CN114793892A - Primary culture method for reducing yellow-heart nocturnal explant pollution and browning - Google Patents
Primary culture method for reducing yellow-heart nocturnal explant pollution and browning Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract
The invention discloses a primary culture method for reducing yellow heart nocturnal explant pollution and browning, which comprises the following steps: selecting terminal buds of the eclipse of yellow heart as explants, pretreating, soaking the terminal buds in 0.01g/L PVP solution, performing dark treatment at 4 ℃ for 4-5 days, and cleaning; sterilizing the pretreated explant by using alcohol and NaClO solution to obtain a sterile explant of the physalis verticillata (L.) Merr; inoculating the sterile explant to a primary culture medium for culture, wherein the primary culture medium is added with anti-browning agents PVP and VC; according to the invention, the explant material taking, the explant pretreatment, the sterilization treatment and the hormone proportion in the culture medium are optimized, so that the pollution rate and the browning rate are effectively reduced to 4.17% and 47.22%, the problems of high pollution rate and browning rate of the primary culture of the night closure of the yellow heart are solved, and the method is simple to operate and low in cost.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a primary culture method for reducing pollution and browning of a nightshade yellow-heart explant.
Background
The Michelia martini (Michelia martini) is a Michelia plant of Magnoliaceae, has straight trunk, full and compact tree shape, bright leaf color, beautiful leaf shape, fragrant flower, light yellow, ripe fruit in autumn and dazzling red color, can be used for urban street trees or parks and courtyard arrangement, can also be used for extracting aromatic oil, and has ornamental value and material value. The yellow heart is naturally distributed in the south of Henan, the west of Hu Bei, the middle and south of Sichuan, Guizhou and the northeast of Yunnan in China. This species is listed in "national book of important protected wild plants (first lot)" approved on 8/4 of 1999: II grade; listed in the red bibliography (IUCN) of endangered species in the world natural protection alliance, namely near-danger (NT).
Magnoliaceae plants, as a typical representative of the multi-carpel plants, are key materials indispensable for exploring angiosperm origin, developing evolution and establishing angiosperm natural systems; meanwhile, the Magnoliaceae plants are important components of evergreen broad-leaved forests and deciduous broad-leaved forests from tropical to temperate zones, have high ecological value, are ornamental trees and industrial materials, medicinal materials and spice trees, and have economic value. However, the magnolia plants widely used at present are limited to a small part of the species of magnolia, michelia and manglietia, and the development and utilization degree is still insufficient compared with the abundant magnolia germplasm resources in China.
Under natural conditions, the magnolia plants have poor natural reproductive capacity due to the reasons of heterogynic plants, amphoteric flowers, early falling of stamens, different flowering phases of the gynoecial flowers and the like; in seed propagation, the germination rate of some species is low, and the distribution area of some species is narrow and the seed source is limited; in conventional vegetative propagation, the problem of difficult rooting in cutting propagation exists, and grafting propagation is influenced by a grafting mode and grafting time, so that the technical requirement on operators is high. Compared with the traditional propagation mode, the tissue culture is less influenced by factors such as seasons, environmental conditions and the like, has the advantages of high propagation speed and high propagation coefficient, can realize industrialized seedling culture, and is an effective way for developing and utilizing magnolia plant resources. At present, there are many reports on the tissue culture of magnolia, but there is no research on the tissue culture of the bulbophyllum flavum.
In the process of tissue culture of magnolia, pollution and browning phenomena generally exist. The browning of explants affects the germination of shoots of various magnolia plants and the proliferation and differentiation of callus (study on callus induction and browning control of stem segments of magnolia liliiflora pall. of Zingiberaceae [ J ] forestry science study, 2004 (06): 757) 762.; Liequly, Mamindong [ J ] Zhejiang forestry science, 2007 (01): 20-23+32.) and results in low efficiency of tissue culture and limits the large-scale production of tissue culture seedlings (Zhouyan, Guoshan, Qinzhiyu, Sumonti. Magnolia alba tissue culture [ J ] The proceedings of Hebei science and technology institute 2008, 22 (04): 19-22.). In the early stage of preliminary experiments on the tissue culture of the amaurosis zakii, the explants have serious pollution and browning phenomena, which hinders the establishment of a rapid propagation system.
