CN116711639A - Culture medium combination for preventing tissue culture browning of stevia rebaudiana and application thereof - Google Patents

Culture medium combination for preventing tissue culture browning of stevia rebaudiana and application thereof Download PDF

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CN116711639A
CN116711639A CN202310769307.7A CN202310769307A CN116711639A CN 116711639 A CN116711639 A CN 116711639A CN 202310769307 A CN202310769307 A CN 202310769307A CN 116711639 A CN116711639 A CN 116711639A
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culture
explant
culture medium
medium
tissue culture
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王冬良
何润琳
陈友根
邹良昊
陈珂畯
丁银媛
张玲
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Anhui Agricultural University AHAU
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Anhui Agricultural University AHAU
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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
    • A01H4/008—Methods for regeneration to complete plants
    • 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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  • Developmental Biology & Embryology (AREA)
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  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

The application belongs to the technical field of stevia rebaudiana tissue culture, and particularly relates to a culture medium combination for preventing stevia rebaudiana tissue culture from browning and application thereof. According to the application, the released phenolic acid substances can be absorbed by adding activated carbon into the induction culture medium, so that the browning of stevia rebaudiana tissue culture seedlings is reduced, the browning of explants is effectively prevented, and strong, wide leaf green and no Huang Shede adventitious buds are obtained; by adding activated carbon into the rooting culture medium, the rooting of the tissue culture seedlings can be promoted, the rooting number and root length of the tissue culture seedlings can be increased, and the regenerated stevia rebaudiana seedlings with good growth vigor, robustness and green leaves can be obtained.

Description

Culture medium combination for preventing tissue culture browning of stevia rebaudiana and application thereof
Technical Field
The application belongs to the technical field of stevia rebaudiana tissue culture, and particularly relates to a culture medium combination for preventing stevia rebaudiana tissue culture from browning and application thereof.
Background
Stevia rebaudiana (Steviare baudianaBertoni) is a perennial herb of the family Compositae, native to the yerba mate highland in south america. Stevia rebaudiana has been widely cultivated in various places since the 20 th century, 70 s. The main component of stevia rebaudiana Bertoni is stevioside, which is not only a natural sweetener, but also has the effects of controlling obesity, relieving nerve fatigue, lowering blood pressure, resisting tumor, etc. Stevia is therefore one of the important raw materials for the food and pharmaceutical industries.
At present, stevia rebaudiana adopts tissue culture technology to carry out breeding work. However, in the tissue culture process, phenolic substances released by the cut of the explant are oxidized by polyphenol oxidase to generate quinone substances, so that the culture medium is brown, the browning and death of the explant are easily caused, and the tissue culture survival rate of stevia rebaudiana is greatly reduced. Therefore, how to effectively prevent tissue culture browning of stevia rebaudiana and improve tissue culture survival rate of stevia rebaudiana is a problem to be solved urgently.
Disclosure of Invention
The application aims to provide a culture medium combination for preventing tissue culture browning of stevia rebaudiana and application thereof, which can effectively prevent tissue culture browning and improve tissue culture survival rate in the process of tissue culture breeding of stevia rebaudiana.
The application provides a culture medium combination for preventing tissue culture browning of stevia rebaudiana, which comprises an induction culture medium and a rooting culture medium; the induction culture takes MS culture medium as basic culture medium, and also comprises the following components in content: 0.5 to 1.5 mg.L -1 6-BA of (2), 0.1-0.5 mg.L -1 NAA of (C) 0.5-1.5 g.L -1 15-35 g.L -1 Sucrose and 4-8 g.L -1 Agar powder of (2); the rooting culture medium takes a 1/2MS culture medium as a basic culture medium and further comprises the following components in percentage by weight: 0 to 0.5 mg.L -1 IAA of (C) 0-0.5 mg.L -1 NAA of (2), 1.0-1.5 g.L -1 15-35 g.L -1 Sucrose and 4-8 g.L -1 Agar powder of (2).
Preferably, the combination of media further comprises a proliferation medium.
Preferably, the proliferation culture is based on MS culture medium, and further comprises the following components in percentage by weight: 0.5 to 1.5 mg.L -1 6-BA of (2), 0.1-0.5 mg.L -1 NAA of (2), 15-35 g.L -1 Sucrose and 4-8 g.L -1 Agar powder of (2).
The application also provides application of the culture medium combination in preventing tissue culture browning of stevia rebaudiana.
In addition, the application also provides a method for preventing tissue culture browning of stevia rebaudiana, which adopts the culture medium combination in the technical scheme, and comprises the following steps: inoculating the sterilized explant into an induction culture medium for induction culture to obtain adventitious buds; inoculating the adventitious buds into a proliferation culture medium for proliferation culture to obtain tissue culture seedlings; inoculating the tissue culture seedlings into a rooting culture medium for rooting culture to obtain regenerated seedlings; transplanting and cultivating the regenerated seedlings to obtain regenerated stevia rebaudiana.
Preferably, the first sterilization, the second sterilization and the third sterilization are sequentially carried out on the explant, so that the sterilized explant is obtained;
the first sterilization includes: mixing the explant with a washing powder aqueous solution with the mass percentage of 0.3% -0.5%, and then carrying out first soaking for 10-15 min, and sequentially cleaning the first soaked explant by flowing water and RO water to obtain a first sterilized explant;
the second sterilizing includes: mixing the first sterilized explant with 75% alcohol by volume percentage, and then soaking for 30-40 s, and cleaning the second soaked explant with sterile water to obtain a second sterilized explant;
the third sterilization includes: mixing the second sterilized explant with 0.1% mercury chloride solution, and then third soaking for 4-6 min, and cleaning the third soaked explant again by using sterile water.
