Tissue Culture Method of Zhongmu No.3

Abstract

The present application belongs to the field of tissue culture, and specifically relates to a tissue culture method of Zhongmu No. 3. It includes obtaining the cotyledons of Zhongmu No. 3 as explants, inoculating the explants into an induction medium for induction culture, preparing callus tissue, then inoculating the callus tissue into a regeneration seedling medium for culture, preparing regeneration seedlings, and finally transplanting the regeneration seedlings into a seedling medium for culture to obtain Zhongmu No. 3 plants. The present application specifically uses cotyledons as explants and cooperates with a specific tissue culture process to make the callus induction rate of Zhongmu No. 3 more than 90% and the budding rate more than 40%.

Description

Technical Field

[0001] The present application belongs to the field of tissue culture, and specifically relates to a tissue culture method of Zhongmu No. 3. Background Art

[0002] Plant tissues and cells cultured in vitro can regenerate new organs or a complete plant under the induction of plant hormones. Improving the regeneration efficiency of crops is an important development direction of plant tissue culture and cell engineering in the future.

[0003] Alfalfa is a perennial leguminous herb. The protein content of high-quality alfalfa hay is as high as over 22%, and the protein digestibility is over 70%. It is one of the forage grasses with the highest digestible protein content and is known as the "king of forage grasses". However, most alfalfa explants have low differentiation rate and regeneration rate, long regeneration cycle, and strong dependence on genes for regeneration ability. When researchers apply the culture procedures provided by previous reports, the repeatability is poor.

[0004] The purple alfalfa variety "Zhongmu No. 3" was obtained through cyclic selection with "Zhongmu No. 1" as the parent material, and is suitable for the light and moderate saline-alkali land in the Huanghuaihai area. Zhongmu No. 3 has the characteristics of well-developed lateral roots, rapid growth, many branches, high yield, early maturity, and salt tolerance. It can be mowed 4 to 5 times a year, and the number of lateral roots accounts for 31.3% of the total number of plants, which is 21.7% higher than that of Zhongmu No. 1 alfalfa and 23.2% higher than that of Baoding alfalfa. Its main advantage lies in its good salt tolerance. The yield in saline-alkali land with a salt content of 0.18% to 0.39% is at least 10% higher than that of its parent Zhongmu No. 1, and the crude protein content of dry matter in the early flowering period reaches 19%. The development of this variety is to adapt to the planting conditions of saline-alkali land, improve the utilization rate of saline-alkali land and the ability to produce high-protein forage, which is of great significance to the sustainable development of agriculture.

[0005] However, the callus induction rate of Zhongmu No. 3 in the traditional technology is low, and the germination rate of the regeneration system prepared is also low. Summary of the invention

[0006] Based on this, an embodiment of the present application provides a tissue culture method of Zhongmu No. 3, and the tissue culture of Zhongmu No. 3 using this method has a higher induction rate and seedling germination rate.

[0007] On the one hand, the present application provides a tissue culture method of Zhongmu No. 3, comprising the following steps:

[0008] Disinfecting the seeds of Zhongmu No. 3, soaking the disinfected seeds in 97% to 99% strong acid for 3 to 5 minutes to break dormancy; and,

[0009] The treated seeds of Zhongmu No. 3 are inoculated into 1 / 2MS basal medium, vernalized and cultured at a temperature of 3.5°C to 4.5°C for 2.5 to 3.5 days, and then cultured at a temperature of 22°C to 26°C for 6 to 8 days to obtain cotyledons of Zhongmu No. 3;

[0010] Inoculating the cotyledons of Zhongmu No. 3 into an induction medium for induction culture to prepare callus tissue;

[0011] Inoculating the callus tissue into a regenerated seedling medium for cultivation to prepare a regenerated seedling; and,

[0012] The regenerated seedlings are transplanted into a seedling culture medium for cultivation to obtain Zhongmu No. 3 plants.

[0013] In one embodiment, sterilization comprises sterilizing the seeds in a sealed container containing chlorine gas for 2 hours to 3 hours.

