CN111448988B

CN111448988B - Culture method and culture medium for inhibiting endophyte pollution in primary culture of magnolia zenii

Info

Publication number: CN111448988B
Application number: CN202010417890.1A
Authority: CN
Inventors: 王姗; 鲍华鹏; 黄帅; 何洪宇; 张永丽; 徐池; 朱碧娴
Original assignee: Jiangsu Polytechnic College of Agriculture and Forestry
Current assignee: Jiangsu Polytechnic College of Agriculture and Forestry
Priority date: 2020-05-18
Filing date: 2020-05-18
Publication date: 2021-08-13
Anticipated expiration: 2040-05-18
Also published as: CN111448988A

Abstract

The invention discloses a culture method and a culture medium for inhibiting endophyte pollution in primary culture of magnolia zenii. The culture method comprises pretreatment, wherein the pretreatment is to immerse an explant in 100-200mg/L neomycin sulfate sterile water solution for 12-36h, and the explant is a current-year shoot of magnolia zenii. The method adopts the annual sprouting branch of magnolia zenii as the explant, inoculates the explant on a primary culture bud induction culture medium after the explant is subjected to aseptic treatment, and cultures the explant under the conditions of proper temperature, humidity and illumination intensity to induce the bud to sprout. The bacteriostatic rate of the invention reaches 100 percent, the bud induction rate is 100 percent, the death rate is 0, the browning rate is 0, and the clustered buds grow robustly.

Description

Culture method and culture medium for inhibiting endophyte pollution in primary culture of magnolia zenii

Technical Field

The invention relates to a plant tissue culture propagation method, in particular to a culture method and a culture medium for inhibiting endophyte pollution in primary culture of magnolia zenii.

Background

Magnolia grandiflora (Magnolia zenii Cheng) is a deciduous tree of the genus Magnolia of the family Magnoliaceae, and belongs to the extremely dangerous (CR) species according to the IUCN latest evaluation criteria. The trunk of the magnolia zenii is straight and can be used as good wood; the flower is fragrant, is a precious ornamental garden tree, can be used as a medicine, and can play a role in resisting inflammation and tumors by using the magnolia zenii seed total extract. The magnolia zenii is difficult to naturally update, and if measures are not taken in time to solve the problem of difficult updating of the magnolia zenii, the magnolia zenii has extinction danger. Although natural protection areas and national forest parks of magnolia zenii have been established at present, less work is carried out aiming at the tissue culture rapid breeding technology of magnolia zenii, the magnolia zenii seeds are utilized for sexual propagation, most wild resources are not effectively developed and utilized due to low natural propagation rate, and the problem of difficult propagation of magnolia plants can be effectively solved by adopting a plant tissue culture method.

The tissue culture research of magnolia plants is just started, but the tissue culture of magnolia zenii has not been reported yet, and the magnolia plants and most of woody plants also face the problem of serious endophyte pollution. Woody plants have long growth years and complex growth environment, and endophytes are ubiquitous. The research shows that endophytes generally exist in the magnolia zenii root system, when the plant tissue in-vitro culture is carried out by adopting the conventional sterilization method, the conventional method can only kill surface germs but cannot kill the endophytes, and the endophytes can cause serious pollution to the explant of the magnolia zenii and directly cause failure of primary generation induction, so that a high-frequency regeneration system for tissue culture of the magnolia zenii can not be established, and further scientific research processes such as rapid propagation of magnolia zenii seedlings and magnolia zenii genetic engineering are influenced. Therefore, the development of a safe and effective endophyte inhibition method is a key link of magnolia zenii tissue culture.

Disclosure of Invention

The purpose of the invention is as follows: the invention provides a culture method for inhibiting endophyte pollution in primary culture of magnolia zenii. The invention also provides a culture medium adopted in the method. The invention solves the problem of the pollution of the endophyte of the exophyte in the primary culture of the magnolia zenii.

The technical scheme is as follows: the culture method for inhibiting the endophyte pollution in the primary culture of the magnolia zenii comprises pretreatment, wherein the pretreatment is to immerse an explant in 100-200mg/L neomycin sulfate sterile aqueous solution, the immersion time is 12-36h, and the explant is a current-year shoot of the magnolia zenii.

The culture method for inhibiting the endophyte pollution in the primary culture of magnolia zenii provided by the invention is characterized in that the annual shoot of magnolia zenii is taken as an explant, the explant is inoculated on a primary culture bud induction culture medium after the explant is subjected to aseptic treatment, and the culture is carried out under the conditions of proper temperature, humidity and illumination intensity, so as to induce the bud to germinate.