Preferably, the first soaking process comprises shaking a container containing the mixture of the explant and the washing powder.
Preferably, the pH values of the culture mediums of the induction culture, the proliferation culture and the rooting culture are respectively 5.8-6.0.
Preferably, the culture conditions of the induction culture, the proliferation culture and the rooting culture respectively comprise: the culture temperature is 22-27 ℃, the relative humidity is 60-70%, the illumination intensity is 1500-2000 lx, and the illumination time is 12-16 h/d.
Preferably, the light source for illumination comprises an LED light source; the LED light source includes red light and blue light.
The beneficial effects are that:
the application provides a culture medium combination for preventing tissue culture browning of stevia rebaudiana, which comprises an induction culture medium and a rooting culture medium; the induction culture takes MS culture medium as basic culture medium, and also comprises the following components in content: 0.5 to 1.5 mg.L -1 6-BA of (2), 0.1-0.5 mg.L -1 NAA of (C) 0.5-1.5 g.L -1 15-35 g.L -1 Sucrose and 4-8 g.L -1 Agar powder of (2); the rooting culture medium takes a 1/2MS culture medium as a basic culture medium and further comprises the following components in percentage by weight: 0 to 0.5 mg.L -1 IAA of (C) 0-0.5 mg.L -1 NAA of (2), 1.0-1.5 g.L -1 15-35 g.L -1 Sucrose and 4-8 g.L -1 Agar powder of (2). The active carbon is added into the induction culture medium to effectively prevent the brown stain of the explant, phenol substances such as phenolic acid substances, e.g. chlorogenic acid, flavonoid and tannin, released in the tissue culture process of the stevia rebaudiana are effectively adsorbed, the occurrence probability of the brown seedlings of the tissue culture seedlings of the stevia rebaudiana is reduced, the strong and wide leaves without Huang Shede adventitious buds are obtained, then the active carbon is added into the rooting culture medium to promote the rooting of the tissue culture seedlings in the rooting stage, and the rooting number and root length of the tissue culture seedlings are improved. Experiments prove that when the induction culture medium and the rooting culture medium provided by the application are adopted, the probability of the brown change rate of the adventitious buds is only 6.7% compared with a rooting culture medium without adding activated carbon; and the regenerated seedlings after rooting culture are good in growth vigor and relatively strong, the rooting number and root length of the regenerated seedlings can be obviously increased by 15.5% and 10.0%, and the occurrence rate of tissue culture browning is greatly reduced.
Based on the advantages, the culture medium combination comprising the induction culture medium and the rooting culture medium provided by the application is combined with any conventional proliferation culture medium in the field, so that the occurrence probability of tissue culture brown seedlings can be effectively reduced, and the growth of the tissue culture seedlings can be promoted.
The application also provides a method for preventing tissue culture browning of stevia rebaudiana, which comprises the following steps: inoculating the sterilized explant into an induction culture medium for induction culture to obtain adventitious buds; inoculating the adventitious buds into a proliferation culture medium for proliferation culture to obtain tissue culture seedlings; inoculating the tissue culture seedlings into a rooting culture medium for rooting culture to obtain regenerated seedlings; transplanting and cultivating the regenerated seedlings to obtain regenerated stevia rebaudiana. The sterilization treatment is carried out on the explant, so that the surface of the explant is thoroughly sterilized, microorganisms and endophytes on the surface of the explant can be killed at the same time, and the pollution rate in the tissue culture process is reduced; the sterilized explant is subjected to induction culture, so that the induction rate of the adventitious buds is improved, the browning rate of the explant in the induction stage is reduced, and the proliferation culture of the adventitious buds is facilitated; the rooting culture of the obtained tissue culture seedlings by proliferation culture further reduces the browning probability of the regenerated seedlings and improves the rooting number and root length of the regenerated seedlings; the survival rate of the regenerated stevia rebaudiana is greatly improved by transplanting the regenerated seedlings to outdoor culture, and good technical support is provided for the tissue culture seedlings of the stevia rebaudiana.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 shows the growth of explants when sterilized with mercuric chloride for 4min in example 1;
FIG. 2 shows the growth of explants when sterilized with mercuric chloride for 5min in example 1;
FIG. 3 shows the growth of adventitious buds after the induction culture with the A3 medium for 14d in the induction culture in example 1;
FIG. 4 shows the growth of tissue culture seedlings cultured in proliferation culture using J2 medium in example 1;
FIG. 5 shows the growth of tissue culture seedlings cultured by the LED light source of example 1 with red light and blue light at a ratio of 3:1;
FIG. 6 shows the growth of tissue culture seedlings cultivated by irradiation with red light from the LED light source in comparative example 2;
FIG. 7 shows the growth of tissue culture seedlings cultured by irradiation with blue light from the LED light source in comparative example 3;
FIG. 8 shows the growth of tissue culture seedlings by irradiation with a white fluorescent lamp as an LED light source in comparative example 6.
Detailed Description
The application provides a culture medium combination for preventing tissue culture browning of stevia rebaudiana, which comprises an induction culture medium and a rooting culture medium; the induction culture takes MS culture medium as basic culture medium, and also comprises the following components in content: 0.5 to 1.5 mg.L -1 6-BA of (2), 0.1-0.5 mg.L -1 NAA of (C) 0.5-1.5 g.L -1 15-35 g.L -1 Sucrose and 4-8 g.L -1 More preferably, the agar powder comprises only 0.5 to 1.5 mg.L -1 6-BA of (2), 0.1-0.5 mg.L -1 NAA of (C) 0.5-1.5 g.L -1 15-35 g.L -1 Sucrose and 4-8 g.L -1 Agar powder of (2); the rooting culture medium takes a 1/2MS culture medium as a basic culture medium and further comprises the following components in percentage by weight: 0 to 0.5 mg.L -1 IAA of (C) 0-0.5 mg.L -1 NAA of (2), 1.0-1.5 g.L -1 15-35 g.L -1 Sucrose and 4-8 g.L -1 More preferably, the agar powder comprises only 0 to 0.5 mg.L -1 IAA of (C) 0-0.5 mg.L -1 NAA of (2), 1.0-1.5 g.L -1 15-35 g.L -1 Sucrose and 4-8 g.L -1 Agar powder of (2).
