CN111149705A - Tissue culture and rapid propagation method for stem segments of rhubarb vines - Google Patents
Tissue culture and rapid propagation method for stem segments of rhubarb vines Download PDFInfo
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- MIJYXULNPSFWEK-UHFFFAOYSA-N Oleanolinsaeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4=CCC3C21C MIJYXULNPSFWEK-UHFFFAOYSA-N 0.000 description 1
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- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
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- HZLWUYJLOIAQFC-UHFFFAOYSA-N prosapogenin PS-A Natural products C12CC(C)(C)CCC2(C(O)=O)CCC(C2(CCC3C4(C)C)C)(C)C1=CCC2C3(C)CCC4OC1OCC(O)C(O)C1O HZLWUYJLOIAQFC-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
- A01H4/001—Culture apparatus 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 relates to a tissue culture method of Chinese medicinal materials, in particular to a tissue culture and rapid propagation method of a stem section of a rhubarb vine, which selects a stem end of the rhubarb vine as an explant, and uses alcohol, bleaching powder solution and mercuric chloride solution to disinfect in a super clean bench in sequence; inoculating the sterilized stem segments into an induction culture medium which takes WPM as a basic culture medium and is added with NAA, TDZ and active carbon; inoculating the induced cluster buds into a multiplication culture medium which takes WPM as a basic culture medium and is added with NAA, 6-BA, KT, GA3 and activated carbon; inducing or proliferating to obtain cluster buds, inoculating to rooting culture medium containing WPM as basic culture medium and NAA, IBA, paclobutrazol and activated carbon. The method has higher cluster bud induction rate, rooting rate and survival rate than the known literature.
Description
Technical Field
The invention relates to a tissue culture method of a traditional Chinese medicinal material, belongs to the field of plant tissue culture and traditional Chinese medicinal material planting, and particularly relates to a tissue culture method of a caulis Fibraureae.
Background
Caulis Fibraurearecae Pierre, also named caulis Fibraureae, herba Aristolochiae, caulis Fibraureae and Coptis chinensis, is perennial woody big vine of Fibrauremia of Menispermaceae, is originally used only by minority nationalities in producing areas and is used as a yellow dye and for treating sores and ulcers, and the medical application of caulis Fibraureae is explored in the last century Chinese herbal medicine sports by Mongolian autonomous military health team and Honghe State hospitals, is popularized and applied in Yunnan province after primary clinical verification, is quickly recorded by 1977 edition of Chinese pharmacopoeia, and is a more typical example for developing modern plant medicines from traditional national medicines.
The caulis Fibraureae is used as a medicine with rattan, has the effects of clearing heat and removing toxicity, and purging fire and relaxing bowels, and is mainly used for treating diseases such as heat toxin exuberance inside, constipation, diarrhea and dysentery, sore throat, conjunctival congestion and red swelling, carbuncle swelling and sore toxicity, and the like. Modern medical research shows that the rhubarb vine contains a plurality of alkaloids such as fibrauretine, jateorhizine, tetrahydropalmatine and the like, lactone compounds such as fibrauretine and the like, terpenoid compounds such as oleanolic acid and the like, and volatile oil such as palmitic acid and the like, wherein the content of the fibrauretine is the highest, and the application is wide. Alkaloids such as fibrauretine have antibacterial, antiinflammatory, antiviral, immunity enhancing, and memory improving effects. At present, 11 traditional Chinese medicine preparations taking a fibraurea stem medicinal material or fibrauretine extracted from the fibraurea stem medicinal material as a raw material medicine comprise compound artemisia apiacea spray, compound artemisia apiacea liniment, swelling and pain relieving tincture, alum-vine hemorrhoid injection, fibrauretine dispersible tablets, fibrauretine injection, fibrauretine tablets, fibrauretine capsules, fibrauretine soft capsules, compound fibrauretine lotion and fibrauretine suppository, wherein the fibrauretine tablets and the fibrauretine capsules are produced by about 45 pharmaceutical enterprises, and the production and market application of the fibrauretine tablets and the fibrauretine capsules are most.
