Induction culture method of pinus khasys embryogenic callus

Abstract

The invention provides an induction culture method for embryonic calluses of pinus khasys, and relates to the technical field of biologication.The method comprises the steps that male flowers of the pinus khasys are collected, and the collected male flowers are subjected to surface disinfection treatment; soaking the male flowers of the pinus khasys subjected to surface disinfection in a detoxification solution, and performing detoxification treatment; cleaning the detoxified male flower explant of pinus khasys, and absorbing moisture; and taking out the explant of the pinus khasys after moisture absorption, separating out a male gametophyte of the explant, and placing the male gametophyte in a culture medium for dark culture until callus is induced. According to the method, the male gametophyte of the pinus khasys is adopted for explant cultivation of the pinus khasys, anther serves as a reproductive organ of the plant, genetic information is stable, and the good characters of the pinus khasys can be kept; by selecting the pinus kesiya anther as a breeding material, stable separation of offspring can be realized as early as possible, and the reliability and accuracy of selection are improved; in addition, the calluses induced by the pollen can maintain the excellent characters of the female parent, and have important significance on subsequent superior plant propagation.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an induction and culture method for embryonic callus of Pinus koraiensis. Background Art

[0002] Simiao pine is an evergreen tree of the genus Pinus in the Pinaceae family. Due to its fast growth rate, good material, strong adaptability and high resin production, Simiao pine has become an important forest resource in my country and has a wide range of application values ​​in many fields such as wood, pulp, and rosin. The breeding and growth cycle of Simiao pine is relatively long, and conventional breeding methods cannot meet the current needs of genetic improvement and germplasm innovation. Therefore, it is necessary to use asexual reproduction technology to achieve large-scale breeding of Simiao pine and accelerate the breeding process. Somatic embryogenesis (somatic embryogenesis) is a highly potential asexual reproduction method with the advantages of short cycle, high regeneration rate and genetic stability. Somatic embryogenesis mainly includes four stages: induction and proliferation of embryonic callus, induction and maturation of somatic embryos, and germination and regeneration of somatic embryos. Among them, the induction of embryonic callus is the key prerequisite for somatic embryogenesis.

[0003] At present, there are few studies on the induction of embryonic callus of Pinus truncatula. The existing pine trees all use zygotic embryos as explants for embryonic callus induction. However, the use of zygotic embryos for induction makes it difficult to give full play to the advantages of haploid breeding, which hinders the pine breeding process. In addition, zygotic embryos are relatively simple in genetics, and long-term use is not conducive to the selection and genetic improvement of excellent pine varieties. The genetic material carried by the anthers of Pinus truncatula is diverse. Inducing callus through anthers can broaden the genetic background and tap into more excellent traits. However, there is currently no research on the induction of embryonic callus using the anthers of Pinus truncatula, which makes the detoxification method of anther explants also blank.

[0004] Therefore, it is urgent to study a method for inducing embryonic callus using anthers of Pinus korshinskii to overcome the defects of the existing asexual reproduction of Pinus korshinskii. Summary of the invention

[0005] In order to overcome the problems existing in the related art, the purpose of the present invention is to provide an induction and culture method for embryonic callus tissue of Simiao pine, which adopts Simiao pine anthers to cultivate Simiao pine explants. Anthers are reproductive organs of plants, and their genetic information is stable, which helps to maintain the excellent traits of Simiao pine. Selecting Simiao pine anthers as breeding materials can realize the stable separation of offspring as early as possible, and improve the reliability and accuracy of selection. In addition, the callus tissue induced by pollen can maintain the excellent traits of the mother plant, which is of great significance for the subsequent propagation of superior plants.

[0006] A method for inducing and culturing embryonic callus of Pinus koraiensis, comprising:

[0007] Collecting male flowers of Pinus koraiensis and performing surface disinfection on the collected male flowers;

[0008] The male flowers of Pinus koraiensis with disinfected surfaces are immersed in a detoxification solution for detoxification treatment;

[0009] The virus-free male flower explants of Simiao pine were washed and dried;

[0010] The explants of Pinus koraiensis were taken out after being dried, and the anthers of the explants were separated and placed in a culture medium for dark culture until callus tissue was induced.

[0011] In a preferred technical solution of the present invention, the step of collecting male flowers of Pinus kornelengensis comprises:

[0012] On a sunny day, young male flowers of Pinus sphenanthera that had not yet shed pollen were collected as explants;

[0013] Wrap the male flowers of Pinus magnoliae with wet newspaper to preserve the explants.

