CN114680044B

CN114680044B - Tissue culture and rapid propagation seedling raising method for wintergreen

Info

Publication number: CN114680044B
Application number: CN202210396217.3A
Authority: CN
Inventors: 许宗亮; 张金渝; 曾祥飞; 杨天梅; 朱新焰; 赵露琴; 王丽; 冯人和; 李绍兴
Original assignee: Lincang Linxiang Genuine Medicinal Materials Research And Planting Association; Yunnan Plant Insect Medicine Biotechnology Co ltd; Institute of Medicinal Plants Yunnan Academy of Agricultural Sciences
Current assignee: Lincang Linxiang Genuine Medicinal Materials Research And Planting Association; Yunnan Plant Insect Medicine Biotechnology Co ltd; Institute of Medicinal Plants Yunnan Academy of Agricultural Sciences
Priority date: 2022-04-15
Filing date: 2022-04-15
Publication date: 2022-11-01
Anticipated expiration: 2042-04-15
Also published as: CN114680044A

Abstract

The invention belongs to the field of traditional Chinese medicinal materials and tissue culture, and particularly discloses a tissue culture seedling raising method of wintergreen.

Description

Tissue culture and rapid propagation seedling raising method for wintergreen

Technical Field

The invention belongs to the field of traditional Chinese medicinal materials and tissue culture, and particularly relates to a tissue culture seedling method of wintergreen.

Background

Pyrola calliantha (Pyrola callianthha) also called Meihua Pyrola, pyrola calliantha, rohan tea, chang Green tea and the like, grow under mountain coniferous forest, needle and broad leaf mixed forest or broad leaf forest with the elevation of 700 to 4100 m, and are mainly distributed in Yunnan, sichuan, guizhou, tibet, shaanxi, qinghai, gansu, shanxi, shandong, hebei, henan, anhui, jiangsu, zhejiang, fujian, hubei, hunan and Jiangxi. The whole herb is used as a medicine, has the effects of expelling wind-damp, strengthening bones and muscles, stopping bleeding and relieving cough, and is mainly used for treating rheumatism pain, kidney deficiency and lumbago, weakness of waist and knees, menorrhagia, chronic cough, overstrain cough and the like.

The wintergreen contains dozens of active ingredients such as crystal orcin, wintergreen glycoside, wintergreen, homoarbutin, hyperin, quercetin, hydroxyl-folium et wintergreen glycoside, gallic acid and the like, wherein the crystal glycoside is the most representative, and the content of the crystal glycoside is the most important index for quality detection of the wintergreen medicinal materials in 2020 edition of Chinese pharmacopoeia. Modern medicine research shows that wintergreen has the functions of resisting bacteria, resisting inflammation, resisting tumor, improving cardiovascular and cerebrovascular circulation, lowering blood pressure, dilating blood vessel, strengthening heart, etc. and is used in treating cardiac and cerebral vascular diseases, cervical spondylosis, chronic dysentery and other bacterial infection, rheumatic arthritis, pulmonary tuberculosis hemoptysis, chronic bronchitis, hyperosteogeny, sciatica, etc. At present, nearly thirty kinds of traditional Chinese medicine preparations taking wintergreen as raw material medicines comprise dizzy-stopping and nerve-soothing granules, jingkang tablets, hyperosteogeny-resisting tablets, breast nodule-dispersing capsules and the like.

With the development of the great health of the traditional Chinese medicine, the demand of the wintergreen decoction pieces and the traditional Chinese medicine preparation taking the wintergreen decoction pieces as the raw material medicine is increased day by day, the wild resources are excavated inexhaustibly, and the storage amount of the wild resources is reduced greatly. In recent years, introduction and domestication of wintergreen wild resources are carried out in provinces and cities such as Yunnan province and Hebei province, but because seeds of the wintergreen wild resources are dormant, the wintergreen wild resources cannot germinate under the prior art, but the 1 plant can only be divided into 2 to 3 plants in the division propagation process, the propagation period is at least 1 year, and the wintergreen wild resources have the defects of low propagation coefficient, long propagation period and the like. The tissue culture researches of the wintergreen are proposed by major papers published by Zhao Yao (introduction research of the wintergreen [ D ]. Baoding City: hebei agriculture university, 2005, 10 to 13) and by Wangwei, lu Jiantao and the like (tissue culture research of the wintergreen [ J ], hebei agriculture science and technology, 2008.

Aiming at the defects of the wintergreen in the prior art, the invention particularly discloses a wintergreen tissue culture technology which comprises explant selection, explant disinfection, primary cluster bud induction culture, subculture multiplication culture and rooting culture.

