Tissue culture method of casuarina equisetifolia

Abstract

The invention provides a tissue culture method of casuarina equisetifolia. Comprising the following steps: taking a casuarina equisetifolia rejuvenized branch as an explant, and sequentially performing disinfection treatment, induction culture, multiplication culture and rooting culture on the explant, wherein a basic culture medium for rooting culture is a 1 / 2WPM culture medium. Casuarina equisetifolia is subjected to propagation culture through a tissue culture method, excellent characteristics of female parents can be kept, and the method has the advantages of being short in propagation period, high in efficiency and the like; meanwhile, a 1 / 2WPM culture medium is adopted as a basic culture medium for rooting culture, root development can be promoted, and the rooting rate is increased.

Description

Technical Field

[0001] The present application relates to the field of plant propagation technology, and in particular to a tissue culture method of Casuarina equisetifolia. Background Art

[0002] Casuarina equisetifolia is an evergreen tall tree of the genus Casuarina in the family Casuarinaceae, native to Australia and the Pacific Islands. Casuarina is drought-resistant, heat-resistant, salt-alkali-resistant, wind-resistant, has a well-developed root system, and has nodule nitrogen fixation. It is a fast-growing and excellent tree species for coastal windbreak and sand fixation. Its wood is hard and rot-resistant, and can be used as sleepers, mine pillars, shipbuilding, and firewood, etc., with good economic value.

[0003] At present, the reproduction of Casuarina is mainly through sowing and cutting, but the reproduction efficiency is low and it is difficult to meet the market demand. Summary of the invention

[0004] Based on this, one or more embodiments of the present application provide a tissue culture method for Casuarina equisetifolia.

[0005] According to a first aspect of an embodiment of the present application, a tissue culture method of Casuarina equisetifolia is provided, comprising the following steps:

[0006] Taking young branches of Casuarina equisetifolia as explants, and sequentially performing disinfection, induction culture, proliferation culture and rooting culture on the explants;

[0007] Among them, the basic culture medium for rooting culture is 1 / 2WPM culture medium.

[0008] In some of the embodiments, the culture medium for rooting culture further includes 1.5 mg / L-2.5 mg / L IBA, 12 g / L-18 g / L sucrose and 5 g / L-7 g / L carrageenan.

[0009] In some embodiments, the disinfection step includes: disinfecting the explant using 0.05 w / v% to 0.5 w / v% mercuric chloride.

[0010] In some of the embodiments, the basic culture medium for induction culture is 1 / 2MS culture medium, and the culture medium for induction culture also includes 0.4 mg / L~0.6 mg / L6-BA, 12 g / L~18 g / L sucrose, 7 g / L~8 g / L carrageenan and 0.4 g / L~0.6 g / L activated carbon.

[0011] In some embodiments, the induction culture step includes: culturing at 22° C. to 28° C. for 16 h to 20 h per day, for 28 to 32 days, with a light intensity of 1800 Lux to 2200 Lux.

[0012] In some of the embodiments, the basic culture medium for proliferation culture is WPM culture medium, and the culture medium for proliferation culture also includes 0.1 mg / L~1 mg / L 6-BA, 24 g / L~26 g / L sucrose and 5 g / L~7 g / L carrageenan.

[0013] In some embodiments, the proliferation culture step includes: culturing at 22° C. to 28° C. for 16 h to 20 h per day, for 28 to 32 days, with a light intensity of 1800 Lux to 2200 Lux.

[0014] In some embodiments, the rooting culture step includes: culturing at 22° C. to 28° C. for 16 h to 20 h per day, for 28 days to 32 days, with a light intensity of 1800 Lux to 2200 Lux.

[0015] In some embodiments, after the rooting culture, the following steps are further included: transferring the rooted seedlings obtained by the rooting culture to a seedling bed for cultivation;

[0016] Optionally, the substrate in the seedbed includes peat soil and perlite.

[0017] In some embodiments, the volume ratio of the peat soil to the perlite is (0.8-1.2): (0.8-1.2).