In the present application, the induction culture is based on MS medium; the concentration of 6-BA in the induction medium is preferably 0.5-1.0mg.L -1 More preferably 0.5 to 0.8 mg.L -1 More preferably 0.5 mg.L -1 The method comprises the steps of carrying out a first treatment on the surface of the The concentration of NAA is preferably 0.1-0.5 mg.L -1 More preferably 0.3 to 0.5 mg.L -1 More preferably 0.5 mg.L -1 The method comprises the steps of carrying out a first treatment on the surface of the The concentration of the activated carbon is preferably 0.5-1.0g.L -1 More preferably 1.0 g.multidot.L -1 The method comprises the steps of carrying out a first treatment on the surface of the The concentration of sucrose is preferably 15-35 g.L -1 More preferably 30 g.L -1 The method comprises the steps of carrying out a first treatment on the surface of the The agar powderThe concentration is preferably 4-8 g.L -1 More preferably 6 g.L -1 The method comprises the steps of carrying out a first treatment on the surface of the The pH of the induction medium is preferably 5.8 to 6.0, more preferably 5.8. By adopting the induction culture medium provided by the application, the browning probability of the explant can be reduced in the induction stage, and the growth vigor of the adventitious bud can be improved.
In the application, the rooting culture medium is based on 1/2MS culture medium; the IAA concentration in the rooting medium is preferably 0.1-0.5mg.L -1 More preferably 0.2 to 0.5 mg.L -1 More preferably 0.1 mg.L -1 The method comprises the steps of carrying out a first treatment on the surface of the The NAA concentration is preferably 0.1 to 0.5 mg.L -1 More preferably 0.2 to 0.5 mg.L -1 More preferably 0.1 mg.L -1 The method comprises the steps of carrying out a first treatment on the surface of the The concentration of the activated carbon is preferably 0.5-1.0g.L -1 More preferably 1.0 g.multidot.L -1 The method comprises the steps of carrying out a first treatment on the surface of the The concentration of sucrose is preferably 15-35 g.L -1 More preferably 30 g.L -1 The method comprises the steps of carrying out a first treatment on the surface of the The concentration of the agar powder is preferably 4-8 g.L -1 More preferably 6 g.L -1 The method comprises the steps of carrying out a first treatment on the surface of the The pH value of the rooting medium is preferably 5.8-6.0, and more preferably 5.8. The rooting medium provided by the application can reduce the occurrence rate of the brown seedlings of the stevia rebaudiana tissue culture seedlings; meanwhile, the rooting of the tissue culture seedlings is promoted, and the rooting number and root length of the tissue culture seedlings are improved.
Based on the advantages of the culture medium combination, the culture medium combination is preferably combined with any conventional proliferation culture medium in the field, so that the occurrence probability of tissue culture brown seedlings can be reduced, and the growth of the tissue culture seedlings can be promoted.
In the present application, the proliferation medium is preferably based on MS medium, and further preferably comprises 0.5 to 1.5 mg.L -1 6-BA of (2), 0.1-0.5 mg.L -1 NAA of (2), 15-35 g.L -1 Sucrose and 4-8 g.L -1 More preferably, the agar powder comprises only 0.5 to 1.5 mg.L -1 6-BA of (2), 0.1-0.5 mg.L -1 NAA of (2), 15-35 g.L -1 Sucrose and 4-8 g.L -1 Agar powder of (2). The concentration of 6-BA in the proliferation medium of the present application is more preferably 0.5 to 0.8 mg.L -1 More preferably 0.5mgL -1 The method comprises the steps of carrying out a first treatment on the surface of the The NAA concentration is more preferably 0.1 to 0.3 mg.L -1 More preferably 0.2 mg.L -1 The method comprises the steps of carrying out a first treatment on the surface of the The concentration of sucrose is further preferably 30 g.L -1 The method comprises the steps of carrying out a first treatment on the surface of the The concentration of the agar powder is more preferably 6 g.L -1 The method comprises the steps of carrying out a first treatment on the surface of the The pH of the proliferation medium is preferably 5.8 to 6.0, more preferably 5.8. After the proliferation medium provided by the application is used for culturing, the proliferation coefficient is as high as 3.3, and the adventitious buds are robust and grow well.
In addition, the application also provides application of the culture medium combination in preventing tissue culture browning of stevia rebaudiana.
The application provides a method for preventing tissue culture browning of stevia rebaudiana, which adopts the culture medium combination in the technical scheme, and comprises the following steps: inoculating the sterilized explant into an induction culture medium for induction culture to obtain adventitious buds; inoculating the adventitious buds into a proliferation culture medium for proliferation culture to obtain tissue culture seedlings; inoculating the tissue culture seedlings into a rooting culture medium for rooting culture to obtain regenerated seedlings; transplanting and cultivating the regenerated seedlings to obtain regenerated stevia rebaudiana.
The present application preferably washes stevia explants with flowing tap water to obtain washed explants. The washing can wash away impurities such as sediment attached to the surface of the explant. In the present application, the stevia rebaudiana explant preferably comprises a stem section which is in a vigorous growth state and has no disease and axillary buds, and the length of the stem section is preferably 2-3 cm. The application has no special requirements on the source of stevia rebaudiana.
