CN113598049A - Chinese herbal medicine pre-cooking liquid for inhibiting tissue culture plant endophyte pollution and application method - Google Patents
Chinese herbal medicine pre-cooking liquid for inhibiting tissue culture plant endophyte pollution and application method Download PDFInfo
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Abstract
The invention relates to the technical field of plant tissue culture, and discloses a Chinese herbal medicine pre-boiling liquid for inhibiting the pollution of endophytes of tissue-cultured plants and an application method thereof.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a Chinese herbal medicine precooking liquid for inhibiting the endophyte pollution of tissue-cultured plants and an application method thereof.
Background
Endophytes refer to fungi, bacteria or actinomycetes that live inside healthy plant tissues and organs, and form a mutual promotion or inhibition relationship with host plants, thereby influencing plant growth and development. In plant tissue culture, most endophyte pollution can cause withering and death of plants, and the seedling rate is reduced. Research shows that the addition of bacteriostatic agent with proper concentration into the culture medium can reduce the growth of endophyte in plant tissue culture, reduce pollution coefficient and improve tissue culture efficiency. In the tissue culture of the anoectochilus formosanus, yellow endophyte is seriously polluted along with the increase of the subculture times of the anoectochilus formosanus, so that the normal operation of tissue culture growth of the anoectochilus formosanus is influenced.
Currently, the commonly used endophyte bacteriostats are mainly chloramphenicol, potassium sorbate, streptomycin sulfate, ampicillin, sodium benzoate and the like, the bacteriostat for the endophytes of the anoectochilus roxburghii is relatively limited, and some of the bacteriostats even have an enhancement effect on the endophytes of the anoectochilus roxburghii.
Prunellae Spica (Prunella vulgaris L.) is a perennial herb of Labiatae, has a height of 1-40 cm, and has effects of lowering blood pressure and dilating blood vessel, and contains flavonoid, rutin, and Rosmarinic acid, which has antiinflammatory, antioxidant, and antiviral effects. The antibacterial spectrum of the selfheal is wide, the water extract has the effect of inhibiting growth of escherichia coli, tubercle bacillus, pneumococcus, dysentery bacillus, staphylococcus and the like, at present, the antibacterial effect of the selfheal is mainly embodied in inhibition of pathogenic bacteria of animals, and no related research report that the selfheal is applied to inhibition of endophytic bacteria pollution of plants exists at present.
Disclosure of Invention
Therefore, the Chinese herbal medicine pre-boiling liquid for inhibiting the tissue culture plant endophyte pollution and the application method thereof are needed to be provided, and the application of the selfheal for inhibiting the plant endophyte, in particular for inhibiting the anoectochilus formosanus endophyte is expanded.
In order to achieve the purpose, the invention provides a Chinese herbal medicine precooking liquid for inhibiting the pollution of endophytes of tissue-cultured plants, which comprises a selfheal precooking liquid.
Further comprises MS, 0.1-0.3mg/L IBA, 5-15% of bananas, 0.5-1.0% of agar powder, 0.1-0.5% of cane sugar, 0.1-0.5% of active carbon and 100-200 g/L of selfheal precooking liquid.
Further comprises MS, 0.1mg/L IBA, 10% of bananas, 0.7% of agar powder, 0.3% of cane sugar, 0.3% of active carbon and 100g/L-200g/L of selfheal precooking liquid.
Further, the preparation process of the selfheal precooking liquid is as follows: adding 15-25 times of water into Prunellae Spica, heating and boiling for 6-8 hr, filtering, and cooling to obtain Prunellae Spica precooked solution.
The application method of the antibacterial culture medium for inhibiting the tissue culture plant endophyte pollution comprises the following use processes: and (3) preparing the antibacterial culture medium, and then inoculating the tissue culture plant to the antibacterial culture medium for culture.
Further, the tissue culture plant is anoectochilus roxburghii.
Further, the culture conditions of anoectochilus formosanus are as follows: the temperature is 25-28 ℃, the humidity is controlled within 35%, the light intensity is 500-1500 lx, and the illumination time is 10 hours per day.
Furthermore, the stem section with buds of the anoectochilus formosanus is selected during the anoectochilus formosanus inoculation.