[0014] In one embodiment, the strong acid comprises concentrated sulfuric acid.

[0015] In one embodiment, the method further comprises removing the edges of the cotyledons of Zhongmu No. 3 before inoculation, and / or;

[0016] During inoculation, the cotyledons of Zhongmu No. 3 were inoculated on the induction medium with the back side of the cotyledons facing upward.

[0017] In one embodiment, the induction medium comprises: 1.8 mg / L~2.2 mg / L 2,4-dichlorophenoxyacetic acid, 0.4 mg / L~0.6 mg / L 6-furfurylaminopurine, 4.2 g / L~4.6 g / L MS salt, 28 g / L~32 g / L sucrose, 6 g / L~10 g / L agar and water, with a pH of 5.6~6.0.

[0018] In one embodiment, the specific steps of inducing culture include:

[0019] The first week is to be cultured in dark conditions; from the second to fourth weeks, the cells are cultured alternately under 1800lx~2200lx light for 14 hours to 18 hours and then in darkness for 6 hours to 10 hours; and / or,

[0020] The culture temperature from the first to the fourth week was 22℃~26℃.

[0021] In one embodiment, the regeneration seedling culture medium includes: 0.4 mg / L~0.6 mg / L 6-benzylaminopurine, 0.8 mg / L~1.2 mg / L 6-furfurylaminopurine, 4.2 g / L~4.6 g / L MS salt, 28 g / L~32 g / L sucrose, 6 g / L~10 g / L agar and water, with a pH of 5.6~6.0.

[0022] In one embodiment, the specific steps of inoculating the callus tissue into a regeneration seedling culture medium for cultivation include:

[0023] The culture is alternately cultured with 2900lx~3100lx light for 14 hours~18 hours and then dark for 6 hours~10 hours; and / or, the culture temperature from the first week to the fourth week is 24~28℃; and,

[0024] Subculturing once every 1.5 to 2.5 weeks until a 1.5 to 2.5 cm regenerated seedling juvenile grows out, and after the callus tissue at the base of the regenerated seedling juvenile is removed, the juvenile is transferred to 1 / 2MS medium for rooting culture to obtain a regenerated seedling;

[0025] The 1 / 2MS culture medium includes 2.0-2.4 g / L MS salt, 28 g / L-32 g / L sucrose, 6 g / L-10 g / L agar and water, and has a pH of 5.6-6.0.

[0026] In one embodiment, the specific steps of transplanting the regenerated seedlings into the seedling culture medium for cultivation include:

[0027] After the regenerated seedlings grow roots, they are transplanted to a seedling culture medium for cultivation to obtain Zhongmu No. 3 plants;

[0028] The seedling raising medium comprises garden soil and vermiculite.

[0029] In one embodiment, the volume ratio of the garden soil to the vermiculite is (0.8-1.2):(0.8-1.2). BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0031] Figure 1 The callus tissue blocks were inoculated into the induction medium. The two explants (cotyledons and hypocotyls) were in the state shown in the figure 10 weeks later.

[0032] Figure 2 The callus induced from the cotyledons was transferred to the redifferentiation medium for 16 weeks to regenerate the seedlings;

[0033] Figure 3 The callus induced from the cotyledons was transferred to the redifferentiation medium for 22 weeks to show the state of the regenerated seedlings;

[0034] Figure 4The seedlings are taken out after rooting in 1 / 2MS medium and transplanted;

[0035] Figure 5 This is the state after transplanting to the soil with a ratio of 1:1 garden soil and vermiculite for one week;

[0036] Figure 6 The callus tissues of Example 1 and Comparative Examples 1 to 3 are in a state after 4 weeks of induction. DETAILED DESCRIPTION

[0037] The present invention will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the present invention, and the equivalent form obtained also falls within the protection scope of the present application. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing implementation modes and embodiments and are not intended to limit the present invention.

[0039] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0040] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0041] In the present invention, "plurality", "multiple", "multiple times", "multiple" and the like, unless otherwise specified, refer to a number greater than or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0042] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0043] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of the present invention, solve the technical problem of the present invention, and achieve the expected technical effect of the present invention.

