CN114680045A - Tissue culture seedling raising method of Yunnan caulis spatholobi - Google Patents
Tissue culture seedling raising method of Yunnan caulis spatholobi Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
Abstract
The invention belongs to the field of traditional Chinese medicinal materials and tissue culture, and particularly discloses a tissue culture seedling method of Yunnan caulis Spatholobi.
Description
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 Yunnan caulis spatholobi.
Background
The caulis Spatholobi plant source recorded in 2020 edition of China pharmacopoeia is Kadsura intercarrier A.C. Smith of Kadsura of Magnoliaceae, and is grown in hillside and forest with elevation below 1000 m, and mainly distributed in Yunnan, Jiangsu, Anhui, Zhejiang, Jiangxi, Fujian, Hubei, Hunan, Guangdong, Guangxi and Sichuan. Caulis Spatholobi Yunnan has effects of promoting blood circulation and replenishing blood, regulating menstruation and relieving pain, and relaxing muscles and tendons and dredging collaterals, and is mainly used for treating menoxenia, dysmenorrhea, numbness and paralysis, rheumatalgia, qi and blood deficiency, etc. The caulis Spatholobi Yunnanensis contains lignin compounds and terpenoids, wherein dibenzocyclooctene type lignanoid represented by heteroschisandrin is the most marked component, and the content of heteroschisandrin is the most core index of quality detection of caulis Spatholobi Yunnanensis. Modern medicine research shows that the caulis Spatholobi has antioxidation, antiaging, antibacterial, tranquilizing, hypnotic, hepatoprotective, anticancer and antivirus effects.
In recent years, because of the reduction of the storage amount of wild resources and the enhancement of the protection of governments on wild environments, the artificial introduction, domestication and cultivation of the Yunnan caulis spatholobi is carried out in the local region of Yunnan, the main mode is to adopt the steps of digging and digging wild plants for artificial domestication and cultivation, and then obtaining seedlings through seeds and cutting propagation, but because the Yunnan caulis spatholobi is a male and female plant, the number of wild living female plants is very small, so that the maturing rate of the living group is extremely low, meanwhile, the seeds also have the condition of low germination rate, and the defects of long cuttage propagation period, low propagation rate and the like exist.
It should be noted that some people can consider the Yunnan caulis Spatholobi material and caulis Spatholobi material as the same or similar species, but actually, the plant source of caulis Spatholobi is the Puccinia Spatholobus suberectus Dunn of the Puccinia of the leguminosae, which is different from the Yunnan caulis Spatholobi of the present invention in the same family and even different from the genus, and the sensitivity degree of each plant growth regulator and culture component may be greatly different during tissue culture.
Aiming at the defects of the prior art, the group specially invents a tissue culture seedling method of Yunnan caulis spatholobi by utilizing the modern biological tissue culture technology.
Disclosure of Invention
The invention mainly aims to provide a tissue culture seedling method of Yunnan caulis spatholobi, which aims to solve the problem of shortage of Yunnan caulis spatholobi germplasm resources.
In order to achieve the above purpose, the invention provides the following technical scheme:
a tissue culture method for culturing the seedlings of Yunnan caulis Spatholobi comprises selecting tender branch of Yunnan caulis Spatholobi as explant, sterilizing, inoculating to the first generation multiple bud induction culture medium to induce multiple buds, transferring to the subculture multiplication culture medium to obtain more multiple buds, and finally inoculating to the rooting culture medium to form rooted seedlings.
The explant sterilization method comprises the steps of 75% alcohol sterilization for 15-20 s, saturated bleaching powder solution sterilization for 10-15 min, 0.1% mercury bichloride solution sterilization for 3-4 min, and 0.05% mercury bichloride solution sterilization for 10-11 min.
The primary cluster bud induction culture medium is WPM + NAA 0.02-0.05 mg/L +6-BA 2.0-3.0 mg/L + PVP (polyvinylpyrrolidone) 0.6-0.8 g/L + active carbon 1.0 g/L.
The subculture multiplication medium for the cluster buds is modified WPM + NAA 0.1-0.2 mg/L + IAA 0.02-0.05 mg/L + TDZ 2.0-2.5 mg/L + BR 0.1-0.2 mg/L + activated carbon 0.4 g/L.
The rooting culture medium for the cluster buds is 1/2WPM, 0.5-0.6 mg/L NAA, 1.0-1.5 mg/L IBA, 50-70 ml/L coconut juice and 0.7g/L active carbon.