After obtaining the washed explant, the application preferably disinfects the washed explant to obtain a disinfected explant. The sterilization according to the present application preferably comprises sequentially performing a first sterilization, a second sterilization and a third sterilization of the implant.
The first sterilization preferably comprises the steps of mixing the explant with a washing powder aqueous solution for first soaking, and further preferably placing the explant in a beaker for first soaking after mixing the explant with the washing powder aqueous solution; the mass percentage of the washing powder in the washing powder water solution is preferably 0.3-0.5%, and more preferably 0.5%; the time of the first soaking is preferably 10-15 min, and more preferably 10min; by limiting the concentration and soaking time of the aqueous solution of the washing powder, the stevia rebaudiana explant is prevented from browning, and the survival rate of the explant is improved. The brand and the source of the washing powder are not particularly limited, and the washing powder is purchased conventionally. In an embodiment of the present application, the first sterilization process is performed using an delicate detergent. During the first soaking, the present application preferably oscillates the container containing the mixture of explant and washing powder sufficiently. As the surface of the stevia rebaudiana explant is not smooth and is long with fluff, bacteria are easy to store, and the explant is promoted to be fully contacted with the washing powder solution for thorough disinfection after vibration, the pollution rate of a inoculated culture medium is reduced, and the probability of tissue culture browning is reduced.
After the first soaking, the application preferably cleans the foam on the first soaked explant with flowing water. The foam cleaning process of the present application is not particularly limited, and may be carried out by techniques well known in the art.
After the foam is cleaned, the application preferably cleans the foam-free explant with flowing water and RO water in sequence to obtain a first sterilized explant. The application preferably transfers the foam-free explant into a tissue culture glass bottle and seals the bottle, and then washes the bottle under flowing water; the material of the seal is preferably gauze; the washing time is preferably 1.5 to 2 hours, more preferably 2 hours. When the RO water is cleaned, the RO water is preferably poured into a tissue culture glass bottle for oscillation; the time of the shaking is preferably 1min; the number of times of the washing is preferably 2.
The second sterilization according to the present application is preferably performed under aseptic conditions, more preferably on an ultra clean bench. The second sterilization according to the present application preferably comprises mixing the first sterilized explant with alcohol for a second soaking. The volume percentage of the alcohol is preferably 70-75%, and more preferably 75%; the second soaking time is preferably 30 to 40 seconds, more preferably 30 seconds.
After the second soaking, the application preferably cleans the second soaked explant with sterile water to obtain a second sterilized explant. The number of times of the sterile water washing after the second soaking in the present application preferably includes 2 to 3 times, and more preferably 3 times.
The third sterilization according to the present application is preferably performed under aseptic conditions, more preferably on an ultra clean bench. The third sterilization according to the present application preferably comprises a third soaking after mixing the second sterilized explant with the mercuric chloride solution. The weight percentage of the mercuric chloride solution is preferably 0.1%; the time of the third soaking is preferably 3 to 6 minutes, more preferably 5 minutes.
After the third soaking, the application preferably cleans the third soaked explant by sterile water to obtain the sterilized explant. The number of times of the sterile water washing after the third soaking in the present application preferably includes 3 to 5 times, and more preferably 5 times. After disinfection, the surface microorganisms and partial endophytes of the explants can be killed, which is beneficial to reducing the pollution rate of the inoculated explants and preventing browning.
After the sterilized explant is obtained, the method preferably carries out induction culture on the sterilized explant to obtain adventitious buds. In the present application, the induction culture is preferably performed in an induction medium in the above technical scheme, and the induction medium is defined and preferred in the above technical scheme, and is not described in detail; the induction culture is preferably terminated when the length of the adventitious bud is 3 to 4cm, and more preferably the length of the adventitious bud is 1 to 2cm.
In the present application, the conditions for the induction culture preferably include: the culture temperature is 22 to 27 ℃, more preferably 22 to 25 ℃, still more preferably 25 ℃; the relative humidity is 60% to 70%, more preferably 65% to 70%, still more preferably 70%; the illumination intensity is 1500-2000 lx, more preferably 1600-2000 lx, still more preferably 2000lx; the illumination time is 12-16 h/d, more preferably 16h/d; the light source for illumination preferably comprises an LED light source, preferably comprising red and blue light, the ratio of the red and blue light qualities preferably being (2-3): 1, more preferably 3:1.
After the adventitious buds are obtained, the method preferably carries out proliferation culture on the adventitious buds to obtain tissue culture seedlings. In the present application, the proliferation culture is preferably performed by cutting off the adventitious bud and inoculating the adventitious bud into the proliferation culture medium in the above technical scheme, wherein the proliferation culture medium is defined and preferred in the above technical scheme, and is not described in detail; the time for the proliferation culture is preferably 14 to 28 days, more preferably 19 to 21 days.
In the present application, the conditions of the proliferation culture preferably include: the culture temperature is 22 to 27 ℃, more preferably 22 to 25 ℃, still more preferably 25 ℃; the relative humidity is 60% to 70%, more preferably 65% to 70%, still more preferably 70%; the illumination intensity is 1500-2000 lx, more preferably 1600-2000 lx, and still more preferably 2000lx; the illumination time is 12-16 h/d, more preferably 16h/d; the light source for illumination preferably comprises an LED light source, preferably comprising red and blue light, the ratio of the red and blue light qualities preferably being (2-3): 1, more preferably 3:1.
After the tissue culture seedlings are obtained, the application preferably carries out rooting culture on the tissue culture seedlings to obtain regenerated seedlings. In the application, the rooting culture is preferably carried out by cutting the tissue culture seedling into single plants and inoculating the single plants into the rooting culture medium in the technical scheme, wherein the rooting culture medium is limited and preferred in the technical scheme and is not repeated; the rooting culture time preferably comprises 14-28 d, more preferably 19-21 d.
