Alpinia oxyphylla culture medium and application thereof

Abstract

The invention discloses a fructus alpiniae oxyphyllae culture medium and application thereof. Belongs to the technical field of tissue culture. The culture medium comprises a callus induction and proliferation culture medium, a cluster bud differentiation and proliferation culture medium and a rooting culture medium. Each culture medium is based on the cheap and easily available MS or 1 / 2 MS culture medium, appropriate exogenous hormones or raw materials are added, each culture medium is scientific and reasonable in formula and can be respectively used for each stage of tissue culture of the alpinia oxyphylla, and the whole culture process is high in propagation speed, short in period and high in survival rate; the excellent hereditary character of the alpinia oxyphylla variety can be well maintained. The culture medium is simple in formula and low in cost, and has good popularization and application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue culture, and more particularly to an intelligence-enhancing culture medium and application thereof. Background Art

[0002] Alpinia oxyphylla is a perennial herb of the genus Alpinia in the Zingiberaceae family. Its capsule is spindle-shaped or spherical and can be used as medicine or made into health food. It has the functions of benefiting the spleen and stomach, regulating vital energy, and replenishing kidney deficiency.

[0003] The propagation of yizhi seedlings usually adopts seed propagation and division propagation. Yizhi is a cross-pollinated plant, and the characteristics of seedlings are severely separated and the individual differences are large. Although division propagation can maintain the excellent characteristics of the mother plant, the propagation coefficient is low and a large number of seedlings cannot be propagated in a short period of time. How to breed a large number of yizhi seedlings with uniform quality and stable genetics in a short period of time has become an important factor restricting the healthy development of the yizhi industry.

[0004] Plant tissue culture technology has the characteristics of accelerating the breeding process, shortening the reproduction time, improving plant quality, saving space, reducing labor, producing all year round, and not being restricted by natural conditions. In recent years, some progress has been made in the research of tissue culture of Alpinia oxyphylla, but there are still some problems, mainly the lack of culture medium suitable for the nutritional requirements of each stage of tissue culture of Alpinia oxyphylla, which greatly limits the application of tissue culture technology in Alpinia oxyphylla.

[0005] In summary, how to provide a phased Alpinia oxyphylla tissue culture medium is a problem that those skilled in the art urgently need to solve. Summary of the invention

[0006] In view of this, the present invention provides an intelligence-enhancing culture medium and application thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] An Alpinia oxyphylla culture medium, comprising a callus induction and proliferation culture medium, a cluster bud differentiation and proliferation culture medium and a rooting culture medium;

[0009] The callus induction and proliferation culture medium includes: MS+6-BA 2-5 mg / L+NAA 0.1-0.3 mg / L;

[0010] The cluster bud differentiation and proliferation culture medium includes: MS+6-BA 5-8 mg / L+TDZ 0.3-0.7 mg / L;

[0011] The rooting culture medium comprises: 1 / 2MS+NAA 0.2-0.5 mg / L+IAA 0.1-0.4 mg / L.

[0012] Beneficial effects achieved: Under in vitro culture conditions, plants have different nutritional requirements at different growth and development stages. Only when their respective special requirements are met can they grow and develop better. Therefore, screening and optimizing the culture medium formula is a key link for successful tissue culture. The culture medium formula of the present invention is obtained through careful and repeated experiments, and different types and proportions of growth hormones are added according to the nutritional requirements of each growth and development period of Alpinia oxyphylla.

[0013] Further, it includes callus induction and proliferation medium, cluster bud differentiation and proliferation medium and rooting medium;

[0014] The callus induction and proliferation medium includes: MS+6-BA 3mg / L+NAA 0.1mg / L;

[0015] The cluster bud differentiation and proliferation culture medium includes: MS+6-BA 8mg / L+TDZ 0.3mg / L;

[0016] The rooting culture medium includes: 1 / 2MS+NAA 0.3mg / L+IAA 0.2mg / L.

[0017] Furthermore, the callus induction and proliferation medium, cluster bud differentiation and proliferation medium and rooting medium also include 30-55 g / L of sucrose.

[0018] Furthermore, the callus induction and proliferation medium, cluster bud differentiation and proliferation medium and rooting medium also include 4-6 g / L of agar or carrageenan.

[0019] Further, it includes callus induction and proliferation medium, cluster bud differentiation and proliferation medium and rooting medium;

[0020] The callus induction and proliferation medium is: MS+6-BA 3mg / L+NAA 0.1mg / L+sucrose 30g / L+carrageenan 5g / L;

[0021] The cluster bud differentiation and proliferation medium is: MS+6-BA 8mg / L+TDZ 0.3mg / L+sucrose 45g / L+agar 5g / L;

[0022] The rooting medium is: 1 / 2MS+NAA 0.3mg / L+IAA 0.2mg / L+sucrose 40g / L+agar 6g / L.