[0044] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of the present invention.

[0045] Herein, “further”, “furthermore”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present invention.

[0046] In this article, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel options of "yes" or "no". If there are multiple "options" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "option" is independent.

[0047] In this document, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0048] In this article, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0049] Herein, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges included therein.

[0050] The temperature parameters herein, if not specifically limited, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0051] In this article, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass volume percentage. The term "callus" refers to the in vitro regeneration system of plants, in which in vitro tissues or organs can be induced by plant hormones to form a cell mass with regeneration ability. It refers to the new tissue on the wound surface after the original plant body is locally stimulated by trauma. It is composed of living thin-walled cells and can originate from living cells of various tissues in any organ of the plant body.

[0052] The term "tissue culture" Plant tissue culture is a new asexual reproduction technology developed in recent decades based on the theory that plant cells are omnipotent. Plant tissue culture is also called in vitro culture in a broad sense, which refers to the separation of tissues, organs or cells, protoplasts, etc. that meet the needs from the plant body, and the use of aseptic operations and inoculation under aseptic conditions on a culture medium containing various nutrients and plant hormones to obtain regenerated complete plants or produce other products with economic value. In a narrow sense, it refers to the use of various parts of plant tissues, such as cambium, parenchyma, mesophyll tissue, endosperm, etc. to obtain regenerated plants. It also refers to the culture of callus tissue produced from various organs during the culture process, and the callus tissue is then redifferentiated to form a regenerated plant.

[0053] The term "seed dormancy" Seed dormancy refers to a biological phenomenon in which seeds that should have signs of vitality within a predetermined time frame fail to germinate normally under the expected combination of environmental factors that are conducive to seed germination. Dormancy is a survival strategy of plants. It not only buys time for the spread of seeds, but also promotes the germination of seeds under the most ideal environmental conditions. For example, many seeds produced in summer or autumn are likely to be frozen to death if they germinate before the arrival of winter, so this mechanism that can ensure that the seeds germinate in spring is still necessary. Seed dormancy has important ecological significance and can effectively regulate the temporal and spatial distribution of seed germination.

[0054] The term "vermiculite" refers to a natural, inorganic, non-toxic mineral that expands under high temperature. It is a relatively rare mineral and belongs to silicate. Its crystal structure is monoclinic and it looks like mica. Vermiculite is produced when certain granites are hydrated. It is usually produced at the same time as asbestos. Because vermiculite has the ability to exchange ions, it has a great effect on soil nutrition.

[0055] The term "cotyledon" Cotyledon is a temporary leaf organ. Their number is quite stable in angiosperms. Plants with only one cotyledon in the mature embryo are called monocotyledons, such as wheat and lily; plants with two cotyledons are called dicotyledons, such as rapeseed and soybean.

[0056] The number of cotyledons in the embryo varies depending on the plant species. Monocots have one cotyledon, such as corn and lily; dicots have two cotyledons, such as soybeans and pears; gymnosperms have a large variation in the number of cotyledons, such as cypress with two, ginkgo with three, and pine with many. The cotyledons of dicot embryos have a variety of shapes and physiological functions. For example, the cotyledons of beans are thick and fleshy, containing a lot of nutrients, but the cotyledons of castor are flat and thin.

[0057] The term "hypocotyl" is a component of the embryo of a seed plant. It is the axis between the cotyledon attachment point and the radicle. After the seed germinates, it develops into a part connecting the stem and the root. The part between the cotyledon and the first true leaf is called the "epicotyl"; the part between the cotyledon and the root is called the "hypocotyl". The hypocotyl refers to the lower cotyledon, which is the embryonic organ of higher plants and the part of the first stem that grows below the cotyledon attachment point.

[0058] The term "root" is a botanical term that generally refers to the underground part of a plant. Its main functions are to hold the plant body, absorb water and minerals dissolved in water, transport water and minerals to the stem, and store nutrients. The underground structure of many plants is essentially a specialized stem (such as a corm or tuber). The main difference between roots and branches is that they lack leaf scars and buds, have a root cap, and branches are produced by internal tissues rather than buds.

