Cultivation method of amomum tsao-ko polyploidy

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a cultivation method of amomum tsao-ko polyploidy, which comprises the following steps: putting cluster buds subjected to tissue culture induction into an induction solution, and shaking; inoculating the buds into a cluster bud proliferation culture medium for culturing; cutting into single buds, inoculating into a rooting culture medium, culturing, screening out seedlings with obviously widened and thickened leaves and obviously thickened roots, carrying out microscopic examination by using a chromosome staining counting method, and screening out rooting seedlings with the chromosome number greater than or equal to 4n. Protruding points of 0.3-0.5 cm are adopted as an induction material, 0.01-0.03% of colchicine and 0.03-0.05% of oryzalin are combined as induction substances, and 0.3% of dimethyl sulfoxide is matched, so that the chromosome doubling rate reaches 40% or above, material damage and death are hardly caused, the chromosome doubling rate is greatly increased, and meanwhile the material is not damaged; nAA, BR and GA3 with proper concentration are combined, so that the germination rate of multiple shoots of the doubled material can 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 at an altitude of 1100-1800 meters in the tropical and subtropical regions, 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, expelling phlegm and arresting malaria, and are mainly used for treating symptoms such as internal obstruction of cold-dampness, abdominal distension and pain, fullness and vomiting, malaria with chills and fever, etc. 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 fruit is 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 and flavoring agents.

[0003] Polyploid is widespread in nature, and is that the chromosome number of the somatic cells of an organism is three groups or more than three groups. Polyploid plants generally show characteristics such as thickened and thickened 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, put them into the induction solution, and shake them in a shaker at a frequency of 50-60 r / min for 24-36 h under the conditions of a temperature of 25±2°C and no light;

[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 grown 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 wider and thicker leaves and significantly thicker roots as the visually doubled seedlings;

[0012] (5) Chromosome identification: Cut the root tips of the visually doubled seedlings and examine them under a microscope using the chromosome staining and counting method to select the rooted seedlings with a chromosome number greater than or equal to 4n.

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

[0014] Further, the induction solution is an aqueous solution mixture 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. Currently, 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. 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 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 tsaoko, when the doubling material is too large, regardless of the 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 drop in the induction rate. The present invention uses a convex 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 appropriate concentration of the induction solution hardly damages the materials.

[0019] 3. During the tissue culture of tsaoko amomum, the conventional proliferation medium or cauloid induction medium for tsaoko amomum tissue culture is used, but it cannot germinate well or has a low germination rate. When inducing cauloids from the doubled materials in the present invention, an appropriate concentration of NAA, BR and GA 3 combination is used, which can make the germination rate of cauloids 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 drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0021] Figure 1 are the tsaoko amomum seedlings without polyploid treatment and with polyploid treatment in the present invention. The left side of the figure shows the seedlings without polyploid treatment, and the right side shows the seedlings treated in Example 1. Detailed 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, not all embodiments. All other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments of the present invention 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 cauloids 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 convex point of 0.4 cm, 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 under the condition of no light at a temperature of 25 ± 2 °C.

[0026] (2) Growth of cauloids: The buds after doubling treatment are rinsed once with sterile water and inoculated into the cauloid 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: Cut the buds growing from the cluster buds into single buds and inoculate them into the rooting medium (MS + NAA 0.15 mg / L + BR 0.01 mg / L + GA3 0.35 mg / L), and culture them in an environment with a temperature of 25 ± 2 °C, a light intensity of 2500 lx, and a single-day light exposure time of 12 h.

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

[0029] (5) Chromosome identification: Cut 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.

[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 cluster 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: Cut the growth point, which is a 0.3 cm convex point, and put it into the 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) Cluster bud growth: Rinse the buds treated by doubling once with sterile water, and inoculate them into the cluster bud proliferation medium (MS + 6-BA 2 mg / L + KT 5.0 mg / L + coconut water 100 ml / L), and culture them in an environment with a temperature of 25 ± 2 °C, a light intensity of 2000 lx, and a single-day light exposure time of 12 h.

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

[0036] (4) Trait screening: After 50 days of culture, select the seedlings with significantly wider and thicker leaves and significantly thicker roots 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 method for cultivating polyploid tsaoko fruits, the steps include:

[0040] (1) Doubling treatment: Take the clustered buds obtained from steps (1) to (4) of Example 1 of a method for tissue-culturing tsaoko fruits with the publication number CN111616049A and the name "A Method for Tissue-Culturing Seedlings of Tsao Kuo", cut off the growth points, where the growth points are convex points of 0.5 cm, and place them in 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 clustered buds: Rinse the buds treated by doubling once with sterile water, and inoculate them in the clustered bud proliferation medium (MS + 6-BA 2 mg / L + KT 5.0 mg / L + coconut water 100 ml / L), and culture them 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: Cut the buds growing from the clustered buds into single buds, and inoculate them in the rooting medium (formula: MS + NAA 0.2 mg / L + BR 0.02 mg / L + GA3 0.4 mg / L), and culture them 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 d, screen out 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] Statistics on the chromosome doubling rate, the mortality rate of buds after induction, and the germination rate of clustered buds were carried out 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 cluster 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, and any modification or partial replacement without departing from the spirit and scope of the present invention should be covered by the scope of the claims of the present invention.

Claims

1. A method for cultivating polyploid Amomum villosum, characterized in that the steps include: (1) Doubling treatment: Take the clustered buds induced by tissue culture, cut the growth point, put it into the induction solution, and shake it at a frequency of 50-60 r / min in a shaker for 24-36 hours at a temperature of 25±2℃ and without 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) Cluster bud growth: The double-treated buds were rinsed once with sterile water, inoculated into cluster bud proliferation medium, and cultured at a temperature of 25±2°C, a light intensity of 2000-3000 lx, and a single-day light duration of 12 h. (3) Rooting culture: The buds growing in clusters are cut into single buds, inoculated into rooting medium, and cultured in an environment with a temperature of 25±2℃, a light intensity of 2000-3000lx, and a single-day light duration of 12h; The rooting medium formula is: MS+NAA 0.1-0.2mg / L+BR 0.01-0.02mg / L +GA3 0.3-0.4mg / L; (4) Character screening: After 50 to 60 days of cultivation, the seedlings with significantly wider and thicker leaves and significantly thicker roots were selected as the appearance doubled seedlings; (5) Chromosome identification: Cut off the root tip of the doubled seedlings, examine them microscopically using the chromosome staining counting method, and select the rooting seedlings with a chromosome number greater than or equal to 4n.

2. The method according to claim 1, characterized in that: The growth point is a raised point of 0.3 to 0.5 cm.

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

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

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