CN109089888B

CN109089888B - Tissue culture and rapid propagation method of irkutsk anemone rhizome

Info

Publication number: CN109089888B
Application number: CN201811259744.XA
Authority: CN
Inventors: 刘红美; 梁大勇; 石建龙
Original assignee: Zunyi Longchi Biotechnology Co ltd
Current assignee: Zunyi Longchi Biotechnology Co ltd
Priority date: 2018-10-26
Filing date: 2018-10-26
Publication date: 2021-11-16
Anticipated expiration: 2038-10-26
Also published as: CN109089888A

Abstract

The invention relates to the technical field of seedling culture of traditional Chinese medicinal materials, in particular to a tissue culture and rapid propagation method of irkutsk anemone rhizome.

Description

Tissue culture and rapid propagation method of irkutsk anemone rhizome

Technical Field

Background

The irkutsk Anemone rhizome is a dried rhizome of Altai Anemone (Anemone altaica fisch.exe C.A.May.) belonging to Ranunculaceae, contains saponins, flavonoids and other components, and is one of the common traditional Chinese medicines for treating neurosis, fullness in chest and abdomen, inappetence and other symptoms. At present, more medicinal and scientific researches on irkutsk anemone rhizome are carried out, and the dosage is greatly increased, so that the existing market supply is seriously insufficient, and therefore, the artificial seedling and planting strength of the irkutsk anemone rhizome is required to be increased. However, in the prior art, researches on irkutsk anemone have mainly focused on the application of irkutsk anemone and the extraction of effective components in irkutsk anemone, such as: extracting saponin components (saponin monomer compounds, total saponins, etc.) from rhizoma anemones Altaicae; such as that disclosed in patent application No. 201210351556.6.

However, the research on artificial seedling and planting of irkutsk anemone rhizome is few, so that the irkutsk anemone rhizome is planted according to the traditional planting method, the planting cost of the irkutsk anemone rhizome is high, the seedling demand is high, the planting cost and the harvesting cost of the wild irkutsk anemone rhizome are greatly increased, and the medicine cost is increased.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention provides a tissue culture and rapid propagation method of irkutsk anemone rhizome.

The method is realized by the following technical scheme:

peeling off 0.05-0.1 cm-sized stem tip of irkutsk anemone rhizome, inoculating in callus induction culture medium, and culturing in dark environment for 40-50 days to obtain callus; inoculating the callus to an embryonic callus induction culture medium, and culturing for 30-35 days or 33 days to obtain embryonic callus; then transferring the embryonic callus into a differential proliferation culture medium, and carrying out differential culture for 30-40 days to obtain adventitious buds; and (3) cutting the adventitious buds into single seedlings, inoculating the single seedlings into a rooting and strong seedling culture medium, carrying out rooting culture for 25 days, and domesticating to obtain the plant seedling. The stem tip of the irkutsk anemone rhizome is used as a tissue culture and rapid propagation raw material, and callus induction culture, differentiation and proliferation culture and rooting and seedling strengthening culture are carried out, so that tissue culture and seedling raising of the irkutsk anemone rhizome are realized, the culture of the irkutsk anemone rhizome is accelerated, the seedling demand in the planting process of the irkutsk anemone rhizome is met, and a feasible scheme is provided for storing the germplasm resources of the irkutsk anemone rhizome.

In order to enable irkutsk anemone rhizome to form callus with better quality, meet the requirements of subsequent differentiation and proliferation and improve the seedling emergence rate, preferably, the callus induction culture medium is MS +6-BA 1.0mg/L + TDZ 0.1mg/L +2, 4-D0.1 mg/L + sucrose 30g/L + agar powder 5 g/L. More preferably, the callus induction medium has a pH of 5.8 to 6.0.

In order to greatly improve the adventitious bud formation rate, improve the inoculation success rate and reduce the propagation and seedling cost, preferably, the embryogenic callus induction culture medium is MS +6-BA 0.5mg/L + TDZ 0.1mg/L + NAA 0.1mg/L + sucrose 30g/L + fructose 5g/L + hydrolyzed peptone 300g/L + agar powder 5 g/L. More preferably, the pH of said embryogenic callus induction medium is 5.8-6.0. The addition of fructose and hydrolyzed peptone can significantly improve the quality of callus and promote the formation of embryogenic callus.

In order to ensure that the nutrient requirement of the callus can be met in the differentiation and proliferation process and the embryogenic callus is promoted to be differentiated and form adventitious buds, preferably, the differentiation and proliferation culture medium is 0.5mg/L of MS +6-BA, 0.1mg/L of NAA, 30g/L of cane sugar and 5g/L of agar powder, and the pH value is 5.8-6.0.

In order to promote the rapid growth of adventitious buds, improve the survival rate of seedling culture and reduce the seedling culture cost, the preferable rooting and strong seedling culture medium is 1/2MS + NAA 0.1mg/L + IBA 0.1mg/L + GA30.1mg/L + sucrose 30g/L + agar powder 5g/L, and the pH value is 5.8-6.0.

The invention is not the most suitable, and the technicians in the field can operate according to the related common knowledge and the conventional technology in the biological tissue culture, particularly can manage and domesticate according to the operation method in the aspect of plant tissue culture, and realize the tissue culture of the irkutsk anemone rhizome.

Detailed Description

The technical solution of the present invention is further defined below with reference to the specific embodiments, but the scope of the claims is not limited to the description.

