CN112913612B - Method for increasing content of saponin VI of paris polyphylla cultivated - Google Patents
Method for increasing content of saponin VI of paris polyphylla cultivated Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/25—Root crops, e.g. potatoes, yams, beet or wasabi
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/10—Mycorrhiza; Mycorrhizal associations
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
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Abstract
The invention discloses a method for improving the content of cultivated paris polyphylla saponin VI, which is used for keeping the field water holding capacity of soil at about 50%; artificial inoculant ivory white saccule enzyme, cryptosaccule enzyme and simple bacillus, and the microorganism and paris polyphylla plant are co-cultured. Different microorganisms are inoculated in the process of cultivating paris polyphylla, and meanwhile, moderate drought treatment is assisted, so that the content of saponin VI is improved, and the quality of paris polyphylla is improved; meanwhile, the chlorophyll content of paris polyphylla seedlings can be improved, and the growth of paris polyphylla seedlings is promoted.
Description
Technical Field
The invention relates to the technical field of Chinese herbal medicine cultivation, in particular to a method for improving the content of saponin VI of paris polyphylla cultivated.
Background
The paris polyphylla belongs to paris genus of lily family, has long medicinal history, and is one of the most representative medicinal plants occupying the dominant position of industrial utilization in western regions of China. Modern pharmacological researches have shown that paris polyphylla has the effects of promoting blood circulation, removing blood stasis, relieving swelling and pain, cooling liver, arresting convulsion, clearing away heat and toxic materials, resisting cancer, resisting tumor and regulating immunity, etc., and is commonly used for treating traumatic injury, snake and insect bite, and clinically applied to tumor, parotitis, tonsillitis, respiratory tract infection, etc. Because of unique curative effect, the Chinese patent medicine and health care products produced by using the Chinese patent medicine as raw materials are as many as nearly hundred. The sum of the effective components of rhizoma paridis saponins I, II, VI and VII in Chinese pharmacopoeia (2015) is not less than 0.6% as the standard of qualified rhizoma paridis.
At present, the demand of Yunnan rhizoma paridis per year is more than 5000 tons, but because the regeneration period of the Yunnan rhizoma paridis is long (8 to 10 years), and seeds have the physiological characteristics of 'secondary dormancy', the emergence rate of seed propagation is lower, and the asexual propagation technology (such as tissue culture) does not make breakthrough progress at present, so that the wild rhizoma paridis resources are increasingly reduced for a long time, whether people or enterprises use the wild rhizoma paridis resources as raw materials.
In order to solve the market situation of the supply and the shortage of the traditional Chinese medicinal materials of the paris polyphylla, the paris polyphylla is planted in the places of the Yunnan province in recent years, and the paris polyphylla planting area of the whole Yunnan province is estimated to be approximately 14 ten thousand mu in a conservation manner. Along with the continuous expansion of the planting area of paris polyphylla, the quality problem is increasingly attracting the attention of enterprises. The paris polyphylla varieties mainly cultivated in Yunnan province almost lack steroidal paris polyphylla saponin VI, and the deficiency of the steroidal paris polyphylla saponin VI greatly reduces the medicinal material quality of the cultivated paris polyphylla and severely restricts the benign development of related pharmaceutical enterprises.
Disclosure of Invention
Aiming at the problems, the invention provides a method for improving the content of saponin VI of paris polyphylla cultivated, which adopts different microorganisms inoculated in the paris polyphylla cultivation process and simultaneously assists in moderate drought treatment, improves the content of saponin VI and improves the quality of paris polyphylla; meanwhile, the chlorophyll content of paris polyphylla seedlings can be improved, and the growth of paris polyphylla seedlings is promoted.