[0014] In a preferred technical solution of the present invention, the step of soaking the surface-disinfected male flowers of Pinus kornelengensis in a detoxification solution for detoxification comprises:

[0015] Soak the male flowers of Pinus kokonisii in alcohol for 30s-60s, then transfer the male flowers of Pinus kokonisii to mercuric chloride solution and soak for 5min-20min;

[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 technical solution of the present invention, the surface disinfection of the collected male flowers further comprises:

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

[0019] In a preferred technical solution of the present invention, the steps of washing the detoxified male flower explants of Pinus magnoliae and drying the water include:

[0020] Take out the anthers soaked in mercuric chloride solution;

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

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

[0023] Separate the anthers of Pinus kesiya var. langbianensis, and inoculate the separated anthers into the culture medium;

[0024] Conduct dark culture in the culture room until callus is induced;

[0025] Among them, the temperature of the culture room is 25±2°C; the humidity is 65%-75%,

[0026] In a preferred technical solution of the present invention,

[0027] The culture medium includes DCR medium, and the following additional components are also present in the DCR medium: 2,4-D, TDZ, 6-BA (6-benzylaminopurine), KT (kinetin), agar;

[0028] Among them, in each liter of DCR medium, 0.2 mg - 0.4 mg of 2,4-D is added, 0.4 mg - 0.7 mg of TDZ is added, 0.3 mg - 0.5 mg of 6-BA is added, 0.5 g - 1 g of KT is added, and 7.0 g - 8.0 g of agar is added.

[0029] In a preferred technical solution of the present invention, the detoxification treatment of the male flowers of Pinus kesiya var. langbianensis includes:

[0030] Soak the explants in a mixed solution containing an antiviral agent and an antibacterial agent for detoxification;

[0031] Among them, the antiviral agent is any one of ribavirin and moroxydine hydrochloride, and the antibacterial agent is any one of penicillin and streptomycin.

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

[0033] The beneficial effects of the present invention are:

[0034] The present invention provides an induction culture method for the embryogenic callus of Pinus kesiya var. langbianensis, which includes: collecting the male flowers of Pinus kesiya var. langbianensis and performing surface disinfection treatment on the collected male flowers; putting the surface-disinfected male flowers of Pinus kesiya var. langbianensis into a detoxification solution for soaking for detoxification treatment; washing the explants of the male flowers of Pinus kesiya var. langbianensis after detoxification and sucking dry the moisture; taking out the explants of Pinus kesiya var. langbianensis after sucking dry the moisture, separating the anthers of the explants and placing the anthers in the culture medium for dark culture until callus is induced. This method uses the anthers of Pinus kesiya var. langbianensis for the cultivation of the explants of Pinus kesiya var. langbianensis. As the reproductive organs of plants, anthers have stable genetic information, which helps to maintain the excellent traits of Pinus kesiya var. langbianensis and has the characteristics of homozygous ploidy. Selecting the anthers of Pinus kesiya var. langbianensis as breeding materials 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 excellent traits of the female parent, which has a positive significance for the subsequent propagation of excellent strains. Brief Description of the Drawings

[0035] Figure 1 is a flowchart of the method for inducing and culturing embryogenic callus of Pinus kesiya var. langbianensis provided by an embodiment of the present application;

[0036] Figure 2 is a flowchart of the detoxification treatment operation of Pinus kesiya var. langbianensis anthers provided by an embodiment of the present application;

[0037] Figure 3 is a schematic diagram of male flowers of Pinus kesiya var. langbianensis provided by an embodiment of the present application;

[0038] Figure 4 is a schematic diagram of an explant of Pinus kesiya var. langbianensis anthers provided by an embodiment of the present application;

[0039] Figure 5 is a schematic diagram of callus induced from Pinus kesiya var. langbianensis anthers provided by an embodiment of the present application;

[0040] Figure 6 is a schematic diagram of callus of Pinus kesiya var. langbianensis provided by an embodiment of the present application;

[0041] Figure 7 is a schematic diagram of the stained callus of Pinus kesiya var. langbianensis provided by an embodiment of the present application. Detailed Description of the Embodiments

[0042] The preferred embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although the preferred embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to make the present invention more thorough and complete, and to fully convey the scope of the present invention to those skilled in the art.

[0043] The terms used in the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The singular forms "a", "the", and "the" used in the present invention and the appended claims are also intended to include the plural forms unless the context clearly dictates otherwise. It should also be understood that the term "and / or" as used herein refers to and encompasses any and 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 the present invention to describe various information, such information should not be limited to these terms. These terms are only used to distinguish the same type of information from each other. For example, without departing from the scope of the present invention, the first information may also be referred to as the second information, and similarly, the second information may also be referred to as the first information. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. In the description of the present invention, the meaning of "a plurality" is two or more, unless otherwise specifically defined.

