quality, strong adaptability, and high resin yield. It has wide applications in various fields such as timber, pulp, and resin production. However, the breeding and growth cycle of *Pinus simonii* is relatively long, and conventional breeding methods cannot meet current needs for genetic improvement and germplasm innovation. Therefore, asexual reproduction technology is needed to achieve large-scale propagation of *Pinus simonii* and accelerate the breeding process. Somatic embryogenesis is a highly promising asexual reproduction method with advantages such as short cycle, high regeneration rate, and genetic stability. Somatic embryogenesis mainly includes four stages: induction and proliferation of embryogenic callus, induction and maturation of somatic embryos, and germination and regeneration of somatic embryos. Among these, the induction of embryogenic callus is a key prerequisite for somatic embryogenesis.

[0003] Currently, research on embryogenic callus induction in *Pinus simonii* is limited. Existing methods for inducing embryogenic callus in pine trees all use zygotic embryos as explants. However, using zygotic embryos for induction makes it difficult to leverage the advantages of haploid breeding, hindering the pine breeding process. Furthermore, zygotic embryos are genetically relatively homogeneous, and long-term use is detrimental to the selection and genetic improvement of superior pine varieties. The anthers of *Pinus simonii* carry diverse genetic material; inducing callus through anthers can broaden the genetic background and uncover more desirable traits. However, there is currently no research on embryogenic callus induction using *Pinus simonii* anthers, leaving a gap in methods for detoxifying anther explants.

[0004] Therefore, there is an urgent need to study a method for inducing embryogenic callus using the anthers of Pinus kesiya to overcome the shortcomings of existing asexual reproduction methods for Pinus kesiya. Summary of the Invention

[0005] To overcome the problems existing in related technologies, the purpose of this invention is to provide a method for inducing and culturing embryogenic callus of Pinus kesiya. This method uses Pinus kesiya anthers for explant cultivation. As the reproductive organ of plants, the anthers have stable genetic information, which helps to maintain the superior traits of Pinus kesiya. Using Pinus kesiya anthers as breeding material can achieve stable segregation of offspring as early as possible, improving the reliability and accuracy of selection. In addition, the callus induced by pollen can maintain the superior traits of the maternal parent, which is of great significance for the subsequent propagation of superior plants.

[0006] A method for inducing and culturing embryogenic callus from Pinus simonii includes:

[0007] Collect male flowers of Pinus simaoensis and disinfect the surface of the collected male flowers;

[0008] After surface disinfection, the male flowers of Pinus kesao-yomi were soaked in a detoxification solution for detoxification treatment.

[0009] Clean the explants of male flowers of *Pinus simaoensis* after detoxification and dry them.

[0010] After the water in the Pinus sylvestris explants were dried, the anthers of the explants were isolated and placed in a culture medium for dark culture until callus tissue was induced.

[0011] In a preferred embodiment of the present invention, the collection of male flowers of Pinus simonii includes:

[0012] Young male flowers of Pinus simaoensis that had not yet shed pollen were collected on a sunny day as explants;

[0013] The male flowers of the Simao pine were wrapped in soaked newspaper to preserve the explants.

[0014] In a preferred embodiment of the present invention, the step of immersing the surface-sterilized male flowers of *Pinus kesiaoensis* in a detoxification solution for detoxification treatment includes:

[0015] Soak the male flowers of Pinus sylvestris in alcohol for 30-60 seconds, then transfer them to mercuric chloride solution and soak for 5-20 minutes.

[0016] The detoxification solution includes any one of a 0.1% mercuric chloride solution, a 0.3% mercuric chloride solution, or a 10% sodium hypochlorite solution.

[0017] In a preferred embodiment of the present invention, the surface disinfection treatment of the collected male flowers further includes:

[0018] Place the male flowers of Pinus simonii in the preparation bottle, cover the bottle mouth with a layer of gauze and tie it tightly, rinse under tap water for 1 hour, soak in a 1% PVP-K30 solution for 1 hour, and rinse twice with sterile water.

[0019] In a preferred embodiment of the present invention, the step of cleaning and detoxifying the male flower explants of *Pinus simaoensis* and drying them includes:

[0020] Remove the anthers after soaking in mercuric chloride solution;

[0021] Wash 4-6 times with sterile water, then blot dry with sterile filter paper.