According to the purpose of the invention, the invention provides the following technical scheme:
a method for increasing the content of saponin VI of paris polyphylla cultivated in the cultivation method comprises the following steps:
s1, propagation and inoculation of arbuscular mycorrhizal fungi
S101, expanding propagation:
selecting ivory white saccule enzyme and hidden saccule enzyme, firstly mixing humus soil and river sand according to a ratio of 1:1, setting an autoclave at 121 ℃ for 1h after uniformly mixing, and cooling for later use after sterilization;
taking a proper amount of wheat seeds, and soaking the wheat seeds in 0.2% potassium permanganate solution for 15min for standby;
placing sterilized mixed sandy soil at 2/3 of flowerpot, weighing 20g of arbuscular mycorrhizal fungus strain, uniformly spreading in the flowerpot, placing wheat seeds on the flowerpot, and covering with sandy soil mixture until the flowerpot is full; finally pouring a proper amount of sterile water;
s102, inoculating:
sterilizing humus soil and loess according to a proportion of 3:1, firstly filling 1/2 of soil into a flowerpot, uniformly laying 40g of arbuscular mycorrhizal fungi weighed in advance on the 1/2 of soil surface, covering a layer of thin soil, orderly placing paris polyphylla seedlings on the soil surface, and finally filling the whole flowerpot with soil and watering fully;
s2, inoculation of growth-promoting bacteria
The growth promoting bacterial strain is simple bacillus separated from paris polyphylla tissue, simple bacillus liquid stored at-20 deg.c is spread onto LB plate culture medium and cultured in 37 deg.c incubator for 24-48 hr;
then picking single colony, placing in LB liquid culture medium, shaking at 37deg.C, 180rpm, shake culturing for 2-3 hr to make OD of bacterial liquid be 0.6-0.8, taking out bacterial liquid, centrifuging at 8000rpm/min for 10 min, collecting bacterial cells, adding sterile water, and re-suspending until bacterial concentration is about 2×10 7 cfu/ml, and irrigating 50ml of heavy suspension once for each pot of paris polyphylla seedlings subjected to the inoculation treatment;
s3, daily management
Maintaining the water holding capacity of soil water at 50%; two layers of 9-needle black shading nets are covered on the planting greenhouse, fertilizer is not applied to paris polyphylla seedlings during the treatment period, and soil moisture is timely watered according to a weighing method to supplement moisture;
s4, harvesting
After the inoculation treatment, the water control treatment and the paris polyphylla co-culture are finished for 60 days, paris polyphylla seedlings are collected, potting soil is gently shaken when the seedlings are collected, and the seedlings are carefully taken out, so that the damage to root systems caused by overlarge actions is avoided.
Further, in S102, the paris polyphylla seedling is selected from the seedlings of paris polyphylla which are developed in root system, disease-free and damage-free and normal in growth.
Further, in S2, LB liquid medium: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride, distilled water to a volume of 1L, regulating the pH to 7.4, and adding 15g/L of agar to prepare a solid culture medium.
Further, in S101, a flowerpot with the caliber of 21cm and the height of 17cm is selected, and is sprayed and disinfected by 75% of acetaldehyde after being washed by clean water.
Compared with the prior art, the invention has the beneficial effects that:
according to the method for improving the content of the cultivated paris polyphylla saponin VI, different microorganisms are inoculated in the paris polyphylla cultivation process, meanwhile, moderate drought treatment is assisted, the content of the saponin VI is improved, the paris polyphylla quality is improved, meanwhile, the chlorophyll content of paris polyphylla seedlings can be improved, and the growth of paris polyphylla seedlings is promoted.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 is a view showing the infection observation of AM in Paris polyphylla rhizome;
FIG. 2 is a graph showing the hormone content of Paris polyphylla;
FIG. 3 is a N, P, K content chart of Paris polyphylla;
FIG. 4 is a graph showing the content of saponins in Paris polyphylla.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
A method for increasing the content of saponin VI of paris polyphylla cultivated in the cultivation method comprises the following steps:
s1, propagation and inoculation of arbuscular mycorrhizal fungi
S101, expanding propagation:
selecting ivory white saccule enzyme and hidden saccule enzyme, firstly mixing humus soil and river sand according to a ratio of 1:1, setting an autoclave at 121 ℃ for 1h after uniformly mixing, and cooling for later use after sterilization;
selecting flowerpots with caliber of 21cm and height of 17cm, washing with clear water, and then spraying and sterilizing with 75% acetaldehyde;
taking a proper amount of wheat seeds, and soaking the wheat seeds in 0.2% potassium permanganate solution for 15min for standby;
placing sterilized mixed sandy soil at 2/3 of flowerpot, weighing 20g of arbuscular mycorrhizal fungus strain, uniformly spreading in the flowerpot, placing wheat seeds on the flowerpot, and covering with sandy soil mixture until the flowerpot is full; finally pouring a proper amount of sterile water;
s102, inoculating:
sterilizing humus soil and loess according to a proportion of 3:1, firstly filling 1/2 of soil into a flowerpot, uniformly laying 40g of arbuscular mycorrhizal fungi weighed in advance on the 1/2 of soil surface, covering a layer of thin soil, orderly placing paris polyphylla seedlings on the soil surface, and finally filling the whole flowerpot with soil and watering fully; the paris polyphylla seedling is selected from the seedlings of the paris polyphylla which are developed in root system, free of diseases and damage and normal in growth.