The improved WPM culture medium is prepared by changing ammonium nitrate to 800mg/L, monopotassium phosphate to 210mg/L, magnesium sulfate to 370mg/L, potassium sulfate to 800mg/L, calcium chloride tetrahydrate to 400mg/L and the rest elements to be unchanged on the basis of the original WPM culture medium.
The invention has the beneficial effects that:
1. the invention relates to a method for producing Yunnan caulis Spatholobi seedlings by tissue culture technology, which belongs to the modern biotechnology, is the factory seedling culture carried out in the 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 caulis Spatholobi Yunnanensis medicinal material takes the content of heteroschisin as the core index of the detection, and the higher the content and the higher the price, while the contents of caulis Spatholobi Yunnanensis in different production areas have great difference even in the same production area or even the same group. The invention uses stem section of Yunnan caulis Spatholobi as explant to carry out tissue culture and rapid propagation, belongs to the category of asexual propagation, can ensure the stable inheritance of female parent characters to the maximum extent, and can produce high-quality seedlings with stable hereditary characters by using high-quality germplasm with high content of heteroschisandrin as the female parent.
3. The invention adopts the disinfection method of short-term disinfection of 75 percent alcohol, saturated bleaching powder solution and high-concentration mercury bichloride solution and long-term disinfection of low-concentration mercury bichloride solution during disinfection, can reduce the disinfection pollution rate and the disinfection death rate to the maximum extent, and is the most suitable disinfection technology for the tissue culture of the Yunnan caulis spatholobi at present.
4. When the Yunnan caulis Spatholobi is induced in the primary cluster buds, because of the influence of the disinfectant, the browning phenomenon is very easy to generate, PVP with a certain concentration is added in the primary induction, the browning can be inhibited in a range without influencing the growth, and meanwhile, the addition of the concentration does not influence the primary cluster bud induction rate of the Yunnan caulis Spatholobi.
5. When the Yunnan caulis Spatholobi is subjected to secondary multiplication of the cluster buds, the situations of slow growth speed, low cluster bud rate, short cluster bud internodes, easy withering and the like caused by long-time culture exist, and when the cluster bud is subjected to secondary multiplication culture, the combination of NAA, IAA, TDZ and BR with certain concentration is adopted, so that a large number of cluster buds can be generated in a short period, the multiplication coefficient is improved, the cluster bud internodes can be increased, the actual production operation is facilitated, the production multiplication coefficient is briefly improved, and meanwhile, the WPM basic culture medium is improved, so that the growth of the Yunnan caulis Spatholobi is facilitated, and the withering situation is almost avoided, and is shown in a figure 1 and a figure 2.
6. According to the invention, the combination of IAA and NAA with certain concentration is adopted, and 50-70 ml/L of coconut juice is added, so that the strong seedling culture link can be reduced, and the rooting rate is improved to about 95%.
Drawings
FIG. 1 is a graph showing the proliferation of multiple shoots of Mucuna yunnanensis after using a modified WPM medium according to the embodiment 1;
FIG. 2 is a graph showing the proliferation of multiple shoots of Mucuna yunnanensis after using WPM medium according to the embodiment 1.
Detailed Description
The present invention will be further described with reference to specific examples.
Example 1
1. Selection and cleaning of explants: selecting tender and non-lignified stem segments at the top end of the caulis Spatholobi as explants, cutting the explants into small segments with buds with the length of about 2-3 cm after removing leaves, rinsing the small segments with saturated soap solution for 10min, washing residual soap solution on the surface by tap water, dropwise adding 3 drops of Tween-80, shaking for 5min, and showering by tap water for 50 min.
2. And (3) disinfection of explants: and transferring the cleaned explant to a super clean workbench, disinfecting the explant for 15s by using a 75% alcohol solution, rinsing the explant by using sterile water for 2 times, disinfecting the explant by using a saturated bleaching powder solution for 10min, rinsing the explant by using sterile water for 3 times, disinfecting the explant by using a 0.1% mercuric chloride solution for 3min, rinsing the explant by using sterile water for 3 times, finally disinfecting the explant by using a 0.05% mercuric chloride solution for 10min, rinsing the explant by using sterile water for 8 times, wherein the disinfection pollution rate is 21.7%, and the disinfection death rate is 28.7%.