[0018] Compared with the traditional technology, this application has the following beneficial effects:

[0019] The present application propagates and cultivates Casuarina equisetifolia through tissue culture method, which can maintain the excellent characteristics of the mother plant and has the characteristics of short reproduction cycle and high efficiency; at the same time, the present application adopts 1 / 2WPM culture medium as the basic culture medium for rooting culture, which can promote root development and improve the rooting rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 This is a schematic diagram of a process for the tissue culture method of Casuarina equisetifolia in one embodiment of the present application;

[0022] Figure 2 Schematic diagram of Casuarina equisetifolia branches with different degrees of lignification in this application. DETAILED DESCRIPTION

[0023] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present application. The terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. Unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present application, etc. can be purchased from the market or can be prepared by existing methods.

[0025] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0026] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0027] In the present application, 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.

[0028] In the present application, 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 1-10, 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.

[0029] The temperature parameters in this application, unless otherwise specified, 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 accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0030] At present, some researchers have tried to propagate Casuarina through tissue culture methods, but during the research process, they found that the offspring seedlings obtained basically had mutations, so further improvements are needed.

[0031] Some embodiments of the present application provide a method for tissue culture of Casuarina equisetifolia, comprising the following steps:

[0032] The young branches of Casuarina equisetifolia were taken as explants, and the explants were sterilized, induced, proliferated and rooted in sequence;

[0033] Among them, the basic culture medium for rooting culture is 1 / 2WPM culture medium.

[0034] In some examples, the Casuarina equisetifolia shoots are derived from robust disease-free cuttings of elite grafted Casuarina equisetifolia clones.

[0035] The inventors of the present application have found through extensive research that, compared with the commonly used 1 / 4MS medium and 1 / 2MS medium, using 1 / 2WPM medium as the basic medium for rooting culture can effectively promote the root development of young branches of Casuarina equisetifolia and significantly improve its rooting rate.

[0036] In some embodiments, the culture medium for rooting culture further includes 1.5 mg / L-2.5 mg / L IBA, 12 g / L-18 g / L sucrose and 5 g / L-7 g / L carrageenan.

[0037] It is understood that the concentrations of IBA, sucrose and carrageenan in the culture medium for rooting culture in the present application are calculated based on the volume of the basal culture medium.

[0038] It should be noted that, in the present application, "IBA" refers to indolebutyric acid, and "6-BA" refers to 6-benzylaminoadenine.

[0039] As an example, the concentration of IBA in the medium for rooting culture can be 1.5 mg / L, 1.6 mg / L, 1.7 mg / L, 1.8 mg / L, 1.9 mg / L, 2.0 mg / L, 2.1 mg / L, 2.2 mg / L, 2.3 mg / L, 2.4 mg / L, 2.5 mg / L, or any value within the range formed by any two of the above values. The concentration of sucrose in the medium for rooting culture can be 12 g / L, 12.5 g / L, 13 g / L, 13.5 g / L, 14 g / L, 14.5 g / L, 15 g / L, 15.5 g / L, 16 g / L, 16.5 g / L, 17 g / L, 17.5 g / L, 18 g / L, or any value within the range formed by any two of the above values. The concentration of carrageenan in the culture medium for rooting culture can be 5g / L, 5.1g / L, 5.2g / L, 5.3g / L, 5.4g / L, 5.5g / L, 5.6g / L, 5.7g / L, 5.8g / L, 5.9g / L, 6.0g / L, 6.1g / L, 6.2g / L, 6.3g / L, 6.4g / L, 6.5g / L, 6.6g / L, 6.7g / L, 6.8g / L, 6.9g / L, 7g / L, or any value within the range formed by any two of the above point values.

[0040] Furthermore, the culture medium for rooting culture also includes 1.8 mg / L to 2.2 mg / L IBA, 14 g / L to 16 g / L sucrose and 5.5 g / L to 6.5 g / L carrageenan.

[0041] Furthermore, the culture medium for rooting culture also includes 2 mg / L IBA, 15 g / L sucrose and 6 g / L carrageenan.

[0042] In some embodiments, the sterilization step includes: sterilizing the explant with 0.05 w / v% to 0.5 w / v% mercuric chloride.

[0043] The inventors of the present application have found through research that Casuarina equisetifolia branches have poor tolerance to alcohol, and that disinfecting Casuarina equisetifolia with mercuric chloride alone has a better induction effect than disinfecting Casuarina equisetifolia with alcohol, and the preservation rate of Casuarina equisetifolia explants after induced culture is significantly higher.