[0022] In a preferred embodiment of the present invention, the step of placing the anthers in a culture medium for dark culture until callus tissue is induced includes:

[0023] The anthers of Pinus simaoides were isolated, and the isolated anthers were inoculated into a culture medium;

[0024] Dark culture was performed in the culture room until callus tissue was induced;

[0025] The temperature in the culture room is 25±2℃; the humidity is 65%-75%.

[0026] In a preferred embodiment of the present invention

[0027] The culture medium includes DCR medium, and the DCR medium also contains the following added components: 2,4-D, TDZ, 6-BA (6-benzylaminopurine), KT (furanomethylaminopurine), and agar;

[0028] Each liter of DCR medium contains 0.2-0.4 mg of 2,4-D, 0.4-0.7 mg of TDZ, 0.3-0.5 mg of 6-BA, 0.5-1 g of KT, and 7.0-8.0 g of agar.

[0029] In a preferred embodiment of the present invention, the detoxification treatment of male flowers of Pinus kesao-yomi includes:

[0030] Explants were detoxified by immersing them in a mixed solution containing antiviral and antibacterial agents;

[0031] Among them, the antiviral agent is either ribavirin or ribavirin, and the antibacterial agent is either penicillin or streptomycin.

[0032] The present invention also provides a callus culture system for Pinus kesiya, which is used to implement the induction culture method of Pinus kesiya embryogenic callus as described above.

[0033] The beneficial effects of this invention are as follows:

[0034] This invention provides a method for inducing and culturing embryogenic callus from *Pinus kesiya*. The method includes: collecting male flowers of *Pinus kesiya* and performing surface disinfection on the collected male flowers; immersing the surface-disinfected male flowers in a detoxification solution for detoxification; washing the detoxified male flower explants and drying them; removing the dried explants, separating the anthers, and culturing the anthers in a culture medium in the dark until callus is induced. This method uses *Pinus kesiya* anthers for explant cultivation. Anthers, as reproductive organs of plants, possess stable genetic information, helping to maintain the superior traits of *Pinus kesiya*, and exhibit homozygous ploidy. Using *Pinus kesiya* anthers as breeding material allows for early and stable segregation of offspring, improving the reliability and accuracy of selection. Furthermore, the callus induced by pollen can maintain the superior traits of the maternal parent, which is beneficial for subsequent propagation of superior plants. Attached Figure Description

[0035] Figure 1 This is a flowchart of the method for inducing and culturing embryogenic callus of Pinus simaoides provided in the embodiments of this application;

[0036] Figure 2 A flowchart illustrating the detoxification process of Pinus simaoides pollen provided for embodiments of this application;

[0037] Figure 3 A schematic diagram of male flowers of Pinus simonii provided for embodiments of this application;

[0038] Figure 4 A schematic diagram of anther explants of Pinus simaoides provided for embodiments of this application;

[0039] Figure 5 A schematic diagram illustrating the induction of callus tissue by *Pinus simaoides* anthers in an embodiment of this application;

[0040] Figure 6 A schematic diagram of callus tissue of *Pinus sapiens* provided for embodiments of this application;

[0041] Figure 7 This is a schematic diagram of stained callus tissue of *Pinus sapiens* provided for an embodiment of this application. Detailed Implementation

[0042] Preferred embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While preferred embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0043] The terminology used in this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The singular forms “a,” “the,” and “the” as used in this invention and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0044] It should be understood that although the terms "first," "second," "third," etc., may be used in this invention to describe various information, this information should not be limited to these terms. These terms are only used to distinguish information of the same type from one another. For example, without departing from the scope of this invention, first information may also be referred to as second information, and similarly, second information may also be referred to as first information. Thus, features defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0045] Traditional seedling propagation of *Pinus kesiya* is through seedling cultivation, which is inefficient and results in unstable genetic traits, failing to meet the demand for the propagation of superior seedlings. Asexual reproduction can improve efficiency and enable large-scale propagation. However, existing traditional methods such as cuttings have low rooting rates and unstable grafting survival rates, making it difficult to meet the needs of large-scale *Pinus kesiya* propagation. Asexual reproduction through somatic embryogenesis (somatic embryogenesis) has advantages such as a short cycle, high regeneration rate, and genetic stability. Somatic embryogenesis mainly consists of four stages: induction of embryogenic callus, proliferation of embryogenic callus, induction and maturation of somatic embryos, and germination and regeneration of somatic embryos. The first step, induction of embryogenic callus, is a crucial prerequisite for somatic embryogenesis. Current methods use cones to cultivate *Pinus kesiya* explants, and all existing methods for inducing embryogenic callus in pine trees use zygotic embryos as explants. There is no research on inducing embryogenic callus using *Pinus kesiya* anthers, leaving a gap in methods for detoxifying anther explants.