3. Inducing the primary cluster buds: absorbing surface moisture of the disinfected explant by using sterile paper, cutting wounds at two ends, cutting the explant into small sections with 1-2 cm buds, inoculating the small sections with the buds into a primary cluster bud induction culture medium of WPM + NAA 0.04mg/L +6-BA 2.0mg/L + PVP0.6g/L + activated carbon 1.0g/L according to polarity, carrying out dark culture at the temperature of 25 +/-2 ℃ for 7 days, keeping the temperature unchanged, illuminating for 12h every day, carrying out light-dark alternate culture with the light intensity of 2000-3000 lx for 50 days, wherein the browning rate is 34.1%, but the browning does not influence the induction and growth of the cluster buds, and the cluster bud induction rate is 67.4%.
4. Subculturing and proliferating the cluster buds: cutting the induced cluster buds into single buds, inoculating the single buds into a cluster bud subculture multiplication medium of improved WPM + NAA0.1 mg/L + IAA0.02 mg/L + TDZ 2.0mg/L + BR 0.1mg/L + activated carbon 0.4g/L according to polarity, illuminating for 12h every day at the temperature of 25 +/-2 ℃, and culturing for 40 days under the light-dark alternation of light intensity of 2000-3000 lx, wherein the multiplication coefficient is 4.6, no withering phenomenon occurs in the culture process, the improved WPM medium is obtained by changing ammonium nitrate to 800mg/L, monopotassium phosphate to 210mg/L, magnesium sulfate to 370mg/L, potassium sulfate to 800mg/L, calcium chloride tetrahydrate to 400mg/L and the rest elements to be unchanged on the basis of the original WPM medium.
5. And (3) rooting culture of cluster buds: cutting the cluster buds subjected to subculture propagation into small segments with 2 or more buds, inoculating the small segments into a rooting culture medium of 1/2WPM + NAA 0.5mg/L + IBA 1.2mg/L + coconut juice 50ml/L + activated carbon 0.7g/L according to polarity, and culturing for 30 days at the temperature of 25 +/-2 ℃ under illumination for 12h every day and under the light and dark alternation of the light intensity of 2000-3000 lx, wherein the rooting rate is 89.3%.
Example 2
1. Selection and cleaning of explants: selecting tender and non-lignified stem segments at the top end of the caulis Spatholobi as explants, cutting the caulis Spatholobi into small segments with buds of about 2-3 cm after removing leaves, rinsing the caulis Spatholobi segments with saturated soap solution for 13min, washing residual soap solution on the surface of the caulis Spatholobi segments with tap water, dropwise adding 3 drops of Tween-80, shaking for 8min, and showering with tap water for 55 min.
2. And (3) disinfection of explants: and transferring the cleaned explant to a super clean workbench, disinfecting the explant for 20s by using a 75% alcohol solution, rinsing the explant by using sterile water for 3 times, disinfecting the explant by using a saturated bleaching powder solution for 13min, rinsing the explant by using sterile water for 4 times, disinfecting the explant by using a 0.1% mercuric chloride solution for 4min, rinsing the explant by using sterile water for 4 times, finally disinfecting the explant by using a 0.05% mercuric chloride solution for 10min, rinsing the explant by using sterile water for 8 times, wherein the disinfection pollution rate is 20.6%, and the disinfection death rate is 30.2%.
3. Inducing the primary cluster buds: absorbing surface moisture of the disinfected explant by using sterile paper, cutting wounds at two ends, cutting the explant into small sections with 1-2 cm buds, inoculating the small sections with the buds into a primary cluster bud induction culture medium of WPM + NAA 0.05mg/L +6-BA 2.5mg/L + PVP0.7g/L + activated carbon 1.0g/L according to polarity, carrying out dark culture at the temperature of 25 +/-2 ℃ for 7 days, keeping the temperature unchanged, irradiating 12 hours every day, and carrying out light-dark alternate culture with the light intensity of 2000-3000 lx for 60 days, wherein the browning rate is 38.7%, but the browning does not influence the induction and growth of the cluster buds, and the cluster bud induction rate is 69.3%.
4. Subculturing and proliferating the cluster buds: cutting the induced cluster buds into single buds, inoculating the single buds into a cluster bud subculture multiplication medium of improved WPM + NAA 0.2mg/L + IAA 0.04mg/L + TDZ 2.3mg/L + BR 0.2mg/L + activated carbon 0.4g/L according to polarity, and culturing for 45 days at the temperature of 25 +/-2 ℃ under illumination for 12h every day and under the light intensity of 2000-3000 lx under light and dark alternation, wherein the multiplication coefficient is 4.8, and the preparation method of the improved WPM medium is the same as that of example 1.