S2, inoculation of growth-promoting bacteria
The growth promoting bacterial strain is simple bacillus separated from paris polyphylla tissue, simple bacillus liquid stored at-20 deg.c is spread onto LB plate culture medium and cultured in 37 deg.c incubator for 24-48 hr; LB liquid medium: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride, distilled water to a volume of 1L, regulating the pH to 7.4, and adding 15g/L of agar to prepare a solid culture medium.
Then picking single bacterial colony, placing the bacterial colony into LB liquid culture medium, shaking and culturing for 2-3 hours at 37 ℃ and 180rpm by a shaking table, taking out the bacterial liquid when the OD of the bacterial liquid is 0.6-0.8, centrifuging for 10 minutes at 8000rpm/min, collecting bacterial cells, adding sterile water to resuspend until the bacterial concentration is about 2X 107cfu/ml, and irrigating 50ml of resuspension once for each pot of paris polyphylla seedlings subjected to the inoculation treatment;
s3, daily management
Maintaining the water holding capacity of soil water at 50%; two layers of 9-needle black shading nets are covered on the planting greenhouse, fertilizer is not applied to paris polyphylla seedlings during the treatment period, and soil moisture is timely watered according to a weighing method to supplement moisture;
s4, harvesting
After the inoculation treatment, the water control treatment and the paris polyphylla co-culture are finished for 60 days, paris polyphylla seedlings are collected, potting soil is gently shaken when the seedlings are collected, and the seedlings are carefully taken out, so that the damage to root systems caused by overlarge actions is avoided.
Example 2
Control test
The method specifically comprises the following steps:
1. expanding propagation and inoculation of standby mycorrhizal fungi (AM)
Expanding propagation: the ivory white saccule enzyme (Diversispora tortuosa) and the cryptosaccule enzyme (Paraglomus occultum) are selected. Firstly, mixing humus soil and river sand according to a proportion of 1:1, and setting the autoclave at 121 ℃ for 1h. Cooling for standby after sterilization; selecting flowerpots with the sizes of 21cm and 17cm in height, washing with clear water, and then spraying and sterilizing with 75% acetaldehyde; taking a proper amount of wheat seeds, and soaking the wheat seeds in 0.2% potassium permanganate solution for 15min for standby; placing sterilized mixed sandy soil at 2/3 of flowerpot, weighing 20g of AM strain, uniformly spreading in the flowerpot, placing wheat seeds thereon, and covering with sandy soil mixture until flowerpot is full; finally pouring a proper amount of sterile water.
Inoculating: mixing sterilized humus soil and loess collected from the rear mountain according to the ratio of (3:1). The AM fungi are inoculated by firstly filling 1/2 of the soil into the flowerpot, uniformly laying 40/gAM fungi which are weighed in advance on the 1/2 of the soil surface, covering a layer of thin soil, orderly placing paris polyphylla seedlings on the soil surface, and finally filling the whole flowerpot with the soil and watering the flowerpot fully. The whole transplanting and inoculation process is completed, and the paris polyphylla seedlings are selected as seedlings from the seedlings of the paris polyphylla which are developed in root system, free of diseases and damage and normal in growth.
2. Inoculation of growth-promoting bacteria (PEPR)
The strain is Bacillus simplex isolated from Paris polyphylla tissue. Coating simple bacillus liquid stored at-20deg.C on LB plate medium, and culturing in incubator at 37deg.C24-48h. Then picking single bacterial colony and placing the single bacterial colony in LB liquid culture medium, wherein the LB liquid culture medium is as follows: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride, distilled water to a volume of 1L, regulating the pH to 7.4, and adding 15g/L of agar to prepare a solid culture medium. Shaking table is set at 37 ℃, 180rpm, shake culture is carried out for 2-3h, when the OD of bacterial liquid is 0.6-0.8, the bacterial liquid is taken out, 8000rpm/min, centrifugation is carried out for 10 minutes, bacterial cells are collected, and sterile water is added for resuspension until the bacterial concentration is about 2X 10 7 cfu/ml, 50ml of heavy suspension is irrigated once for each pot of paris polyphylla seedlings subjected to inoculation treatment. Sterile water is not inoculated for Comparison (CK), and seedlings are prevented from being burnt by contact with leaves.