[0046] Based on this, this application provides a method for inducing and culturing embryogenic callus from Pinus simaoides.

[0047] Example 1

[0048] like Figures 1-7 As shown in this embodiment, a method for inducing and culturing embryogenic callus from Pinus simonii is provided, comprising:

[0049] S100. Collect male flowers of Pinus simaoensis and disinfect the surface of the collected male flowers.

[0050] Specifically, the collection of male flowers of *Pinus simaoense* includes:

[0051] Young male flowers of Pinus simaoensis that had not yet shed pollen were collected on a sunny day as explants;

[0052] The male flowers of the Simao pine were wrapped in soaked newspaper to preserve the explants.

[0053] In a more specific implementation, young male flowers (male cones) that have not yet shed pollen are collected as explants during the peak flowering period of Pinus simaoensis, usually in mid-to-late February.

[0054] The anthers of young male flowers of *Pinus kesao-ko*, used as explants, possess a unique growth stage and physiological state, providing favorable conditions for callus induction. Cells in the anthers of young male flowers can dedifferentiate and regain differentiation capacity, thus forming callus. This callus has potential regenerative capacity, which is beneficial to subsequent plant growth. Using young male flowers without pollen shedding as explants for induction yields embryogenic callus with a high induction rate.

[0055] More preferably, to maintain the activity and quality of the anthers, collection should be carried out on sunny, dry days with moderate temperatures. In a more specific embodiment, the relative humidity of the day for collecting explants should be 60%-75%. Before collection, select robust, disease-free Pinus kesua plants with abundant flower spikes. Then collect the male explants of Pinus kesua, wrapping them in newspaper moistened with distilled water to maintain their activity.

[0056] In one specific embodiment, the surface disinfection treatment of the collected male flowers further includes:

[0057] Place the male flowers of Pinus simonii in the preparation bottle, cover the bottle mouth with a layer of gauze and tie it tightly, rinse under tap water for 1 hour, soak in a 1% PVP-K30 solution for 1 hour, and rinse twice with sterile water.

[0058] Specifically, a PVP-K30 solution needs to be prepared first. The preparation process is as follows: Dissolve an appropriate amount of PVP-K30 powder in distilled or deionized water, stirring until completely dissolved. It is crucial to ensure the PVP-K30 powder used is pure; therefore, the PVP-K30 used should be pharmaceutical-grade or food-grade to guarantee its purity and safety. During soaking, the film-forming and moisturizing properties of PVP-K30 help protect the anthers and reduce damage during subsequent operations. Rinsing the anthers twice with sterile water ensures the removal of excess PVP-K30 solution and any adhering impurities.

[0059] S200. After surface disinfection, the male flowers of Pinus kesao-yomi are soaked in a detoxification solution for detoxification treatment.

[0060] Specifically, the step of soaking the surface-sterilized male flowers of *Pinus kesao-ko* in a detoxification solution for detoxification treatment includes:

[0061] Soak the male flowers of Pinus sylvestris in alcohol for 30-60 seconds, then transfer them to mercuric chloride solution and soak for 5-20 minutes.

[0062] The detoxification solution includes any one of a 0.1% mercuric chloride solution, a 0.3% mercuric chloride solution, or a 10% sodium hypochlorite solution.

[0063] Specifically, the male flowers can be soaked in alcohol for 30s, 40s, 50s or 60s, and in mercuric chloride solution for 5min, 10min, 15min or 20min.

[0064] S300, clean the virus-free male flower explants of Pine simaoense and dry them;

[0065] Specifically, the male flower explants soaked in mercuric chloride solution were removed, rinsed 4-5 times with sterile water, and then dried with sterile filter paper.

[0066] S400. Remove the dried Pinus sylvestris explants, separate the anthers (male gametophytes) from the explants, and place the anthers in a culture medium for dark culture until callus tissue is induced.

[0067] Specifically, the male flowers of Pinus sylvestris var. spp., after being dried, were placed in sterile culture dishes. The anthers were separated using tweezers and dissecting needles and then inoculated into callus induction medium. Each bottle of medium was inoculated with 15 anthers and cultured in the dark in a culture room at (25±2)℃ until callus was induced.