[0059] The term "vernalization" generally refers to the phenomenon that plants must experience a period of sustained low temperatures before they can transition from the vegetative growth stage to the reproductive stage. We call this phenomenon vernalization. For example, cold-resistant flowers from temperate regions, with longer winters and moderate cold, can better meet their requirements for low temperatures during the vernalization stage. The effect of low temperature treatment on promoting flowering of flowers varies depending on the type of flower.

[0060] The term "subculture" means that after the callus tissue has grown on the culture medium for a period of time, the nutrients are exhausted, the water is lost, and some metabolites have accumulated. At this time, these tissues need to be transferred to a new culture medium. This transfer is called subculture or subculture.

[0061] It is understandable that the current method of using alfalfa leaves as explants to induce callus regeneration is not suitable for all alfalfa varieties. This application uses cotyledons as explants and cooperates with a specific tissue culture process to make the callus induction rate of Zhongmu No. 3 more than 90% and the budding rate more than 40%.

[0062] On the one hand, the present application provides a tissue culture method of Zhongmu No. 3, comprising the following steps:

[0063] Disinfecting the seeds of Zhongmu No. 3, soaking the disinfected seeds in 97% to 99% strong acid for 3 to 5 minutes to break dormancy; and,

[0064] The treated seeds of Zhongmu No. 3 are inoculated into 1 / 2MS culture medium, vernalized at a temperature of 3.5°C to 4.5°C for 2.5 to 3.5 days, and then cultured at a temperature of 22°C to 26°C for 6 to 8 days to obtain cotyledons of Zhongmu No. 3;

[0065] Inoculating the cotyledons of Zhongmu No. 3 into an induction medium for induction culture to prepare callus tissue;

[0066] Inoculating the callus tissue into a regenerated seedling medium for cultivation to prepare a regenerated seedling; and,

[0067] The regenerated seedlings are transplanted into a seedling culture medium for cultivation to obtain Zhongmu No. 3 plants.

[0068] There are many specific methods to break dormancy, depending on the cause of seed dormancy. For seeds that have poor seed coat permeability and become dormant, mechanical friction, heating, and strong acid treatment can be used to break the seed coat and increase its air and water permeability.

[0069] In one specific example, sterilization includes sterilizing the seeds in a sealed container containing chlorine gas for 2 hours to 3 hours.

[0070] For example, sodium hypochlorite is mixed with hydrochloric acid to produce chlorine gas, which can be used to sterilize seeds in a sealed container for 2 to 3 hours.

[0071] Optionally, before inoculation, the step of removing the edges of the cotyledons of Zhongmu No. 3 is also included, for example, by cutting off the edges with a scalpel.

[0072] Further optionally, during inoculation, the cotyledons of Zhongmu No. 3 are inoculated into the induction medium with the back side facing upwards.

[0073] In a specific example, the induction medium contains: 1.8 mg / L~2.2 mg / L 2,4-dichlorophenoxyacetic acid, 0.4 mg / L~0.6 mg / L 6-furfurylaminopurine, 4.2 g / L~4.6 g / L MS salts, 28 g / L~32 g / L sucrose, 6 g / L~10 g / L agar and water, with a pH of 5.6~6.0.

[0074] Reagent MS salt refers to MS culture medium. It is characterized by high concentration of inorganic salts and ions, and is a relatively stable ion balance solution. It has a high nitrate content, and the quantity and proportion of its nutrients are appropriate, which can meet the nutritional and physiological needs of plant cells. Therefore, it has a wide range of applications. Most plant tissue cultures use it as the basic culture medium for rapid propagation.

[0075] In a specific example, the induction culture specific steps include:

[0076] The first week is to be cultured in dark conditions; from the second to fourth weeks, the cells are cultured in alternating conditions of 1800 lx to 2200 lx light for 14 hours to 18 hours and then 6 hours to 10 hours in darkness; and / or,

[0077] The culture temperature from the first to the fourth week was 22℃~26℃.

