WO2013164835A1 - Novel method for the detection of microbial contaminants in plant tissue cell cultures - Google Patents

Novel method for the detection of microbial contaminants in plant tissue cell cultures Download PDF

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WO2013164835A1
WO2013164835A1 PCT/IN2013/000059 IN2013000059W WO2013164835A1 WO 2013164835 A1 WO2013164835 A1 WO 2013164835A1 IN 2013000059 W IN2013000059 W IN 2013000059W WO 2013164835 A1 WO2013164835 A1 WO 2013164835A1
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detection
novel method
microbial contaminants
plant
indicator
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PCT/IN2013/000059
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French (fr)
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Gayatri Ashwinkumar DAVE
Rucha Harishkumar MEHTA
Niralee Gautambhai PATEL
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Dave Gayatri Ashwinkumar
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/045—Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/22—Testing for sterility conditions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2304/00—Chemical means of detecting microorganisms
    • C12Q2304/20—Redox indicators

Definitions

  • the present invention relates to novel method for the detection of microbial contaminants in plant tissue cultures.
  • the present invention also relates to the use of indicators for the detection of contaminants in plant tissue cultures.
  • Microbial contamination can be defined as growth of harmful microorganisms. Epiphytic and entophytic organisms can cause severe losses to micropropagated plants at each stage of growth.
  • Contaminated plants may have no visible symptoms, reduced multiplication and rooting rates, or may die.
  • microbes Bacteria, molds and yeasts are found virtually everywhere and are able to quickly colonize and flourish in the rich and relatively undefended environment provided by plant tissue culture medium. Because of their size and fast growth rates, these microbes are the most commonly plant tissue culture contaminants. In the absence of antibiotics, microbes can usually be readily detected in a culture within a few days of becoming contaminated, either by direct microscopic observation or by the effects they have on the culture (pH shifts, turbidity, and cell destruction). However, when antibiotics are routinely used in culture, resistant organisms may develop into slow growing, low level infections that are very difficult to detect by direct visual observation. Similar detection problems can occur with naturally slow growing organisms or very small or intracellular bacteria that are difficult to see during routine microscopic culture observation.
  • Our invention is also based on introduction of chemical indicator compound in plant tissue culture media, which detects very small concentration of contaminant and in very short duration. This is the hallmark of the invention that facilitates early detection of such contaminants which are generally not seen until growth is seen.
  • novel method for detecting contaminants in plant tissue cultures by the use of chemical indicators at a very low concentration.
  • novel method for detecting contaminants in plant tissue cultures by using chemical indicators that helps in detecting even minute quantities of microbial contaminants.
  • the present invention relates to novel method for the detection of microbial contaminants in plant tissue cultures by the use of chemical indicators which work independent of pH and temperature conditions in plant tissue cultures. These chemical indicators work at various ranges of concentrations and are able to detect microbial contaminants at the earliest possible period of time once the plant tissue cultures are incubated for the plant growth.
  • Fig. 1 Flow chart of method of detection of microbial contaminants in plant tissue cultures
  • Fig.2 Addition of various concentrations of Resozurine / Resorufin (RZ) in plant tissue culture media containing bacterial cultures
  • Fig.3 Addition of various concentrations of Methylene Blue (MB) in plant tissue culture media containing bacterial cultures
  • Fig.4 Addition of MB and RZ at different stages of plant tissue culture media (Micropropagation and Multiplication stages)
  • Fig. 5 Effect of indicators on seed germination
  • Fig.6 Effect of indicators on pH changes during plant growth
  • novel method for the detection of microbial contamination in plant cell cultures employs class of chemical compounds which are used as growth index belongs from pH dependent and pH independent redox indicators. A handful of such compounds are listed in Table 1 and Table 2. Both pH dependent and pH independent redox indicators are equally effective against microbial contamination at plant tissue culture pH range. Table: 1 pH independent redox indicator