In one embodiment of the invention, the shoot induction medium comprises neomycin sulfate at a concentration of 300-500 mg/L.

In one embodiment of the present invention, the shoot induction medium is an MS medium, further comprising the following components: the content of 6-BA is 0.5-2mg/L, the content of NAA is 0.1-0.5mg/L, the content of active carbon is 2-5mg/L, the content of neomycin sulfate is 500mg/L, the content of agar is 7g/L, the content of sucrose is 30g/L, and the pH value of the culture medium is adjusted to 5.8-6.0.

In one embodiment of the invention, the culture method comprises an explant sterilization treatment, wherein the explant sterilization treatment comprises the steps of cutting off branches and leaves of the magnolia zenii subjected to the preliminary treatment, cutting long strips into 5-8cm short branches, washing the short branches with washing powder, washing the short branches with running water for 2-4 hours, transferring the explant to a sterile operating platform, sterilizing the short branches with 75% alcohol for 30-40 seconds, then sterilizing the short branches with 0.1% mercuric chloride for 8-10 minutes, washing the short branches with sterile water for 6-8 times, and then cutting the long branches into 1-2cm long-strip bud stem sections.

In one embodiment of the invention, the culturing method comprises inoculation of the budded stem segments onto a bud induction medium.

In one embodiment of the invention, the culture method comprises blue light induction culture, wherein the blue light induction culture is to irradiate the inoculated explants under 400-440nm blue light for 7-10d, and the culture condition is that the temperature is 23-27 ℃.

In one embodiment of the invention, the culture method comprises sunlight-induced culture, wherein the sunlight-induced culture is to transfer explants subjected to blue light-induced culture into a fluorescent lamp with the light intensity of 1500-.

The invention relates to a bud induction culture medium for inhibiting endophyte pollution in primary culture of magnolia zenii, which comprises the following components: the content of 6-BA (6-benzylaminopurine) is 0.5-2mg/L, the content of NAA (naphthylacetic acid) is 0.1-0.5mg/L, the content of active carbon is 2-5mg/L, the content of neomycin sulfate is 300-500mg/L, the content of agar is 7g/L, the content of sucrose is 30g/L, and the pH value of the culture medium is adjusted to 5.8-6.0.

Has the advantages that: according to the existing cluster bud induction technology, the invention overcomes the problem of the pollution of the endophyte in the explant in the bud induction process, improves the survival rate of the regeneration bud, obtains cluster buds with large quantity and good quality, meets the requirement of subculture multiplication and promotes the establishment of a rapid propagation system.

Detailed Description

Selection of culture conditions

1.1 selection of culture Medium and explant of Magnolia Baphicacanthus

Selecting an explant: selecting one year upper branch in crown of magnolia zenii (explant source used in the treatment 1 and the treatment 3) and a rhizome sprout (explant source used in the treatment 2 and the treatment 4) as explants, soaking the bottom of the cut branch in a sterile aqueous solution containing 200mg/L neomycin sulfate, and pretreating for 24 h;

explant disinfection: cutting leaves of pretreated magnolia zenii branches, cutting long strips into 5-8cm short branches, washing with washing powder, washing with running water for 2-4h, transferring explants onto a sterile operating table, sterilizing with 75% alcohol for 30-40 s, sterilizing with 0.1% mercuric chloride for 8-10 min, washing with sterile water for 6-8 times, and cutting into 1-2cm long-strip bud stem segments.

Inoculation: inoculating the stem segments with buds on a bud induction culture medium, inoculating one stem segment per glass bottle, wherein the components of the bud induction culture medium are MS culture medium, the content of added 6-BA is 1mg/L, the content of NAA is 0.5mg/L, the content of activated carbon is 5mg/L, the content of neomycin sulfate is 500mg/L, the content of agar is 7g/L, the content of sucrose is 30g/L, and the pH value of the culture medium is adjusted to 5.8 to be used as the bud induction culture medium (the culture medium for processing 1-2). Control Medium 1 (medium used in treatments 3-4) was prepared by replacing 500mg/L of neomycin sulfate with 500mg/L of cefamycin.

And (3) sunlight induction culture: the inoculated explants were placed under a fluorescent lamp with a light intensity of 2000lux for continuous culture for 30 d.