In the present application, the rooting culture conditions preferably include: the culture temperature is 22 to 27 ℃, more preferably 22 to 25 ℃, still more preferably 25 ℃; the relative humidity is 60% to 70%, more preferably 65% to 70%, still more preferably 70%; the illumination intensity is 1500-2000 lx, more preferably 1600-2000 lx, still more preferably 2000lx; the illumination time is 12-16 h/d, more preferably 16h/d; the light source for illumination preferably comprises an LED light source, preferably comprising red and blue light, the ratio of the red and blue light qualities preferably being (2-3): 1, more preferably 3:1. The culture conditions provided by the application are favorable for reducing phenolic substances, preventing tissue culture from browning and enabling regenerated seedlings to be more robust.
After the regenerated seedlings are obtained, the application preferably carries out seedling hardening treatment on the regenerated seedlings. In the present application, the seedling hardening method preferably includes: when the root length of the regenerated seedling is preferably 3-5 cm, and is further preferably 5cm, opening the bottle mouth of the cultured tissue culture seedling, and hardening the seedling under the scattered light of the culture chamber; the seedling hardening time is preferably 5 to 10 days, more preferably 5 days. After hardening treatment, the probability of bacteria infection of the root of the tissue culture seedling is reduced, and the activity of the root is improved. In the present application, it is preferable to clean the culture medium remaining at the roots of the regenerated seedlings after the seedling hardening is completed. The application has no special requirement on the mode of hardening and cleaning, and adopts the technology well known in the field.
After the seedling hardening treatment, the application preferably carries out transplanting culture on the regenerated seedlings after the seedling hardening treatment to obtain regenerated stevia rebaudiana. In the present application, the transplanting cultivation preferably includes indoor transplanting and outdoor cultivation. The indoor transplanting mode preferably comprises transplanting regenerated seedlings with roots cleaned to a substrate for cultivation under greenhouse cultivation conditions. The substrate of the application preferably comprises peat soil, vermiculite and perlite; the mass ratio of the peat soil to the vermiculite to the perlite preferably comprises 2:1:1; the greenhouse culture conditions preferably include: the temperature is preferably 20 to 25 ℃, and more preferably 22 to 25 ℃; the relative humidity is preferably 70% to 80%, more preferably 70%. Preferably, the method is to transplant the regenerated seedlings transplanted indoors into the outdoor for culture after the regenerated seedlings completely survive. The method has no special requirements on the mode and the culture condition of the outdoor culture, and the method is known by adopting the technology known in the field. The method is not particularly limited to the criterion of the complete survival of the regenerated seedlings, and the conventional determination method in the field is adopted.
For further explanation of the present application, a culture medium combination for preventing tissue culture browning of stevia rebaudiana and its application are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present application.
In the following examples and comparative examples, steps 2) to 4), conditions of induction culture, proliferation culture and rooting culture are as follows, unless otherwise specified: the culture temperature is 22-25 ℃, the relative humidity is 60-70%, the illumination intensity is 2000lx, the illumination time is 16h/d, the illumination light source is an LED light source, wherein the light quality ratio of red light and blue light in the LED light source is 3:1, and the pH value of the used culture medium is 5.8.
Example 1
A method for preventing tissue culture browning of stevia rebaudiana comprises the following specific steps:
step 1) selection and sterilization of explants
Selecting tender stem segments of stevia rebaudiana in vigorous growth state and without diseases, removing leaves at two sides of the stem segments, keeping the length of the stem segments to be 2-3 cm as an explant, placing the explant in a beaker, and flushing away impurities such as sediment attached to the surface of the material by flowing tap water.
When the method is used for disinfection, the explant is mixed with an aqueous solution of the delicate washing powder with the mass percentage concentration of 0.5%, then the mixture is placed in a beaker for first soaking for 10min, the surface of the explant needs to be subjected to sufficient oscillation, sterilization treatment is carried out on the surface of the explant, then the soaked explant is washed by flowing water, after the foam is washed cleanly, the explant is replaced in a tissue culture glass bottle, the tissue culture glass bottle is sealed by gauze, the tissue culture glass bottle is placed under flowing water for 2h, and the water flow is not easy to be excessively large in the washing process so as to avoid damage to the explant. After the washing is finished, the tissue culture glass bottle is washed for 2 times by RO water, and the tissue culture glass bottle is fully vibrated in the washing process for 1min each time. Sterilizing the surface of the explant with 75% alcohol by volume for 30s in an ultra clean bench under aseptic conditions, rinsing with sterile water 3 times after sufficient shaking, and then rinsing with HgC1 by mass of 0.1% 2 After sterilization, the sterilized explant is obtained by washing 5 times with sterile water. Meanwhile, the experimental groups are divided into 4 experimental groups, and only HgC1 exists among different experimental groups 2 The sterilization times were varied and are shown in Table 1.
Table 1HgC1 2 Effects of different sterilization times on explants
As can be seen from Table 1, hgC1 having a mass percentage concentration of 0.1% was used 2 Sterilizing for 3-6 min, the survival rate of the explant is up to 90-100% and the pollution rate of the culture medium is low, wherein HgC1 2 The explant growth for 4min of sterilization is shown in FIG. 1. The pollution rate and the induction rate of the explant are combined, so that the effect of inducing the stem sections of stevia rebaudiana to generate adventitious buds is inhibited by prolonging the disinfection time. At 0.1% HgC1 2 When the stevia rebaudiana stem segment is soaked for 6min, the induction rate of the adventitious buds is 90.6%. Therefore, the most suitable method for sterilizing the stem with the axillary buds of stevia rebaudiana comprises the steps of sterilizing the surface of the stem for 30s by using alcohol with the concentration of 75%, soaking the stem in a mercuric chloride solution with the concentration of 0.1% for 5min (see figure 2), and achieving a better sterilizing effect, wherein the pollution rate of the stem is only 21.1%, the generation of adventitious buds is facilitated, and the induction rate can reach 100%.