5. And (3) rooting culture of cluster buds: cutting the cluster buds subjected to subculture propagation into small segments with 2 or more buds, inoculating the small segments into a rooting culture medium of 1/2WPM, NAA 0.6mg/L, IBA 1.5mg/L, coconut juice 60ml/L and active carbon 0.7g/L according to polarity, and culturing for 35 days at the temperature of 25 +/-2 ℃ under illumination for 12h every day and under the light and dark alternation of the light intensity of 2000-3000 lx, wherein the rooting rate is 93.2%.
Example 3
1. Selection and cleaning of explants: selecting tender and non-lignified stem segments at the top end of the caulis Spatholobi as explants, cutting the explants into small segments with buds with the length of about 2-3 cm after removing leaves, rinsing the small segments with saturated soap solution for 15min, washing residual soap solution on the surface by tap water, dropwise adding 3 drops of Tween-80, shaking for 10min, and showering by tap water for 60 min.
2. And (3) disinfection of explants: and transferring the cleaned explant to a super clean workbench, disinfecting the explant for 20s by using a 75% alcohol solution, rinsing the explant by using sterile water for 3 times, disinfecting the explant by using a saturated bleaching powder solution for 15min, rinsing the explant by using sterile water for 4 times, disinfecting the explant by using a 0.1% mercuric chloride solution for 4min, rinsing the explant by using sterile water for 4 times, finally disinfecting the explant by using a 0.05% mercuric chloride solution for 11min, rinsing the explant by using sterile water for 8 times, wherein the disinfection pollution rate is 19.8%, and the disinfection death rate is 29.3%.
3. Inducing the primary cluster buds: absorbing surface moisture of the disinfected explant by using sterile paper, cutting wounds at two ends, cutting the explant into small sections with 1-2 cm buds, inoculating the small sections with the buds into a primary cluster bud induction culture medium of WPM + NAA 0.02mg/L +6-BA 3.0mg/L + PVP 0.8g/L + activated carbon 1.0g/L according to polarity, carrying out dark culture at the temperature of 25 +/-2 ℃ for 7-10 days, keeping the temperature unchanged, illuminating for 12h every day, carrying out light-dark alternate culture at the light intensity of 2000-3000 lx for 60 days, wherein the browning rate is 36.6%, but the browning does not influence the induction and growth of the cluster buds, and the cluster bud induction rate is 67.0%.
4. Subculture multiplication culture of the cluster buds: cutting the induced cluster buds into single buds, inoculating the single buds into a cluster bud subculture multiplication medium of improved WPM + NAA 0.2mg/L + IAA 0.05mg/L + TDZ 2.5mg/L + BR 0.2mg/L + activated carbon 0.4g/L according to polarity, culturing for 50 days at 25 +/-2 ℃ under illumination for 12h every day and under the light intensity of 2000-3000 lx in light and dark alternately, wherein the multiplication coefficient is 5.2, and the configuration method of the improved WPM medium is the same as that of the improved WPM medium in example 1
5. And (3) rooting culture of cluster buds: cutting the cluster buds subjected to subculture propagation into small segments with 2 or more buds, inoculating the small segments into a rooting culture medium of 1/2WPM, NAA 0.6mg/L, IBA 1.0mg/L, coconut juice 70ml/L and active carbon 0.7g/L according to polarity, and culturing for 40 days at the temperature of 25 +/-2 ℃ under the condition of 12h of illumination per day and the light intensity of 2000-3000 lx under the condition of light and dark alternation, wherein the rooting rate is 91.9%.
Caulis Spatholobi Dianthi part experimental record
1. The influence of the disinfection treatment on the disinfection pollution rate, the disinfection death rate and the browning rate
The following disinfection treatments were performed and inoculated into WPM + NAA 0.04mg/L +6-BA 2.5mg/L + PVP0.7g/L + activated carbon 1.0g/L, and the disinfection contamination rate, disinfection death rate and browning rate were counted.