Disclosure of Invention

The invention mainly aims to provide a method for tissue culture and rapid propagation of wintergreen to solve the problems in the background technology.

The invention is realized by the following scheme:

1. selection of explants: transplanting wintergreen in a room, spraying stem leaves with 1000 times of 75% carbendazim solution after green turning, spraying once every 7 days for 3 times continuously, digging the whole plant, removing roots and leaves under the soil surface, and taking stem segments with axillary buds and axillary buds at the base parts as explants.

2. Cleaning explants: dipping a saturated soap aqueous solution by using a brush, repeatedly brushing the surface until no soil residue exists, then adding a proper amount of water into a beaker, dropwise adding 2-3 drops of Tween-80, shaking for 10-15min, and showering with tap water for 60-70min.

3. And (3) disinfection of explants: and transferring the cleaned explant to an ultraclean workbench, disinfecting the explant with 75% alcohol for 10 to 15s, rinsing with sterile water for 2 to 3 times, disinfecting with a saturated bleaching powder solution for 10 to 15nin, rinsing with sterile water for 3 to 4 times, disinfecting with an 8% hydrogen peroxide solution for 4 to 5min, rinsing with sterile water for 3 to 4 times, disinfecting with a 0.025% mercuric chloride solution dropwise added with 2 drops of Tween-80 for 4 to 6min, and rinsing with sterile water for 6 to 8 times.

4. Inducing primary cluster buds: and (2) absorbing surface water of the disinfected explant by using sterile paper, cutting wounds at two ends, inoculating the explant into a tufted bud induction culture medium consisting of MS +2, 4-D0.02-0.05mg/L, CPPU 0.4-0.5mg/L, 2-IP 0.1-0.3mg/L, silver nitrate 0.1-0.2 mu g/L and active carbon 0.5g/L, carrying out dark culture at the temperature of 25 +/-2 ℃ for 7 days, keeping the temperature unchanged, carrying out light illumination for 10 hours every day, and carrying out light-dark culture at the light intensity of 2000-3000lx for 60-70 days.

5. Subculture multiplication culture of cluster buds: cutting the induced cluster buds into single buds, inoculating the single buds into a MS + NAA 0.05-0.1mg/L + CPPU 0.5-0.8mg/L + TIBA 0.01-0.02mg/L + banana mud 50-60g/L cluster bud subculture multiplication medium, and culturing for 30-40 days under the light-dark alternation of 25 +/-2 ℃, 10 hours of illumination per day and 2000-3000lx of light intensity.

6. Rooting culture of cluster buds: cutting the subcultured and propagated clustered shoots into single shoots, inoculating the single shoots into 1/2MS + NAA 0.2 to 0.4mg/L, IBA 0.6 to 0.8mg/L, KT 0.2 to 0.5mg/L, banana mud 50 to 60g/L and active carbon 0.5g/L rooting medium, and culturing the single shoots under the conditions of illumination for 12 hours per day and light intensity of 2000 to 3000lx at the temperature of 25 +/-2 ℃ for 30 to 40 days in an alternating dark environment with the light intensity of 2000 to 3000lx.

The invention has the beneficial effects that

1. The invention relates to a method for producing wintergreen seedlings by using a tissue culture technology, belongs to the modern biotechnology, is used for factory seedling culture in an artificial controllable environment, is not limited by climate and season, and can provide a large amount of seedlings with stable related quality in a short time.

2. The wintergreen medicinal material takes the content of the crystal orcein as a core index for detecting the wintergreen medicinal material, and partial enterprises simultaneously take the content of active substances such as the wintergreen and the wintergreen as quality indexes to determine whether the wintergreen medicinal material reaches Chinese pharmacopoeia or the purchasing standard of the medicinal materials in the enterprises. Different production areas or different colonies in the same production area or even the same colony have larger content difference of each component, and each type of high-quality germplasm can be screened out according to each evaluation index when artificial domestication cultivation introduction is carried out. The invention uses wintergreen axillary buds as explants for tissue culture and rapid propagation, uses mother somatic cells for propagation, belongs to the category of asexual propagation, can ensure the stable inheritance of female parent traits to the maximum extent, and can produce seedlings of high-quality germplasm with stable hereditary traits.