[0045] The traditional seedling raising method of Pinus kesiya var. langbianensis is the reproduction of seedlings from seeds. This method has low seedling raising efficiency and unstable genetic traits of seedlings, and cannot meet the needs of the propagation of superior strains. By means of asexual reproduction, the seedling raising efficiency can be improved and large-scale breeding can be achieved. However, in the existing traditional seedling raising methods, such as the rooting rate of cutting technology is relatively low, and the survival rate of grafting is unstable. Therefore, it is difficult to meet the needs of large-scale breeding of Pinus kesiya var. langbianensis. Asexual reproduction by somatic embryogenesis has the advantages of short cycle, high regeneration rate and genetic stability. Somatic embryogenesis is mainly divided into four stages: induction of embryogenic callus, proliferation of embryogenic callus, induction and maturation of somatic embryos, and germination and regeneration of somatic embryos. Among them, the first step, the induction of embryogenic callus, is an important prerequisite for somatic embryogenesis. In the existing method of culturing explants of Pinus kesiya var. langbianensis using cones, when inducing embryogenic callus in existing pine trees, zygotic embryos are all selected as explants, and there is no research on inducing embryogenic callus using anthers of Pinus kesiya var. langbianensis, which makes the detoxification method of anther explants also in a blank state.

[0046] Based on this, the present application provides a method for inducing and culturing embryogenic callus of Pinus kesiya var. langbianensis.

[0047] Example 1

[0048] As Figures 1 - 7 shown, this example provides a method for inducing and culturing embryogenic callus of Pinus kesiya var. langbianensis, including:

[0049] S100. Collect male flowers of Pinus kesiya var. langbianensis and perform surface disinfection treatment on the collected male flowers;

[0050] Specifically, the collection of male flowers of Pinus kesiya var. langbianensis includes:

[0051] Collect young and tender male flowers of Pinus kesiya var. langbianensis that have not shed pollen on a sunny day as explants;

[0052] And wrap the male flowers of Pinus kesiya var. langbianensis with wet newspapers to preserve the explants.

[0053] In a more specific embodiment, young and tender male flowers (male cones) that have not shed pollen are collected as explants during the full bloom period of Pinus kesiya var. langbianensis, usually in the middle and late February.

[0054] The anthers of young male flowers of Pinus kesiya var. langbianensis serve as explants, which have special growth stages and physiological states, providing good conditions for the induction of callus. The cells in the anthers of young male flowers can dedifferentiate and regain the ability to differentiate, and then form callus. This kind of callus has potential regenerative ability, which is beneficial to subsequent plant growth. Using young male flowers that have not shed pollen as explants for induction can obtain embryogenic callus with a high induction rate.

[0055] Preferably, in order to maintain the activity and quality of the anthers, choose a sunny, dry and moderately warm weather for collection. In a more specific embodiment, the relative humidity of the weather for collecting explants is preferably 60%-75%. Before collection, first select Pinus kesiya var. langbianensis plants that are robust in growth, free from pests and diseases and have plump flower spikes. Then collect the male flower explants of Pinus kesiya var. langbianensis and wrap the male flowers of Pinus kesiya var. langbianensis with newspaper moistened with distilled water to maintain the activity of the explants.

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

[0057] Place the male flowers of Pinus kesiya var. langbianensis in a culture bottle, cover a layer of gauze on the bottle mouth and tie it tightly, rinse under tap water for 1 hour, soak in a 1% pvp-K30 solution for 1 hour, and wash with sterile water twice.

[0058] Specifically, it is necessary to prepare the pvp-K30 solution first. The preparation process is as follows: Dissolve an appropriate amount of PVP-K30 powder in distilled water or deionized water and stir evenly until completely dissolved. It is necessary to ensure that the PVP-K30 powder used in the preparation process is pure, so the PVP-K30 used should be of pharmaceutical grade or food grade to ensure its purity and safety. During the soaking process, the film-forming property and moisture retention property of PVP-K30 help to protect the anthers and reduce their damage in subsequent operations. Washing the anthers twice with sterile water is to ensure the removal of excess pvp-K30 solution and possible attached impurities.

[0059] S200. Put the surface-disinfected male flowers of Pinus kesiya var. langbianensis into a detoxification solution and soak them for detoxification treatment;

[0060] Specifically, the step of putting the surface-disinfected male flowers of Pinus kesiya var. langbianensis into a detoxification solution and soaking them for detoxification treatment specifically includes:

[0061] Put the male flowers of Pinus kesiya var. langbianensis into alcohol and soak for 30s - 60s, and then transfer the male flowers of Pinus kesiya var. langbianensis into a mercuric chloride solution and soak for 5min - 20min;

[0062] Wherein 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 soaking time of male flowers in alcohol can be 30 s, 40 s, 50 s or 60 s, and the soaking time in mercuric chloride solution can be 5 min, 10 min, 15 min, 20 min.