[0044] As an example, the concentration of mercuric chloride can be 0.05w / v%, 0.06w / v%, 0.07w / v%, 0.08w / v%, 0.09w / v%, 0.10w / v%, 0.11w / v%, 0.12w / v%, 0.13w / v%, 0.14w / v%, 0.15w / v%, 0.16w / v%, 0.17w / v%, 0.18w / v%, 0.19w / v%, 0.20w / v%, 0.21w / v%, 0.22w / v%, 0.23w / v%, 0.24w / v%, 0.25w / v%, 0.26w / v%, 0.27w / v%, 0.28 w / v%, 0.29w / v%, 0.30w / v%, 0.31w / v%, 0.32w / v%, 0.33w / v%, 0.34w / v%, 0.35w / v%, 0.36w / v%, 0.37w / v%, 0.38w / v%, 0.39w / v%, 0.40w / v%, 0.41w / v%, 0.42w / v%, 0.43w / v%, 0.44w / v%, 0.45w / v%, 0.46w / v%, 0.47w / v%, 0.48w / v%, 0.49w / v%, 0.50w / v%, or any value within the range formed by any two of the above point values.

[0045] Furthermore, the concentration of mercuric chloride is 0.1 w / v% to 0.5 w / v%.

[0046] In some of the embodiments, the basic medium for induction culture is 1 / 2MS medium, and the medium for induction culture also includes 0.4 mg / L~0.6 mg / L 6-BA, 12 g / L~18 g / L sucrose, 7 g / L~8 g / L carrageenan and 0.4 g / L~0.6 g / L activated carbon.

[0047] It is to be understood that the concentrations of 6-BA, sucrose, carrageenan and activated carbon in the culture medium for induction culture in the present application are calculated based on the volume of the basal culture medium.

[0048] As an example, the concentration of 6-BA in the culture medium for induced culture can be 0.4 mg / L, 0.41 mg / L, 0.42 mg / L, 0.43 mg / L, 0.44 mg / L, 0.45 mg / L, 0.46 mg / L, 0.47 mg / L, 0.48 mg / L, 0.49 mg / L, 0.50 mg / L, 0.51 mg / L, 0.52 mg / L, 0.53 mg / L, 0.54 mg / L, 0.55 mg / L, 0.56 mg / L, 0.57 mg / L, 0.58 mg / L, 0.59 mg / L, 0.6 mg / L, or any value within the range formed by any two of the above point values. The concentration of sucrose in the culture medium for induction culture can be 12g / L, 12.5g / L, 13g / L, 13.5g / L, 14g / L, 14.5g / L, 15g / L, 15.5g / L, 16g / L, 16.5g / L, 17g / L, 17.5g / L, 18g / L, or any value within the range formed by any two of the above values. The concentration of carrageenan in the culture medium for induction culture can be 7g / L, 7.1g / L, 7.2g / L, 7.3g / L, 7.4g / L, 7.5g / L, 7.6g / L, 7.7g / L, 7.8g / L, 7.9g / L, 8g / L, or any value within the range formed by any two of the above values. The concentration of activated carbon in the induction medium can be 0.4g / L, 0.41g / L, 0.42g / L, 0.43g / L, 0.44g / L, 0.45g / L, 0.46g / L, 0.47g / L, 0.48g / L, 0.49g / L, 0.50g / L, 0.51g / L, 0.52g / L, 0.53g / L, 0.54g / L, 0.55g / L, 0.56g / L, 0.57g / L, 0.58g / L, 0.59g / L, 0.6g / L, or any value within the range formed by any two of the above point values.

[0049] Furthermore, the induction culture medium also includes 0.45 mg / L~0.55 mg / L 6-BA, 14 g / L~16 g / L sucrose, 7.3 g / L~7.8 g / L carrageenan and 0.45 g / L~0.55 g / L activated carbon.

[0050] Furthermore, the culture medium for induction culture also includes 0.5 mg / L 6-BA, 15 g / L sucrose, 7.5 g / L carrageenan and 0.5 g / L activated carbon.

[0051] In some embodiments, the induction culture step includes: culturing at 22° C. to 28° C. for 16 h to 20 h per day, for 28 to 32 days, with a light intensity of 1800 Lux to 2200 Lux.

