Breeding method for cunninghamia lanceolata stem tip tissue culture

Abstract

The invention discloses a breeding method for fir stem tip tissue culture, and belongs to the technical field of plant tissue culture. Comprising the following steps: (1) selecting a stem tip on an excellent seed tree as an explant, sterilizing, inoculating to a callus induction culture medium, and culturing and inducing to obtain a callus; then inoculating to a callus proliferation culture medium to carry out callus proliferation; (2) carrying out hot air treatment on the proliferated callus; and (3) sequentially inoculating the callus treated by the hot air to an adventitious bud induction culture medium and a rooting culture medium to form tissue culture seedlings. According to the breeding method for cedarwood stem tip tissue culture, the culture period is remarkably shortened, operation is easy, cost is low, the method is not limited by seasons, and the survival rate is greatly increased.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a breeding method for Chinese fir stem tip tissue culture. Background Art

[0002] Cunninghamia lanceolata (Lamb.) Hook. is an evergreen tree of the genus Cunninghamia in the family Taxaceae. The tree can reach a height of 30 meters; the bark is gray-brown and cracked into long strips, and the inner bark is light red; the large branches are flat, the small branches are opposite or whorled, the young branches are green, smooth and hairless, the winter buds are nearly spherical, with small leaf-like bud scales; the leaves are lanceolate or narrow; the male cones are conical, green; the cones are oval, brown-yellow; the seeds are flat, long oval or rectangular, dark brown; the flowering period of Cunninghamia is April; the cones mature in late October. Because of its tall and upright tree shape and elegant crown, it is named Cunninghamia. Cunninghamia is one of the unique tree species in my country, and it also plays an important role in my country's artificial forestry. Because it has obvious advantages in growth rate and material texture, it is also widely used as a timber forest species.

[0003] The development of Chinese fir forests in my country is relatively slow, and the distribution of Chinese fir forests in various places is uneven. Relying solely on seedling cultivation can no longer meet the demand for Chinese fir seedlings. Compared with the cultivation of seedlings, the cultivation of Chinese fir seedlings through tissue culture and rapid propagation technology has the advantages of stable genetics, less impact from natural disasters, and fast and economical. Therefore, it is an urgent problem to use tissue culture and rapid propagation technology for Chinese fir and provide a standardized production process for high-quality tissue culture seedlings of Chinese fir. Summary of the invention

[0004] The purpose of the present invention is to provide a method for propagating Chinese fir stem tip tissue culture to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is a method for propagating Chinese fir stem tip tissue culture, comprising the following steps:

[0007] (1) selecting the stem tip of an excellent mother tree as an explant, sterilizing it, inoculating it into a callus induction medium for induction of callus, and then inoculating it into a callus proliferation medium for callus proliferation;

[0008] (2) subjecting the proliferated callus to hot air treatment;

[0009] (3) The callus tissue treated with hot air is inoculated into adventitious bud induction medium and rooting medium in sequence to form tissue culture seedlings.

[0010] Based on the above technical solution, the present invention has the following technical effects:

[0011] The invention discloses a method for propagating Chinese fir stem tip tissue culture, which can effectively promote the growth of adventitious buds and rooting culture of callus tissue by hot air treatment and adding onion extract to rooting culture medium before adventitious bud induction, making the plants stronger and growing better. The method for propagating Chinese fir stem tip tissue culture provided by the invention significantly shortens the culture cycle, is simple to operate, has low cost, is not restricted by seasons, and greatly improves the survival rate.

[0012] The method of the present invention can efficiently breed a large number of high-quality Chinese fir plants with consistent genetic traits. This tissue culture technology not only shortens the breeding cycle of Chinese fir, but also improves the breeding efficiency and quality, which is of great significance for the protection and utilization of Chinese fir germplasm resources. At the same time, the method also provides a useful reference and reference for the breeding of other tree species. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0014] Figure 1 The rooting culture of Example 1 of the present invention was carried out for 20 days.

[0015] Figure 2 The rooting culture was carried out for 20 days in Comparative Example 1 of the present invention.

[0016] Figure 3 The rooting culture was carried out for 20 days in comparative example 2 of the present invention. DETAILED DESCRIPTION

[0017] Now, various exemplary embodiments of the present invention are described in detail. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention.

[0018] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0019] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0020] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0021] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0022] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0023] The embodiment of the present invention provides a method for propagating Chinese fir stem tip tissue culture, comprising the following steps:

[0024] (1) selecting the stem tip of an excellent mother tree as an explant, sterilizing it, inoculating it into a callus induction medium for induction of callus, and then inoculating it into a callus proliferation medium for callus proliferation;

[0025] (2) subjecting the proliferated callus to hot air treatment;

[0026] (3) The callus tissue treated with hot air is inoculated into adventitious bud induction medium and rooting medium in sequence to form tissue culture seedlings.

[0027] In some specific embodiments, the callus induction medium includes: MS+6-BA 1.0 mg / L+2,4-D 0.2-0.5 mg / L+3% sucrose+0.5% agar powder, and the pH is adjusted to 6.0-6.5.

[0028] In some specific embodiments, the callus proliferation medium includes: WPM medium + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 3% sucrose + 0.5% agar powder.

[0029] In some specific embodiments, the temperature of the hot air treatment is 35-40° C., and the time is 30-60 min.

[0030] In some specific embodiments, the adventitious bud induction medium includes: MS+6-BA 0.5-1.0 mg / L+NAA 0.1-0.5 mg / L+3% sucrose+0.5% agar powder.