TABLE 1 Effect of different culture conditions on Primary culture of Magnolia Baphicacanthus

From the results shown in table 1, under the same culture conditions, when the rhizome shoot is selected as the explant, the culture effect of the primary culture of magnolia zenii is superior to that of the one-year-old shoot at the middle upper part of the crown as the explant. As can be seen from the culture effects of treatments 1-2 and 3-4, the use of neomycin sulfate in the induction medium of the present invention can greatly reduce the mortality and browning rate of the primary culture of magnolia zenii.

1.2 selection of light quality and culture Medium for Primary culture of Magnolia Baphicacanthus

According to the 1.1 part of the culture method, the treatment 5 is to place the inoculated explant under the blue light of 400-440nm for 7d, the culture condition is that the temperature is 25 ℃ and 2 ℃, and then the explant is transferred to a fluorescent lamp with the light intensity of 2000lux for continuous culture for 25 d.

Treatment 6 was continued for 32 days under 2000lux daylight lamp according to the cultivation method of part 1.1.

According to the cultivation method of part 1.1, treatment 7 is to remove neomycin sulfate from the bud induction medium, and the inoculated explants are irradiated under blue light of 400-440nm for 32 d.

According to the cultivation method of part 1.1, the treatment 8 is to remove neomycin sulfate from the bud induction medium, and the inoculated explant is placed under a 2000lux fluorescent lamp for continuous cultivation for 32 days. The results are shown in Table 2.

TABLE 2 influence of different light qualities and culture media on the primary culture of Magnolia grandiflora

As can be seen from the results in Table 2, the pollution of endophyte to the induced bud in the primary culture of Magnolia grandiflora is solved by the combination of the blue light and the sunlight.

Through the above experimental results, the following method is adopted in this example to perform the primary culture of magnolia zenii.

Selecting an explant: selecting a magnolia zenii current-year sprouting branch (rhizome sprouting branch) as an explant, soaking the bottom of the cut branch in a sterile aqueous solution containing 200mg/L neomycin sulfate, and pretreating for 24 hours;

Inoculation: inoculating the stem segments with buds on a bud induction culture medium, inoculating one stem segment per glass bottle, wherein the components of the bud induction culture medium are MS culture medium, adding 1mg/L of 6-BA, 0.5mg/L of NAA, 5mg/L of activated carbon, 500mg/L of neomycin sulfate, 7g/L of agar and 30g/L of cane sugar, and adjusting the pH value of the culture medium to 5.8.

And (3) blue light induction culture: the inoculated explants were placed under 400-440nm blue light for 7 days at 25 ℃ and 2 ℃.

And (3) sunlight induction culture: and transferring the culture medium to a fluorescent lamp with the light intensity of 2000lux for continuous culture for 25 d.

Through the culture by the method, the bacteriostasis rate of the primary culture of magnolia zenii reaches 100%, the inductivity reaches 100%, the primary budding time is advanced to 12d, the death rate of the explant is 0, the browning rate is 0, and the clustered buds grow robustly.

Claims

1. A culture method for inhibiting endophyte pollution in primary culture of magnolia zenii is characterized by comprising the following steps:

(1) pretreatment: the pretreatment is to immerse the explant in 100-200mg/L sterile water solution of neomycin sulfate, the immersion time is 12-36h, and the explant is the current-year sprouting branch of magnolia zenii;

(2) sterilizing explants; the explant sterilization treatment comprises the steps of cutting off branches and leaves of the magnolia zenii subjected to pretreatment, cutting long strips into 5-8cm short branches, washing with washing powder, washing with running water for 2-4h, transferring the explant to a sterile operating table, sterilizing with 75% alcohol for 30-40 seconds, then sterilizing with 0.1% mercuric chloride for 8-10 minutes, washing with sterile water for 6-8 times, and cutting into 1-2cm long-strip bud stem sections;

(3) inoculation: the inoculation is to inoculate the stem with the bud on a bud induction culture medium; the bud induction culture medium is an MS culture medium and comprises the following components: the content of 6-BA is 0.5-2mg/L, the content of NAA is 0.1-0.5mg/L, the content of active carbon is 2-5mg/L, the content of neomycin sulfate is 500mg/L, the content of agar is 7g/L, the content of sucrose is 30g/L, and the pH value of the culture medium is adjusted to 5.8-6.0;

(4) and (3) blue light induction culture: the blue light induction culture is to place the inoculated explant under 400-440nm blue light for irradiation for 7-10d, and the culture condition is 23-27 ℃;

(5) and (3) sunlight induction culture: the sunlight induction culture is to transfer the explants subjected to the blue light induction culture into a fluorescent lamp with the light intensity of 1500-.