Further, the length of the stem section with buds of the anoectochilus formosanus is 1-2 cm.
The technical scheme has the following beneficial effects:
according to the invention, the Chinese herbal medicine precooking liquid is used as an antibacterial culture medium of the tissue culture plant, when the precooking liquid of the selfheal is applied to the culture medium of the tissue culture plant creatively, the precooking liquid of the selfheal has certain antibacterial activity, and can inhibit the reproduction of endophytes by influencing the activity of specific endophytes, so that the pollution of the endophytes of the tissue culture plant, especially the endophytes of anoectochilus formosanus is effectively inhibited, and the antibacterial effect can reach 100% along with the increase of the using amount of the precooking liquid of the selfheal in the culture medium.
Drawings
FIG. 1 is a graph showing the culture results of test groups 1 to 6 according to the embodiment.
FIG. 2 is a graph showing the culture results of test groups 7 to 12 according to the embodiment.
FIG. 3 is a graph showing the culture results of test groups 13 to 18 according to the embodiment.
FIG. 4 is a graph showing the results of the culture of test groups 19 to 21 and a blank control group according to the embodiment.
Detailed Description
To explain technical contents, structural features, and objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments.
Example 1
Antibacterial test of pre-cooked liquid of Prunellae Spica.
Firstly, experimental preparation:
1. preparing a selfheal (code number XKC) precooking liquid: 500g of selfheal is put into 10L of pure water (the volume of the selfheal is larger) and heated and boiled for 6-8h, and is slowly concentrated to 1L to obtain 500g/L of selfheal pre-boiling liquid mother liquor, and the 500g/L of selfheal pre-boiling liquid mother liquor is diluted to the corresponding concentration according to the adding amount when being prepared into a culture medium;
2. preparation of a bidens pilosa (code number GZX) precooking liquid: 500g of bidens pilosa is put into 5L of pure water to be boiled and slowly chelated to 1L, and the bidens pilosa precooking liquid mother liquor is diluted into the corresponding concentration according to the adding amount when being prepared into a culture medium;
3. streptomycin.
Second, grouping experiments
Different experimental culture media are prepared according to different concentrations of streptomycin, bidens bipinnata (code GZX) precooking liquid and selfheal (code XKC) precooking liquid, and the experimental groups are as follows:
test 1: MS + IBA0.1mg/L + banana 10% + agar powder 0.7% + cane sugar 0.3% + active carbon 0.1% + streptomycin 0.1 g/L;
test 2: MS + IBA0.1mg/L + banana 10% + agar powder 0.7% + cane sugar 0.3% + active carbon 0.1% + streptomycin 0.5 g/L;
test 3: MS + IBA0.1mg/L + banana 10% + agar powder 0.7% + cane sugar 0.3% + active carbon 0.1% + streptomycin 1.0 g/L;
test 4: MS + IBA0.1mg/L + banana 10% + agar powder 0.7% + cane sugar 0.3% + active carbon 0.1% + streptomycin 2.0 g/L;
test 5: MS + IBA0.1mg/L + banana 10% + agar powder 0.7% + sucrose 0.3% + active carbon 0.1% + streptomycin 5.0 g/L;
test 6: MS + IBA0.1mg/L + banana 10% + agar powder 0.7% + cane sugar 0.3% + active carbon 0.1% + streptomycin 10.0 g/L;
test 7: MS + IBA0.1mg/L + banana 10% + agar powder 0.7% + sucrose 0.3% + active carbon 0.1% + XKC 0.1 g/L;
test 8: MS + IBA0.1mg/L + banana 10% + agar powder 0.7% + sucrose 0.3% + active carbon 0.1% + XKC 0.5 g/L;
test 9: MS + IBA0.1mg/L + banana 10% + agar powder 0.7% + sucrose 0.3% + active carbon 0.1% + XKC 1.0 g/L;
test 10: MS + IBA0.1mg/L + banana 10% + agar powder 0.7% + sucrose 0.3% + active carbon 0.1% + XKC 2.0 g/L;
test 11: MS + IBA0.1mg/L + banana 10% + agar powder 0.7% + sucrose 0.3% + active carbon 0.1% + XKC 5.0 g/L;