[0052] As an example, the temperature of induction culture can be 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, or any value within the range formed by any two of the above point values; the daily illumination culture time can be 16h, 17h, 18h, 19h, 20h, or any value within the range formed by any two of the above point values; the culture days can be 28 days, 29 days, 30 days, 31 days, 32 days, or any value within the range formed by any two of the above point values; the light intensity can be 1800Lux, 1900Lux, 2000Lux, 2100Lux, 2200Lux, or any value within the range formed by any two of the above point values.

[0053] Furthermore, the induction culture step includes: culturing at 25° C. for 18 hours per day, for 30 days, with a light intensity of 2000 Lux.

[0054] In some of the embodiments, the basic culture medium for proliferation culture is WPM culture medium, and the culture medium for proliferation culture also includes 0.1 mg / L~1 mg / L 6-BA, 24 g / L~26 g / L sucrose and 5 g / L~7 g / L carrageenan.

[0055] The inventors of the present application have found through extensive research that if MS medium is used as the basic medium for proliferation culture, the cultured Casuarina equisetifolia will be short and easy to vitrify; if 1 / 2MS medium is used as the basic medium for proliferation culture, although the plants are bright green, they basically do not proliferate; and if WPM medium is used as the basic medium, the cultured Casuarina equisetifolia plants grow well and cluster buds occur, so the present application uses WPM medium as the basic medium for proliferation culture.

[0056] It is to be understood that the concentrations of 6-BA, sucrose and carrageenan in the culture medium for proliferation culture in the present application are calculated based on the volume of the basal culture medium.

[0057] As an example, the concentration of 6-BA in the culture medium for proliferation culture can be 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1 mg / L, or any value within the range formed by any two of the above values. The concentration of sucrose in the culture medium for proliferation culture can be 24 g / L, 24.5 g / L, 25 g / L, 25.5 g / L, 26 g / L, or any value within the range formed by any two of the above values. The concentration of carrageenan in the culture medium for proliferation culture can be 5 g / L, 5.2 g / L, 5.4 g / L, 5.6 g / L, 5.8 g / L, 6.0 g / L, 6.2 g / L, 6.4 g / L, 6.6 g / L, 6.8 g / L, 7 g / L, or any value within the range formed by any two of the above point values.

[0058] Furthermore, the concentration of 6-BA in the proliferation culture medium is 0.1 mg / L~0.5 mg / L.

[0059] Furthermore, the concentration of 6-BA in the proliferation culture medium is 0.25 mg / L~0.5 mg / L.

[0060] It can be understood that the concentration of 6-BA in the culture medium has a significant effect on the proliferation of Casuarina equisetifolia. In the range of 0.1 mg / L~1 mg / L, the proliferation rate first increases and then decreases with the concentration of 6-BA. If the concentration of 6-BA is too high, the bud clusters may be short and vitrified. If the concentration of 6-BA is too low, the number of bud clusters may be small.

[0061] Furthermore, the culture medium for proliferation culture also includes 0.1 mg / L to 0.5 mg / L 6-BA, 24.5 g / L to 25.5 g / L sucrose and 5.5 g / L to 6.5 g / L carrageenan.

[0062] Furthermore, the culture medium for proliferation culture also includes 0.5 mg / L 6-BA, 25 g / L sucrose and 6 g / L carrageenan.

[0063] In some embodiments, the proliferation culture step includes: culturing at 22° C. to 28° C. for 16 h to 20 h per day, for 28 days to 32 days, and the light intensity is 1800 Lux to 2200 Lux.

[0064] As an example, the temperature of proliferation culture can be 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, or any value within the range formed by any two of the above point values; the daily illumination culture time can be 16h, 17h, 18h, 19h, 20h, or any value within the range formed by any two of the above point values; the culture days can be 28 days, 29 days, 30 days, 31 days, 32 days, or any value within the range formed by any two of the above point values; the light intensity can be 1800Lux, 1900Lux, 2000Lux, 2100Lux, 2200Lux, or any value within the range formed by any two of the above point values.

[0065] Furthermore, the rooting culture step includes: culturing at 25° C. for 18 hours per day, for 30 days, with a light intensity of 2000 Lux.