3. Generally, 0.1% mercuric chloride solution is the best disinfectant for explant disinfection, but wintergreen axillary buds are extremely young and tender, the disinfection purpose cannot be achieved when the 0.1% mercuric chloride solution is used for disinfection for a short time, the disinfection time is prolonged, the disinfection pollution rate is reduced, but the explants are dead greatly, and the non-dead explants are seriously browned and can not induce cluster buds. According to the invention, 75% alcohol, saturated bleaching powder solution and 8% hydrogen peroxide solution are adopted for early disinfection during disinfection, most of microorganisms in the explant can be killed, and meanwhile, the mercury bichloride solution with ultralow concentration is matched, so that the microorganisms carried by the explant can be basically killed, the pollution rate is reduced to the lowest, the death of the explant is avoided, and the browning rate of the explant is reduced.

4. The wintergreen explant is extremely difficult to induce cluster buds, and is limited by a disinfection technology on one hand and a culture medium formula on the other hand. The invention adopts the low-concentration combination of the low-concentration 2,4-D and the ultrahigh-activity CPPU and 2-IP, can effectively induce the generation of the cluster buds, and can effectively inhibit the browning of the explant without influencing the growth of the cluster buds by adding a proper concentration of silver nitrate.

5. When the cluster bud induction culture medium is adopted for the subculture multiplication of the wintergreen cluster buds, the conditions of slow growth speed, low cluster bud rate, short cluster bud internodes and the like exist, and the 2-IP price is extremely high. In the invention, during the subculture multiplication culture of the cluster buds, on the basis of a cluster bud induction culture medium, the auxin is changed into NAA, the 2-IP with extremely high price is removed from cytokinin, the concentration of CPPU is adjusted, the TIBA with moderate price and capable of well adjusting the multiplication of wintergreen is creatively added, and meanwhile, banana mud with certain concentration is added, so that a large number of cluster buds can be generated in a short period, the multiplication coefficient is improved, and the internode of the cluster buds can be increased without causing the thin and weak seedlings.

Drawings

FIG. 1 is a schematic representation of the induction of primary multiple shoots under the protocol of example 1;

FIG. 2 is a graph showing the proliferation of multiple shoots under the protocol of example 1;

FIG. 3 shows that the sterilization technique under the embodiment 1 does not cause death and browning of the explant, and is effective in inducing multiple shoots;

FIG. 4 another illustration of the efficient induction of clumping buds without explant death, browning, of the sterilization technique of example 1.

Detailed Description

The present invention will be further described with reference to specific examples, in which plant growth regulating hormones such as 2-IP (isopentenyladenine), CPPU (forchlorfenuron) and TIBA (triiodobenzoic acid) are commercially available.

Example 1

2. Cleaning explants: dipping a saturated soap aqueous solution by a brush, repeatedly scrubbing the surface until no soil residue exists, then adding a proper amount of water into a beaker, dropwise adding 3 drops of Tween-80, shaking for 10min, and showering by tap water for 60min.

3. And (3) disinfection of explants: and transferring the cleaned explant to a clean bench, disinfecting for 10s by using 75% alcohol, rinsing for 3 times by using sterile water, disinfecting for 10nin by using a saturated bleaching powder solution, rinsing for 3 times by using the sterile water, disinfecting for 4min by using 8% hydrogen peroxide solution, rinsing for 3 times by using the sterile water, dropwise adding 2 drops of Tween-80 and 0.025% mercuric chloride solution, disinfecting for 4min, rinsing for 8 times by using the sterile water, wherein the disinfection pollution rate is 14.9%, and the explant is not disinfected and dies.

4. Inducing the primary cluster buds: the sterilized explant is absorbed by sterile paper to remove surface moisture, wounds at two ends are cut off, the explant is inoculated into MS +2, 4-D0.02 mg/L + CPPU 0.4mg/L +2-IP 0.2mg/L + silver nitrate 0.1 mu g/L + activated carbon 0.5g/L cluster bud induction culture medium, dark culture is carried out for 7 days at the temperature of 25 +/-2 ℃, the subsequent temperature is unchanged, illumination is carried out for 10 hours every day, and culture is carried out for 60 days under the light-dark alternation of light intensity ranging from 2000 to 3000lx, the cluster buds can be effectively induced, the induction condition is shown in figure 1, the induction rate is 48.9%, the browning rate is 13.7%, the growth of the cluster buds cannot be influenced by browning, and particularly shown in figure 3 or figure 4, the cluster buds still grow even if the callus has a browning phenomenon.

