CN112167058A

CN112167058A - Rapid propagation method for improved plant tissue culture

Info

Publication number: CN112167058A
Application number: CN202011050750.1A
Authority: CN
Inventors: 杨静; 何素芬
Original assignee: Sichuan Yunchen Garden Technology Co ltd
Current assignee: Sichuan Yunchen Garden Technology Co ltd
Priority date: 2020-09-29
Filing date: 2020-09-29
Publication date: 2021-01-05

Abstract

The invention discloses a rapid propagation method for improved plant tissue culture, which comprises the following steps: in the rapid propagation stage, an antibacterial culture medium, a differentiation culture medium and a strong seedling culture medium are adopted to sequentially carry out tissue culture seedling cultivation; in the rooting stage, the tissue culture seedlings cultured in the culture medium are planted in the matrix and then sprayed with the sterilized rooting mixed solution; the antibacterial agent used in the antibacterial culture medium comprises the following components in parts by weight: streptomycin 1.8-2.5 mg/L, gentamycin 2.5-4 mg/L, penicillin 0.8-1.5 mg/L, allicin 4.5-6 mg/L; the disinfection and rooting mixed solution comprises the following components in parts by weight: 90-110 mL/L of oxacor 2000 times, 3-8 mg/L of catechol and 7-12 mg/L of indoleacetic acid. The tissue culture seedling cultured by the method has low pollution rate, the transplanted tissue culture seedling has a thick and strong root system and high rooting rate and survival rate.

Description

Rapid propagation method for improved plant tissue culture

Technical Field

The invention relates to the technical field of agriculture, in particular to a rapid propagation method for improved plant tissue culture.

Background

Chrysanthemum (Dendranthema morifolium (Ramat) Tzvel) is a perennial root herbaceous plant of the compositae, is a traditional famous flower in China, is also one of the four cut flowers in the world, and is an important flower which is widely cultivated. At present, chrysanthemum is mainly used for greening and appreciation of gardens, such as manufacturing bouquets, wreaths, ornamental potted flowers, autumn flower beds, potted flower groups and the like, and is popular with people. The traditional chrysanthemum propagation method mainly comprises cuttage and grafting, but has the defects of aggravated generation-by-generation accumulation of plant viruses, degraded quality, unpleasant propagation speed, susceptibility to seasonal changes and external environmental conditions, high pest and disease occurrence frequency and the like, and in order to solve the problems, one of the most effective technical measures at present is to adopt a tissue culture technology.

The plant tissue culture technology develops rapidly from the exploration in the beginning of the 20 th century to the present, plays a great role in the aspects of rapid breeding of good varieties of agricultural, forestry and horticultural flower plants, industrialized production of virus-free seedlings and the like, brings considerable economic and social benefits to some large-scale tissue culture seedling production and management companies, and becomes one of the most vital subjects in the modern bioscience. The conventional plant tissue culture technology needs to be carried out in a very harsh sterile environment, and various problems of high requirements on equipment conditions, complex operation procedures, fine technology, high energy consumption, high culture cost, high pollution rate, difficult acquisition of explant sterile systems, low transplanting survival rate and the like exist all the time, so that the popularization and the application of the plant tissue culture technology are influenced and limited. Moreover, the tissue culture technology aiming at the chrysanthemum mainly relates to the relationship among hormone, chrysanthemum proliferation amount and rooting amount, the rooting amount is not ideal, and the comprehensive quality of seedlings is hardly related. Therefore, the establishment of a perfect rapid propagation technical system for chrysanthemum tissue culture has important significance for chrysanthemum detoxification and rejuvenation, industrialized rapid seedling propagation, germplasm resource preservation and the like.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a rapid propagation method for improved plant tissue culture, the pollution rate of the tissue culture seedling of chrysanthemum propagated by the method is low, the rooting rate and the survival rate after transplantation are both improved, and the root system is robust.

In order to solve the problems, the inventor improves the tissue culture operation procedure, simplifies the tissue culture breeding procedure and equipment conditions, and particularly provides the technical scheme that:

a rapid propagation method for improving plant tissue culture comprises the following steps:

a rapid propagation stage: sequentially carrying out tissue culture seedling cultivation by adopting an antibacterial culture medium, a differentiation culture medium and a strong seedling culture medium;

a rooting stage: planting the cultured tissue culture seedling in the culture medium into the matrix, and spraying the matrix with the sterilized rooting mixed solution;

the antibacterial agent used in the antibacterial culture medium comprises the following components in parts by weight: streptomycin 1.8-2.5 mg/L, gentamycin 2.5-4 mg/L, penicillin 0.8-1.5 mg/L, allicin 4.5-6 mg/L;

the disinfection and rooting mixed solution comprises the following components in parts by weight: 100-150 mg/L of 2-fold liquid of oxacor, 3-8 mg/L of catechol and 7-12 mg/L of indoleacetic acid.