[0066] In some embodiments, the rooting culture step includes: culturing at 22° C. to 28° C. for 16 h to 20 h per day, for 28 days to 32 days, with a light intensity of 1800 Lux to 2200 Lux.

[0067] As an example, the temperature of rooting culture can be 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, or any value within the range formed by any two of the above point values; the daily light culture time can be 16h, 17h, 18h, 19h, 20h, or any value within the range formed by any two of the above point values; the culture days can be 28 days, 29 days, 30 days, 31 days, 32 days, or any value within the range formed by any two of the above point values; the light intensity can be 1800Lux, 1900Lux, 2000Lux, 2100Lux, 2200Lux, or any value within the range formed by any two of the above point values.

[0068] In some embodiments, after the rooting culture, the following steps are further included: transferring the rooted seedlings obtained by the rooting culture to a seedling bed for cultivation;

[0069] Optionally, the substrate in the seedbed includes peat moss and perlite.

[0070] In some examples, the volume ratio of peat moss to perlite is (0.8~1.2):(0.8~1.2).

[0071] As an example, the volume ratio of peat soil and perlite can be 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 0.9:0.8, 1:0.8, 1.1:0.8, 1.2:0.8, 0.8:0.9, 1:0.9, 1.1:0.9, 1.2:0.9, 0.8:1.1, 0.9:1.1, 1:1.1, 1.2:1.1, 0.8:1.2, 0.9:1.2, 1:1.2, 1.1:1.2, or any value within the range formed by any two of the above ratios.

[0072] The present application adopts the above method to carry out tissue culture on Casuarina equisetifolia to obtain rooted seedlings, which can maintain the excellent characteristics of the mother plant and have the advantages of short cycle and high efficiency.

[0073] The present application will be further described below in conjunction with specific examples and comparative examples, but they should not be construed as limiting the scope of protection of the present application. The raw materials involved in the following specific examples, unless otherwise specified, can all be sourced from commercial sources, the instruments used, unless otherwise specified, can all be sourced from commercial sources, and the processes involved, unless otherwise specified, are all conventionally selected by those skilled in the art.

[0074] Example 1

[0075] Refer to Figure 1 The specific steps are as follows:

[0076] (1) Cultivation of cuttings: Select a flat, sunny, turtle-back-shaped land to build a micro-vegetable nursery of 2 mu, and select excellent grafted asexual Casuarina as the material; use a planting bag with a diameter of 40 cm as the container, use yellow heart soil as the cultivation medium, apply 0.25 kg of compound fertilizer as the base fertilizer, supplemented by automatic drip irrigation facilities, regular weeding and pruning, and cultivate young cuttings of Casuarina.

[0077] (2) Explant selection and disinfection: Select healthy, disease-free, tender young spikelets, clean the surface of the spikelets with detergent, rinse with water for 30 minutes, and cut into 5 cm long segments containing buds. Disinfect with 0.1% mercuric chloride in a clean bench for 5 minutes.

[0078] like Figure 2 As shown, the degree of lignification of young cuttings is divided according to the following standards: the green and thinner branches newly sprouted from the main trunk are classified as "young and tender", the yellow-brown and thicker branches are classified as "medium", and the yellow and thick branches are classified as "old".

[0079] (3) Induction culture: After disinfection, the culture was induced in a culture medium containing 1 / 2MS medium + 0.5 mg / L 6-BA + 15 g / L sucrose + 7.5 g / L carrageenan + 0.5 g / L activated carbon. The culture temperature was 25 °C, the light duration was 18 h per day, the total culture period was 30 days, and the light intensity was 2000 Lux.

[0080] (4) Proliferation culture: Proliferation culture was carried out in a culture medium containing WPM medium + 0.5 mg / L 6-BA + 25 g / L sucrose + 6 g / L carrageenan; the culture temperature was 25°C, the light duration was 18 h per day, the total culture period was 30 days, and the light intensity was 2000 Lux.

[0081] (5) Rooting culture: Rooting culture was carried out in a culture medium containing 1 / 2WPM medium + 2 mg / L IBA + 15 g / L sucrose + 6 g / L carrageenan. The culture temperature was 25 °C, the light duration was 18 h per day, the total culture time was 30 days, and the light intensity was 2000 Lux.