[0068] The aforementioned method for inducing and culturing embryogenic callus from *Pinus kesiya* utilizes *Pinus kesiya* anthers for explant culture. Anthers, as reproductive organs of plants, possess stable genetic information, which helps maintain the superior traits of *Pinus kesiya* and exhibits homozygous ploidy. Using *Pinus kesiya* anthers as breeding material allows for early and stable segregation of offspring, improving the reliability and accuracy of selection. Furthermore, due to the low virus content in anthers, using anthers for explant culture helps reduce the difficulty of virus elimination and improves the effectiveness. The callus induced by pollen can maintain the superior traits of the maternal parent, which is beneficial for the subsequent propagation of superior plants.

[0069] In a preferred embodiment, the step of placing the anthers in a culture medium for dark culture until callus tissue is induced includes:

[0070] The anthers of Pinus simaoides were isolated, and the isolated anthers were inoculated into a culture medium;

[0071] Dark culture was performed in the culture room until callus tissue was induced;

[0072] The temperature in the culture room is 25±2℃; the humidity is 65%-75%.

[0073] Specifically, a temperature range of 25±2℃ helps maintain the activity of callus tissue and promotes its normal growth and differentiation. Appropriate humidity helps maintain the moisture level of the culture medium, providing the necessary growth environment for the callus tissue while avoiding the adverse effects of excessive dryness or moisture.

[0074] This embodiment also provides the specific composition of the culture medium, as follows: the culture medium includes DCR medium, and the DCR medium also contains the following added components: 2,4-D, TDZ, 6-BA (6-benzylaminopurine), KT (furanomethylaminopurine), and agar;

[0075] Each liter of DCR medium contains 0.2-0.4 mg of 2,4-D, 0.4-0.7 mg of TDZ, 0.3-0.5 mg of 6-BA, 0.5-1 g of KT, and 7.0-8.0 g of agar.

[0076] Specifically, the DCR culture medium of this application mainly includes macroelements, microelements, iron salts, and organic matter. Among them, macroelements include: ammonium nitrate: 400 mg / L; potassium nitrate: 340 mg / L; calcium nitrate tetrahydrate: 556 mg / L; potassium dihydrogen phosphate: 170 mg / L; magnesium sulfate heptahydrate: 370 mg / L; and calcium chloride dihydrate: 85 mg / L. Trace elements include: boric acid: 6.2 mg / L; manganese sulfate hydrate: 22.3 mg / L; zinc sulfate heptahydrate: 8.6 mg / L; copper sulfate pentahydrate: 0.25 mg / L; potassium iodide: 0.83 mg / L; cobalt chloride hexahydrate: 0.025 mg / L; lithium chloride: 0.025 mg / L; sodium molybdate dihydrate: 0.25 mg / L; iron salts include: ferrous sulfate heptahydrate: 27.8 mg / L; disodium EDTA: 37.3 mg / L; organic compounds include: thiamine hydrochloride (vitamin B1): 1.0 mg / L; pyridoxine hydrochloride (vitamin B6): 0.5 mg / L; niacin: 0.5 mg / L; glycine: 2.0 mg / L; inositol: 200 mg / L.

[0077] In addition, this application also adds 2,4-D, TDZ, 6-BA (6-benzylaminopurine), KT (furanomethylaminopurine), agar and other components to the DCR medium.

[0078] Each liter of DCR medium contains 0.2-0.4 mg of 2,4-D, 0.4-0.7 mg of TDZ, 0.3-0.5 mg of 6-BA, 0.5-1 g of KT, and 7.0-8.0 g of agar.

[0079] 2,4-D is 2,4-dichlorophenoxyacetic acid, used to promote callus formation.

[0080] TDZ (thiadiazophenylurea) promotes cell division in tissue culture.

[0081] In addition, sucrose is added to the culture medium. As a carbon source, sucrose can be added at a concentration of 30 g / L. During cell culture, sucrose is broken down into monosaccharides, providing the cells with essential carbon and energy. These monosaccharides can be further converted into energy substances such as ATP through cellular metabolic pathways, supporting cell growth and division. Sucrose also plays a role in regulating osmotic pressure in the culture medium. Appropriate osmotic pressure is crucial for maintaining normal cell physiological functions and morphology. The addition of sucrose helps simulate the osmotic conditions of cells in their natural environment, maintaining the water balance inside and outside the cell. The addition of sucrose also helps maintain the stability of the culture medium, preventing precipitation or stratification during long-term culture.

