A method for cultivating polyploid tsaoko fruits

Abstract

The present invention relates to the technical field of plant tissue culture, and specifically relates to a method for cultivating polyploid tsaoko fruits. The clustered buds induced by tissue culture are taken and shaken in an induction solution; the buds are inoculated in a clustered bud proliferation medium for cultivation; then they are cut into single buds and inoculated in a rooting medium for cultivation, and the seedlings with significantly widened and thickened leaves and significantly thickened roots are screened out, and chromosome staining and counting method is used for microscopic examination to screen out the rooting seedlings with chromosome number greater than or equal to 4n. The present invention uses 0.3-0.5 cm convex points as induction materials in combination with 0.01-0.03% colchicine + 0.03-0.05% oryzalin as induction substances, and cooperates with 0.3% dimethyl sulfoxide, so that the chromosome doubling rate reaches more than 40%, and almost no material damage and death will be caused, and while the chromosome doubling rate is greatly improved, the material will not be damaged; the combination of appropriate concentrations of NAA, BR and GA3 can make the germination rate of the clustered buds of the doubled materials reach more than 80%.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue culture, and specifically relates to a method for cultivating polyploid tsaoko amomum fruits. Background Art

[0002] Tsao-ko amomum fruit (Amomum tsao-ko Crevost et Lemaire) is a perennial herb of the genus Amomum in the Zingiberaceae family. It grows in the shaded and humid sparse forests in the tropical and subtropical regions at an altitude of 1100-1800 meters, and is mainly distributed in Yunnan, Guangxi, Guizhou and other places. Its dried and mature fruits are used as medicine, with the effects of drying dampness and warming the middle-jiao, and eliminating phlegm and arresting malaria. It is mainly used for symptoms such as damp-cold stagnation in the interior, abdominal distension and pain, fullness and vomiting, and malaria with chills and fever. At the same time, tsaoko amomum fruit belongs to the medicinal and edible homologous herbs, and is widely used as a flavoring raw material for food in the folk. Its whole plant (usually the fruits are the most) contains aromatic substances such as 1,8-cineole, trans-2-decenal, citral-b, etc., and is often used to extract volatile essential oils as perfume fragrances.

[0003] Polyploid exists widely in nature, and is that the number of somatic chromosomes in an organism is three or more groups. Polyploid plants generally show characteristics such as thickening of stems and leaves, enhanced stress resistance, and increased secondary metabolites, making polyploid breeding an important way of crop breeding. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for cultivating polyploid tsaoko amomum fruits.

[0005] To achieve the above purpose, the technical scheme adopted by the present invention is: a method for cultivating polyploid tsaoko amomum fruits, the steps include:

[0006] (1) Doubling treatment: Take the cluster buds induced by tissue culture, cut the growth points, and put them into the induction solution. Under the condition of no light at a temperature of 25±2°C, shake in a shaker at a frequency of 50-60 r / min for 24-36 h;

[0007] The induction solution is a mixed aqueous solution of 0.03% colchicine + 0.05% oryzalin + 0.3% dimethyl sulfoxide;

[0008] (2) Growth of cluster buds: The buds treated by doubling are rinsed once with sterile water and inoculated into the cluster bud proliferation medium, and cultured in an environment with a temperature of 25±2°C, a light intensity of 2000-3000 lx, and a single-day light time of 12 h;

[0009] (3) Rooting culture: The buds growing from the cluster buds are cut into single buds and inoculated into the rooting medium, and cultured in an environment with a temperature of 25±2°C, a light intensity of 2000-3000 lx, and a single-day light time of 12 h;

[0010] The rooting medium formula is: MS + NAA 0.1 - 0.2 mg / L + BR 0.01 - 0.02 mg / L + GA3 0.3 - 0.4 mg / L;

[0011] (4) Trait screening: After culturing for 50 - 60 days, select the seedlings with significantly widened and thickened leaves and significantly thickened roots as the visually doubled seedlings;

[0012] (5) Chromosome identification: Cut off the root tips of the visually doubled seedlings, and use the chromosome staining and counting method for microscopic examination to select the rooted seedlings with a chromosome number greater than or equal to 4n.

[0013] Further, the growth point is a raised point of 0.3 - 0.5 cm.

[0014] Further, the induction solution is a mixed aqueous solution of 0.02% colchicine + 0.04% oryzalin + 0.3% dimethyl sulfoxide.

[0015] Further, the rooting medium formula is: MS + NAA 0.15 mg / L + BR 0.01 mg / L + GA3 0.35 mg / L.

[0016] The beneficial technical effects of the present invention are:

[0017] 1. The composition and concentration of the induction solution are the key to the success of chromosome doubling. At present, most chromosome doubling techniques use a single colchicine as the induction solution, and a small number use one of oryzalin, trifluralin, pendimethalin, propyzamide, etc. as the induction solution, and there are even fewer research reports using two or more substances as the induction solution. After repeated induction experiments in the present invention, among the current induction substances, a single induction solution has extremely poor effects on chromosome doubling of Amomum tsaoko. The present invention finally uses an induction solution prepared by compounding multiple induction substances for induction experiments and finds that an induction solution composed of 0.01 - 0.03% colchicine + 0.03 - 0.05% oryzalin as the induction substances and 0.3% dimethyl sulfoxide can achieve a chromosome doubling rate of over 40% and hardly cause damage and death to the materials.