  • redox indicator in the tissue culture medium which alters with the growth of microorganism (for some compound color to colourless and for some compound colourless to color).
  • the chemical indicators as mentioned in above tables 1 and 2 are prepared in different concentrations, ranging from 0.0001 % to 0.001 %.
  • the chemical compounds are added to plant tissue culture medium either in test tube or conical flask or bottles in different concentrations, ranging from 0.0001 % to 0.001 %. Before adding, the chemical indicators are autoclaved and changes in colour before and after autoclave are noted.
  • the above media are incubated at desired conditions (temperature, pH, light and humidity) for 7 days to 8 weeks based on type of plant growth. These media were periodically checked for presence or absence of contamination, beginning as early as 30 minutes post incubation and then hourly basis. Changes in colour of chemical indicators are noted at every interval of time. Absence of any change in colour indicates absence of microbial contamination and any change in colour as mentioned for each chemical indicator in Tables 1 and 2 indicates presence of microbial contamination (change from oxidized to reduced form). Control samples were run along with the experimental samples. Control samples were only chemical indicator solutions prepared at the desired concentrations.
  • the beneficial and harmful effects that a chemical has on an organism depend, in part, on the amount of the chemical that gets into the organism.
  • the total amount of chemical administered to, or taken by, an organism is called a dose
  • the effect a chemical has on a living organism is called the response.
  • the effect a chemical has on a living organism is related to its dose and the resultant concentration of chemical in the organism. Toxicity tests enable toxicologists to learn about responses of living organisms, especially humans, to doses of chemicals.
  • Methylene blue (MB) is efficient at all working concentration range (in experimental range of 0.0001 % to 0.001 %). Change of colour in the culture medium indicates presence of contamination. This has been shown in enclosed figures. Control samples did not show any colour change during incubation.
  • Resozurine/Resorufin is effective over wide concentration range (experimental range; 0.0001% to 0.001%). Change of colour in the culture medium indicates presence of contamination. This has been shown in enclosed figures. The initial colour of the indicator is blue which is Resozurine. After autoclaving, the solution becomes pink which is Resorufin compound and it is this compound which was added to the plant tissue culture. Control samples did not show any colour change during incubation.
  • the indicators are eco-friendly, not toxic to growing plants, confirmed by seed germination assay, and as shown in enclosed figure 5. In this figure the highest concentration of chemical shows no harmful effect on seed germination i.e. all seeds were germinated.
  • the color reactions are stable at entire range of temperature which is normally used for cultivation of plants in plant tissue culture unit.
  • the indicators are compatible for different types of plant tissue culture medium.
  • the indicators are used for all different types of plant variety; it can be used for any plant variety.
  • pH changes are observed during plant growth, but the chemical indicators are independent to these specific pH changes, only microbial growth can reduce the compound. This is represented in the enclosed figure 6.
  • pH after plant growth is generally 6.0, 5.8; de-colorization is independent to pH, only microbes can decolorize (reduce) the indicator, i.e. pH shift is not responsible for de-colorization.
  • the method uses chemical indicators to identify the contaminants which are easy to use, non-toxic to plants.
  • the invention helps identify the contaminants at the earliest possible time post- incubation of culture media thereby saving enormous amount of time.
  • the chemical indicators used in the present invention works independent of pH and temperature conditions of plant tissue culture media.
  • the chemical indicators used in the present invention woks in a wide range of concentration, ranging from 0.0001 % to 0.001 %.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
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  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The present invention relates to novel method for the detection of microbial contaminants in plant tissue cultures by the use of chemical indicators which work independent of pH and temperature conditions in plant tissue cultures. These chemical indicators work at various ranges of concentrations and are able to detect microbial contaminants at the earliest possible period of time once the plant tissue cultures are incubated for the plant growth.