Function identification of AM and PEPR mixed inoculation
Since there is no strict specificity between AM and PEPR for the host plant, symbiosis of two bacteria in the same plant is a common phenomenon in nature and plays a great important role in the ecological system. However, most studies are conducted by inoculating AM or endophyte alone to plants, ignoring the potential synergistic effect of the combination of inoculation, and the synergistic effect between the two microorganisms helps to increase the tolerance of plants to growth in a stress environment and also helps to mitigate the negative effects on microorganisms in a stress environment.
The plant growth promoting bacteria can promote plant growth through substances such as nitrogen fixation, potassium decomposition, phosphorus dissolution, secretion of plant hormone, synthesis of siderophores and the like. AM fungi infects plant root systems to enlarge the contact area of the root systems and soil, increase the absorption of P elements by plants, and even inhibit some pathogenic bacteria, and can also produce plant hormones to promote plant growth. The main interaction mechanism of the two existing in the plant root system is as follows: a. the interaction of AM and PGPR promotes the absorption of nutrients by plants, especially improves the utilization of N, P by plants, thereby promoting the growth of plants; b. the two microorganisms can synergistically inhibit the growth of pathogenic bacteria; c. AM inoculation can synthesize plant hormone to promote plant growth and accumulation of active substances; d. AM hypha grows continuously in soil, so that the spread of surrounding bacteria is promoted, and the colonization of partial bacteria in rhizosphere can be increased; e. bacteria also contribute to the colonization of AM, promoting the growth of AM hyphae and extending their life.
4. Treatment of different moisture
Water stress treatment: soil moisture is normal moisture (W): 80% of the maximum water holding capacity of the soil field, and moderate drought treatment (D): the test was controlled by 50% of the field capacity of the soil. Three treatments were set under the same moisture conditions: inoculating 2 AM fungi and simple bacillus mixed inoculation; control treatments with AM alone and without bacteria. There were 6 treatments, 10 replicates each, 60 pots total, randomly placed test pots. The water content is controlled by a method of weighing and supplementing water, and water is supplemented every two days, so that the soil water content of each paris polyphylla is set in a corresponding control range (directly measured by a water content measuring instrument). And recording the weight of each basin under the changed state by using a balance, and firstly weighing the water consumed by each basin when the water is replenished for the second time, and correspondingly replenishing the water to each basin. After 90 days of moisture control, the corresponding index of paris polyphylla was determined.
5. Daily management
The planting greenhouse is covered with two layers of 9-needle black shading nets, fertilizer is not applied to paris polyphylla seedlings during treatment, soil moisture is timely watered according to a weighing method to supplement moisture, sterile water cooled by the water is watered due to the specificity of the test, a tray is placed at the bottom of the planting pot, the moisture is prevented from being lost rapidly, and cross contamination is prevented between treatments. The sunshade rate of the greenhouse suitable for the growth of paris polyphylla is adjusted, the plant diseases and insect pests are prevented and treated, the greenhouse is kept clean and in a good ventilation state, timely weeding is carried out, potting management is carried out regularly, and the sequence of each treatment and various planting pots in the same treatment is exchanged every two weeks.
6. Harvesting
After the inoculation treatment, the water control treatment and the paris polyphylla co-culture are finished for 60 days, paris polyphylla seedlings are collected, potting soil is gently shaken when the seedlings are collected, the seedlings are carefully taken out, and experimental errors caused by overlarge actions to damage root systems are avoided. The following indices were measured:
1. infection observation of AM in paris polyphylla rhizomes is shown in fig. 1:
it was found that, under normal water or moderate drought treatment, the AM fungus inoculated paris polyphylla root system was seen to have a cluster or vesicle structure, hyphae formed invasion points from the root surface (shown as a in fig. 1), and mycelium was continuously grown in (shown as B in fig. 1) to form a tree structure (shown as C in fig. 1).
2. The hormone content of paris polyphylla is shown in figure 2:
under moderate drought, ABA and IAA content is significantly increased compared to normal moisture treatment; compared with inoculated AM and CK, the ABA content of the double inoculation AM+PEPR is respectively increased by 11.55 percent and 23.7 percent; IAA content was doubled compared to CK in the double inoculation am+pepr treatment.
3. N, P, K content of paris polyphylla, see fig. 3:
the content of N, P, K in the paris polyphylla rhizome is reduced by moderate drought, and the content of N, P is not influenced by the treatment of double inoculation AM+PEPR and single inoculation AM; but the content of K tends to increase significantly. Inoculation of am+pepr treatment under normal moisture treatment significantly increased N, P, K content compared to CK.