[0082] Agar: As a solidifying agent, the amount added depends on the desired hardness of the culture medium, typically between 7-8 g / L. Adding agar to the culture medium transforms the liquid medium into a solid or semi-solid state, which is beneficial for the fixation and support of plant tissues. For plants requiring tissue or cell culture, solid media provide a better growth environment. The gelling properties of agar give the culture medium a certain degree of structural stability. This stability is crucial for the growth and differentiation of plant tissues because it ensures that nutrients, hormones, and other additives in the culture medium remain uniformly distributed during culture, without being altered by flow or mixing. The gel-like structure of agar can also protect plant tissues from mechanical damage to some extent, while reducing the risk of contamination.

[0083] Furthermore, the detoxification treatment of the male flowers of *Pinus kesiaoensis* includes:

[0084] Explants were detoxified by immersing them in a mixed solution containing antiviral and antibacterial agents;

[0085] The antiviral agent is either ribavirin or amantadine, and the antibacterial agent is either penicillin or streptomycin. Penicillin and streptomycin have inhibitory effects on a variety of bacteria, including Gram-positive and Gram-negative bacteria. By eliminating bacteria on the surface of the anthers, penicillin or streptomycin treatment can significantly improve the health of the anthers and reduce the occurrence and spread of bacterial diseases.

[0086] Ribavirin, a broad-spectrum antiviral agent for plants, exhibits inhibitory activity against plant viruses. Virazole, similarly, is a highly effective antiviral agent, combining rapid and long-lasting effects to significantly prevent and treat various stubborn viral diseases that are difficult to cure with conventional agents. This application involves immersing anther explants in a solution containing ribavirin or virazole, which effectively removes viruses from the explants, thereby improving anther health and providing healthy, disease-free germplasm resources for subsequent propagation. Anther explants treated with ribavirin or virazole may show significantly enhanced growth vigor and reproductive capacity, facilitating larger-scale propagation and improving propagation efficiency.

[0087] Example 2

[0088] This embodiment provides a callus culture system for Pinus kesiya, which is used to implement the induction and culture method of Pinus kesiya embryogenic callus as described above.

[0089] The process of performing plant tissue culture of Pinus yunnanensis in this system is as follows:

[0090] Healthy male flower explants of Pinus sylvestris were collected, disinfected, and the anthers of the male flower explants were removed.

[0091] The anthers obtained from the dissection were inoculated into a culture medium containing specific nutrients and growth regulators to induce and culture callus tissue.

[0092] Example 3

[0093] This embodiment provides a detailed explanation of how to obtain callus tissue using Pinus simaoides anthers:

[0094] Select healthy, disease-free Pinus simonii plants and collect young male flowers that have not yet shed pollen on a sunny day as explants.

[0095] The male flowers of Pinus sylvestris were soaked in 75% alcohol for 30 seconds, and then soaked in 0.1% mercuric chloride for 5 minutes.

[0096] After soaking, the explants are removed, rinsed 4-5 times with sterile water, and then dried with sterile filter paper.

[0097] After disinfection, the male flowers of Pinus sylvestris were placed in sterile petri dishes, and the anthers were separated using tweezers and dissecting needles. They were then inoculated into callus induction medium, with 15 anthers inoculated into each bottle of medium. The culture was carried out in the dark in a culture room at (25±2)℃ until callus was induced.

[0098] Example 4

[0099] This embodiment provides a detailed explanation of how to obtain callus tissue using Pinus simaoides anthers:

[0100] Select healthy, disease-free Pinus simonii plants and collect young male flowers that have not yet shed pollen on a sunny day as explants.

[0101] The male flowers of Pinus sylvestris were soaked in 75% alcohol for 30 seconds, and then soaked in 0.1% mercuric chloride for 10 minutes.

[0102] After soaking, the explants are removed, rinsed 4-5 times with sterile water, and then dried with sterile filter paper.

[0103] After disinfection, the male flowers of Pinus sylvestris were placed in sterile petri dishes, and the anthers were separated using tweezers and dissecting needles. They were then inoculated into callus induction medium, with 15 anthers inoculated into each bottle of medium. The culture was carried out in the dark in a culture room at (25±2)℃ until callus was induced.

[0104] Example 5

[0105] This embodiment provides a detailed explanation of how to obtain callus tissue using Pinus simaoides anthers:

[0106] Select healthy, disease-free Pinus simonii plants and collect young male flowers that have not yet shed pollen on a sunny day as explants.

