CN114680045B - Tissue culture seedling raising method for Kadsura intercer A.C. Smith of Schisandra sphenanthera - Google Patents
Tissue culture seedling raising method for Kadsura intercer A.C. Smith of Schisandra sphenanthera 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
-
- 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
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention belongs to the field of traditional Chinese medicinal materials and tissue culture, and particularly discloses a tissue culture seedling method of Kadsura interceror A.C.Smith of Kadsura heteroclita.
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 Schisandra 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 Yunnan caulis Spatholobi has antioxidation, antiaging, antibacterial, tranquilizing, hypnotic, liver protecting, 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 sterilization method of the explant comprises the steps of sterilizing 15 to 20s with 75% alcohol, sterilizing 10 to 15min with a saturated bleaching powder solution, sterilizing 3 to 4min with a 0.1% mercury bichloride solution, and sterilizing 10 to 111min with a 0.05% mercury bichloride solution.
The primary cluster bud induction culture medium is WPM + NAA 0.02 to 0.05mg/L +6-BA 2.0 to 3.0mg/L + PVP (polyvinylpyrrolidone) 0.6 to 0.8g/L + activated carbon 1.0g/L.
The culture medium for the cluster bud subculture propagation is improved WPM + NAA0.1 to 0.2mg/L + IAA0.02 to 0.05mg/L + TDZ 2.0 to 2.5mg/L + BR 0.1 to 0.2mg/L + activated carbon 0.4g/L.
The rooting culture medium for the cluster buds is 1/2WPM + NAA 0.5-0.6 mg/L + IBA 1.0-1.5 mg/L + coconut juice 50-70ml/L + activated carbon 0.7g/L.
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 uses the content of heteroclitic Schisandrin D as the core index of the detection, the content is higher, the price is higher, the content of caulis Spatholobi in each production area is different, and even in the same production area or even the same group, the content is greatly different. 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 75 percent alcohol, saturated bleaching powder solution and high-concentration mercury bichloride solution for short-term disinfection and low-concentration mercury bichloride solution for long-term disinfection 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. When the caulis Spatholobi Yunnan adopts NAA, IBA, IAA, 2,4-D categories and auxin with various concentrations, the rooting rate is lower, and when strong seedling culture is carried out later, the rooting rate can be improved to a certain extent, but the rooting rate is not high.
Drawings
FIG. 1 is a diagram showing the proliferation of multiple shoots of caulis Spatholobi Yunnanensis after using modified WPM medium in the technical scheme of example 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 caulis Spatholobi into small segments with buds with the length of about 2-3cm after leaf removal, rinsing the caulis Spatholobi segments with saturated soap solution for 10min, washing residual soap solution on the surface with tap water, dropwise adding 3 drops of Tween-80, shaking for 5min, and showering with tap water for 50min.
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 buds ranging from 1 to 2cm, inoculating the small sections into an initial 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 12 hours every day, and culturing for 50 days under light-dark alternation with the light intensity of 2000-3000 lx, 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: shearing 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, and culturing for 40 days at the temperature of 25 +/-2 ℃ under the condition of illumination for 12 hours every day and light intensity of 2000-3000 lx with the multiplication coefficient of 4.6 and no withering phenomenon in the culture process, wherein the improved WPM 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 other elements to be unchanged on the basis of the original WPM medium.
5. And (3) rooting culture of cluster buds: cutting the cluster buds propagated by successive transfer into small segments with 2 or more buds, inoculating the small segments into a rooting culture medium with the polarity of 1/2WPM + NAA 0.5mg/L + IBA 1.2mg/L + coconut juice 50ml/L + activated carbon 0.7g/L, and culturing for 30 days under the light-dark alternation of illumination for 12h and light intensity of 2000-3000lx every day at the temperature of 25 +/-2 ℃, 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 with the length of about 2-3cm after leaf removal, rinsing the caulis Spatholobi segments with saturated soap solution for 13min, washing residual soap solution on the surface with tap water, dropwise adding 3 drops of Tween-80, shaking for 8min, and showering with tap water for 55min.