[0018] 2. During the chromosome doubling process of Amomum tsaoko, when the doubling material is too large, regardless of the type of induction solution used, the induction rate is relatively low. When the doubling material is too small and the concentration of the induction solution is too high, it is easy to cause a large number of deaths of the materials, and reducing the concentration of the induction solution will result in a significant decrease in the induction rate. The present invention uses a raised point of 0.3 - 0.5 cm as the induction material. When using an appropriate induction solution, on the one hand, the chromosome doubling rate can be significantly increased, and on the other hand, the induction solution with an appropriate concentration hardly damages the materials.

[0019] 3. During the tissue culture of tsaoko amomum, the conventional tsaoko amomum tissue culture proliferation medium or cluster bud induction medium is used, but it cannot sprout well or has a low sprouting rate. When inducing cluster buds from the doubled materials in the present invention, a combination of NAA, BR, and GA3 at appropriate concentrations is used, which can make the germination rate of cluster buds from the doubled materials reach more than 80%. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0021] Figure 1 It is the tsaoko amomum seedlings of the present invention without polyploid treatment and with treatment. On the left side of the figure is the one without polyploid treatment, and on the right side is the seedling treated in Example 1. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0023] Example 1

[0024] A method for cultivating polyploid tsaoko amomum, the steps include:

[0025] (1) Doubling treatment: Take the cluster buds obtained from steps (1) to (4) of Example 1 of a method for tissue culture and seedling raising of tsaoko amomum with the publication number CN111616049A and the name "A Method for Tissue Culture and Seedling Raising of tsaoko amomum", cut the growth point, the growth point is a 0.4 cm convex point, and put it into the induction solution (the induction solution is a mixed aqueous solution of 0.02% colchicine + 0.04% oryzalin + 0.3% dimethyl sulfoxide), and shake it at a frequency of 55 r / min in a shaker for 30 h at a temperature of 25 ± 2 °C under no light conditions.

[0026] (2) Growth of cluster buds: The buds after doubling treatment are rinsed once with sterile water and inoculated into the cluster bud proliferation medium (MS + 6-BA 2 mg / L + KT 5.0 mg / L + coconut water 100 ml / L), and cultured in an environment with a temperature of 25 ± 2 °C, a light intensity of 2500 lx, and a single-day light time of 12 h.

[0027] (3)Rooting culture: The clustered buds growing from the shoots are cut into single buds and inoculated into a rooting medium (MS + NAA 0.15 mg / L + BR 0.01 mg / L + GA3 0.35 mg / L), and cultured in an environment with a temperature of 25 ± 2°C, a light intensity of 2500 lx, and a single-day light duration of 12 h.

[0028] (4)Trait screening: After 55 days of culture, the seedlings with significantly wider and thicker leaves and significantly thicker roots are selected as the visually doubled seedlings.

[0029] (5)Chromosome identification: For the visually doubled seedlings, the root tips are cut off and examined under a microscope by the chromosome staining and counting method to screen out the rooted seedlings with a chromosome number greater than or equal to 4n.

[0030] As Figure 1 shown, after chromosome doubling culture on the right, compared with the non-doubled culture on the left, its stem is significantly wider and thicker, and the leaves are significantly wider.

[0031] Example 2

[0032] A method for cultivating Amomum tsaoko polyploid, the steps include:

[0033] (1)Doubling treatment: Take the clustered buds obtained from steps (1) to (4) of Example 1 of a method for tissue culture and seedling raising of Amomum tsaoko with the publication number CN111616049A and the name "A method for tissue culture and seedling raising of Amomum tsaoko", cut the growth point, where the growth point is a 0.3 cm convex point, and put it into an induction solution (the induction solution is a mixed aqueous solution of 0.01% colchicine + 0.03% oryzalin + 0.3% dimethyl sulfoxide), and shake it at a frequency of 50 r / min in a shaker for 24 h under the conditions of a temperature of 25 ± 2°C and no light.

[0034] (2)Growth of clustered buds: The buds treated by doubling are rinsed once with sterile water and inoculated into a clustered bud proliferation medium (MS + 6-BA 2 mg / L + KT 5.0 mg / L + coconut water 100 ml / L), and cultured in an environment with a temperature of 25 ± 2°C, a light intensity of 2000 lx, and a single-day light duration of 12 h.

[0035] (3)Rooting culture: The buds growing from the clustered buds are cut into single buds and inoculated into a rooting medium (formula: MS + NAA 0.1 mg / L + BR 0.01 mg / L + GA3 0.3 mg / L), and cultured in an environment with a temperature of 25 ± 2°C, a light intensity of 2000 lx, and a single-day light duration of 12 h.