The disinfection of the magnolia explants is more than that of the growing seasons, the apical bud and stem explant is disinfected by 0.1% mercuric chloride solution. However, mercury bichloride has a large environmental pollution, and it is difficult to recover and treat the waste liquid. Meanwhile, the mercury ions can kill microorganisms and damage explants at the same time. The longer the disinfection time, the lower the rate of explant contamination, but the higher the rate of explant browning.
It is currently generally accepted that browning in plant tissue culture is an enzymatic browning, that enzymes, substrates and oxygen are essential conditions for enzymatic browning, that phenolics are substrates for enzymatic browning reactions in explants, and that polyphenol oxidase (PPO) is a commonly accepted enzyme for inducing browning. In normal tissue cells, phenolic substances are distributed in vacuoles and PPO is distributed in various plastids or cytoplasms, without contact between the two. When the explant is established, cells near the incision are injured, the separation of phenols and PPO is broken, the phenols and the PPO are contacted, and the phenols are quickly oxidized into brown quinones and water under the catalysis of the PPO, so that the explant is damaged. The browning of the explant is inhibited by a plurality of technologies such as explant type, selection of picking season, pretreatment mode of the explant, antioxidant addition, dark culture and the like.
When the tissue of the yellow heart nocturnal emission is used for in vitro culture, how to control pollution and inhibit browning of the tissue of the yellow heart nocturnal emission so as to improve the success rate and efficiency of in vitro culture becomes a technical problem to be solved urgently in the development and utilization process of the wild resource of the yellow heart nocturnal emission.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a primary culture method for reducing the pollution and browning of the yellow heart nocturnal emission.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a primary culture method for reducing pollution and browning of a nightshade yellow-heart explant comprises the following steps:
(1) taking materials of explants and pretreating: selecting healthy and disease-free semi-lignified branches with terminal buds growing in the current year with yellow hearts and nights in 12-1 months in winter, wrapping the base parts of the semi-lignified branches with terminal buds by using wet absorbent cotton, transporting the semi-lignified branches in the direction of natural growth, and pretreating the semi-lignified branches for later use;
because stem segment pollution and browning of the yellow-heart nocturnal emission are serious, and a sterile system is difficult to establish for the stem segment explants, the application does not select stem segment explants commonly used by other magnolia plants, but selects top bud explants in the dormancy stage of the yellow-heart nocturnal emission.
Pretreatment of explants: soaking the top bud explant of the yellow-heart nocturnal emission in the dormancy stage in 0.01g/L PVP solution, performing dark treatment at 4 ℃ for 4-5 days, washing the top bud explant for 0.5h by using tap water, dipping the top bud explant with a cleaning solution by using a brush pen, slightly brushing the surface of the bud of the yellow-heart nocturnal emission explant, soaking the bud in the cleaning solution for 5min after brushing, and washing the bud with tap water for 1-2 h after soaking.
(2) Surface sterilization of explants: and (3) disinfecting and sterilizing the pretreated explant by using alcohol and NaClO solution to obtain the aseptic explant of the bulbophyllum flavum.
The method specifically comprises the following steps: on a clean bench, firstly using 75% alcohol with volume percentage for disinfection for 25s, washing with sterile water for 3-4 times, then adding 0.1 wt.% Tween 80 into 5 wt.% NaClO solution for disinfection for 12-14 min, washing with sterile water for 5-7 times to disinfect the treated explant, and fully contacting the disinfected and sterilized solution with the explant in the disinfection and sterilization process.
(3) Primary culture: inoculating a sterile explant to a primary culture medium for culture, removing outer-layer scales of the sterile explant before inoculating the sterile explant to the primary culture medium for culture, cutting off an injured part at the end of the sterile explant, and taking a glass can bottle with the caliber of 7cm as a culture container, wherein each bottle contains 40-50 mL of the culture medium. Inoculating 1 explant per bottle, and primary culture medium including MS +0.5 mg/L6-BA +0.1mg/L NAA + GA 3 50mg/L +0.5g/L PVP +0.5g/L VC +30g/L sucrose +7.5g/L agar; the culture conditions were: the culture temperature is 24 +/-1 ℃, the illumination time is 10-16 h/d, and the illumination intensity is 1000-3000 lx.
Further, adjusting pH of the primary culture medium to 5.80 before sterilization under 121 deg.C for 20min, adding PVP into the primary culture medium before high temperature sterilization, cooling the primary culture medium to below 60 deg.C after high temperature sterilization, and filtering sterilized VC and GA 3 Adding primary culture medium.