[0082] (6) Transplantation: Transplant the rooted seedlings obtained from the rooting culture into the seedbed, where the substrate is a mixture of peat soil and perlite in a volume ratio of 1:1; cover with film and shade net after transplanting, and ventilate and water regularly; new roots sprout at the base after 1 week, remove the film after 1 month, and remove the shade net after 2 months when the seedlings grow to about 5 cm in height.

[0083] Example 2

[0084] The method is basically the same as Example 1, except that the degree of lignification of the young cuttings in step (2) is medium.

[0085] Example 3

[0086] The method is basically the same as Example 1, except that the degree of lignification of the young cuttings in step (2) is old.

[0087] Example 4

[0088] The method is basically the same as Example 1, except that the disinfection method in step (2) is different, specifically: first use 75% alcohol to treat for 5 seconds, and then use 0.1% mercuric chloride to treat for 5 minutes.

[0089] Example 5

[0090] The method is basically the same as Example 2, except that the disinfection method in step (2) is different, specifically: first use 75% alcohol to treat for 5 seconds, and then use 0.1% mercuric chloride to treat for 5 minutes.

[0091] Example 6

[0092] The method is basically the same as Example 3, except that the disinfection method in step (2) is different, specifically: first use 75% alcohol to treat for 5 seconds, and then use 0.1% mercuric chloride to treat for 5 minutes.

[0093] Example 7

[0094] It is basically the same as Example 1, except that the mass volume concentration of mercuric chloride is different; specifically, the mass volume concentration of mercuric chloride in Example 7 is 0.05%.

[0095] Example 8

[0096] It is basically the same as Example 1, except that the mass volume concentration of mercuric chloride is different; specifically, the mass volume concentration of mercuric chloride in Example 8 is 0.2%.

[0097] Example 9

[0098] It is basically the same as Example 1, except that the mass volume concentration of mercuric chloride is different; specifically, the mass volume concentration of mercuric chloride in Example 9 is 0.5%.

[0099] The preservation rates of the spikelets after one week of induction culture in Examples 1 to 9 were statistically analyzed, and the results are shown in Table 1. Wherein, preservation rate = number of spikelets that survived and were in good condition after one week of induction culture / number of original spikelets × 100%.

[0100] Table 1

[0101]

[0102] As shown in Table 1, the preservation rate of young branches as explants was the highest when disinfected in 0.1% mercuric chloride for 5 minutes, reaching 92.11%; as the concentration of disinfectant increased, the preservation rate of Casuarina increased, and the stem segments were bright green without browning. When the concentration was 0.5%, the preservation rate was the highest, reaching 96.63%. Therefore, the disinfection effect of Casuarina using 0.5% mercuric chloride for 5 minutes was good. The above results show that Casuarina branches are intolerant to alcohol, and it is better to disinfect with mercuric chloride alone; at the same time, the higher the degree of lignification of the branches, the higher the browning rate within 1 week of induction culture (the lower the preservation rate), and the worse the induction effect.

[0103] Example 10

[0104] The method is basically the same as Example 1, except that the concentration of 6-BA in the culture medium for proliferation culture in step (4) is different; specifically, the concentration of 6-BA in Example 10 is 1 mg / L.

[0105] Embodiment 11

[0106] The method is basically the same as Example 1, except that the concentration of 6-BA in the culture medium for proliferation culture in step (4) is different; specifically, the concentration of 6-BA in Example 11 is 0.25 mg / L.

[0107] Example 12

[0108] The method is basically the same as Example 1, except that the concentration of 6-BA in the culture medium for proliferation culture in step (4) is different; specifically, the concentration of 6-BA in Example 12 is 0.1 mg / L.

[0109] The proliferation rates of the spikelets cultured in Examples 1 and 11-12 were statistically analyzed, and the results are shown in Table 2.

[0110] Table 2

[0111]

[0112] It should be noted that the data in the above tables are all mean values ​​± standard deviations; different lowercase letters after the data in the same column in the table indicate significant differences among the treatments at the a=0.05 level.