[0107] The male flowers of Pinus sylvestris were soaked in 75% alcohol for 30 seconds, and then soaked in 0.1% mercuric chloride for 15 minutes.

[0108] After soaking, the explants are removed, rinsed 4-5 times with sterile water, and then dried with sterile filter paper.

[0109] After disinfection, the male flowers of Pinus sylvestris were placed in sterile petri dishes, and the anthers were separated using tweezers and dissecting needles. They were then inoculated into callus induction medium, with 15 anthers inoculated into each bottle of medium. The culture was carried out in the dark in a culture room at (25±2)℃ until callus was induced.

[0110] Example 6

[0111] This embodiment provides a detailed explanation of how to obtain callus tissue using Pinus simaoides anthers:

[0112] Select healthy, disease-free Pinus simonii plants and collect young male flowers that have not yet shed pollen on a sunny day as explants.

[0113] The male flowers of Pinus sylvestris were soaked in 75% alcohol for 30 seconds, and then soaked in 0.1% mercuric chloride for 20 minutes.

[0114] After soaking, the explants are removed, rinsed 4-5 times with sterile water, and then dried with sterile filter paper.

[0115] After disinfection, the male flowers of Pinus sylvestris were placed in sterile petri dishes, and the anthers were separated using tweezers and dissecting needles. They were then inoculated into callus induction medium, with 15 anthers inoculated into each bottle of medium. The culture was carried out in the dark in a culture room at (25±2)℃ until callus was induced.

[0116] Example 7

[0117] This embodiment provides a detailed explanation of how to obtain callus tissue using Pinus simaoides anthers:

[0118] Select healthy, disease-free Pinus simonii plants and collect young male flowers that have not yet shed pollen on a sunny day as explants.

[0119] The male flowers of Pinus sylvestris were soaked in 75% alcohol for 1 minute, and then soaked in 0.1% mercuric chloride for 5 minutes.

[0120] After soaking, the explants are removed, rinsed 4-5 times with sterile water, and then dried with sterile filter paper.

[0121] After disinfection, the male flowers of Pinus sylvestris were placed in sterile petri dishes, and the anthers were separated using tweezers and dissecting needles. They were then inoculated into callus induction medium, with 15 anthers inoculated into each bottle of medium. The culture was carried out in the dark in a culture room at (25±2)℃ until callus was induced.

[0122] Example 8

[0123] This embodiment provides a detailed explanation of how to obtain callus tissue using Pinus simaoides anthers:

[0124] Select healthy, disease-free Pinus simonii plants and collect young male flowers that have not yet shed pollen on a sunny day as explants.

[0125] The male flowers of Pinus sylvestris were soaked in 75% alcohol for 1 minute, and then soaked in 0.1% mercuric chloride for 10 minutes.

[0126] After soaking, the explants are removed, rinsed 4-5 times with sterile water, and then dried with sterile filter paper.

[0127] After disinfection, the male flowers of Pinus sylvestris were placed in sterile petri dishes, and the anthers were separated using tweezers and dissecting needles. They were then inoculated into callus induction medium, with 15 anthers inoculated into each bottle of medium. The culture was carried out in the dark in a culture room at (25±2)℃ until callus was induced.

[0128] Example 9

[0129] This embodiment provides a detailed explanation of how to obtain callus tissue using Pinus simaoides anthers:

[0130] Select healthy, disease-free Pinus simonii plants and collect young male flowers that have not yet shed pollen on a sunny day as explants.

[0131] The male flowers of Pinus sylvestris were soaked in 75% alcohol for 1 minute, and then soaked in 0.1% mercuric chloride for 15 minutes.

[0132] After soaking, the explants are removed, rinsed 4-5 times with sterile water, and then dried with sterile filter paper.

[0133] After disinfection, the male flowers of Pinus sylvestris were placed in sterile petri dishes, and the anthers were separated using tweezers and dissecting needles. They were then inoculated into callus induction medium, with 15 anthers inoculated into each bottle of medium. The culture was carried out in the dark in a culture room at (25±2)℃ until callus was induced.

[0134] Example 10

[0135] This embodiment provides a detailed explanation of how to obtain callus tissue using Pinus simaoides anthers:

[0136] Select healthy, disease-free Pinus simonii plants and collect young male flowers that have not yet shed pollen on a sunny day as explants.

[0137] The male flowers of Pinus sylvestris were soaked in 75% alcohol for 1 minute, and then soaked in 0.1% mercuric chloride for 20 minutes.