Further, the antibacterial agent used in the antibacterial culture medium comprises the following components in parts by weight: streptomycin 2mg/L, gentamicin 3mg/L, penicillin 1mg/L and allicin 5 mg/L;

the disinfection and rooting mixed solution comprises the following components in parts by weight: 100mL/L of 2-fold liquid of oncomelania, 5mg/L of catechol and 10mg/L of indoleacetic acid.

The disinfection rooting mixed solution has an obvious synergistic effect on promoting rooting, and the oncomelania and the catechol can effectively inhibit the bioactivity of the indole acetic acid oxidase in vivo, so that the indole acetic acid treated by an external source can fully exert the effect of inducing rooting, and the effect is obvious.

Further, the differentiation medium is: MS culture medium +6-BA 0.8-1.2 mg/L + NAA 0.08-0.1 mg/L + agar 4.5-5.5 g/L + sugar 24-28 g/L, preferably MS culture medium +6-BA1.0mg/L + NAA0.1mg/L + agar 5g/L + sugar 26 g/L.

Further, the strong seedling culture medium is as follows: MS culture medium + agar 5g/L + sugar 26 g/L.

Further, the antibacterial culture medium also comprises a plant growth regulator.

Furthermore, the plant growth regulator is 0.5-1.5 mg/L6-BA, 0.08-0.12 mg/L NAA, and the purities of 6-BA and NAA are all 99.0%.

In a specific embodiment of the invention, the MS culture medium comprises macroelements, microelements, 10-20 mg/L of iron salt and 5-10 mg/L of organic matter;

the organic matter comprises the following components in parts by weight: 0.5-1.0 mg/L of nicotinic acid, 0.2-0.5 mg/L of pyridoxine hydrochloride, 0.1-0.3 mg/L of folic acid, 100-150 mg/L of inositol and 2-5 mg/L of glutamic acid, and dissolving the components of the organic matters and then mixing or dissolving after mixing to prepare a solution with the concentration of 5-10 mg/L;

the macroelements comprise 1200-1500 mg/L of ammonium nitrate, 1600-1800 mg/L of potassium nitrate, 400-420 mg/L of calcium chloride dihydrate, 350-365 mg/L of magnesium sulfate heptahydrate and 160-175 mg/L of monopotassium phosphate;

the trace elements comprise 7.5-8.5 mg/L of potassium iodide, 50-60 mg/L of boric acid, 200-250 mg/L of manganese sulfate tetrahydrate, 75-85 mg/L of zinc sulfate heptahydrate, 2.0-2.5 mg/L of sodium molybdate dihydrate, 0.2-0.25 mg/L of copper sulfate pentahydrate and 0.2-0.25 mg/L of cobalt chloride hexahydrate.

Further, the matrix is 1:1 perlite and vermiculite.

Further, the spraying frequency of the disinfection and rooting mixed solution is 2-3 days/time.

In a specific embodiment of the present invention, the tissue culture seedling inoculation in the rapid propagation stage is performed on an open type workbench.

The invention has the following beneficial effects:

(1) the operation process can be carried out only on the disinfected working table, the operation is convenient and fast, only 4-5 hours are needed for inoculating 100 explants, and the working efficiency is improved by 50%.

(2) The invention does not need ultraviolet irradiation and high-pressure sterilization, and utilizes natural light culture to replace the sterile indoor artificial light culture, thereby reducing the threshold and the production cost of the plant tissue culture technology and being beneficial to popularization and application.

(3) The invention combines rooting and seedling hardening, simplifies the rooting culture link of the test-tube plantlet in the bottle, saves labor and time, saves the rooting culture time by 20 days, has the rooting rate which is averagely higher than 20 percent, has more and more robust root systems and has the transplanting survival rate which is higher than 28 percent.