[0023] Application of the above culture medium in Alpinia oxyphylla tissue culture.

[0024] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects achieved by the present invention are:

[0025] Each culture medium in the present invention is based on cheap and easily available MS or 1 / 2MS culture medium, and is further formed by adding appropriate exogenous hormones or raw materials. The formula of each culture medium is scientific and reasonable, and can be used for each stage of tissue culture of Alpinia oxyphylla. The whole cultivation process has a fast reproduction speed, a short cycle, and a high survival rate, and can well maintain the excellent genetic characteristics of the Alpinia oxyphylla variety. In addition, the culture medium formula of the present invention is simple, the cost is low, and it has a good promotion and application prospect. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] The drugs required for the present invention are conventional experimental drugs, which are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0028] Example 1

[0029] An Alpinia oxyphylla culture medium, comprising a callus induction and proliferation culture medium, a cluster bud differentiation and proliferation culture medium and a rooting culture medium;

[0030] The callus induction and proliferation medium includes: MS+6-BA 2mg / L+NAA 0.1mg / L+sucrose 30g / L+agar 4g / L;

[0031] The cluster bud differentiation and proliferation culture medium includes: MS+6-BA 5mg / L+TDZ 0.3mg / L+sucrose 30g / L+agar 4g / L;

[0032] The rooting medium comprises: 1 / 2MS+NAA 0.2mg / L+IAA 0.1mg / L+sucrose 30g / L+agar 4g / L.

[0033] Example 2

[0034] An Alpinia oxyphylla culture medium, comprising a callus induction and proliferation culture medium, a cluster bud differentiation and proliferation culture medium and a rooting culture medium;

[0035] The callus induction and proliferation medium includes: MS+6-BA 5mg / L+NAA 0.3mg / L+sucrose 55g / L+carrageenan 6g / L;

[0036] The cluster bud differentiation and proliferation culture medium includes: MS+6-BA 8mg / L+TDZ 0.7mg / L+sucrose 55g / L+carrageenan 6g / L;

[0037] The rooting medium comprises: 1 / 2MS+NAA 0.5mg / L+IAA 0.4mg / L+sucrose 55g / L+carrageenan 6g / L.

[0038] Example 3

[0039] An Alpinia oxyphylla culture medium, comprising a callus induction and proliferation culture medium, a cluster bud differentiation and proliferation culture medium and a rooting culture medium;

[0040] The callus induction and proliferation medium is: MS+6-BA 3mg / L+NAA 0.1mg / L+sucrose 30g / L+carrageenan 5g / L;

[0041] The cluster bud differentiation and proliferation medium is: MS+6-BA 8mg / L+TDZ 0.3mg / L+sucrose 45g / L+agar 5g / L;

[0042] The rooting medium is: 1 / 2MS+NAA 0.3mg / L+IAA 0.2mg / L+sucrose 40g / L+agar 6g / L.

[0043] Comparative Example 1

[0044] An Alpinia oxyphylla culture medium, comprising a callus induction and proliferation culture medium, a cluster bud differentiation and proliferation culture medium and a rooting culture medium;

[0045] The callus induction and proliferation medium is: MS+6-BA 7mg / L+NAA 0.4mg / L+sucrose 30g / L+carrageenan 5g / L;

[0046] The cluster bud differentiation and proliferation medium is: MS+6-BA 3mg / L+TDZ 0.9mg / L+sucrose 45g / L+agar 5g / L;

[0047] The rooting medium is: 1 / 2MS+NAA 0.6mg / L+IAA 0.5mg / L+sucrose 40g / L+agar 6g / L.

[0048] Comparative Example 2

[0049] An Alpinia oxyphylla culture medium, comprising a callus induction and proliferation culture medium, a cluster bud differentiation and proliferation culture medium and a rooting culture medium;

[0050] The callus induction and proliferation medium is: MS+6-BA 1mg / L+NAA 0.1mg / L+sucrose 30g / L+carrageenan 5g / L;

[0051] The cluster bud differentiation and proliferation medium is: MS+6-BA 4mg / L+NAA 0.1mg / L+sucrose 45g / L+agar 5g / L;

[0052] The rooting medium is: MS+NAA 0.5 mg / L+IBA 0.3 mg / L+sucrose 60 g / L+agar 6 g / L.

[0053] Test 1

[0054] The culture medium of Examples 1 to 3 and Comparative Examples 1 to 2 was used to carry out tissue culture of Alpinia oxyphylla, and the specific steps were as follows:

[0055] (1) Collection and processing of explants:

[0056] The collection site is the Alpinia oxyphylla garden of the Institute of Tropical Crops Variety Resources, Chinese Academy of Tropical Agricultural Sciences. The Alpinia oxyphylla shoots were collected as explants. The mother plants of the collected shoots are strong, compact, with dense fruit clusters, no diseases and pests, and full side buds.