[0064] S300. Wash the explants of male flowers of Pinus szemois after detoxification and blot dry the water.

[0065] Specifically, take out the soaked male flower explants from the mercuric chloride solution, wash them 4 - 5 times with sterile water, and then blot dry the water with sterile filter paper.

[0066] S400. Take out the explants of Pinus szemois after blotting dry the water, separate the anthers (male gametophytes) of the explants and place the anthers in the culture medium for dark culture until callus is induced.

[0067] Specifically, place the male flowers of Pinus szemois after blotting dry the water in a sterile culture dish, use forceps and dissecting needles to separate the anthers, and then inoculate them into the callus induction medium. 15 anthers are inoculated into each bottle of the medium, and dark culture is carried out in an incubator at (25 ± 2) °C until callus is induced.

[0068] The above - mentioned method for inducing and culturing embryogenic callus of Pinus szemois uses the anthers of Pinus szemois flowers for culturing the explants of Pinus szemois. As the reproductive organs of plants, anthers have stable genetic information, which helps to maintain the excellent traits of Pinus szemois and has the characteristics of homozygous ploidy. Selecting the anthers of Pinus szemois flowers as breeding materials can achieve stable segregation of offspring as early as possible, improve the reliability and accuracy of selection. In addition, because the anthers have a low virus content, using anthers for culturing explants helps to reduce the difficulty of detoxification and improve the detoxification effect. The callus induced from pollen can maintain the excellent traits of the female parent, which has a positive significance for the subsequent propagation of excellent strains.

[0069] In a better implementation manner, the step of placing the anthers in the culture medium for dark culture until callus is induced includes:

[0070] Separate the anthers of Pinus szemois flowers and inoculate the separated anthers into the culture medium;

[0071] Carry out dark culture in the incubator until callus is induced;

[0072] where the temperature of the incubator is 25 ± 2 °C; the humidity is 65% - 75%.

[0073] Specifically, the temperature range of 25 ± 2 °C helps to maintain the activity of callus, promote its normal growth and differentiation. Appropriate humidity helps to maintain the moisture of the culture medium, provides a necessary growth environment for callus, and at the same time avoids the adverse effects caused by over - dryness or over - wetness on callus.

[0074] This embodiment also provides the specific composition of the culture medium, which is as follows: The culture medium includes DCR medium, and the following additives are also included in the DCR medium: 2,4-D, TDZ, 6-BA (6-benzylaminopurine), KT (kinetin), and agar.

[0075] Among them, in each liter of DCR medium, 0.2 mg - 0.4 mg of 2,4-D is added, 0.4 mg - 0.7 mg of TDZ is added, 0.3 mg - 0.5 mg of 6-BA is added, 0.5 g - 1 g of KT is added, and 7.0 g - 8.0 g of agar is added.

[0076] Specifically, the DCR medium of this application mainly includes macronutrients, micronutrients, iron salts, and organic substances. Among them, the macronutrients 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; calcium chloride dihydrate: 85 mg / l. The micronutrients include: boric acid: 6.2 mg / l; manganese sulfate monohydrate: 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. The iron salts include: ferrous sulfate heptahydrate: 27.8 mg / l; EDTA disodium: 37.3 mg / l. The organic substances include: thiamine hydrochloride (vitamin B1): 1.0 mg / l; pyridoxine hydrochloride (vitamin B6): 0.5 mg / l; nicotinic acid: 0.5 mg / l; glycine: 2.0 mg / l; inositol: 200 mg / l.

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

[0078] Among them, in each liter of DCR medium, 0.2 mg - 0.4 mg of 2,4-D is added, 0.4 mg - 0.7 mg of TDZ is added, 0.3 mg - 0.5 mg of 6-BA is added, 0.5 g - 1 g of KT is added, and 7.0 g - 8.0 g of agar is added.

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

[0080] TDZ (thidiazuron), which helps cell division in tissue culture.

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

[0082] Agar: As a solidifying agent, the addition amount is determined according to the required hardness of the culture medium, usually between 7 - 8 g / L. Adding agar to the culture medium can transform the liquid culture medium into a solid or semi-solid state, which is beneficial for the fixation and support of plant tissues. For plants that need tissue culture or cell culture, a solid culture medium can provide a better growth environment. The gel properties of agar endow the culture medium with a certain structural stability. This stability is very important for the growth and differentiation of plant tissues because it can ensure that nutrients, hormones, and other additives in the culture medium are evenly distributed during the culture process and will not change due to the flow or mixing of the culture medium. The gel-like structure of agar can, to a certain extent, protect plant tissues from mechanical damage and also reduce the risk of contamination.