[0138] After soaking, the explants are removed, rinsed 4-5 times with sterile water, and then dried with sterile filter paper.

[0139] After disinfection, the male flowers of Pinus sylvestris were placed in sterile petri dishes, and the anthers were separated using tweezers and dissecting needles. They were then inoculated into callus induction medium, with 15 anthers inoculated into each bottle of medium. The culture was carried out in the dark in a culture room at (25±2)℃ until callus was induced.

[0140] In Examples 3-10 of this application, the soaking time of male flowers of Pinus kesiao during the detoxification process was changed to obtain the effects of different detoxification methods on the contamination and survival of anther explants, as shown in Table 1.

[0141] Table 1. Effects of different virus elimination methods on anther explant contamination and survival.

[0142]

[0143] Table 1 shows that the alcohol disinfection time significantly affects the contamination rate of *Pinus kesiaoensis* anther explants. When the mercuric chloride disinfection time is 5 min, the highest contamination rate (66.67%) is observed when the alcohol disinfection time is 30 s. When the alcohol disinfection time is extended to 1 min, the contamination rate decreases to 37.78%. The concentration of mercuric chloride and the disinfection time significantly affect the survival rate of explants. The highest survival rate (84.44%) is observed when alcohol disinfection is performed for 30 s followed by 0.1% mercuric chloride disinfection for 10 min. The lowest survival rate (36.67%) is observed when alcohol disinfection is performed for 30 s followed by 0.3% mercuric chloride disinfection for 15 min. Therefore, the disinfection combination of 30 s of alcohol and 10 min of 0.1% mercuric chloride is most effective for disinfecting *Pinus kesiaoensis* anther explants. Furthermore, the combined disinfection method of 30 s of 75% alcohol and 10 min of 0.1% mercuric chloride achieves a good detoxification effect and a high anther survival rate (84.44%) with a contamination rate of 0%. This method can be used to obtain sterile anthers and successfully induce callus tissue.

[0144] In addition, this application also uses sodium hypochlorite and alcohol to disinfect the explants of Pinus kesao-yomi flowers, as detailed below:

[0145] Table 2. Effects of sodium hypochlorite on explant detoxification

[0146]

[0147] Table 2 shows that the combined use of sodium hypochlorite and alcohol has a poor disinfection effect on the explants of *Pinus kesiaoensis*, with an explant survival rate of 0% and high contamination rates of 81.11% and 77.78%, respectively. When mercuric chloride is used in combination with alcohol, the contamination rate is 0%, and the explant survival rate can reach as high as 84.44%. Therefore, sodium hypochlorite is not suitable for the disinfection of *Pinus kesiaoensis* explants.

[0148] Example 11

[0149] This embodiment investigates the effects of different basic culture media types on the induction of embryogenic callus from *Pinus yew*, as well as the effects of different hormone types and concentrations, and biotin, on the induction of embryogenic callus from *Pinus yew* under the same culture medium. Details are as follows:

[0150] (1) Effects of different basic culture media types on the induction of embryogenic callus from Pinus simonii:

[0151] Table 3. Effects of different basic culture media on the induction of embryogenic callus from Pinus simaoense anthers.

[0152]

[0153]

[0154] Table 3 shows that the type of basal culture medium has a significant impact on the induction of embryogenic callus from *Pinus kesiya* anthers. Embryogenic callus could not be induced in WPM and 1 / 2 MS basal media, while the induction rate in 1 / 2 DCR and LP basal media was extremely low, at 2.67%. DCR medium showed the best effect on embryogenic callus in *Pinus kesiya* anthers, with an induction rate reaching 12.00%.

[0155] (2) Effects of different hormones on the induction of embryogenic callus from Pinus sylvestris:

[0156] Table 4. Effects of different hormones on the induction of embryogenic callus from Pinus simonii.

[0157]

[0158] Table 4 shows that different hormone types and concentrations significantly affected the induction of embryogenic callus in *Pinus kesiya*. The lowest embryogenic callus induction rate was observed in medium 1 (4.00%), while the highest rates were observed in media 10 and 11 (14.67%). Specifically, the combined use of 2,4-D, KT, and TDZ significantly improved the induction of embryogenic callus compared to 2,4-D, 6-BA, and TDZ alone. A 2.0 mg / L concentration of 2,4-D resulted in a higher induction rate than a 1.0 mg / L concentration. A 1.0 mg / L concentration of TDZ also resulted in a relatively high callus rate. A 0.5 mg / L concentration of KT consistently showed high induction rates, for example, media 10, 11, and 12 had induction rates of 10.67%, 14.67%, and 10.67%, respectively. In summary, medium 11 with DCR + 2,4-D 2.0 mg / L + TDZ 1.0 mg / L + KT 1.0 mg / L is the optimal medium for inducing embryogenic callus in Pinus kesiya.