(4) The components in the antibacterial agent are synergistic, so that the antibacterial agent has different degrees of inhibition and killing effects on various pathogenic microorganisms such as bacteria, viruses, fungi and the like, has an obvious sterilization effect, and can reduce the pollution rate of tissue culture seedlings.

Detailed Description

The technical solutions of the present invention are described clearly and completely below, and it is obvious that the described embodiments are some, not all embodiments of the present invention. Based on the embodiments of the present invention, other embodiments obtained by a person of ordinary skill in the art without any creative effort belong to the protection scope of the present invention.

Example 1

First, preparation work

1. Preparation of antibacterial culture medium

Weighing 5g of agar and 26g of cane sugar, adding water, boiling, pouring a macroelement solution prepared from 1450mg/L of ammonium nitrate, 1700mg/L of potassium nitrate, 400mg/L of calcium chloride dihydrate, 350mg/L of magnesium sulfate heptahydrate and 170mg/L of potassium dihydrogen phosphate after melting, a microelement solution prepared from 0.85mg/L of potassium iodide, 5.5mg/L of boric acid, 25mg/L of 4-water manganese sulfate, 8.0mg/L of zinc sulfate heptahydrate, 0.2.5mg/L of sodium molybdate dihydrate, 0.025mg/L of copper sulfate pentahydrate and 0.025mg/L of cobalt chloride hexahydrate, 10mg/L of iron salt and 8.0mg/L of organic matter (the organic matter component comprises 0.8mg/L of nicotinic acid, 0.3mg/L of pyridoxine hydrochloride, 0.2mg/L of folic acid, 125mg/L of inositol and 4mg/L of glutamic acid), mixing after dissolving, mixing and boiling 1.0mg/L6-BA and 0.1mg/L NAA as plant growth regulators, fixing the volume, adjusting the pH value to 5.8-6.0, adding 2mg/L streptomycin, 3mg/L gentamicin, 1mg/L penicillin and 5mg/L allicin, boiling for 2 minutes, subpackaging and solidifying.

2. Formula and preparation of differentiation culture medium and seedling strengthening culture medium

The preparation is carried out according to the following formula:

differentiation medium: MS +6-BA1.0mg/L + NAA0.1mg/L + agar 5g/L + sugar 26g/L

Strong seedling culture medium: MS + agar 5g/L + sugar 26g/L

3. Soaking and sterilizing culture bottle and inoculation tool

Unscrewing the culture bottle and the cover, scissors, tweezers and other tools, putting the culture bottle and the cover into an aqueous solution containing 2% benzalkonium bromide, soaking the culture bottle and the cover into a 10% Oxetaz disinfection and sterilization solution for 30 minutes, taking out the culture bottle, standing upside down on a disinfected working table for later use, and putting the scissors and the tweezers into a 75% alcohol bottle for later use.

4. Room disinfection

Closing the door and the window to seal the room, fumigating for two hours by using 1 percent of potassium permanganate and 15mL of formaldehyde according to the volume of 20-25 mL per square meter, opening the door and the window, ventilating, and spraying 75 percent of alcohol for dust reduction.

Secondly, cleaning and disinfecting the surface of the explant

1. Explant selection

Selecting the tender stems of the current-year chrysanthemum which is strong and free from disease, insect and pest infection, cutting off the leaves, and then cutting into stem sections with two leaf nodes.

2. Surface cleaning and disinfecting

And (3) dropwise adding a few drops of liquid detergent into water, wherein the amount of the water can submerge the materials, washing the materials for 5 to 6 minutes by oscillation, then washing the materials for 20 minutes by using tap water, soaking the materials for 8 seconds by using 70 percent of alcohol after washing the materials completely, soaking the materials for 10 minutes by using 0.1 percent of mercuric chloride, and finally washing the materials for 5 times by using sterile water in an oscillation mode for 5 minutes each time.

Thirdly, inoculation and culture

1. Inoculation method

Cleaning both hands with soap, wiping the table and both hands with 75% alcohol, opening the bottle cap, removing the tool from the 75% alcohol bottle, inoculating the sterilized explant into the antimicrobial culture medium, and closing the bottle cap.

2. Indoor culture

Culturing in an antibacterial culture medium for 3 days, transferring a pollution-free explant to a differentiation culture medium, selecting a room with good illumination conditions and windows on the east and west, placing the inoculated tissue culture seedling on a culture shelf, regulating the indoor temperature to be 25 +/-2 ℃, culturing under natural light by using an outdoor light source, forming callus on the base of the explant after one week, growing axillary buds at leaf nodes, beginning to differentiate cluster buds after 10 days, and differentiating 10-20 slave buds of each explant.