[0083] Furthermore, the detoxification treatment of Pinus kesiya var. langbianensis male flowers includes:

[0084] Immersing the explants in a mixed solution containing antiviral agents and antibacterial agents for detoxification;

[0085] Among them, the antiviral agent can be any one of ribavirin and moroxydine hydrochloride, and the antibacterial agent can be any one of penicillin and streptomycin. Penicillin and streptomycin have inhibitory effects on a variety of bacteria, including Gram-positive bacteria and Gram-negative bacteria. By eliminating bacteria on the surface of anthers, penicillin or streptomycin treatment can significantly improve the health of anthers and reduce the occurrence and spread of bacterial diseases.

[0086] Ribavirin, as a broad-spectrum anti-plant virus agent, has inhibitory activity against plant viruses; Moroxydine is also an efficient virucide, with the characteristics of combining quick-acting and long-acting, and can significantly prevent and treat various intractable virus diseases that are difficult to cure with conventional agents. In this application, the anther explants are immersed in a solution containing ribavirin or moroxydine, which can effectively remove the viruses on the anther explants, thereby improving the health of the anthers and providing healthy and disease-free germplasm resources for subsequent propagation. The growth potential and reproductive ability of the anther explants treated with ribavirin or moroxydine may be significantly improved, which is beneficial to expanding the propagation scale and improving the propagation efficiency.

[0087] Example 2

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

[0089] The process of plant tissue culture of Pinus kesiya var. langbianensis performed by this system is as follows:

[0090] Collect healthy male flower explants of Pinus kesiya var. langbianensis, disinfect the explants, and dissect the anthers of the male flower explants;

[0091] Inoculate the dissected anthers into a culture medium containing specific nutrients and growth regulators for the induction and culture of callus

[0092] Example 3

[0093] This example specifically describes how to obtain callus from the anthers of Pinus kesiya var. langbianensis:

[0094] Select a Pinus kesiya var. langbianensis plant with good growth and no pests and diseases, and collect young and tender male flowers that have not shed pollen on a sunny day as explants.

[0095] Soak the male flowers of Pinus kesiya var. langbianensis in 75% alcohol for 30 s, and then soak them in 0.1% mercuric chloride for 5 min respectively.

[0096] Take out the soaked explants, wash them 4 - 5 times with sterile water, and dry them with sterile filter paper.

[0097] Place the disinfected male flowers of Pinus kesiya var. langbianensis in a sterile culture dish, separate the anthers using forceps and dissecting needles, and then inoculate them into the callus induction medium. Inoculate 15 anthers into each bottle of the medium and perform dark culture in a culture room at (25 ± 2) °C until callus is induced.

[0098] Example 4

[0099] This example specifically describes how to obtain callus from the anthers of Pinus kesiya var. langbianensis:

[0100] Select Pinus kesiya var. langbianensis plants with good growth and no pests and diseases, and collect young and immature male flowers that have not shed pollen on sunny days as explants.

[0101] Soak the male flowers of Pinus kesiya var. langbianensis in 75% alcohol for 30 s, and then soak them in 0.1% mercuric chloride for 10 min respectively.

[0102] Take out the soaked explants, wash them 4 - 5 times with sterile water, and dry them with sterile filter paper.

[0103] Place the male flowers of Pinus kesiya var. langbianensis after disinfection treatment in a sterile petri dish, separate the anthers using forceps and dissecting needles, and then inoculate them into the callus induction medium. Inoculate 15 anthers in each bottle of medium, and conduct dark culture in an incubator at (25 ± 2)°C until callus is induced.

[0104] Example 5

[0105] This example specifically describes how to obtain callus from the anthers of Pinus kesiya var. langbianensis:

[0106] Select Pinus kesiya var. langbianensis plants with good growth and no pests and diseases, and collect young and immature male flowers that have not shed pollen on sunny days as explants.

[0107] Soak the male flowers of Pinus kesiya var. langbianensis in 75% alcohol for 30 s, and then soak them in 0.1% mercuric chloride for 15 min respectively.

[0108] Take out the soaked explants, wash them 4 - 5 times with sterile water, and dry them with sterile filter paper.

[0109] Place the male flowers of Pinus kesiya var. langbianensis after disinfection treatment in a sterile petri dish, separate the anthers using forceps and dissecting needles, and then inoculate them into the callus induction medium. Inoculate 15 anthers in each bottle of medium, and conduct dark culture in an incubator at (25 ± 2)°C until callus is induced.