[0036] (4)Trait screening: After 50 days of culture, the seedlings with significantly wider and thicker leaves and significantly thicker roots are selected as the visually doubled seedlings.

[0037] (5)Chromosome identification: Cut off the root tips of the visually doubled seedlings, and use the chromosome staining and counting method for microscopic examination to screen out the rooted seedlings with a chromosome number greater than or equal to 4n.

[0038] Example 3

[0039] A cultivation method for Amomum tsaoko polyploid, the steps include:

[0040] (1)Doubling treatment: Take the cluster buds obtained from steps (1) to (4) of Example 1 of a method for tissue culture seedlings of Amomum tsaoko with the publication number CN111616049A and the name "A Method for Tissue Culture Seedlings of Amomum tsaoko", cut off the growth points, where the growth points are convex points of 0.5 cm, and put them into the induction solution (the induction solution is a mixed aqueous solution of 0.03% colchicine + 0.05% oryzalin + 0.3% dimethyl sulfoxide), and shake at a frequency of 60 r / min in a shaker for 36 h under the condition of a temperature of 25 ± 2 °C and no light.

[0041] (2)Growth of cluster buds: The buds after doubling treatment are rinsed once with sterile water and inoculated into the cluster bud proliferation medium (MS + 6-BA 2 mg / L + KT 5.0 mg / L + coconut water 100 ml / L), and cultured in an environment with a temperature of 25 ± 2 °C, a light intensity of 3000 lx, and a single-day light time of 12 h.

[0042] (3)Rooting culture: The buds growing from the cluster buds are cut into single buds and inoculated into the rooting medium (formula: MS + NAA 0.2 mg / L + BR 0.02 mg / L + GA3 0.4 mg / L), and cultured in an environment with a temperature of 25 ± 2 °C, a light intensity of 3000 lx, and a single-day light time of 12 h.

[0043] (4)Trait screening: After culturing for 60 days, select the seedlings with significantly wider and thicker leaves and significantly thicker roots as the visually doubled seedlings.

[0044] (5)Chromosome identification: Cut off the root tips of the visually doubled seedlings, and use the chromosome staining and counting method for microscopic examination to screen out the rooted seedlings with a chromosome number greater than or equal to 4n.

[0045] The chromosome doubling rate, the mortality rate of buds after induction, and the germination rate of cluster buds were statistically analyzed for Examples 1 - 3, and the results are shown in the following table.

[0046] Table 1 Statistical results

[0047] Chromosome doubling rate Bud mortality rate after induction Germination rate of clustered buds Example 1 45.1% 0.2% 84.2% Example 2 43.3% 0.1% 81.6% Example 3 42.8% 0.3% 83.2%

[0048] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that the present invention can still be modified or equivalently replaced. Any modification or partial replacement without departing from the spirit and scope of the present invention shall be covered by the scope of the claims of the present invention.

Claims

1. A method for cultivating polyploid tsaoko fruits, characterized in that the steps are as follows Including: (1) Doubling treatment: Take the clustered buds induced by tissue culture, cut the growth points, put them into the induction solution, and shake them at a frequency of 50 - 60 r / min in a shaker for 24 - 36 h under the condition of a temperature of 25 ± 2°C and no light; The induction solution is a mixed aqueous solution of 0.01% - 0.03% colchicine + 0.03% - 0.05% oryzalin + 0.3% dimethyl sulfoxide; (2) Growth of clustered buds: The buds after doubling treatment are rinsed once with sterile water and inoculated into the clustered bud proliferation medium, and cultured in an environment with a temperature of 25 ± 2°C, a light intensity of 2000 - 3000 lx, and a single-day light time of 12 h; (3) Rooting culture: The buds growing from the clustered buds are cut into single buds and inoculated into the rooting medium, and cultured in an environment with a temperature of 25 ± 2°C, a light intensity of 2000 - 3000 lx, and a single-day light time of 12 h; The formula of the rooting medium is: MS + NAA 0.1 - 0.2 mg / L + BR 0.01 - 0.02 mg / L + GA3 0.3 - 0.4 mg / L; (4) Character screening: After 50 - 60 days of culture, select the seedlings with significantly widened and thickened leaves and significantly thickened roots as the externally doubled seedlings; (5) Chromosome identification: Cut off the root tips of the externally doubled seedlings, and examine them under a microscope by the chromosome staining and counting method to select the rooted seedlings with a chromosome number greater than or equal to 4n.

2. The method according to claim 1, wherein The growth point is a convex point of 0.3 - 0.5 cm.

3. The method according to claim 1, wherein The induction solution is a mixture of 0.02% colchicine + 0.04% oryzalin + 0.3% dimethyl sulfoxide.

4. The method according to claim 1, wherein The formula of the rooting medium is: MS + NAA 0.15 mg / L + BR 0.01 mg / L + GA3 0.35 mg / L.

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