Four, strong seedling culture

The method comprises the steps of cutting off side buds from the fact that the living buds grow to the fact that the height of the seedlings is about 1.5-2 cm, transferring the cut side buds to a sound seedling culture medium, and regulating and controlling the indoor temperature to be 25 +/-2 ℃ and the height of the seedlings to be 5-7 cm at 10-15 days.

Fifthly, ex vitro rooting and transplanting are combined

The method comprises the following steps of 1:1, preparing a planting matrix by perlite and vermiculite, pouring the matrix by clear water, then filling the matrix into a seedling raising hole tray with 50 holes, taking out a rootless tissue culture seedling from a culture bottle, washing off the culture medium attached to the seedling by clear water, planting the seedling exercising hole tray with the matrix with tight pressure, and spraying a sterilized rooting mixed solution prepared by 100mg/L of 2-fold liquid of oxazaline, 5mg/L of catechol and 10mg/L of indoleacetic acid for 2-3 days/time.

Comparative example 1

In contrast to example 1, in the preparation of an antibacterial medium, 1mg/L of streptomycin, 2mg/L of gentamicin and 3mg/L of penicillin were added to the medium.

Comparative example 2

In contrast to example 1, when preparing an antibacterial medium, streptomycin 3mg/L, gentamicin 1mg/L, and penicillin 2mg/L were added to the medium.

Comparative example 3

Compared with example 1, conventional tissue culture procedure was performed.

Firstly, preparing a culture medium

1. Culture medium formula

Preparing a primary culture medium, a secondary culture medium and a rooting culture medium according to the formula.

Primary culture medium: MS +6-BA1.0mg/L + NAA0.1mg/L + agar 7g/L + sugar 30g/L

Subculture medium: MS +6-BA1.5mg/L + NAA0.1mg/L + agar 7g/L + sugar 30g/L

Rooting culture medium: 1/2MS + NAA0.5mg/L + agar 7g/L + sugar 30g/L

2. Autoclaving of culture media

Placing the subpackaged culture bottles and inoculating tools into an autoclave, sterilizing at 121 deg.C under 0.105MPa for 21min, and taking out for use.

Secondly, cleaning and disinfecting the surface of the explant

The same procedure as in example 1 was used for cleaning and sterilizing the surface of the explant.

Thirdly, sterile inoculation and culture

1. And (2) sterile inoculation, wherein an ultraviolet lamp and a superclean workbench are started in advance 20 minutes in a sterile room, an alcohol lamp is ignited on superclean work, an inoculation tool is placed into a 75% alcohol bottle, the inoculation tool is taken out and poured into the alcohol lamp for 30-50 seconds, and after cooling, the explant is inoculated onto the culture medium subjected to high-pressure sterilization according to a conventional sterile operation procedure.

2. And (3) performing sterile culture, namely placing a culture bottle inoculated with chrysanthemum materials on a culture rack in a sterile culture room, controlling the indoor temperature to be 25 +/-2 ℃, the illumination intensity to be 2000Ix, and the illumination time to be 12-14 hours every day, and growing 1-3 lateral buds after 25 days.

Fourth, subculture

The lateral bud grows to 1-2 cm, the lateral bud is cut and transferred to a fresh differentiation culture medium, 2-5 lateral buds are differentiated from each stem section after 25 days, the lateral bud is cut down to be connected to a successive generation culture medium after the lateral bud grows to 5 cm, and a successive generation period is 25-28 days.

Fifthly, rooting culture and hardening seedling transplantation

1. Rooting culture: and (3) transferring the rootless seedlings into a rooting culture medium (1/2MS + NAA0.5mg/L), regulating and controlling the indoor temperature to be 25 +/-2 ℃, the illumination intensity to be 2000-2500 Ix, the illumination time to be 12-14 hours per day, and starting rooting in 15 days.

2. Hardening and transplanting seedlings: and (3) moving the test tube bottle seedlings out of the culture room one week before transplanting, placing the test tube bottle seedlings in the room under the conditions of natural temperature and illumination, opening the bottle caps, and exposing the test tube seedlings in the air for 5-7 days. Mixing perlite and vermiculite according to the proportion of 1:1, taking out tissue culture seedlings after being showered by clear water, washing off the culture medium attached to the seedlings by the clear water, planting the seedlings in the matrix, properly compacting the matrix, atomizing and spraying the wet seedlings by a sprayer after the matrix is planted, building a small arched shed, and keeping the matrix moist in the shed to ensure that the relative humidity of air is 90 percent.