Step 2) Induction culture of explants
In step 1), hgC1 is used 2 And (5) sterilizing for 5min to obtain the sterilized explant, and performing induction culture.
A. The concentration of hormone in the induction medium was first determined: on a sterile operating table, the sterilized explant is cut into about 1cm, the explant is placed on filter paper by forceps to suck the water, and then the explant is inoculated on an induction culture medium for induction culture. In the induction culture process, 9 treatments were set up, and 6g.L was added on the basis of MS medium -1 Is 30 g.L -1 In addition, 6-BA and NAA were added at different concentrations and a two-factor completely random test was performed. After 3 weeks of induction culture, the induction rate was counted, and the influence of induction culture with different hormones on adventitious buds was shown in Table 2.
TABLE 2 influence of different hormone ratios on adventitious bud Induction
As can be seen from Table 2, when the concentration of the medium is 0.5 to 1.5 mg.L -1 6-BA of (2) and 0.1-0.5 mg.L -1 All are beneficial to induction during NAAAnd (3) generation of adventitious buds. When A3 medium is used, the induction rate of adventitious buds is up to 100%, and the adventitious buds grow better (see FIG. 3).
B. Determination of the kind of browning agent in the induction medium: and (3) screening the type and the concentration of the browning agent on the premise of determining the optimal hormone concentration. In this process, A3 medium was selected: MS+0.5mg.L -1 6-BA+0.5mg.L of (C) -1 NAA of (2) as basal medium, and adding different kinds of browning agents at different concentrations, to obtain HgC1 in example 1 2 The sterilized explants obtained after 5min of sterilization were subjected to an anti-browning agent treatment test to determine specific components of the induction medium, and the addition types and concentrations of the browning agents are shown in table 3.
TABLE 3 effects of addition of different anti-browning on the browning of stevia adventitious buds
From Table 3, it can be seen that, after adding activated carbon AC, polyvinylpyrrolidone PVP and ascorbic acid VC to the A3 medium, the brown of the stem can be reduced to different degrees, and the growth condition of adventitious buds can be improved. However, when 0.5 to 1.5 g.L is added -1 The effect of preventing browning of adventitious buds of stevia rebaudiana is preferable when activated carbon is added, particularly when 1.0 g.L -1 When the active carbon is treated, the tissue culture Miao Hebian rate of the stevia rebaudiana is only 6.7 percent at the lowest, the adventitious bud has good growth vigor, the plant is strong and fast to grow, the leaves are green and wide, and no yellow leaves exist.
Step 3) proliferation culture
The medium in step 2) is: MS+0.5mg.L -1 6-BA+0.5mg.L of (C) -1 NAA+1.0g·L -1 Activated carbon +6g.L -1 Agar of (2) and 30 g.L -1 When the adventitious bud length on sucrose reaches 1-2 cm, lateral buds are cut off and proliferation culture is carried out.
In the proliferation culture process, 9 kinds of proliferation culture mediums are arranged, and 6 g.L of proliferation culture mediums are added on the basis of MS culture mediums -1 Is 30 g.L -1 In addition, 6-BA and NAA were added at different concentrations and a two-factor completely random test was performed. After 3 weeks of proliferation culture, the proliferation coefficient was counted, the growth vigor of adventitious buds was observed, and the effect of different proliferation culture media on proliferation culture is shown in Table 4.
TABLE 4 Effect of different proliferation Medium on proliferation culture
As can be seen from Table 4, when 0.5 to 1.5 mg.multidot.L is added during the proliferation culture -1 6-BA+0.1-0.5mg.L -1 All are beneficial to the improvement of proliferation coefficients during NAA. Wherein, in J2 medium: MS+0.5mg.L -1 6-BA+0.2mg.L of (C) -1 The NAA of (C) had a best effect of proliferation culture, a proliferation factor of 3.3, and a large number of adventitious buds, a strong and good growth (see FIG. 4).
Step 4) rooting culture
Cutting when the cluster buds cultured by the culture medium with the number J2 in the table 4 in the step 3) grow to 2-3 cm, carrying out rooting culture, and growing regenerated stevia seedlings after rooting culture, wherein the growth vigor of the regenerated stevia seedlings is shown in figure 5.
In the rooting culture process, according to the early verification of a laboratory, 0 to 0.5 mg.L of the culture medium is added on a 1/2MS culture medium -1 IAA of (C) and 0 to 0.5 mg.L -1 The NAA of (C) is favorable for rooting culture of cluster buds. To determine the addition concentration of activated carbon, the concentration of activated carbon was determined at 1/2MS+0.1mg.L -1 IAA+0.1 mg.L of (A) -1 NAA+6g.L of (C) -1 Agar of (2) and 30 g.L -1 Activated carbon of different concentrations is added on the basis of sucrose. After rooting culture for 4 weeks, the rooting rate, root length and root number of the regenerated seedlings are counted, and the influence of different rooting culture media on rooting culture is shown in table 5.
TABLE 5 influence of different rooting media on rooting culture
As can be seen from Table 5, when 1.0 to 1.5 g.L of the medium was added -1 When the activated carbon is used, the average root length of the regenerated seedlings reaches 5.04-5.26 cm, the average root number reaches 5.17-5.21, and the rooting rate of the regenerated seedlings can reach 85% at most.