Compared with the prior art, the invention has the beneficial effects that:
1) the operating method of the primary culture stage of the tissue culture of the yellow-heart nocturnal emission is optimized, the approaches of explant material taking, explant pretreatment, sterilization treatment, hormone proportion in a culture medium and the like are optimized, the pollution rate and the browning rate are respectively and effectively reduced to 4.17 percent and 47.22 percent, the problems of high pollution rate and browning rate of the primary culture of the yellow-heart nocturnal emission are solved, and the operating method is simple to operate and low in cost.
2) According to the invention, the top bud explant with the yellow-heart nightshade in the dormant period is soaked in 0.01g/n of PVP solution and is treated in the dark at 4 ℃ for 4-5 d before sterilization treatment, so that the pollution rate and browning rate are effectively reduced.
3) The method adopts sodium hypochlorite sterilization with proper concentration to replace mercury bichloride, solves the problems that the mercury bichloride is harmful to the environment and heavy metal pollution, and the explant browning caused by improper mercury bichloride sterilization concentration and time, and can effectively reduce the browning rate and the pollution rate.
4) Cytokinin has regulating effect on the generation of phenolic substances in the buds of Magnoliaceae plants, and high-concentration cytokinin 6-BA can stimulate the activity of PPO, promote the synthesis of phenolic compounds and promote browning. In the invention, the proper 6-BA concentration of 0.5mg/L is selected, so that the browning caused by the concentration of cytokinin is reduced.
5) The invention simultaneously adopts two anti-browning agents of PVP (polyvinylpyrrolidone) as an adsorbent and VC (vitamin C) as an antioxidant to be added into the culture medium, and the phenolic substances released by the explants and quinone substances formed after oxidation are adsorbed, so that the formation of browning substances can be reduced through the synergistic effect of the two agents, and the browning prevention effect is achieved.
Drawings
FIG. 1 is a yellow heart nocturnal emission;
FIG. 2 is a apical bud, stem segment explant contaminated with eclipse of yellow heart;
FIG. 3 is terminal bud of the evening primrose without treatment browning;
fig. 4 is a top bud explant after treatment of pelteobagrus fulvidraco to prevent browning.
Detailed Description
The invention is further described with reference to specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. In the following examples, unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
The test plants of the following examples are Megalobaria; the collection place is Guizhou province agricultural science institute; the collection time is 2020, 12 months and 16 days; the test materials were: semi-lignified robust young shoots harvested in the field.
Example 1
A primary culture method capable of reducing pollution and browning of a yellow heart nocturnal explant comprises the following steps:
(1) selecting and pretreating explants: on a sunny day of 12-1 month in winter, selecting a healthy and disease-free semi-lignified branch with terminal buds growing in the current year with a yellow heart and a night, wherein the length of the branch is about 10-12 cm, and the base part of the branch is wrapped with wet absorbent cotton and placed in a foam box containing an ice bag according to a natural growth direction to be transported to a laboratory.
And (2) respectively carrying out pretreatment such as dark culture, low-temperature pretreatment and PVP solution soaking on branches, then cutting off terminal buds on the branches, washing the terminal buds with tap water for 0.5h, slightly brushing the surfaces of buds with a cleaning solution dipped by a brush pen, soaking in the cleaning solution for 5min after brushing, and washing with tap water for 1-2 h after soaking. The cells were sterilized and inoculated on MS medium without any hormones, and the browning rates are shown in Table 1. As can be seen from the table, the inhibition of browning is promoted by PVP soaking, dark culture and low-temperature pretreatment. The browning rate of 0.01g/L PVP soaking is lower than that of clear water soaking; the browning rate is not obviously reduced when the culture is carried out for 2-3 days in a dark environment, and the browning rate is reduced to a certain extent when the culture is carried out for 4-5 days in a dark environment. The browning rate can be reduced by low-temperature treatment for 2-5 days, wherein the browning rate is the lowest by low-temperature treatment for 4 days. The browning rate of the composite pretreatment was lower than that of the single pretreatment, and when 0.01g/L PVP immersion, low temperature treatment and dark treatment were combined, the browning rate was significantly reduced. Therefore, in different pretreatments, the inhibition effect on browning of the branches is the best after the branches are inserted into 0.01g/L PVP solution in a bottle for 4-5 d (the PVP solution is changed once in the midway) at 4 ℃.