[0078] Optionally, the regeneration seedling culture medium comprises:

[0079] 0.4mg / L~0.6mg / L6-benzylaminopurine, 0.8mg / L~1.2mg / L6-furfurylaminopurine, 4.2g / L~4.6g / LMS salt, 28g / L~32g / L sucrose, 6g / L~10g / L agar and water, pH 5.6~6.0.

[0080] In a specific example, the specific steps of inoculating the callus tissue into a regeneration seedling culture medium for cultivation include:

[0081] Subculture is performed every 1.5 to 2.5 weeks until a regenerated seedling of 1.5 to 2.5 cm is grown, and callus tissue at the base of the regenerated seedling is removed and rooting culture is performed to obtain a regenerated seedling.

[0082] In a specific example, the specific steps of transplanting the regenerated seedlings into a seedling culture medium for cultivation include:

[0083] After the regenerated seedlings grow roots, they are transplanted to a seedling culture medium for cultivation, covered with a moisture-retaining plastic cover for 7 days, and then uncovered to obtain Zhongmu No. 3 plants;

[0084] Among them, the seedling medium includes garden soil and vermiculite.

[0085] In a specific example, the volume ratio of the garden soil to the vermiculite is (0.8~1.2):(0.8~1.2).

[0086] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0087] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0088] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0089] Example 1

[0090] This work was supported by the National Key Research and Development Program of China (Grant No. 2023YFD1200600), the CAS Project for Young Scientists in Basic Research (Grant No. YSBR-011), and the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA24010205; Grant No. XDA26030105).

[0091] 1. Disinfect the seeds of Zhongmu No. 3. Mix sodium hypochlorite with hydrochloric acid to produce chlorine gas. Disinfect the seeds in a sealed container for 2 to 3 hours.

[0092] 2. Soak the seed coat in concentrated sulfuric acid for 4 minutes to break dormancy, and then wash 5 times with sterile water.

[0093] 3. Sow seeds in 1 / 2MS culture medium, vernalize in the dark and low temperature for 3 days, take out and place under light conditions of 24℃, and use the expanded cotyledons as explants after 7 days.

[0094] 4. Use a scalpel to cut off the edges of the cotyledons and inoculate them with the back side facing up into the induction medium.

[0095] The induction medium formula is: 2 mg / L 2,4-dichlorophenoxyacetic acid, 0.5 mg / L 6-furfurylaminopurine, 4.4 g / L MS salt, 30 g / L sucrose, 8 g / L agar, and pH is 5.8.

[0096] The callus induction method is: dark culture in the first week, and culture at 16 hours of light / 8 hours of darkness from the second to the fourth week. 24±2℃.

[0097] 5. After 4 weeks of callus induction, transfer the callus pieces to the redifferentiation medium

[0098] The formula of the regeneration seedling culture medium is: 0.5 mg / L 6-benzylaminopurine, 1 mg / L 6-furfurylaminopurine, 4.4 g / L MS salt, 30 g / L sucrose, 8 g / L agar, and the pH is 5.8.

[0099] Methods of regeneration seedling cultivation:

[0100] Subculture once every 2 weeks until the regenerated seedlings grow to about 2 cm with independent growth points. Excise the callus tissue at the base and transfer to 1 / 2MS medium for rooting culture.

[0101] 6. Transplantation: When the regenerated seedlings grow roots in the tissue culture bottle, take them out of the tissue culture bottle, clean the residual culture medium at the base, and transfer them to the soil with a ratio of 1:1 garden soil: vermiculite. Cover with a moisturizing plastic cover for 7 days and then remove the cover.

[0102] 7. Result verification:

[0103] The induction rates and callus status of different parts are shown in Table 2:

[0104] Table 2

[0105]

[0106] The callus block of Example 1 was inoculated into two explants (cotyledons and hypocotyls) in the redifferentiation medium. Among them, the callus induced by "roots" and "true leaves" was "non-embryonic callus", and no buds were successfully induced in the subsequent process.