test 12: MS + IBA0.1mg/L + banana 10% + agar powder 0.7% + sucrose 0.3% + active carbon 0.1% + XKC 10.0 g/L;
test 13: MS + IBA0.1mg/L + banana 10% + agar powder 0.7% + sucrose 0.3% + active carbon 0.1% + GZC 0.1 g/L;
test 14: MS + IBA0.1mg/L + banana 10% + agar powder 0.7% + sucrose 0.3% + active carbon 0.1% + GZC 0.5 g/L;
test 15: MS + IBA0.1mg/L + banana 10% + agar powder 0.7% + sucrose 0.3% + active carbon 0.1% + GZC 1.0 g/L;
test 16: MS + IBA0.1mg/L + banana 10% + agar powder 0.7% + sucrose 0.3% + active carbon 0.1% + GZC 2.0 g/L;
test 17: MS + IBA0.1mg/L + banana 10% + agar powder 0.7% + sucrose 0.3% + active carbon 0.1% + GZC 5.0 g/L;
test 18: MS + IBA0.1mg/L + banana 10% + agar powder 0.7% + sucrose 0.3% + active carbon 0.1% + GZC 10.0 g/L;
test 19: MS + IBA0.1mg/L + banana 10% + agar powder 0.7% + cane sugar 0.3% + active carbon 0.1% + XKC100.0 g/L;
test 20: MS + IBA0.1mg/L + banana 10% + agar powder 0.7% + cane sugar 0.3% + active carbon 0.1% + XKC150.0 g/L;
test 21: MS + IBA0.1mg/L + banana 10% + agar powder 0.7% + cane sugar 0.3% + active carbon 0.1% + XKC200.0 g/L;
blank Control (CK): MS + IBA0.1mg/L + 10% of banana + 0.7% of agar powder + 0.3% of cane sugar + 0.1% of active carbon;
10 parallel tests are set in each test group, and the culture conditions are as follows: after the culture medium of each experimental group is prepared at the temperature of 18-25 ℃, the humidity is controlled within 35%, the light intensity is 500-1500 lx, the illumination time is 10 hours per day, 1-2cm stem sections with buds, which are seriously polluted by endophytes, are cut off and inoculated into the culture medium added with different experimental groups, the time and the pollution condition of each culture medium are observed, and the experimental results are shown in table 1 and figures 1-4.
Table 1, effect of culture medium of different experimental groups on growth of budded stem segments with severe endophyte contamination.
The culture results of the experimental groups 1 to 21 and the blank control group are shown in FIGS. 1 to 4.
Third, experimental analysis
Test groups 1-6: the common bacterial bacteriostatic agent streptomycin has no inhibition effect on the endophytes of the anoectochilus roxburghii, and the material is inoculated for 2 days to cause serious pollution which is earlier than the endophyte pollution time of a blank control,
test groups 7-12: the selfheal precooking liquid in the concentration range has no control effect on anoectochilus roxburghii endophytes, but has a certain bacteriostatic action, the endophyte appears in a later time, and the endophyte appears in a bottle in a slightly light range.
Test groups 13-18: the bidens pilosa pre-boiling liquid has no bacteriostatic effect on the endophytes of anoectochilus formosanus, the serious endophyte pollution is caused after the material is inoculated for 4 days, the bidens pilosa extracting solution is also a traditional Chinese medicine with the antibacterial effect, and the bidens pilosa extracting solution also has the inhibitory effect on pathogenic bacteria of animals and human bodies. As shown in experiments 13-18, not all the herbs known to have bacteriostatic action on human body can be used for inhibiting the endophytes of anoectochilus formosanus, and the invention creatively develops the action of the selfheal precooking liquid in inhibiting the anoectochilus formosanus
Test groups 19-21: the pre-boiling liquid of the selfheal, which has the concentration range of 100g/L-200g/L, has certain bacteriostatic action on the endophytes, and the bacteriostatic effect is more obvious along with the increase of the concentration.
Therefore, the selfheal precooking liquid has certain control effect on the endophytes of the anoectochilus roxburghii when being prepared and diluted to the concentration of 100g/L-200g/L when being prepared with the bacteriostatic culture medium, and the bacteriostatic rate can reach 100% when the consumption is 200 g/L.