TABLE 1 Effect of different pretreatments on explant browning
Note: different letters indicate that the levels differed significantly at the P < 0.05 level, as shown in the table below.
(2) Explant surface sterilization: and (3) disinfecting the pretreated explant with 75% alcohol for 25s on an ultra-clean workbench, washing with sterile water for 3-4 times, disinfecting with 5 wt.% to 10 wt.% NaClO solution added with 0.1% Tween 80 for 8-15 min, and washing with sterile water for 5-7 times. In the process of sterilizing the liquid medicine, the beaker is vibrated ceaselessly so that the sterilizing liquid is in full contact with the explant.
The contamination and browning rates for the different sterilization treatments are shown in table 2.
From the explant species, in different mortisesUnder the treatment of the microbial inoculum, the pollution rate of the stem section is obviously higher than that of the terminal bud, the pollution rate of the terminal bud can reach 4.17 percent at least in different sterilization treatments, and the pollution rate of the stem section is still 70.83 percent at least, so the terminal bud is more suitable to be used as an explant compared with the stem section; the difference between the optimal sterilization treatments for sodium hypochlorite and mercuric chloride sterilization is not significant in terms of the type, concentration and sterilization time of the sterilization solution, 5 wt.% NaClO sterilization for 15min and 0.1 wt.% HgCl sterilization 2 The pollution rate can be reduced to 4.17% after sterilization for 12min, and the browning rate of mercuric chloride after disinfection is slightly higher than that of sodium hypochlorite. Therefore, sodium hypochlorite can replace the common mercury bichloride as a sterilizing agent, and the sterilizing effect is not very different. From the view of the sterilization concentration and time of sodium hypochlorite, the sterilization effect of 10 wt.% NaClO is not obviously better than that of 5 wt.% NaClO, but the browning rate of 10 wt.% NaClO sterilization is higher, and the sterilization effect of 5 wt.% NaClO is optimal in combination. In the aspect of the sterilization time length, the 10-15 min sterilization time can effectively reduce the pollution rate, and the browning rate gradually increases along with the increase of the sterilization time. When 5 wt.% of NaClO solution is sterilized for 10-15 min, the comprehensive browning rate and the pollution rate are relatively optimal.
TABLE 2 Effect of different sterilization treatments on explant culture
(3) Primary culture and addition of browning inhibitor:
placing the sterile explant in a culture dish, removing outer-layer scales, cutting off injured parts at the end parts, taking a glass can bottle with the caliber of 7cm as a culture container, and inoculating 1 explant to each bottle of 40-50 mL primary culture medium. The primary culture medium is MS +0.5 mg/L6-BA (6-benzylamino adenine) +0.1mg/L NAA (1-naphthylacetic acid) +0.05mg/L GA 3 (gibberellin), 30g/L of cane sugar is added, and anti-browning agents CA (citric acid), AC (activated carbon), VC (vitamin C) and PVP (polyvinyl pyrrolidone) are added into a primary culture medium of 7.5g/L of agar, wherein the concentration of the PVP is 0.5g/L, 1g/L and 2 g/L; the CA concentration is 0.1g/L, 0.2g/L, 0.5 g/L; the AC concentration is 0.5g/L, 1g/L and 2 g/L; VC concentration is 0.1g/L, 0.2g/L, 0.5 g/L.
Adjusting pH of the primary culture medium to 5.80 before sterilization, wherein the sterilization condition is at 121 deg.C for 20min, and adding AC and PVP into the primary culture medium before high temperature sterilization; CA. VC, GA 3 Filtering for sterilization, sterilizing at high temperature, cooling the culture medium to below 60 deg.C, and adding CA, VC and GA 3 The medium was added. The temperature of the culture room is 24 +/-1 ℃, the illumination time is 10-16 h/d, and the illumination intensity on the surface of the culture is about 1000-3000 lx.