In the following examples, the Acorus tatarinowii adopted is to peel off the stem tip under a dissecting mirror, and the size of the peeled stem tip is between 0.05 and 0.1 cm.

Example 1

Inoculating the stem tip of irkutsk anemone rhizome into a callus induction culture medium, and culturing for 40-50 days in a dark environment to obtain a callus;

transferring the callus into a differential proliferation culture medium, and performing differential proliferation culture for 30-40 days to obtain adventitious buds;

cutting the adventitious bud into single seedlings, inoculating the single seedlings into a rooting and strong seedling culture medium, carrying out rooting culture for 25 days, and domesticating to obtain the plant seedling;

the callus induction culture medium is MS +6-BA 1.0mg/L + TDZ 0.1mg/L +2, 4-D0.1 mg/L + sucrose 30g/L + agar powder 5g/L, and the pH value is 5.8-6.0;

the differentiation and proliferation culture medium is MS +6-BA 0.5mg/L + NAA 0.1mg/L + sucrose 30g/L + agar powder 5g/L, and the pH value is 5.8-6.0;

the rooting and seedling strengthening culture medium is 1/2MS, NAA 0.1mg/L, IBA 0.1mg/L, GA30.1mg/L, cane sugar 30g/L, agar powder 5g/L and has the pH value of 5.8-6.0.

Example 2

On the basis of example 1, after callus is formed by culturing in a callus culture medium, the callus is inoculated in an embryonic callus induction culture medium for culturing for 10 to 30 days, and then inoculated in a differentiation and proliferation culture medium for culturing; the embryogenic callus induction culture medium comprises MS +6-BA 0.5mg/L + TDZ 0.1mg/L + NAA 0.1mg/L + sucrose 30g/L + fructose 5g/L + hydrolyzed peptone 300g/L + agar powder 5g/L, and the pH value is 5.8-6.0. The rest of the procedure was the same as in example 1.

Example 3

On the basis of example 2, the embryogenic callus induction medium was not supplemented with sucrose, fructose, hydrolyzed peptone components, and other components and amounts were unchanged, and the other contents were the same as in example 2.

The results of the example 1-3 tissue culture including the formation rate of callus formed at the stem tip and the formation rate of adventitious bud formed by differentiation of callus are shown in Table 1 below:

TABLE 1

	Callus formation Rate (%)	Rate of formation of adventitious bud (%)
			Example 1	87.4	90.4
Example 2	86.9	93.5
			Example 3	87.5	79.7

As shown in the data in table 1, the embryogenic callus induction medium culture is helpful for promoting the formation of adventitious buds, and can improve the adventitious bud formation rate to a certain extent; the components and the composition of the components in the culture medium can greatly influence the formation rate of the adventitious bud, and the treatment created by the invention is beneficial to improving the formation rate of the adventitious bud. The treatment method provided by the invention can maintain the formation rate of callus formed by the shoot tips of the irkutsk anemone rhizome to be more than 86%, and greatly reduces the seedling raising cost by using the shoot tips of the irkutsk anemone rhizome. The emergence rate is improved.

Furthermore, the researchers performed the transplantation treatment of the irkutsk anemone obtained from the seedlings of the embodiments 1-3, and counted the transplantation survival rate, the results are shown in the following table 2:

TABLE 2

	Survival Rate of transplantation (%)
		Example 1	93.2
Example 2	93.5
		Example 3	86.5

The data in table 2 show that the method is helpful for improving the transplanting survival rate of the irkutsk anemone seedlings and reducing the planting cost of the irkutsk anemone during the tissue culture and seedling culture process of the irkutsk anemone.

Claims

1. A tissue culture and rapid propagation method of irkutsk anemone rhizome is characterized in that a stem tip of 0.05-0.1cm of irkutsk anemone rhizome is stripped off and inoculated in a callus induction culture medium, and the irkutsk anemone rhizome is cultured for 40-50 days in dark environment to obtain callus; inoculating the callus to an embryonic callus induction culture medium, and culturing for 30-35 days to obtain embryonic callus; then transferring the embryonic callus into a differential proliferation culture medium, and carrying out differential culture for 30-40 days to obtain adventitious buds; cutting the adventitious bud into single seedlings, inoculating the single seedlings into a rooting and strong seedling culture medium, carrying out rooting culture for 25 days, and domesticating to obtain the plant seedling; the callus induction culture medium is MS +6-BA 1.0mg/L + TDZ 0.1mg/L +2, 4-D0.1 mg/L + sucrose 30g/L + agar powder 5 g/L; the pH value is 5.8-6.0; the embryogenic callus induction culture medium comprises MS, 6-BA 0.5mg/L, TDZ 0.1mg/L, NAA 0.1mg/L, sucrose 30g/L, fructose 5g/L, hydrolyzed peptone 300g/L and agar powder 5 g/L; the pH value is 5.8-6.0; the differentiation and proliferation culture medium is MS +6-BA 0.5mg/L + NAA 0.1mg/L + sucrose 30g/L + agar powder 5g/L, and the pH value is 5.8-6.0; the rooting and seedling strengthening culture medium is 1/2MS, NAA 0.1mg/L, IBA 0.1mg/L, GA30.1mg/L, sucrose 30g/L, agar powder 5g/L and has the pH value of 5.8-6.0.

2. The tissue culture and rapid propagation method of irkutsk anemone rhizome as claimed in claim 1, wherein the callus is formed by culturing in callus culture medium, then inoculated into embryogenic callus induction culture medium for 33 days, and then inoculated into differentiation proliferation culture medium for culturing.