[0107] The germination rate of callus induced by "hypocotyl" is also lower than that of callus from "cotyledon". In addition, considering that one seed can obtain 2 cotyledons and 1 hypocotyl after sowing (because the hypocotyl is short, there is only one part that meets the length (0.8cm±2mm) when taking).

[0108] After 10 weeks, the status Figure 1 As shown, the budding rate from 2 mg / L 2,4-D + 0.5 mg / L KT medium transferred to the redifferentiation medium was calculated according to the following formula:

[0109] Germination rate = number of germinated callus / number of callus inoculated on redifferentiation medium.

[0110] Cotyledon: 8 / 17=0.470588235;

[0111] Hypocotyl: 9 / 21=0.428571429

[0112] That is, the germination rate is 47.06% (cotyledon) 42.86% (hypocotyl).

[0113] After another 10 weeks, tissue culture seedlings can be obtained. Cut it from the base, remove the callus tissue, transfer it to 1 / 2MS medium without hormones, and transfer the callus tissue induced by the cotyledons to the redifferentiation medium. The state of the regenerated seedlings after 16 weeks is as follows Figure 2 As shown in Figure 2, the callus induced from the cotyledons was transferred to the redifferentiation medium. The state of the regenerated seedlings after 22 weeks was as follows: Figure 3 As shown, it was cut off from the base, the callus tissue was removed, and transferred to 1 / 2MS medium without hormones. The transplanting status was as shown Figure 4 shown.

[0114] The data are the total data from two culture media: 2 mg / L 2,4-D + 0.5 mg / L KT and 4 mg / L 2,4-D + 0.2 mg / L 6-BA, and two explants: cotyledon and hypocotyl.

[0115] Among them, the numerator 92 means that out of the 198 callus tissues in the denominator, a total of 92 seedlings were produced.

[0116] Seedling emergence rate: number of seedlings / number of callus tissues induced to redifferentiate, 92 / 198 (46.4%), the emergence situation is as follows Figure 5 shown.

[0117] Comparative Example 1

[0118] The induction culture medium formula of Comparative Example 1 is: 2 mg / L 2,4-dichlorophenoxyacetic acid, 4.4 g / L MS salt, 30 g / L sucrose, 8 g / L agar, pH is 5.8, and the remaining culture steps are the same as those of Example 1.

[0119] Comparative Example 2

[0120] The induction medium formula of Comparative Example 2 is: 4 mg / L 2,4-dichlorophenoxyacetic acid, 0.2 mg / L 6-furfurylaminopurine, 4.4 g / L MS salt, 30 g / L sucrose, 8 g / L agar, pH 5.8, and the remaining culture steps are the same as Example 1.

[0121] Comparative Example 3

[0122] The induction medium formula of Comparative Example 3 is: 4 mg / L 2,4-dichlorophenoxyacetic acid, 4.4 g / L MS salt, 30 g / L sucrose, 8 g / L agar, pH is 5.8, and the remaining culture steps are the same as Example 1.

[0123] The induction rates, inoculation numbers and callus states of Comparative Examples 1 to 3 are shown in Table 3:

[0124] Table 3

[0125]

[0126] Comparative Examples 1 and 3 were not transferred to differentiation medium because callus could not be induced / the induced callus was in poor condition.

[0127] The germination rates of comparative example 2 are as follows: cotyledon: 4 / 13 (30.8%), hypocotyl: 5 / 38 (13.16%)

[0128] The callus tissues of Example 1 and Comparative Examples 1 to 3 were in the following state after 4 weeks of induction: Figure 6 shown.