[0031] In some specific embodiments, the rooting medium comprises: 1 / 2MS+IBA 2.0 mg / L+onion extract 0.5 mg / L+1.5% sucrose+0.5% agar powder.

[0032] The onion extract used in the present invention is purchased from Shanyang Lianfeng Biotechnology Co., Ltd.

[0033] Example 1

[0034] 1. Induction of callus and callus proliferation

[0035] Cut the stem tips of healthy branches that have grown in the current year and are free of pests and diseases as explants. The stem tips are the growth points of plants, where cell division is active, the plants have high omnipotence, and are easy to induce callus formation.

[0036] Put the stem tip into clean water and scrub it carefully with a brush, then rinse it with running water for 30 minutes, and transfer it to a clean bench. Disinfect it with 75% alcohol by volume for 30 seconds, rinse it with sterile water three times, wipe it dry, inoculate it into callus induction medium, and culture it in the dark for 10 days to obtain callus. Callus induction medium: MS+6-BA 1.0mg / L+2,4-D0.2~0.5mg / L+3% sucrose+0.5% agar powder, pH adjusted to 6.0~6.5.

[0037] Use forceps to divide the callus into small pieces, inoculate on callus proliferation medium, and subculture once every 30 days. Callus proliferation medium includes: WPM medium + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 3% sucrose + 0.5% agar powder.

[0038] 2. Hot air treatment of the proliferated callus

[0039] The prepared callus tissue was placed in a culture bottle (MS culture medium), the culture bottle was sealed and placed in a heating device for heat treatment, and the temperature of the incubator was set at 37° C. for 40 minutes.

[0040] 3. Adventitious bud induction and rooting culture

[0041] After the heat treatment is completed, the callus tissue is taken out of the incubator and inoculated into an adventitious bud induction medium for induction culture. The adventitious bud induction medium includes: MS + 6-BA 0.5 mg / L + NAA 0.2 mg / L + 3% sucrose + 0.5% agar powder. When the adventitious buds are about 4 cm long, they are transferred to a rooting medium for rooting culture to obtain tissue culture seedlings. The rooting medium includes: 1 / 2MS + IBA 2.0 mg / L + onion extract 0.5 mg / L + 3% sucrose + 0.5% agar powder.

[0042] After 20 days of rooting culture, the number of roots, root length, and plant height were counted, and the survival rate was measured after acclimatization and transplanting.

[0043] Comparative Example 1

[0044] The only difference from Example 1 is that the hot air treatment step is omitted, and the remaining steps are exactly the same as those in Example 1.

[0045] After 20 days of rooting culture, the number of roots, root length, and plant height were counted, and the survival rate was measured after acclimatization and transplanting.

[0046] Comparative Example 2

[0047] The only difference from Example 1 is that no onion extract is added to the rooting medium, and the remaining steps are exactly the same as those in Example 1.

[0048] After 20 days of rooting culture, the number of roots, root length, and plant height were counted, and the survival rate was measured after acclimatization and transplanting.

[0049] Experimental results:

[0050] A control experiment was conducted on the tissue cultured Chinese fir seedlings of Example 1 and Comparative Examples 1-2, with 10 replicates in each group. The results are shown in Table 1.

[0051] Table 1 Effects of different tissue culture methods on Chinese fir tissue culture seedlings

[0052] Number of roots / roots Root length / cm Height / cm Growth Survival rate / % Example 1 7.5 2.16 5.8 Strong and growing well 93.5 Comparative Example 1 4.4 1.29 4.5 Average growth 86.6 Comparative Example 2 4.6 1.12 4.7 Average growth 83.9

[0053] It can be seen from the data in Table 1 that hot air treatment and addition of onion extract to the rooting medium before adventitious bud induction can effectively promote the adventitious bud growth and rooting culture of callus tissue, making the plants stronger and growing better. The breeding method of Chinese fir stem tip tissue culture provided by the present invention significantly shortens the culture cycle, is simple to operate, has low cost, is not restricted by seasons, and greatly improves the survival rate.

[0054] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for breeding Chinese fir stem tip tissue culture, characterized in that: The following steps are involved: (1) selecting the stem tip of an excellent mother tree as an explant, sterilizing it, inoculating it into a callus induction medium for induction of callus, and then inoculating it into a callus proliferation medium for callus proliferation; (2) subjecting the proliferated callus to hot air treatment; (3) The callus tissue treated with hot air is inoculated into adventitious bud induction medium and rooting medium in sequence to form tissue culture seedlings.

2. The breeding method according to claim 1, characterized in that: The callus induction culture medium comprises: MS+6-BA 1.0 mg / L+2,4-D 0.2-0.5 mg / L+3% sucrose+0.5% agar powder, and the pH is adjusted to 6.0-6.

5.

3. The breeding method according to claim 1, characterized in that: The callus proliferation culture medium comprises: WPM culture medium+1.0 mg / L 6-BA+0.1 mg / L NAA+3% sucrose+0.5% agar powder.

4. The breeding method according to claim 1, characterized in that: The temperature of the hot air treatment is 35-40° C. and the time is 30-60 minutes.

5. The breeding method according to claim 1, characterized in that: The adventitious bud induction culture medium comprises: MS+6-BA 0.5-1.0 mg / L+NAA 0.1-0.5 mg / L+3% sucrose+0.5% agar powder.

6. The breeding method according to claim 1, characterized in that: The rooting medium comprises: 1 / 2MS+IBA 2.0 mg / L+onion extract 0.5 mg / L+1.5% sucrose+0.5% agar powder.

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