[0110] Example 6

[0111] This example specifically describes how to obtain callus from the anthers of Pinus kesiya var. langbianensis:

[0112] Select Pinus kesiya var. langbianensis plants with good growth and no pests and diseases, and collect young and immature male flowers that have not shed pollen on sunny days as explants.

[0113] Soak the male flowers of Pinus kesiya var. langbianensis in 75% alcohol for 30 s, and then soak them in 0.1% mercuric chloride for 20 min respectively.

[0114] Take out the soaked explants, wash them 4 - 5 times with sterile water, and dry them with sterile filter paper.

[0115] After the disinfection treatment, the male flowers of Pinus kesiya var. langbianensis were placed in a sterile Petri dish. The anthers were separated using forceps and a dissecting needle, and then inoculated into the callus induction medium. 15 anthers were inoculated into each bottle of the medium, and dark culture was carried out in an incubator at (25±2)°C until callus was induced.

[0116] Example 7

[0117] This example specifically describes how to obtain callus from the anthers of Pinus kesiya var. langbianensis:

[0118] Select healthy Pinus kesiya var. langbianensis plants without pests and diseases, and collect young male flowers that have not shed pollen on sunny days as explants.

[0119] Soak the male flowers of Pinus kesiya var. langbianensis in 75% alcohol for 1 min, and then soak them in 0.1% mercuric chloride for 5 min respectively.

[0120] Take out the soaked explants, wash them 4 - 5 times with sterile water, and dry them with sterile filter paper.

[0121] After the disinfection treatment, the male flowers of Pinus kesiya var. langbianensis were placed in a sterile Petri dish. The anthers were separated using forceps and a dissecting needle, and then inoculated into the callus induction medium. 15 anthers were inoculated into each bottle of the medium, and dark culture was carried out in an incubator at (25±2)°C until callus was induced.

[0122] Example 8

[0123] This example specifically describes how to obtain callus from the anthers of Pinus kesiya var. langbianensis:

[0124] Select healthy Pinus kesiya var. langbianensis plants without pests and diseases, and collect young male flowers that have not shed pollen on sunny days as explants.

[0125] Soak the male flowers of Pinus kesiya var. langbianensis in 75% alcohol for 1 min, and then soak them in 0.1% mercuric chloride for 10 min respectively.

[0126] Take out the soaked explants, wash them 4 - 5 times with sterile water, and dry them with sterile filter paper.

[0127] After the disinfection treatment, the male flowers of Pinus kesiya var. langbianensis were placed in a sterile Petri dish. The anthers were separated using forceps and a dissecting needle, and then inoculated into the callus induction medium. 15 anthers were inoculated into each bottle of the medium, and dark culture was carried out in an incubator at (25±2)°C until callus was induced.

[0128] Example 9

[0129] This example specifically describes how to obtain callus from the anthers of Pinus kesiya var. langbianensis:

[0130] Select vigorous Pinus kesiya var. langbianensis plants without diseases and pests, and collect young and tender male flowers that have not shed pollen on sunny days as explants.

[0131] Soak the male flowers of Pinus kesiya var. langbianensis in 75% alcohol for 1 minute, and then soak them in 0.1% mercuric chloride for 15 minutes respectively.

[0132] Take out the soaked explants, wash them 4 - 5 times with sterile water, and dry them with sterile filter paper.

[0133] Place the male flowers of Pinus kesiya var. langbianensis after disinfection treatment in a sterile petri dish, separate the anthers using forceps and dissecting needles, and then inoculate them into the callus induction medium. Inoculate 15 anthers in each bottle of medium, and conduct dark culture in an incubator at (25 ± 2)°C until callus is induced.

[0134] Example 10

[0135] This example specifically describes how to obtain callus from the anthers of Pinus kesiya var. langbianensis:

[0136] Select vigorous Pinus kesiya var. langbianensis plants without diseases and pests, and collect young and tender male flowers that have not shed pollen on sunny days as explants.

[0137] Soak the male flowers of Pinus kesiya var. langbianensis in 75% alcohol for 1 minute, and then soak them in 0.1% mercuric chloride for 20 minutes respectively.

[0138] Take out the soaked explants, wash them 4 - 5 times with sterile water, and dry them with sterile filter paper.

[0139] Place the male flowers of Pinus kesiya var. langbianensis after disinfection treatment in a sterile petri dish, separate the anthers using forceps and dissecting needles, and then inoculate them into the callus induction medium. Inoculate 15 anthers in each bottle of medium, and conduct dark culture in an incubator at (25 ± 2)°C until callus is induced.

[0140] In Examples 3 - 10 of this application, the soaking treatment time of alcohol and mercuric chloride on the male flowers of Pinus kesiya var. langbianensis 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 specifically.