2. Effect of media additives on browning
Disinfecting according to 15s of 75% alcohol, 15min of 0.1% mercuric chloride and 3min of 0.05% mercuric chloride for 10min, inoculating the disinfected mercuric chloride into a WPM + NAA 0.02-0.05 mg/L and 6-BA 2.0-3.0 mg/L culture medium added with a certain concentration of browning inhibitor,
the browning inhibitors such as Vc and silver nitrate are screened in the early stage, the Vc is found to have no obvious improvement on the browning of Yunnan suberect spatholobus stem, and then a test is carried out by adopting silver nitrate, the lower concentration of the silver nitrate is found to have no obvious inhibition on the browning of Yunnan suberect spatholobus stem, and the high concentration of the silver nitrate has a certain inhibition effect on the browning of Yunnan suberect spatholobus stem, but has a toxic effect on the Yunnan suberect spatholobus stem. And finally, PVP + active carbon with proper concentration is screened out to have obvious inhibition effect on browning of Yunnan suberect spatholobus stem, and the growth is not influenced.
3. Effect of individual hormones on proliferation factor
When the plant growth regulator of NAA or IBA, 6-BA, KT and other conventional plant growth regulators is used, the clustered shoots can grow slowly but hardly proliferate in a short period, and then discovered that Yunnan caulis Spatholobi is extremely sensitive to TDZ and BR, and can grow and proliferate to a certain extent when being matched with NAA alone at proper concentration, and then NAA is combined with the Yunnan caulis Spatholobi and the NAA to discover that the proliferation and the growth are obviously accelerated, and after IAA with a certain concentration is added, the proliferation coefficient can reach the best.
Claims (6)
1. A tissue culture seedling method of Yunnan caulis Spatholobi is characterized in that tender branches of Yunnan caulis Spatholobi are selected as explants, sterilized and inoculated to a primary multiple bud induction culture medium to induce multiple buds, then transferred to a secondary multiplication culture medium to obtain more multiple buds, and finally inoculated to a rooting culture medium to form rooted seedlings.
2. The tissue culture seedling raising method of Yunnan caulis spatholobi, according to claim 1, characterized in that the disinfection method of the explant is 75% alcohol disinfection for 15-20 s, saturated bleaching powder solution disinfection for 10-15 min, 0.1% mercuric chloride solution disinfection for 3-4 min, 0.05% mercuric chloride solution disinfection for 10-11 min.
3. The tissue culture seedling raising method of Yunnan caulis spatholobi, according to claim 1, characterized in that the primary cluster bud induction culture medium is WPM + NAA 0.02-0.05 mg/L +6-BA 2.0-3.0 mg/L + PVP (polyvinylpyrrolidone) 0.6-0.8 g/L + activated carbon 1.0 g/L.
4. The tissue culture seedling method of Yunnan caulis Spatholobi according to claim 1, wherein the secondary propagation culture medium of the cluster buds is modified WPM + NAA 0.1-0.2 mg/L + IAA 0.02-0.05 mg/L + TDZ 2.0-2.5 mg/L + BR 0.1-0.2 mg/L + activated carbon 0.4 g/L.
5. The tissue culture seedling raising method of Yunnan caulis Spatholobi according to claim 1, wherein the rooting culture medium of the cluster buds is 1/2WPM + NAA 0.5-0.6 mg/L + IBA 1.0-1.5 mg/L + coconut juice 50-70 ml/L + activated carbon 0.7 g/L.
6. The tissue culture seedling raising method of Yunnan caulis Spatholobi according to claim 4, wherein the improved WPM culture medium is prepared by changing ammonium nitrate to 800mg/L, monopotassium phosphate to 210mg/L, magnesium sulfate to 370mg/L, potassium sulfate to 800mg/L, calcium chloride tetrahydrate to 400mg/L and the rest elements are not changed on the basis of the original WPM culture medium.
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Effective date of registration: 20240111 Address after: 673200 CCCC Green Spice Industrial Park, Shangjiang Town, Lushui City, Nujiang Lisu Autonomous Prefecture, Yunnan Province Patentee after: Nujiang Angda Biotechnology Development Co.,Ltd. Address before: 2 / F, scientific research office building, Yunnan Academy of Agricultural Sciences, 2238 Beijing Road, Kunming, Yunnan 650000 Patentee before: INSTITUTE OF MEDICINAL PLANTS, YUNNAN ACADEMY OF AGRICULTURAL SCIENCES Patentee before: Yunnan plant insect Medicine Biotechnology Co.,Ltd. |