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 buds ranging from 1 to 2cm, inoculating the small sections with buds to a primary cluster bud induction culture medium of WPM + NAA 0.05mg/L +6-BA 2.5mg/L + PVP0.7g/L + active 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 12 hours every day, and carrying out light-dark alternate culture at the light intensity of 2000 to 3000lx 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. 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 0.04mg/L + TDZ 2.3mg/L + BR 0.2mg/L + activated carbon 0.4g/L according to polarity, and culturing the cluster buds at the temperature of 25 +/-2 ℃ for 45 days under the light-dark alternation of light intensity of 2000 to 3000lx and illumination time of 12h per day, 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. Rooting culture of the cluster buds: cutting the cluster buds propagated by subculture into small segments with 2 or more buds, inoculating the small segments into a rooting culture medium with 1/2WPM + NAA of 0.6mg/L, IBA of 1.5mg/L, coconut juice of 60ml/L and active carbon of 0.7g/L according to polarity, and culturing the small segments under the conditions of illumination for 12 hours every day at the temperature of 25 +/-2 ℃ and light intensity of 2000 to 3000lx for 35 days with the rooting rate of 93.2 percent.
Example 3
1. Selection and cleaning of explants: selecting tender and non-lignified stem segments at the top end of the Yunnan caulis Spatholobi as an explant, removing leaves, cutting into small segments with buds with the length of about 2-3 cm, washing for 15min by using saturated soap solution, washing residual soap liquid on the surface by using tap water, dropwise adding 3 drops of Tween-80, shaking for 10min, and showering for 60min by using tap water.
2. And (3) disinfection of explants: and transferring the cleaned explant to a clean bench, disinfecting with 75% alcohol solution for 20s, rinsing with sterile water for 3 times, disinfecting with saturated bleaching powder solution for 15min, rinsing with sterile water for 4 times, disinfecting with 0.1% mercuric chloride solution for 4min, rinsing with sterile water for 4 times, disinfecting with 0.05% mercuric chloride solution for 11min, rinsing with 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 buds ranging from 1 to 2cm, inoculating the small sections with buds to a primary cluster bud induction culture medium of WPM, NAA 0.02mg/L, 6-BA 3.0mg/L, PVP 0.8g/L and active carbon 1.0g/L according to polarity, carrying out dark culture at the temperature of 25 +/-2 ℃ for 7 to 10 days, keeping the temperature unchanged, illuminating for 12 hours every day, and carrying out light-dark alternate culture at the light intensity of 2000 to 3000lx 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. 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 0.05mg/L + TDZ 2.5mg/L + BR 0.2mg/L + activated carbon 0.4g/L according to polarity, and culturing the cluster buds at the temperature of 25 +/-2 ℃ for 50 days under the light and dark alternation of light intensity of 2000-3000 lx and illumination time of 12h per day with the multiplication coefficient of 5.2, wherein the preparation method of the improved WPM medium is the same as that of the embodiment 1
5. Rooting culture of the cluster buds: cutting the cluster buds after subculture propagation into small segments with 2 or more buds, inoculating the small segments into a rooting culture medium with 1/2WPM + NAA 0.6mg/L + IBA 1.0mg/L + coconut juice 70ml/L + active carbon 0.7g/L according to polarity, and culturing for 40 days under the conditions of illumination for 12h every day and light intensity of 2000-3000 lx under the condition of 25 +/-2 ℃ and light-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
Sterilizing by using 75% alcohol 15s + saturated bleaching powder solution 15min0.1% mercury bichloride 3min +0.05% mercury bichloride 10min, inoculating to WPM + NAA 0.02-0.05mg/L +6-BA 2.0-3.0 mg/L culture medium added with browning inhibitor with a certain concentration,
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 (1)
1. A tissue culture seedling method of Kadsura intercer A.C. Smith of Kadsura longifolia is characterized in that young stem segments of Kadsura intercer are selected as explants, and cut into small segments with buds of 2-3cm after leaves are removed; sterilizing, inoculating to the first generation multiple bud induction culture medium to induce multiple buds, transferring to the multiple bud subculture multiplication culture medium to obtain more multiple buds, inoculating to the multiple bud rooting culture medium to form rooted seedling,
the disinfection method of the explant comprises the steps of disinfecting for 15-20 s by 75% alcohol, disinfecting for 10-15min by saturated bleaching powder solution, disinfecting for 3-4min by 0.1% mercury bichloride solution and disinfecting for 10-11 min by 0.05% mercury bichloride solution;
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 0.6-0.8 g/L + active carbon 1.0g/L;
the cluster bud subculture multiplication medium 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 + active carbon 0.4g/L;
the improved WPM culture medium is characterized in that on the basis of the original WPM culture medium, ammonium nitrate is changed to 800mg/L, monopotassium phosphate is changed to 210mg/L, magnesium sulfate is changed to 370mg/L, potassium sulfate is changed to 800mg/L, calcium chloride tetrahydrate is changed to 400mg/L, and other elements are not changed;
rooting culture medium for 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 and active carbon 0.7g/L.
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