5. Subculture multiplication culture of the cluster buds: cutting the induced cluster buds into single buds, inoculating the single buds into a MS + NAA 0.05mg/L + CPPU 0.5mg/L + TIBA 0.01mg/L + banana mud 50g/L cluster bud subculture multiplication medium, and culturing for 30 days at the temperature of 25 +/-2 ℃, under the condition of illumination for 10 hours every day and under the light intensity of 2000 to 3000lx alternately in dark, wherein the multiplication coefficient is 4.1, and the multiplication condition is shown as figure 2.

6. Rooting culture of the cluster buds: cutting the subcultured and propagated cluster buds into single buds, inoculating the single buds into a 1/2MS + NAA 0.2mg/L + IBA 0.6mg/L + KT 0.2mg/L + banana paste 50g/L + activated carbon 0.5g/L cluster bud rooting culture medium, and culturing the single buds for 30 days under the condition of illumination for 12 hours every day and light intensity of 2000-3000lx alternately in the dark at the temperature of 25 +/-2 ℃, wherein the rooting rate is 97.6%.

Example 2

2. Cleaning explants: dipping a saturated soap water solution by a brush, repeatedly scrubbing the surface until no soil residue exists, then adding a proper amount of water into a beaker, dropwise adding 3 drops of Tween-80, shaking for 13min, and showering by tap water for 65min.

3. And (3) disinfection of explants: the cleaned explants are transferred to a clean bench, sterilized by 75% alcohol for 13s, rinsed 3 times by sterile water, sterilized by saturated bleaching powder solution for 13nin, rinsed 4 times by sterile water, sterilized by 8% hydrogen peroxide solution for 5min, rinsed 4 times by sterile water, sterilized by dropping 2 drops of 0.025% mercuric chloride solution of tween-80 for 5min, rinsed 8 times by sterile water, the sterilization pollution rate is 13.1%, and the sterilization mortality rate is 1.2%.

4. Inducing primary cluster buds: the sterilized explant is absorbed with sterile paper to remove surface moisture, wounds at two ends are cut off, the explant is inoculated into MS +2, 4-D0.03 mg/L + CPPU 0.5mg/L +2-IP 0.3mg/L + silver nitrate 0.2 mu g/L + active carbon 0.5g/L cluster bud induction culture medium, dark culture is carried out for 7 days at the temperature of 25 +/-2 ℃, the subsequent temperature is unchanged, illumination is carried out for 10 hours every day, and culture is carried out for 65 days under light-dark alternation of light intensity of 2000 to 3000lx, the induction rate is 50.8%, the browning rate is 15.9%, and the browning does not influence the growth of the cluster buds.

5. Subculture multiplication culture of cluster buds: cutting the induced cluster buds into single buds, inoculating the single buds into MS + NAA 0.08mg/L + CPPU 0.5-0.8mg/L + TIBA 0.02mg/L + banana paste 55g/L cluster bud subculture multiplication medium, and culturing for 40 days at the temperature of 25 +/-2 ℃, the illumination time of 10h every day and the light intensity of 2000-3000lx under the light-dark alternation, wherein the multiplication coefficient is 4.6.

6. Rooting culture of cluster buds: cutting the subcultured and propagated cluster buds into single buds, inoculating the single buds into a 1/2MS + NAA 0.3mg/L + IBA 0.7mg/L + KT 0.4mg/L + banana paste 55g/L + activated carbon 0.5g/L cluster bud rooting culture medium, and culturing for 35 days under the condition of illumination for 12 hours every day and light intensity of 2000-3000lx alternately in darkness at the temperature of 25 +/-2 ℃, wherein the rooting rate is 98.4%.

Example 3

2. Cleaning explants: dipping a saturated soap aqueous solution by using a brush, repeatedly brushing the surface until no soil remains, then adding a proper amount of water into a beaker, dropwise adding 3 drops of Tween-80, shaking for 15min, and flushing with tap water for 70min.

3. And (3) disinfection of explants: the cleaned explants are transferred to a clean bench, sterilized by 75% alcohol for 15s, rinsed by sterile water for 3 times, sterilized by saturated bleaching powder solution for 15nin, rinsed by sterile water for 4 times, sterilized by 8% hydrogen peroxide solution for 5min, rinsed by sterile water for 4 times, sterilized by dropping 2 drops of Tween-80 in 0.025% mercuric chloride solution for 6min, rinsed by sterile water for 8 times, the sterilization pollution rate is 10.6%, and the sterilization mortality rate is 2.1%.