4. The content of paris polyphylla saponin is shown in figure 4:
in paris polyphylla rhizome, the content of paris polyphylla saponin, whether paris polyphylla saponin I, II, VI, VII or total saponin, is increased under moderate drought compared with water treatment. The content of paris polyphylla saponin VI is found to exist in AM+PEPR treatment under the moisture treatment, and the content of paris polyphylla saponin VI is found to be up to 0.052% and 0.06% in single inoculation and double inoculation treatment under moderate drought. The moderate drought double inoculation treatment is increased by 12.3% compared with inoculation AM.
Double inoculation of arbuscular mycorrhizal fungi and growth promoting bacteria under moderate drought treatment is beneficial to accumulation of hormone in paris polyphylla rhizomes, absorption and utilization of K content, and increase of paris polyphylla saponin VI content and total saponin in paris polyphylla.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (3)
1. A method for improving the content of saponin VI of paris polyphylla cultivated in a cultivation way, which is characterized by comprising the following steps:
s1, propagation and inoculation of arbuscular mycorrhizal fungi
S101, expanding propagation:
selecting ivory white saccule enzyme and hidden saccule enzyme, firstly mixing humus soil and river sand according to a ratio of 1:1, setting an autoclave at 121 ℃ for 1h after uniformly mixing, and cooling for later use after sterilization;
taking a proper amount of wheat seeds, and soaking the wheat seeds in 0.2% potassium permanganate solution for 15min for standby;
placing sterilized mixed sandy soil at 2/3 of flowerpot, weighing 20g of arbuscular mycorrhizal fungus strain, uniformly spreading in the flowerpot, placing wheat seeds on the flowerpot, and covering with sandy soil mixture until the flowerpot is full; finally pouring a proper amount of sterile water;
s102, inoculating:
sterilizing humus soil and loess according to a proportion of 3:1, firstly filling 1/2 of soil into a flowerpot, uniformly laying 40g of arbuscular mycorrhizal fungi weighed in advance on the 1/2 of soil surface, covering a layer of thin soil, orderly placing paris polyphylla seedlings on the soil surface, and finally filling the whole flowerpot with soil and watering fully;
s2, inoculation of growth-promoting bacteria
The growth promoting bacterial strain is simple bacillus separated from paris polyphylla tissue, simple bacillus liquid stored at-20 deg.c is spread onto LB plate culture medium and cultured in 37 deg.c incubator for 24-48 hr;
then picking single bacterial colony, placing the bacterial colony into LB liquid culture medium, shaking and culturing for 2-3 hours at 37 ℃ and 180rpm by a shaking table, taking out the bacterial liquid when the OD of the bacterial liquid is 0.6-0.8, centrifuging for 10 minutes at 8000rpm/min, collecting bacterial cells, adding sterile water to resuspend until the bacterial concentration is about 2X 107cfu/ml, and irrigating 50ml of resuspension once for each pot of paris polyphylla seedlings subjected to the inoculation treatment;
LB liquid medium: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride, distilled water to 1L, regulating pH to 7.4, and adding 15g/L of agar into the solid culture medium;
s3, daily management
Maintaining the water holding capacity of soil water at 50%; two layers of 9-needle black shading nets are covered on the planting greenhouse, fertilizer is not applied to paris polyphylla seedlings during the treatment period, and soil moisture is timely watered according to a weighing method to supplement moisture;
s4, harvesting
After the inoculation treatment, the water control treatment and the paris polyphylla co-culture are finished for 60 days, paris polyphylla seedlings are collected, potting soil is gently shaken when the seedlings are collected, and the seedlings are carefully taken out, so that the damage to root systems caused by overlarge actions is avoided.
2. The method for increasing the content of saponin VI of paris polyphylla cultivated according to claim 1, wherein in S102, paris polyphylla seedlings are selected from the seedlings of paris polyphylla which are developed in root system, free of diseases and damage and normal in growth.
3. The method for increasing the content of saponin VI of paris polyphylla cultivated according to claim 1, wherein in S101, a flowerpot with a caliber of 21cm and a height of 17cm is selected, and is sprayed and disinfected by 75% acetaldehyde after being washed by clean water.
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| CN109392641A (en) * | 2018-12-19 | 2019-03-01 | 兴文县人民医院 | A kind of imitating wild planting process of Paris polyphylla |
| CN110521510A (en) * | 2019-09-06 | 2019-12-03 | 云南农业大学 | A kind of method that paris polyphylla endogenetic bacteria promotes paris polyphylla growth of seedling |
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