Description

FIELD OF THE INVENTION
The present invention relates to novel method for the detection of microbial contaminants in plant tissue cultures. The present invention also relates to the use of indicators for the detection of contaminants in plant tissue cultures.
BACKGROUND OF THE INVENTION
Microbial contamination can be defined as growth of harmful microorganisms. Epiphytic and entophytic organisms can cause severe losses to micropropagated plants at each stage of growth.
Bacterial contaminants are often difficult to detect because they remain mostly within the plant tissue. Contaminated plants may have no visible symptoms, reduced multiplication and rooting rates, or may die.
No plant cell culture problem is as universal as that of culture loss due to contamination. All plant cell culture laboratories and cell culture workers have experienced it. Culture contaminants may be biological or chemical, seen or unseen, destructive or seemingly benign, but in all cases they adversely affect both the use of your cell cultures and the quality of your research. Contamination problems can be divided into three classes:
Minor annoyances— when up to several plates or flasks are occasionally lost to contamination; Serious problems — when contamination frequency increases or entire experiments or cell cultures are lost;
Major catastrophes— contaminants are discovered that call into doubt the validity of your past or current work.
Some of the consequences of contamination are:
• Loss of time, money, and effort
• Adverse effects on the cultures
• Inaccurate or erroneous experimental results
• Loss of valuable products
· Personal embarrassment
Bacteria, molds and yeasts are found virtually everywhere and are able to quickly colonize and flourish in the rich and relatively undefended environment provided by plant tissue culture medium. Because of their size and fast growth rates, these microbes are the most commonly plant tissue culture contaminants. In the absence of antibiotics, microbes can usually be readily detected in a culture within a few days of becoming contaminated, either by direct microscopic observation or by the effects they have on the culture (pH shifts, turbidity, and cell destruction). However, when antibiotics are routinely used in culture, resistant organisms may develop into slow growing, low level infections that are very difficult to detect by direct visual observation. Similar detection problems can occur with naturally slow growing organisms or very small or intracellular bacteria that are difficult to see during routine microscopic culture observation. These cryptic contaminants may persist indefinitely in cultures causing subtle but significant alterations in their behavior. By the time these cryptic contaminants are discovered, many experiments and cultures may have been compromised. Secondly, it is very time consuming to examine each and every vessel every day, lots of manpower and time is required for these tasks, moreover the small concentration cannot be visually detected at early stage at micropropagation and multiplication stage of plant tissue culture.
Current processes that provide incomplete solution(s) for the same problem:
> Use of good aseptic techniques
> Reduce accidents
> Keeping the laboratory clean
> Routinely visual monitoring of each and every bottle/plate for contamination
> Use frozen cell repository strategically
> Use antibiotics sparingly if at all
These are not the solutions of addressed problem, these are the just preventive measure which minimizes the chances of contamination, and however for detection of the contamination only one method is there, i.e. routine visual examination of culture vessel.
The above problems of identification of microbial contaminants are overcome by a novel method for detecting such contaminants in plant tissue cultures by using chemical indicators which detects the contamination of bacteria, mould and yeast at its lowest concentration.
Our invention is also based on introduction of chemical indicator compound in plant tissue culture media, which detects very small concentration of contaminant and in very short duration. This is the hallmark of the invention that facilitates early detection of such contaminants which are generally not seen until growth is seen. EMBODIMENTS OF THE INVENTION
In an embodiment of the present invention is disclosed novel method for detection of contaminants in plant tissue cultures.
In another embodiment of the present invention is enclosed novel method for detection of contaminants in plant tissue cultures by using chemical indicators.
In yet another embodiment is disclosed novel method for detecting contaminants in plant tissue cultures by using chemical indicators which are temperature and pH independent.
In still yet another embodiment of the present invention is disclosed novel method for detecting contaminants in plant tissue cultures at a very early stage of growth by the use of chemical indicators.
In yet another embodiment is disclosed novel method for detecting contaminants in plant tissue cultures by the use of chemical indicators at a very low concentration. In a further embodiment of the present invention is disclosed novel method for detecting contaminants in plant tissue cultures by using chemical indicators that helps in detecting even minute quantities of microbial contaminants. SUMMARY OF THE INVENTIO
The present invention relates to novel method for the detection of microbial contaminants in plant tissue cultures by the use of chemical indicators which work independent of pH and temperature conditions in plant tissue cultures. These chemical indicators work at various ranges of concentrations and are able to detect microbial contaminants at the earliest possible period of time once the plant tissue cultures are incubated for the plant growth. BRIEF DESCRIPTION OF DRAWINGS
Some of the features of the present invention may be better explained by way of figures enclosed:
Fig. 1 : Flow chart of method of detection of microbial contaminants in plant tissue cultures
Fig.2: Addition of various concentrations of Resozurine / Resorufin (RZ) in plant tissue culture media containing bacterial cultures
Fig.3: Addition of various concentrations of Methylene Blue (MB) in plant tissue culture media containing bacterial cultures
Fig.4: Addition of MB and RZ at different stages of plant tissue culture media (Micropropagation and Multiplication stages)
Fig. 5: Effect of indicators on seed germination
Fig.6: Effect of indicators on pH changes during plant growth
Fig.7: No effect of temperature on indicators action in plant tissue culture