Step 5) hardening off and washing the seedlings
Observing step 4) Medium numbered 3 in Table 5 (1/2MS+0.1 mg.L -1 IAA+0.1 mg.L of (A) -1 NAA+6g.L of (C) -1 Agar of (2) and 30 g.L -1 sucrose+1.0g.L -1 Activated carbon) on the tissue culture seedling, and hardening the seedling when the root length of the tissue culture seedling is about 5 cm. Hardening off the seedlings under the condition of scattered light of a culture room, and opening the bottle mouth of the tissue culture seedlings when hardening off the seedlings. The effect of different seedling hardening times on the survival rate of the tissue culture seedlings is shown in table 6. And (3) after seedling hardening, washing the seedlings, and slightly taking out the tissue culture seedlings subjected to seedling hardening treatment from the bottle by using forceps during seedling washing, and slightly cleaning the culture medium remained at the root.
TABLE 6 influence of different hardening-seedling times on survival rate of tissue culture seedlings
As can be seen from Table 6, the survival rate of transplanting is remarkably improved after hardening off of seedlings in different time, the survival rate of tissue culture seedlings is increased along with the extension of hardening off time, and the survival rates of 5d, 10d and 20d are respectively 96.93%, 92.31% and 90.77% after hardening off of the stevia rebaudiana tissue culture seedlings for 5 days. But the survival rate is reduced beyond the proper seedling hardening time, and the survival rate of the tissue culture seedlings hardened by 6d is reduced by 90%, 81.66% and 78.33% respectively at the 5d, 10d and 20d transplanting time. The reason why the seedling hardening time is too long and the transplanting survival rate of the tissue culture seedlings is reduced may be as follows: the seedling hardening time is too long, so that the root of the tissue culture seedling is infected with a large amount of bacteria, and the activity of the root is reduced.
Step 6) transplanting culture
Mixing peat soil, vermiculite and perlite in advance according to a mass ratio of 2:1:1 to obtain a matrix suitable for greenhouse transplanting. And 5d, transplanting and culturing the tissue culture seedlings which are subjected to seedling hardening in the step 5) and have basically consistent growth vigor. During transplanting, the culture medium at the root of the tissue culture seedling is washed, transplanted into a matrix and watered with fixed root. And a sunshade net is covered outside the greenhouse, and the temperature in the greenhouse is kept at 20-25 ℃ and the relative humidity is 70-80%. After the seedlings survive completely, the seedlings are transferred to outdoor natural conditions for culture.
Comparative example 1
Step 1) selection and sterilization of explants
Selecting tender stem segments of stevia rebaudiana in vigorous growth state and without diseases, removing leaves at two sides of the stem segments, keeping the length of the stem segments to be 2-3 cm as an explant, placing the explant in a beaker, and flushing away impurities such as sediment attached to the surface of the material by flowing tap water.
When the method is used for disinfection, the explant is mixed with an aqueous solution of the delicate washing powder with the mass percentage concentration of 0.5%, then the mixture is placed in a beaker for first soaking for 10min, the surface of the explant needs to be subjected to sufficient oscillation, sterilization treatment is carried out on the surface of the explant, then the soaked explant is washed by flowing water, after the foam is washed cleanly, the explant is replaced in a tissue culture glass bottle, the tissue culture glass bottle is sealed by gauze, the tissue culture glass bottle is placed under flowing water for 2h, and the water flow is not easy to be excessively large in the washing process so as to avoid damage to the explant. After the washing is finished, the tissue culture glass bottle is washed for 2 times by RO water, and the tissue culture glass bottle is fully vibrated in the washing process for 1min each time. Sterilizing the surface of the explant with 75% alcohol by volume for 30s in an ultra clean bench under aseptic conditions, rinsing with sterile water 3 times after sufficient shaking, and then rinsing with HgC1 by mass of 0.1% 2 Sterilizing for 5min, and washing with sterile water for 5 times to obtain sterilized explant.
Step 2) Induction culture of explants
And (3) performing induction culture on the explant sterilized in the step (1) until adventitious buds grow.
The induction culture medium is as follows: MS+0.5mg.L -1 6-BA+0.5mg.L of (C) -1 NAA+1.0g·L -1 Activated carbon +6g.L -1 Is of the formula (I)Fat +30g.L -1 Is a sucrose of (2).
Step 3) proliferation culture
When the length of the adventitious bud in the induction culture reaches 1-2 cm, lateral buds are cut off, and proliferation culture is carried out.
Proliferation culture is as follows: MS+0.5mg.L -1 6-BA+0.2mg.L of (C) -1 NAA+6g.L of (C) -1 Agar of (2) and 30 g.L -1 Is a sucrose of (2).
Step 4) rooting culture
After the proliferation culture, cutting off the cluster buds when the cluster buds grow to 2-3 cm, and carrying out rooting culture.
Rooting culture is as follows: 1/2MS+0.1mg.L -1 IAA+0.1 mg.L of (A) -1 NAA+6g.L of (C) -1 Agar of (2) and 30 g.L -1 sucrose+1.0g.L -1 Is an activated carbon of (a).
Step 5) hardening off and washing the seedlings
Transplanting and culturing the tissue culture seedlings which are acclimatized for 5d and have basically consistent growth vigor.
Step 6) transplanting culture
During transplanting, the culture medium at the root of the tissue culture seedling is washed, transplanted into a matrix and watered with fixed root. And a sunshade net is covered outside the greenhouse, and the temperature in the greenhouse is kept at 20-25 ℃ and the relative humidity is 70-80%. After the seedlings survive completely, the seedlings are transferred to outdoor natural conditions for culture.
Wherein, the culture conditions of the step 2) to the step 4) are as follows: the conditions of induction culture, proliferation culture and rooting culture are as follows: the culture temperature is 22-25 ℃, the relative humidity is 60-70%, the illumination intensity is 2000lx, the illumination time is 16h/d, the illumination light source is an LED light source, wherein the light quality ratio of red light and blue light in the LED light source is 2:1, and the pH value of the used culture medium is 5.8.