[0057] Cut the newly-sprouted shoots from the mother plant of Alpinia oxyphylla, wash the debris on the shoots with clean water, soak them in detergent water for 10 minutes, brush them clean and rinse them with tap water for 3 hours, then disinfect them with 75% ethanol for 5 seconds and then rinse them with sterile water, then disinfect them with 0.1% mercuric chloride solution for 10 minutes, rinse them with sterile water and set them aside.

[0058] (2) Callus induction

[0059] Cut the 1-cm-long shoot tip and inoculate it on the callus induction and proliferation medium. The culture temperature is 27±1℃, the light intensity is 1500lx, the light time is 12h / d, and the culture time is 30d.

[0060] The culture medium of Examples 1 to 3 had the best effect, with the explants significantly expanded and the callus having a compact texture. The culture medium of Comparative Examples 1 and 2 produced callus, but the callus was whitish in color and loose in texture.

[0061] (3) Differentiation culture

[0062] The callus tissue was transferred to the cluster bud differentiation and proliferation medium. After inoculation, it was first cultured in total darkness at 27±1°C for 9 days, and then cultured under the conditions of 14 hours of light per day and a light intensity of 3000lx until cluster buds were formed.

[0063] The culture medium of Examples 1 to 3 had the best effect, with robust buds, uniform size, and bud clusters. The culture medium of Comparative Examples 1 and 2 had dwarfed buds that were densely clustered.

[0064] (4) Rooting culture

[0065] The clustered buds were cut into single buds and inoculated into rooting medium. After inoculation, they were first cultured in total darkness at 27±1°C for 9 days, and then cultured under conditions of 18 hours of light per day and a light intensity of 2000lx until roots formed.

[0066] The culture medium of Examples 1 to 3 has the best effect, and the root system is complete and strong. The culture medium of Comparative Examples 1 and 2 has sparse and thin root systems.

[0067] (5) Hardening and transplanting

[0068] After hardening the seedlings in a greenhouse with 50% shade for one week, take the seedlings out of the test tubes, clean the culture medium attached to the roots with clean water, soak them in 0.2% carbendazim for 20 minutes, plant them in a nutrient cup filled with nutrient soil, water them thoroughly to allow them to take root, and cover them with 50% shade nets. After about one week, the seedlings resume growth and are then managed according to conventional seedling raising methods.

[0069] The rooting rate and transplanting survival rate of each group were statistically analyzed and the results are shown in the following table.

[0070] Table 1 Rooting rate and transplant survival rate results

[0071]

[0072]

[0073] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0074] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A brain culture medium, characterized in that It includes callus induction and proliferation medium, cluster bud differentiation and proliferation medium and rooting medium; The callus induction and proliferation culture medium includes: MS+6-BA 2-5 mg / L+NAA 0.1-0.3 mg / L; The cluster bud differentiation and proliferation culture medium includes: MS+6-BA 5-8 mg / L+TDZ 0.3-0.7 mg / L; The rooting culture medium comprises: 1 / 2MS+NAA 0.2-0.5 mg / L+IAA 0.1-0.4 mg / L.

2. The culture medium according to claim 1, characterized in that It includes callus induction and proliferation medium, cluster bud differentiation and proliferation medium and rooting medium; The callus induction and proliferation medium includes: MS+6-BA 3mg / L+NAA 0.1mg / L; The cluster bud differentiation and proliferation culture medium includes: MS+6-BA 8mg / L+TDZ 0.3mg / L; The rooting culture medium includes: 1 / 2MS+NAA 0.3mg / L+IAA 0.2mg / L.

3. The culture medium according to any one of claims 1 or 2, characterized in that The callus induction and proliferation culture medium, cluster bud differentiation and proliferation culture medium and rooting culture medium also include 30-55 g / L of sucrose.

4. The culture medium according to any one of claims 1 or 2, characterized in that The callus induction and proliferation culture medium, cluster bud differentiation and proliferation culture medium and rooting culture medium also include 4-6 g / L of agar or carrageenan.

5. The culture medium according to claim 1, characterized in that It includes callus induction and proliferation medium, cluster bud differentiation and proliferation medium and rooting medium; The callus induction and proliferation medium is: MS+6-BA 3mg / L+NAA 0.1mg / L+sucrose 30g / L+carrageenan 5g / L; The cluster bud differentiation and proliferation medium is: MS+6-BA 8mg / L+TDZ 0.3mg / L+sucrose 45g / L+agar 5g / L; The rooting medium is: 1 / 2MS+NAA 0.3mg / L+IAA 0.2mg / L+sucrose 40g / L+agar 6g / L.

6. Use of the culture medium according to any one of claims 1 to 2 in Alpinia oxyphylla tissue culture.

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