[0159] (3) Effects of different biotin on the induction of embryogenic callus from Pinus simonii:

[0160] Table 5. Effects of biotin on embryogenic callus induction from Pinus simaoense anthers.

[0161]

[0162] Table 5 shows that biotin promotes the induction of embryogenic callus from *Pinus simaoides* anthers. The induction rate of embryogenic callus increases with increasing biotin concentration. When the biotin concentration is 0.75 mg / L, the induction rates of embryogenic callus in media 15 and 18 are 22.67% and 25.33%, respectively. Further analysis revealed that the combined use of biotin and KT is more effective than that of biotin and 6-BA. With a constant biotin concentration, the induction rate of embryogenic callus when used in combination with KT is consistently higher than when used in combination with 6-BA. The induction rates of embryogenic callus in media 16, 17, and 18 are higher than those in media 13, 14, and 15, respectively. The biotin used in this application is Biotin.

[0163] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for inducing embryogenic callus of Pinus massoniana, characterized by, include: Collect male flowers of Pinus simaoensis and disinfect the surface of the collected male flowers; After surface disinfection, the male flowers of Pinus kesao-yomi were soaked in a detoxification solution for detoxification treatment. Clean the explants of male flowers of *Pinus simaoensis* after detoxification and dry them. After the water was absorbed, the explants of Pinus kesao-pina were removed, the anthers of the explants were isolated, and the anthers were placed in a culture medium and cultured in the dark until callus tissue was induced. The process of placing the anthers in a culture medium and culturing them in the dark until callus tissue is induced includes: The anthers of Pinus simaoides were isolated, and the isolated anthers were inoculated into a culture medium; Dark culture was performed in the culture room until callus tissue was induced; The temperature in the culture room was 25±2℃; the humidity was 65%-75%. The culture medium uses DCR as the basic medium, wherein each liter of DCR medium contains 2.0 mg of 2,4-D, 1.0 mg of TDZ, 0.5 mg of KT, 0.75 mg of biotin, and 7.0 g-8.0 g of agar.

2. The method for inducing and culturing embryogenic callus of *Pinus simaoides* according to claim 1, characterized in that: The collection of male flowers of Pinus simaoides includes: Young male flowers of Pinus simaoensis that had not yet shed pollen were collected on a sunny day as explants; The male flowers of the Simao pine were wrapped in soaked newspaper to preserve the explants.

3. The method for inducing and culturing embryogenic callus of *Pinus simaoides* according to claim 1, characterized in that: The step of immersing the surface-sterilized male flowers of Pinus kesao-yima into a detoxification solution for detoxification treatment includes: Soak the male flowers of Pinus sylvestris in alcohol for 30-60 seconds, then transfer them to a detoxification solution and soak for 5-20 minutes. The detoxification solution includes any one of the following: a 0.1% mercuric chloride solution, a 0.3% mercuric chloride solution, or a 10% sodium hypochlorite solution.

4. The method for inducing and culturing embryogenic callus of *Pinus simaoides* according to claim 1, characterized in that: The surface disinfection treatment of the collected male flowers also includes: Place the male flowers of Pinus simaoensis in the preparation bottle, cover the bottle mouth with a layer of gauze and tie it tightly, rinse under tap water for 1 hour, soak in a 1% PVP-K30 solution for 1 hour, and rinse twice with sterile water.

5. The method for inducing and culturing embryogenic callus of *Pinus simaoides* according to claim 3, characterized in that: The process of cleaning and detoxifying the male flower explants of *Pinus simaoensis* and drying them includes: Remove the anthers after soaking in mercuric chloride solution; Wash 4-6 times with sterile water, then blot dry with sterile filter paper.

6. The method for inducing and culturing embryogenic callus of *Pinus simaoides* according to claim 1, characterized in that: The detoxification treatment of male flowers of Pinus kesao-yomi includes: Explants were detoxified by immersing them in a mixed solution containing antiviral and antibacterial agents; Among them, the antiviral agent is either ribavirin or ribavirin, and the antibacterial agent is either penicillin or streptomycin.

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