4. Inducing primary cluster buds: absorbing surface moisture of a sterilized explant by using sterile paper, cutting wounds at two ends, inoculating the explant into MS +2, 4-D0.05 mg/L + CPPU 0.5mg/L +2-IP 0.1mg/L + silver nitrate 0.2 mu g/L + activated carbon 0.5g/L cluster bud induction culture medium, carrying out dark culture at the temperature of 25 +/-2 ℃ for 7 days, keeping the temperature unchanged, illuminating for 10 hours every day, and carrying out light-dark alternate culture at the light intensity of 2000 to 3000lx for 70 days, wherein the induction rate is 47.3%, the browning rate is 17.7%, and the growth of cluster buds cannot be influenced by browning.

5. Subculture multiplication culture of cluster buds: cutting the induced cluster buds into single buds, inoculating the single buds into a MS + NAA 0.1mg/L + CPPU 0.8mg/L + TIBA 0.02mg/L + banana mud 60g/L cluster bud subculture multiplication medium, and culturing for 40 days under the conditions of 25 +/-2 ℃, 10 hours of illumination per day and 2000-3000lx light and dark alternation, wherein the multiplication coefficient is 4.7.

6. Rooting culture of cluster buds: cutting the subcultured and propagated cluster buds into single buds, inoculating the single buds into a 1/2MS + NAA 0.4mg/L + IBA 0.8mg/L + KT 0.5mg/L + banana paste 60g/L + activated carbon 0.5g/L cluster bud rooting culture medium, and culturing the single buds for 40 days under the light-dark alternation of light intensity of 2000-3000lx and illumination time of 12h per day at the temperature of 25 +/-2 ℃, wherein the rooting rate is 98.2%.

Wintergreen test data

1. The influence of the disinfection treatment on the disinfection pollution rate, the disinfection death rate and the browning rate

After the following disinfection treatment, the seedlings are inoculated into MS +2, 4-D0.02 mg/L + CPPU 0.5mg/L +2-IP 0.1mg/L + silver nitrate 0.1 mu g/L + activated carbon 0.5g/L cluster bud induction culture medium, and the disinfection pollution rate, the disinfection death rate and the browning rate are counted.

2. Primary induction culture

According to the conventional single combination of cytokinins such as NAA, IBA or 2,4-D, 6-BA, KT, CPPU, ZT, TDZ, TIBA or 2-IP and the like, no cluster bud can be induced, and then the compound combination of various auxins and various cytokinins is continuously carried out, so that the combination of low-concentration 2,4-D, low-concentration CPPU and 2-IP can have a certain induction success rate, and then the compound combination of 2,4-D, CPPU and 2-IP is repeatedly carried out for a plurality of times, so that the optimal species and concentration collocation can be adjusted, the wintergreen cluster buds can be effectively induced, and the collocation test data at the later stage is shown in the following table 3.

In the test, 15s of 75% alcohol and 15s of saturated bleaching powder solution are disinfected for 10min +8% hydrogen peroxide for 5min, 5min of 0.025% mercury bichloride, the test solution is inoculated into MS culture media added with 0.1 to 0.2 mu g/L silver nitrate, 2,4-D, CPPU and 2-IP with different concentrations are added into each culture medium, and part of test data are shown in the following table 3:

Claims

1. a tissue culture and rapid propagation seedling method of wintergreen is characterized in that wintergreen axillary buds are selected as explants, the explants are inoculated to a primary multiple bud induction culture medium to induce multiple buds after being sterilized, then the multiple buds are transferred to a secondary multiplication culture medium to obtain more multiple buds, and the multiple buds are finally inoculated to a rooting culture medium to form rooted seedlings;

the disinfection method comprises the steps of 10 to 15s of 75% alcohol, 10 to 15nin of saturated bleaching powder solution, 4 to 5min of 8% hydrogen peroxide, and dropwise adding 2 drops of 0.025% mercuric chloride solution of Tween-80 for 4 to 6min;

the primary cluster bud induction culture medium is MS +2, 4-D0.02 to 0.05mg/L + CPPU 0.4 to 0.5mg/L +2-IP 0.1 to 0.3mg/L + silver nitrate 0.1 to 0.2 mu g/L + active carbon 0.5g/L;

the cluster bud subculture multiplication medium comprises MS + NAA 0.05-0.1mg/L + CPPU 0.5-0.8mg/L + TIBA (triiodobenzoic acid) 0.01-0.02mg/L + banana mud 50-60g/L;

rooting culture medium for cluster buds is 1/2MS + NAA 0.2 to 0.4mg/L, IBA 0.6 to 0.8mg/L, KT 0.2 to 0.5mg/L, banana mud 50 to 60g/L and active carbon 0.5g/L.