[0141] Table 1 Effects of different detoxification methods on the contamination and survival of anther explants

[0142]

[0143] It can be obtained from Table 1 that the alcohol disinfection time has a significant effect on the contamination rate of the explants of Pinus kesiya var. langbianensis. When the mercuric chloride disinfection time is 5 min, the contamination rate of the explants with an alcohol disinfection time of 30 s is the highest, reaching 66.67%; when the alcohol disinfection time is extended to 1 min, the contamination rate drops to 37.78%. The concentration and disinfection time of mercuric chloride significantly affect the survival rate of the explants. When the alcohol disinfection is 30 s and 0.1% mercuric chloride is used for disinfection for 10 min, the survival rate of the explants is the highest, reaching 84.44%. When the alcohol disinfection is 30 s and 0.3% mercuric chloride is used for disinfection for 15 min, the survival rate of the explants is the lowest, being 36.67%. Therefore, the disinfection combination of 30 s of alcohol + 10 min of 0.1% mercuric chloride has the best disinfection effect on the explants of Pinus kesiya var. langbianensis anthers. Moreover, the combined disinfection method of disinfecting with 75% alcohol for 30 s and 0.1% mercuric chloride for 10 min can achieve a good detoxification effect, and the survival rate of the anthers is relatively high. At this time, the survival rate of the anthers is 84.44% and the contamination rate is 0%. Using this method, sterile anthers can be obtained and callus can be successfully induced.

[0144] In addition, the present application also uses sodium hypochlorite and alcohol to disinfect the explants of Pinus kesiya var. langbianensis anthers, specifically as follows:

[0145] Table 2 Influence of sodium hypochlorite on the detoxification of explants

[0146]

[0147] It can be obtained from Table 2 that the combined use of sodium hypochlorite and alcohol has a poor disinfection effect on the explants of Pinus kesiya var. langbianensis anthers. The survival rate of the explants is 0, and the contamination rates are relatively high, being 81.11% and 77.78% respectively. When mercuric chloride and alcohol are used in combination, the contamination rate is 0, and the highest survival rate of the explants can reach 84.44%. Therefore, sodium hypochlorite is not suitable for disinfecting the explants of Pinus kesiya var. langbianensis anthers.

[0148] Example 11

[0149] In this example, the effects of different types of basic media on the induction of embryogenic callus of Pinus kesiya var. langbianensis and the effects of different hormone types and concentrations and biotin in the same medium on the induction of embryogenic callus of Pinus kesiya var. langbianensis were explored respectively, specifically as follows:

[0150] (1) Effects of different types of basic media on the induction of embryogenic callus of Pinus kesiya var. langbianensis:

[0151] Table 3 Effects of different basic media on the induction of embryogenic callus of Pinus kesiya var. langbianensis anthers

[0152]

[0153]

[0154] It can be seen from Table 3 that the type of basic medium has a significant impact on the induction of embryogenic callus from Pinus kesiya var. langbianensis anthers. Embryogenic callus could not be induced in the WPM and 1 / 2MS basic media, and the induction rate of embryogenic callus in the 1 / 2DCR and LP basic media was extremely low, at 2.67%. The DCR medium had a better effect on the induction of embryogenic callus from Pinus kesiya var. langbianensis anthers, and the induction rate could reach 12.00%.

[0155] (2) Effects of different hormones on the induction of embryogenic callus from Pinus kesiya var. langbianensis:

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

[0157]

[0158] It can be seen from Table 4 that different hormone types and concentrations have a significant impact on the induction of embryogenic callus from Pinus kesiya var. langbianensis. The induction rate of embryogenic callus in Medium 1 was the lowest, at 4.00%, and the induction rates of embryogenic callus in Medium 10 and Medium 11 were the highest, at 14.67%. Among them, when 2,4-D, KT, and TDZ were used in combination, the effect on the induction of embryogenic callus was significantly better than that of 2,4-D, 6-BA, and TDZ; when the concentration of 2,4-D was 2.0 mg / L, the induction rate of embryogenic callus was higher than that when the concentration was 1.0 mg / L; when the concentration of TDZ was 1.0 mg / L, the callus rate was relatively high; when the concentration of KT was 0.5 mg / L, the induction rates of embryogenic callus were relatively high. For example, the induction rates of Medium 10, Medium 11, and Medium 12 were 10.67%, 14.67%, and 10.67% respectively. In summary, Medium 11 (DCR + 2,4-D 2.0 mg / L + TDZ 1.0 mg / L + KT 1.0 mg / L) is the optimal medium for the induction of embryogenic callus from Pinus kesiya var. langbianensis.