BEST MODE FOR CARRYING OUT THE INVENTION
In accordance with the present there are provided novel method for the detection of microbial contamination in plant cell cultures. The method employs class of chemical compounds which are used as growth index belongs from pH dependent and pH independent redox indicators. A handful of such compounds are listed in Table 1 and Table 2. Both pH dependent and pH independent redox indicators are equally effective against microbial contamination at plant tissue culture pH range. Table: 1 pH independent redox indicator
Figure imgf000007_0001
The respiratory activity of a growing microbial culture is associated with a decreasing redox potential; here incorporated herein is a redox indicator in the tissue culture medium which alters with the growth of microorganism (for some compound color to colourless and for some compound colourless to color). Preparation of chemical indicators:
The chemical indicators as mentioned in above tables 1 and 2 are prepared in different concentrations, ranging from 0.0001 % to 0.001 %.
Procedure:
The chemical compounds are added to plant tissue culture medium either in test tube or conical flask or bottles in different concentrations, ranging from 0.0001 % to 0.001 %. Before adding, the chemical indicators are autoclaved and changes in colour before and after autoclave are noted.
The above media are incubated at desired conditions (temperature, pH, light and humidity) for 7 days to 8 weeks based on type of plant growth. These media were periodically checked for presence or absence of contamination, beginning as early as 30 minutes post incubation and then hourly basis. Changes in colour of chemical indicators are noted at every interval of time. Absence of any change in colour indicates absence of microbial contamination and any change in colour as mentioned for each chemical indicator in Tables 1 and 2 indicates presence of microbial contamination (change from oxidized to reduced form). Control samples were run along with the experimental samples. Control samples were only chemical indicator solutions prepared at the desired concentrations.
The chemical indicators used in plant tissue culture media were subsequently tested for presence or absence of toxicity to plants by seed germination assay and for temperature stability at different temperature ranges from 20 °C to 37 °C. In addition, the impact of chemical indicators on shift in pH during plant growth was tested. Seed germination assay and toxicity study:
The beneficial and harmful effects that a chemical has on an organism depend, in part, on the amount of the chemical that gets into the organism. The total amount of chemical administered to, or taken by, an organism is called a dose, and the effect a chemical has on a living organism is called the response. The effect a chemical has on a living organism is related to its dose and the resultant concentration of chemical in the organism. Toxicity tests enable toxicologists to learn about responses of living organisms, especially humans, to doses of chemicals.
50 ml solutions of each % concentration of chemicals were prepared. Sterile distilled water was used as control. 20 seeds were counted. The seeds were soaked in control and test concentration overnight. The seeds were carefully placed on the moist paper napkins in the control plate, making sure to space them evenly (do not clump them in one spot). The plates were incubated for around two days at 27 °C and observed for budding.
The above novel process for the detection of microbial contaminants can be represented by a suitable flow diagram (Fig.1 ). The present invention is not to be limited in scope by the specific embodiments describe herein by way of examples provided below. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. EXAMPLE 1
METHYLENE BLUE INDICATOR
Methylene blue (MB) is efficient at all working concentration range (in experimental range of 0.0001 % to 0.001 %). Change of colour in the culture medium indicates presence of contamination. This has been shown in enclosed figures. Control samples did not show any colour change during incubation.
EXAMPLE 2
RESORUFIN INDICATOR
Resozurine/Resorufin is effective over wide concentration range (experimental range; 0.0001% to 0.001%). Change of colour in the culture medium indicates presence of contamination. This has been shown in enclosed figures. The initial colour of the indicator is blue which is Resozurine. After autoclaving, the solution becomes pink which is Resorufin compound and it is this compound which was added to the plant tissue culture. Control samples did not show any colour change during incubation.
EXAMPLE 3
GERMINATION ASSAY FOR METHYLENE BLUE AND
RESOZURINE/RESORUFIN INDICATORS
The indicators are eco-friendly, not toxic to growing plants, confirmed by seed germination assay, and as shown in enclosed figure 5. In this figure the highest concentration of chemical shows no harmful effect on seed germination i.e. all seeds were germinated. EXAMPLE 4
TEMPERATURE STABILITY OF CHEMICAL INDICATORS
As shown in enclosed figure 7, the color reactions are stable at entire range of temperature which is normally used for cultivation of plants in plant tissue culture unit. The indicators are compatible for different types of plant tissue culture medium. The indicators are used for all different types of plant variety; it can be used for any plant variety. EXAMPLE 5
EFFECT OF pH CHANGES IN PLANT CULTURE ON CHEMICAL INDICATORS pH changes are observed during plant growth, but the chemical indicators are independent to these specific pH changes, only microbial growth can reduce the compound. This is represented in the enclosed figure 6. In this, pH after plant growth is generally 6.0, 5.8; de-colorization is independent to pH, only microbes can decolorize (reduce) the indicator, i.e. pH shift is not responsible for de-colorization.
The present invention has the following advantages:
1 ) The novel method of detection of microbial contamination is more precise to identify contaminants in plant tissue culture medium as compared to conventional methods.
2) The method uses chemical indicators to identify the contaminants which are easy to use, non-toxic to plants.
3) The invention helps identify the contaminants at the earliest possible time post- incubation of culture media thereby saving enormous amount of time. ) The chemical indicators used in the present invention works independent of pH and temperature conditions of plant tissue culture media.
) The chemical indicators used in the present invention woks in a wide range of concentration, ranging from 0.0001 % to 0.001 %.