[0113] It can be seen from the above table that 6-BA has a significant effect on the proliferation effect of Casuarina equisetifolia. The proliferation rate first increases and then decreases with the increase of concentration. When the 6-BA concentration in Example 10 is too high, although there are many clusters of buds, they are short and vitrified; when the 6-BA concentration in Example 11 is too low, there are fewer clusters of buds, affecting the proliferation efficiency; when the 6-BA concentration in Example 1 is 0.5 mg / L, there are many clusters of buds, which are strong, with less base callus, and the concentration is more appropriate.

[0114] Comparative Example 1

[0115] It is basically the same as Example 1, except that the basal culture medium of the rooting culture medium in step (5) is different; specifically, the basal culture medium of the rooting culture medium in Comparative Example 1 is 1 / 4MS culture medium.

[0116] Comparative Example 2

[0117] It is basically the same as Example 1, except that the basal culture medium of the rooting culture medium in step (5) is different; specifically, the basal culture medium of the rooting culture medium in Comparative Example 2 is 1 / 2MS culture medium.

[0118] Table 3

[0119]

[0120] As can be seen from the table above, the inorganic salt content in 1 / 4MS medium is low, the plants grow low and are yellowish; the roots cultured in 1 / 2MS medium are thick and short; the plants cultured in 1 / 2WPM medium are strong, with bright green stems, 2 to 3 fibrous roots, a well-developed root system, and a rooting rate of 66.7%. Therefore, this application selects 1 / 2WPM medium as the basic medium for rooting medium.

[0121] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0122] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the attached claims.

Claims

1. A tissue culture method for Casuarina equisetifolia, characterized in that: The steps include: Taking young branches of Casuarina equisetifolia as explants, and sequentially performing disinfection, induction culture, proliferation culture and rooting culture on the explants; Among them, the basic culture medium for rooting culture is 1 / 2WPM culture medium.

2. The tissue culture method of Casuarina equisetifolia according to claim 1, characterized in that, The rooting culture medium also includes 1.5 mg / L~2.5 mg / L IBA, 12 g / L~18 g / L sucrose and 5 g / L~7 g / L carrageenan.

3. The tissue culture method of Casuarina equisetifolia according to any one of claims 1 to 2, characterized in that, The disinfection step includes: using 0.05w / v% to 0.5w / v% mercuric chloride to disinfect the explant.

4. The tissue culture method of Casuarina equisetifolia according to any one of claims 1 to 2, characterized in that, The basic culture medium for induction culture is 1 / 2MS culture medium, and the culture medium for induction culture also includes 0.4mg / L~0.6mg / L 6-BA, 12g / L~18g / L sucrose, 7g / L~8g / L carrageenan and 0.4g / L~0.6g / L activated carbon.

5. The tissue culture method of Casuarina equisetifolia according to any one of claims 1 to 2, characterized in that, The induction culture step includes: culturing at 22°C to 28°C for 16 hours to 20 hours per day, culturing for 28 days to 32 days, and the light intensity is 1800Lux to 2200Lux.

6. The tissue culture method of Casuarina equisetifolia according to any one of claims 1 to 2, characterized in that, The basic culture medium for proliferation culture is WPM culture medium, and the culture medium for proliferation culture also includes 0.1 mg / L~1 mg / L 6-BA, 24 g / L~26 g / L sucrose and 5 g / L~7 g / L carrageenan.

7. The tissue culture method of Casuarina equisetifolia according to any one of claims 1 to 2, characterized in that, The steps of proliferation culture include: culturing at 22°C to 28°C for 16 hours to 20 hours per day, for 28 days to 32 days, with a light intensity of 1800 Lux to 2200 Lux.

8. The tissue culture method of Casuarina equisetifolia according to any one of claims 1 to 2, characterized in that, The rooting culture steps include: culturing at 22°C to 28°C for 16 hours to 20 hours per day, for 28 days to 32 days, with a light intensity of 1800 Lux to 2200 Lux.

9. The tissue culture method of Casuarina equisetifolia according to any one of claims 1 to 2, characterized in that, After the rooting culture, the method further comprises the following steps: transferring the rooted seedlings obtained by the rooting culture to a seedling bed for cultivation; Optionally, the substrate in the seedbed includes peat soil and perlite.

10. The tissue culture method of Casuarina equisetifolia according to claim 9, characterized in that: The volume ratio of the peat soil to the perlite is (0.8-1.2):(0.8-1.2).

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