[0159] (3) Effects of different biotin on the induction of embryogenic callus from Pinus kesiya var. langbianensis:

[0160] Table 5 Effects of biotin on the induction of embryogenic callus from Pinus kesiya var. langbianensis anthers

[0161]

[0162] As can be seen from Table 5, biotin has a promoting effect on the induction of embryogenic callus from the anthers of Pinus kesiya var. langbianensis. As the concentration of biotin increases, the induction rate of embryogenic callus shows an upward trend. When the biotin concentration is 0.75 mg / L, the induction rates of embryogenic callus on Medium No. 15 and Medium No. 18 are 22.67% and 25.33% respectively. Further analysis reveals that the effect of using biotin in combination with KT is better than that of using biotin in combination with 6-BA. When the biotin concentration remains unchanged, the induction rate of embryogenic callus when it is used in combination with KT is higher than that when it is used in combination with 6-BA. The induction rates of embryogenic callus on Medium No. 16, Medium No. 17 and Medium No. 18 are higher than those on Medium No. 13, Medium No. 14 and Medium No. 15 respectively. The biotin in this application is Biotin biotin.

[0163] The embodiments of the present invention have been described above. The above description is exemplary and not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of the terms used herein is intended to best explain the principles of the embodiments, practical applications or improvements to the technologies in the market, or to enable other ordinary skill in the art in this technical field to understand the embodiments disclosed herein.

Claims

1. A method for inducing and culturing embryonic callus of Pinus koraiensis, characterized in that: include: Collecting male flowers of Pinus koraiensis and performing surface disinfection on the collected male flowers; The male flowers of Pinus koraiensis with disinfected surfaces are immersed in a detoxification solution for detoxification treatment; The virus-free male flower explants of Simiao pine were washed and dried; The explants of Pinus koraiensis were taken out after being dried, and the anthers of the explants were separated and placed in a culture medium for dark culture until callus tissue was induced.

2. The method for inducing embryonic callus of Pinus koraiensis according to claim 1, characterized in that: The collecting of male flowers of Pinus kornelengensis comprises: On a sunny day, young male flowers of Pinus sphenanthera that had not yet shed pollen were collected as explants; Wrap the male flowers of Pinus magnoliae with wet newspaper to preserve the explants.

3. The method for inducing embryonic callus of Pinus koraiensis according to claim 1, characterized in that: The step of placing the surface-disinfected male flowers of Pinus kornelengensis into a detoxification solution for immersion for detoxification treatment comprises: Soak the male flowers of Pinus kornelengensis in alcohol for 30s-60s, and then transfer the male flowers of Pinus kornelengensis to detoxification solution and soak for 5min-20min; 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.

4. The method for inducing embryonic callus of Pinus koraiensis according to claim 1, characterized in that: The surface disinfection of the collected male flowers also includes: Place the male flowers of Pinus koraiensis in a combination bottle, cover the bottle mouth with a layer of gauze and tie it tightly, rinse under tap water for 1 hour, soak in 1% pvp-K30 solution for 1 hour, and wash twice with sterile water.

5. The method for inducing embryonic callus of Pinus koraiensis according to claim 3, characterized in that: The method of washing the virus-free pine cone male flower explants and drying the water comprises: Take out the anthers after soaking in mercuric chloride solution; Wash 4-6 times with sterile water and then dry with sterile filter paper.

6. The method for inducing embryonic callus of Pinus koraiensis according to any one of claims 1 to 5, characterized in that: The step of placing the anthers in a culture medium for dark culture until callus tissue is induced comprises: separating anthers of Pinus koraiensis, and inoculating the separated anthers into a culture medium; Carry out dark culture in a culture room until callus tissue is induced; The temperature of the culture room is 25±2°C and the humidity is 65%-75%.

7. The method for inducing embryonic callus of Pinus koraiensis according to claim 6, characterized in that: The culture medium includes a DCR culture medium, and the DCR culture medium also has the following additional components: 2,4-D, TDZ, 6-BA (6-benzylaminopurine), KT (furantoin), and agar; In each liter of DCR culture medium, add 0.2mg-0.4mg of 2,4-D, 0.4mg-0.7mg of TDZ, 0.3mg-0.5mg of 6-BA, 0.5g-1g of KT, and 7.0g-8.0g of agar.

8. The method for inducing embryonic callus of Pinus koraiensis according to claim 1, characterized in that: The method of detoxifying male flowers of Pinus koraiensis comprises: The explants are detoxified by soaking them in a mixed solution containing antiviral and antibacterial agents; The antiviral agent is any one of ribavirin and virolin, and the antibacterial agent is any one of penicillin and streptomycin.

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