Example 2
A Chinese herbal medicine pre-cooking liquid for inhibiting pollution of anoectochilus formosanus endophytes comprises MS +0.3mg/L IBA + 5% banana + 0.5% agar powder + 0.1% cane sugar + 0.1% active carbon +100g/L selfheal pre-cooking liquid. .
Example 3
A Chinese herbal medicine pre-cooking liquid for inhibiting pollution of anoectochilus formosanus endophytes comprises MS +0.2mg/L IBA + 15% banana + 1.0% agar powder + 0.5% cane sugar + 0.5% active carbon +200g/L selfheal pre-cooking liquid. .
The Chinese herbal medicine pre-cooking liquor in the embodiment 2 and the embodiment 3 is used as an antibacterial culture medium to culture the anoectochilus formosanus, and can also play a certain protection role in preventing pollution of endophytes of the anoectochilus formosanus. The antibacterial culture medium can be applied to tissue culture of anoectochilus roxburghii and can also be used for inhibiting endophytes of other tissue culture plants.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal. Without further limitation, an element defined by the phrases "comprising … …" or "comprising … …" does not exclude the presence of additional elements in a process, method, article, or terminal that comprises the element. Further, herein, "greater than," "less than," "more than," and the like are understood to exclude the present numbers; the terms "above", "below", "within" and the like are to be understood as including the number.
Although the embodiments have been described, once the basic inventive concept is obtained, other variations and modifications of these embodiments can be made by those skilled in the art, so that the above embodiments are only examples of the present invention, and not intended to limit the scope of the present invention, and all equivalent structures or equivalent processes using the contents of the present specification and drawings, or any other related technical fields, which are directly or indirectly applied thereto, are included in the scope of the present invention.
Claims (9)
1. A Chinese herbal medicine precooking liquid for inhibiting the pollution of endophyte of tissue-cultured plants is characterized by comprising a selfheal precooking liquid.
2. The Chinese herbal medicine precooking liquid for inhibiting the endophyte pollution of tissue culture plants as claimed in claim 1, which comprises MS +0.1-0.3mg/L IBA + 5-15% of bananas + 0.5-1.0% of agar powder + 0.1-0.5% of cane sugar + 0.1-0.5% of active carbon +100g/L-200g/L of selfheal precooking liquid.
3. The Chinese herbal medicine precooking liquid for inhibiting the endophyte pollution of tissue culture plants as claimed in claim 1, which comprises MS +0.1mg/L IBA + 10% banana + 0.7% agar powder + 0.3% sucrose + 0.3% active carbon +100g/L-200g/L selfheal precooking liquid.
4. The Chinese herbal medicine precooking liquid for inhibiting the endophyte pollution of tissue culture plants as claimed in claim 1, wherein the preparation process of the selfheal precooking liquid is as follows: adding 15-25 times of water into Prunellae Spica, heating and boiling for 6-8 hr, filtering, and cooling to obtain Prunellae Spica precooked solution.
5. The application method of the Chinese herbal medicine precooking liquid for inhibiting the endophyte pollution of the tissue culture plants as claimed in any one of claims 1 to 4, which is characterized in that the application process is as follows: preparing the bacteriostatic culture medium according to any one of claims 1 to 4, and then inoculating the tissue culture plants to the bacteriostatic culture medium for culture.
6. The method of use according to claim 5, wherein the tissue-cultured plant is Anoectochilus roxburghii.
7. The application method of claim 6, wherein the Anoectochilus formosanus is cultured under the conditions of 25-28 deg.C, humidity within 35%, light intensity of 500-1500 lx, and illumination time of 10 hr per day.
8. The use of claim 6, wherein the stem of Anoectochilus formosanus with buds is selected for the inoculation of Anoectochilus formosanus.
9. The use of claim 8, wherein the budded stem segment of Anoectochilus roxburghii is 1-2cm in length.
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| CN117502232A (en) * | 2023-11-23 | 2024-02-06 | 福建省中科生物股份有限公司 | Chinese herbal medicine precooking liquid and method for preventing and treating endophytes by using Chinese herbal medicine precooking liquid |
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