Comparative example 2
The difference from comparative example 1 is that: in the culture conditions of step 2) to step 4), only red light exists in the LED light source, wherein the growth vigor of stevia rebaudiana regenerated seedlings is shown in fig. 6.
Comparative example 3
The difference from comparative example 1 is that: in the culture conditions of the steps 2) to 4), only blue light exists in the LED light source, wherein the growth vigor of the stevia rebaudiana regenerated seedlings is shown in fig. 7.
Comparative example 4
The difference from comparative example 1 is that: in the culture conditions of the steps 2) to 4), the light quality ratio of the red light and the blue light in the LED light source is 1:2.
Comparative example 5
The difference from comparative example 1 is that: in the culture conditions of the steps 2) to 4), the light quality ratio of the red light and the blue light in the LED light source is 1:3.
Comparative example 6
The difference from comparative example 1 is that: in the tissue culture condition in the step 2), the light source is a white fluorescent lamp, wherein the growth vigor of the regenerated stevia seedlings is shown in fig. 8, and the influence of different light sources on the tissue culture stevia is shown in table 7.
TABLE 7 influence of different light sources on tissue-cultured stevia rebaudiana
As can be seen from Table 7, single red light treatment is beneficial to the proliferation of adventitious buds, the proliferation multiple reaches the maximum value of 14.6, but the adventitious buds are weak in growth vigor; the single blue light treatment is obviously increased compared with the fluorescent lamp treatment, but the proliferation multiple is smaller than that of red light. In the light quality combination treatment, when the red light and the blue light occupy the same proportion, the proliferation multiple of the adventitious bud of the stevia rebaudiana is larger than that of the blue light treatment, so that the red light is more beneficial to the proliferation of the adventitious bud of the stevia rebaudiana compared with the blue light. When the optimal LED light source suitable for the proliferation and growth of the adventitious buds is that the light quality ratio of red light to blue light is (2-3): 1, the proliferation multiple of the adventitious buds is 12.8-13.9, and the adventitious buds are thicker, so that the browning probability of the tissue culture seedlings is reduced.
Although the foregoing embodiments have been described in some, but not all, embodiments of the application, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the application.

Claims (10)

1. A culture medium combination for preventing tissue culture browning of stevia rebaudiana, which is characterized by comprising an induction culture medium and a rooting culture medium;
the induction culture takes MS culture medium as basic culture medium, and also comprises the following components in content: 0.5 to 1.5 mg.L -1 6-BA of (2), 0.1-0.5 mg.L -1 NAA of (C) 0.5-1.5 g.L -1 15-35 g.L -1 Sucrose and 4-8 g.L -1 Agar powder of (2);
the rooting culture medium takes a 1/2MS culture medium as a basic culture medium and further comprises the following components in percentage by weight: 0 to 0.5 mg.L -1 IAA of (C) 0-0.5 mg.L -1 NAA of (2), 1.0-1.5 g.L -1 15-35 g.L -1 Sucrose and 4-8 g.L -1 Agar powder of (2).
2. The combination of media of claim 1, wherein the combination of media further comprises a proliferation medium.
3. The combination of media of claim 2, wherein the propagation medium is based on MS medium, further comprising the following components in amounts: 0.5 to 1.5 mg.L -1 6-BA of (2), 0.1-0.5 mg.L -1 NAA of (2), 15-35 g.L -1 Sucrose and 4-8 g.L -1 Agar powder of (2).
4. Use of a combination of media according to any one of claims 1 to 3 for preventing tissue browning of stevia rebaudiana.
5. A method for preventing tissue culture browning of stevia rebaudiana, characterized in that the combination of culture mediums of any one of claims 1 to 3 is adopted, comprising the following steps:
inoculating the sterilized explant into the induction culture medium for induction culture to obtain adventitious buds;
inoculating the adventitious buds to the proliferation culture medium for proliferation culture to obtain tissue culture seedlings;
inoculating the tissue culture seedling into the rooting culture medium for rooting culture to obtain regenerated seedling;
transplanting and cultivating the regenerated seedlings to obtain regenerated stevia rebaudiana.
6. The method of claim 5, wherein the first sterilization, the second sterilization and the third sterilization are performed on the explant in sequence to obtain the sterilized explant;
the first sterilization includes: mixing the explant with a washing powder aqueous solution with the mass percentage of 0.3% -0.5%, and then carrying out first soaking for 10-15 min, and sequentially cleaning the first soaked explant by flowing water and RO water to obtain a first sterilized explant;
the second sterilizing includes: mixing the first sterilized explant with 75% alcohol by volume percentage, and then soaking for 30-40 s, and cleaning the second soaked explant with sterile water to obtain a second sterilized explant;
the third sterilization includes: mixing the second sterilized explant with 0.1% mercury chloride solution, and then third soaking for 4-6 min, and cleaning the third soaked explant again by using sterile water.
7. The method of claim 6, wherein the first soaking step comprises shaking a container containing the mixture of the explant and the washing powder.
8. The method according to claim 5, wherein the pH values of the culture medium for induction culture, proliferation culture and rooting culture are 5.8 to 6.0, respectively.
9. The method according to claim 5, wherein the culture conditions of the induction culture, proliferation culture and rooting culture comprise: the culture temperature is 22-27 ℃, the relative humidity is 60-70%, the illumination intensity is 1500-2000 lx, and the illumination time is 12-16 h/d.
10. The method of claim 9, wherein the light source for illumination comprises an LED light source; the LED light source includes red light and blue light.
CN202310769307.7A 2023-06-26 2023-06-26 Culture medium combination for preventing tissue culture browning of stevia rebaudiana and application thereof Pending CN116711639A (en)

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