As shown in Table 3, comparing the effect of different treatments on explant browning, it can be found that the concentration of 6-BA has a certain effect on explant browning, when the concentration of 6-BA exceeds 0.5mg/L, the browning rate is significantly increased, and when the concentration of 6-BA is 0.5mg/L, the concentration is more appropriate. Comparing different anti-browning agents CA, AC, PVP and VC, it can be found that different anti-browning agents can inhibit browning to a certain extent, and the inhibition effect on browning is different due to different mechanisms of anti-browning. When a certain browning agent is singly used, the effects of the reducing agent type CA and VC are obviously superior to those of the adsorbent type PVP and AC; the anti-browning effect of PVP in the adsorbent is obviously better than that of AC. The anti-browning effect of different reducing agents VC and CA is not obvious in difference. The adsorbent PVP and a reducing agent are selected to be combined into a compound anti-browning agent formula, so that the anti-browning effect of the compound formula is better than that of a single formula, and the combination of the PVP and the VC has the best anti-browning effect. MS +6-BA 0.5mg/L + NAA 0.1mg/L + GA 3 50mg/L + VC 0.5g/L + PVP 0.5g/L is a better anti-browning culture medium formula in the primary culture stage, and the browning rate can be reduced to 47.22%.
TABLE 3 Effect of different treatments on explant browning
Claims (9)
1. Initial for reducing yellow heart nocturnal explant pollution and browningThe subculture method is characterized by comprising the following steps: selecting semi-lignified branches with top buds as a yellow-heart nocturnal explant, pretreating, soaking the explant in 0.01g/L PVP solution, performing dark treatment at 4 ℃ for 4-5 days, and cleaning; sterilizing the pretreated explant by using alcohol and NaClO solution to obtain a sterile explant of the physalis verticillata (L.) Merr; inoculating the sterile explant to a primary culture medium for culture, wherein the primary culture medium is MS +0.5 mg/L6-BA +0.1mg/L NAA + GA 3 50mg/L +0.5g/L anti-browning agent +30g/L sucrose +7.5g/L agar; the anti-browning agent comprises PVP and VC, and the concentration of the PVP and the concentration of the VC are both 0.5 g/L.
2. The primary culture method for reducing the pollution and browning of the yellow-heart nocturnal explant according to claim 1, characterized in that in the morning of 12-1 month sunny days in winter, the annual healthy and disease-free semi-lignified shoot with top buds of the yellow-heart nocturnal explant is picked as the yellow-heart nocturnal explant.
3. The primary culture method for reducing yellow heart nocturnal explant contamination and browning according to claim 2, wherein the base of the semi-lignified shoot with apical bud is wrapped with wet absorbent cotton and transported in the direction of natural growth.
4. The primary culture method for reducing the pollution and browning of the yellow-heart nocturnal explant according to claim 1, wherein the yellow-heart nocturnal explant is soaked in 0.1g/L PVP solution and is treated in the dark at 4 ℃ for 4-5 days, then is washed with tap water for 0.5h, then is dipped in a cleaning solution by a brush pen to brush the surface of the bud of the yellow-heart nocturnal explant, is soaked in the cleaning solution for 5min after being brushed, and is washed with tap water for 1-2 h after being soaked.
5. The primary culture method for reducing yellow heart nocturnal explant contamination and browning of claim 1, wherein the pretreated explants are sterilized with 75% volume percent ethanol and 5 wt.% NaClO solution containing 0.1 wt.% tween 80.
6. The primary culture method for reducing the pollution and browning of the nightly yellow-heart explant according to claim 5, wherein the treated explant is sterilized on a clean bench by using 75% alcohol by volume percentage for 25s, and then washed with sterile water for 3-4 times, and then sterilized with 5 wt.% NaClO solution added with 0.1 wt.% Tween 80 for 12-14 min, and then washed with sterile water for 5-7 times.
7. The primary culture method for reducing the pollution and browning of the yellow heart nocturnal explant according to claim 1, wherein the primary culture medium is cultured under the following conditions: the culture temperature is 24 +/-1 ℃, the illumination time is 10-16 h/d, and the illumination intensity is 1000-3000 lx.
8. The primary culture method for reducing the pollution and browning of the yellow-heart nocturnal explant according to claim 1, wherein 1 sterile explant is inoculated into 40-50 mL of primary culture medium, and before the sterile explant is inoculated onto the primary culture medium for culture, the outer layer scale of the sterile explant is removed, and the wound part at the end part of the sterile explant is cut off.
9. The primary culture method for reducing the contamination and browning of a yellow-heart nocturnal explant according to claim 1, wherein the pH of the primary culture medium is adjusted to 5.80 before sterilization under 121 ℃ for 20min, PVP is added to the primary culture medium before high-temperature sterilization, after high-temperature sterilization, the primary culture medium is cooled to below 60 ℃, and VC and GA which are sterilized by filtration are added 3 Adding primary culture medium.
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