[0129] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A tissue culture method of Zhongmu No. 3, characterized in that: The following steps are involved: The seeds of Zhongmu No. 3 are disinfected, and the disinfected seeds are treated by soaking in 97% to 99% strong acid for 3 to 5 minutes to break dormancy; as well as, The treated seeds of Zhongmu No. 3 are inoculated into 1 / 2MS basal medium, vernalized and cultured at a temperature of 3.5°C to 4.5°C for 2.5 to 3.5 days, and then cultured at a temperature of 22°C to 26°C for 6 to 8 days to obtain cotyledons of Zhongmu No. 3; Inoculating the cotyledons of Zhongmu No. 3 into an induction medium for induction culture to prepare callus tissue; Inoculating the callus tissue into a regenerated seedling medium for cultivation to prepare a regenerated seedling; and, Transplanting the regenerated seedlings into a seedling culture medium for cultivation to obtain Zhongmu No. 3 plants; The induction medium is composed of 1.8 mg / L to 2.2 mg / L 2,4-dichlorophenoxyacetic acid, 0.4 mg / L to 0.6 mg / L 6-furfurylaminopurine, 4.2 g / L to 4.6 g / L MS salt, 28 g / L to 32 g / L sucrose, 6 g / L to 10 g / L agar and water, and has a pH of 5.6 to 6.0; The regeneration seedling culture medium consists of 0.4 mg / L to 0.6 mg / L 6-benzylaminopurine, 0.8 mg / L to 1.2 mg / L 6-furfurylaminopurine, 4.2 g / L to 4.6 g / L MS salt, 28 g / L to 32 g / L sucrose, 6 g / L to 10 g / L agar and water, and has a pH of 5.6 to 6.

0.

2. The tissue culture method of Zhongmu No. 3 according to claim 1, characterized in that: Sterilization involves sterilizing the seeds in a sealed container containing chlorine for 2 to 3 hours.

3. The tissue culture method of Zhongmu No. 3 according to claim 1, characterized in that: The strong acid includes concentrated sulfuric acid.

4. The tissue culture method of Zhongmu No. 3 according to claim 1, characterized in that: The method also includes the step of removing the edge parts of the cotyledons of Zhongmu No. 3 before inoculation.

5. The tissue culture method of Zhongmu No. 3 according to claim 4, characterized in that: During inoculation, the cotyledons of Zhongmu No. 3 were inoculated on the induction medium with the back side of the cotyledons facing upward.

6. The tissue culture method of Zhongmu No. 3 according to claim 5, characterized in that: The specific steps of induction culture include: The first week was cultured in dark conditions; from the second to the fourth week, the culture was alternating between 14 hours to 18 hours of light at 1800 lx to 2200 lx and 6 hours to 10 hours of darkness.

7. The tissue culture method of Zhongmu No. 3 according to claim 6, characterized in that: The culture temperature from the first to the fourth week was 22℃~26℃.

8. The tissue culture method of Zhongmu No. 3 according to any one of claims 1 to 4 and 6, characterized in that: The specific steps of inoculating the callus tissue into a regeneration seedling culture medium for cultivation include: The culture is alternately cultured with 2900lx to 3100lx light for 14 to 18 hours and then dark for 6 to 10 hours; and / or, the culture temperature from the first week to the fourth week is 24 to 28°C; and, Subculturing once every 1.5 to 2.5 weeks until a 1.5 to 2.5 cm regenerated seedling juvenile grows out, and after removing the callus tissue at the base of the regenerated seedling juvenile, transferring the juvenile to a 1 / 2 MS medium for rooting culture to obtain a regenerated seedling; The 1 / 2MS culture medium includes 2.0-2.4 g / L MS salt, 28 g / L-32 g / L sucrose, 6 g / L-10 g / L agar and water, and has a pH of 5.6-6.

0.

9. The tissue culture method of Zhongmu No. 3 according to claim 8, characterized in that: The specific steps of transplanting the regenerated seedlings into the seedling medium for cultivation include: After the regenerated seedlings grow roots, they are transplanted to a seedling culture medium for cultivation to obtain Zhongmu No. 3 plants; The seedling raising medium comprises garden soil and vermiculite.

10. The tissue culture method of Zhongmu No. 3 according to claim 9, characterized in that: The volume ratio of the garden soil to the vermiculite is (0.8-1.2):(0.8-1.2).

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