Results were counted in the screening experiments of the antibacterial compositions in example 1 and comparative examples 1 and 2.

TABLE 1 statistics of screening experiments for different antibacterial compositions

As is clear from Table 1, the rate of contamination of the tissue culture seedlings was lower with the antibacterial agent in example 1 than with comparative examples 1 and 2.

The tissue culture seedlings and rooting and transplanting conditions in example 1 (improved tissue culture) and comparative example 3 (conventional tissue culture) were compared.

TABLE 2 rooting of tissue culture seedlings outside the bottles

As can be seen from table 2, compared with comparative example 3, the tissue culture seedling of example 1 had a rooting rate of 50% after 7 days, a rooting rate of 100% on day 15, a short root-delaying time, a short and robust root system, a fast growth, a high survival rate, and a strong seedling, whereas comparative example 3 had a conventional inoculation culture, in-bottle rooting, seedling hardening and transplanting, a rooting culture for 20 days in a bottle, and a long and slender root-delaying time, a long seedling growth and a slow seedling growth on day 15.

TABLE 3 comparison of modified and conventional tissue culture

As can be seen from Table 3, compared with comparative example 3, the contamination rate of the tissue culture seedling inoculated in example 1 is 0, the rooting of the tissue culture seedling outside the bottle is only 15 days, the rooting rate is 100%, the survival rate reaches 98%, and the hardening seedling transplantation is carried out after the rooting in the bottle of comparative example 3, 35 days are needed in total, the time is long, the rooting rate after the transplantation is only 80%, and the survival rate is only 70%.

Therefore, as can be seen from tables 1-3, the rooting culture time can be saved by 20 days by using the antibacterial agent and the sterilized rooting mixed solution, the rooting rate is averagely increased by 20%, the root system grows more and robustly, and the transplanting survival rate is increased by 28%.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. A rapid propagation method for improving plant tissue culture is characterized by comprising the following steps:

the disinfection and rooting mixed solution comprises the following components in parts by weight: 100-150 mg/L of 2 times of oxacor solution, 90-110 mL/L of oxacor solution, 3-8 mg/L of catechol and 7-12 mg/L of indoleacetic acid.

2. The rapid propagation method according to claim 1, wherein the antibacterial agent used in the antibacterial culture medium comprises the following components in parts by weight: streptomycin 2mg/L, gentamicin 3mg/L, penicillin 1mg/L and allicin 5 mg/L;

3. The rapid propagation method according to claim 1, wherein the differentiation medium is: MS culture medium +6-BA 0.8-1.2 mg/L + NAA 0.08-0.1 mg/L + agar 4.5-5.5 g/L + sugar 24-28 g/L, preferably MS culture medium +6-BA1.0mg/L + NAA0.1mg/L + agar 5g/L + sugar 26 g/L.

4. The rapid propagation method according to claim 1, wherein the strong seedling culture medium is: MS culture medium + agar 5g/L + sugar 26 g/L.

5. The rapid propagation method according to claim 1, wherein the antibacterial medium further comprises a plant growth regulator.

6. The rapid propagation method according to claim 5, wherein the plant growth regulator is 0.5 to 1.5mg/L6-BA, 0.08 to 0.12mg/L NAA.

7. The rapid propagation method according to claim 3 or 4, wherein the MS culture medium comprises macroelements, trace elements, iron salts 10-20 mg/L and organic substances 5-10 mg/L;

the organic matter comprises the following components in parts by weight: 0.5-1.0 mg/L of nicotinic acid, 0.2-0.5 mg/L of pyridoxine hydrochloride, 0.1-0.3 mg/L of folic acid, 100-150 mg/L of inositol and 2-5 mg/L of glutamic acid;

8. The rapid propagation method according to claim 1, wherein the matrix is 1:1 perlite, vermiculite.

9. The rapid propagation method according to claim 1, wherein the frequency of spraying the mixed solution for disinfection and rooting is 2-3 days/time.

10. The rapid propagation method according to claim 1, wherein the inoculation of the tissue culture seedling at the rapid propagation stage is performed on an open bench.