Claims

Claims :
1. A novel method for the detection of microbial contaminants in plant tissue culture media comprising of steps:
a. Addition of indicator into plant tissue culture media and incubating the media at desired conditions like temperature, pH, light and humidity for 7 days to 8 weeks based on type of plant growth,
b. Checking for change in colour of the media at regular time intervals.
2. A novel method for the detection of microbial contaminants as claimed in claim 1 wherein optionally, the step further comprises of:
a. Checking the toxicity of indicator on plant growth,
b. Checking the effect of indicator on pH and temperature changes in plant tissue culture media.
3. A novel method for the detection of microbial contaminants as claimed in claims 1-2 wherein the pH independent redox indicator is selected from Resozurine / Resorufin, 2,2-bypyridine, Nitrophenathrolin, N-phenyl anthranillic acid, 1-10 phenenthrolin, N-ethoxychrysodine, 2-2' Bipyridine, 5,6, dimethyl phenethroline, o-Diansidine, Sodium Diphenylamine sulphonate, Diphenylbenzidine, diphenylamine, Viologen and the like.
4. A novel method for the detection of microbial contaminants as claimed in claims 1-2 wherein the pH dependent redox indicator is selected from Methylene Blue, Sodium 2,6-Dibromophenol-indophenol, sodium o-cresol indophenol, Thionine, Indigo tetrasulfonic acid, Indigo carmine, Indigo monosulfonic acid, Phenosafranin, Safranin T, Neutral Red, Tetrazolium chloride, Melechite green and the like.
5. A novel method for the detection of microbial contaminants as claimed in claims 3-4 wherein the concentration of indicator is in the range of 0.0001 % to 0.001 %.
6. A novel method for the detection of microbial contaminants as claimed in claim 2 wherein the indicator added to plant culture media is independent of pH changes of plant growth in tissue culture.
7. A novel method for the detection of microbial contaminants as claimed in claim 2 wherein the indicator added to plant culture media is stable to temperature conditions of plant growth in tissue culture.
8. A novel method for the detection of microbial contaminants as claimed in claim 2 wherein the indicator added to plant culture media is free of toxic effects on plant growth.
9. A novel method for the detection of microbial contaminants in plant tissue cultures as claimed in any of the preceding claims and exemplified with working examples.
PCT/IN2013/000059 2012-04-30 2013-01-29 Novel method for the detection of microbial contaminants in plant tissue cell cultures WO2013164835A1 (en)

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CN114085885A (en) * 2021-10-15 2022-02-25 天津农学院 Detection method and application of tissue culture seedling degerming effect

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Publication number Priority date Publication date Assignee Title
CN114085885A (en) * 2021-10-15 2022-02-25 天津农学院 Detection method and application of tissue culture seedling degerming effect

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