The rhubard is mainly distributed in southeast Yunnan, southwest Guangxi and southwest Guangxi, wherein the Honghe, Xishuangbanna, Pu' er and lincang cities in Yunnan are widely distributed, mainly in tropical and subtropical jungles and mountain valley with the elevation of 180-1000 meters, and Vietnam, Laos and Cambodia are also distributed. The distribution area of the rhubarb vine is narrow, the growth period is long, so that the wild resource storage amount is low, and the distribution is dispersed. Meanwhile, the continuous development of modern Chinese medicinal preparations and the acceptance of medicinal effects are higher and higher, the market application of the rhubarb vine is more and more extensive, the wild plant resources are greatly consumed by artificial disordered mining, and the wild resources with low inherent reserves are more deficient. After 1993, due to shortage of raw materials, prices have risen year by year, and mining has expanded to peripheral countries such as Laos, Vietnam and the like. Since 2003, the Yunnan Honghe state starts to try artificial domestication planting of the rhubarb vine, the Yunnan Lincang city, Dehong state and Xishuangbanna state also try artificial domestication planting of the rhubarb vine successively, the planting technology, the planting adaptability and the like of the rhubarb vine have been preliminarily completed, meanwhile, a certain scale has been preliminarily formed in the Honghe Pingbiang county and the Lincang Shuangjiang county, but the seedling breeding technology in the current planting adopts the traditional seed breeding and the cutting breeding, and has the defects of slow breeding speed, long breeding period and low breeding rate.
A comparison document, namely a research on rapid propagation of the stem tip of a rhubarb vine, discloses a tissue culture method of the rhubarb vine, the stem tip of the rhubarb vine is taken as an explant, the stem tip is directly induced and differentiated into seedlings without callus through a tissue culture method, and the method is proved by a plurality of comparison experiments that the optimal culture medium combination is MS +6-BA 0.5mg/L + LAA 0.5mg/L + ZT 0.1 mg/L, and the optimal culture medium combination for rooting is 1/2 MS + IBA 1.0mg/L + NAA 1.0 mg/L. The invention aims to obtain a tissue culture method of the rhubarb vine with better experimental effect by researching and updating the formula of a culture medium on the basis of the document.
Disclosure of Invention
Aiming at the defects of long propagation period, low propagation speed, low propagation rate and the like of the existing seedling propagation technology of the rhubarb vine, the invention utilizes the modern biotechnology to invent a tissue culture method which has simple components, simple and convenient operation, short production period and high propagation rate and can provide sufficient and high-quality seedlings for large-scale planting in a short period.
The invention is realized by the following technical scheme:
1) selection of explants: selecting a robust rhubarb vine plant, and cutting 10-20 cm of stem tip which is free from diseases and insect pests and is not lignified as an explant;
2) cleaning explants: removing leaves of the selected explants, cutting the explants into stem sections with buds with the size of about 2-3 cm, rinsing the stem sections with a detergent solution for 10-20 min, rinsing the stem sections with tap water for 3-4 times until no detergent remains, and then rinsing the stem sections with tap water for 6-12 h;
3) and (3) disinfection of explants: pouring out all water from the cleaned explant, transferring the water into a clean bench, sucking surface moisture by using sterile paper, placing the explant into a sterile bottle, adding 75% alcohol, shaking for 20-30 s, rinsing with sterile water for 3-4 times, adding a saturated bleaching powder solution, shaking for 20-30 min, rinsing with sterile water for 3-4 times, finally adding a 0.05% mercuric chloride solution and 1 drop of Tween-80, shaking for 10-12 min, rinsing with sterile water for 7-8 times, sucking the surface moisture of the explant by using the sterile paper, wherein the pollution rate in 30 days is 20-30%;
4) and (3) induction culture: cutting wounds at two ends of a sterilized explant to form stem sections with single buds of 1.0-1.5 cm, obliquely inserting the stem sections into an induction culture medium of WPM + NAA 0.2-0.5 mg/L + TDZ 1.0-2.0 mg/L + activated carbon 0.5-1.0 g/L according to polarity, carrying out dark culture at the temperature of 23-27 ℃ for the first 20 days, and then carrying out culture for 60-70 days under the conditions that the illumination intensity is 1000-2000 lux and the single-day illumination time is 10h, wherein the induction rate of the cluster buds is 70-80%;
5) and (3) proliferation culture: after the stem section is induced to generate cluster buds, the cluster buds are cut into small sections with single buds according to internodes, and are obliquely inserted into WPM + NAA 0.2-0.5 mg/L +6-BA 0.2-0.5 mg/L + KT 2.0-4.0 mg/L + GA according to polarity30.5-1.0 mg/L + 0.5-1.0 g/L of activated carbon, performing dark culture at the temperature of 23-27 ℃ for the first 5 days, and performing dark culture for the later 40-45 days under the conditions that the illumination intensity is 1000-2000 lux and the single-day illumination time is 12 hours, wherein the proliferation coefficient is 6.0;
6) strong seedling and rooting culture: cutting cluster buds obtained by induction culture or propagation culture into stem sections with 2-3 buds, vertically inserting the stem sections into a rooting culture medium consisting of WPM, NAA 1.0-2.0 mg/L, IBA 0.5-1.0 mg/L, PP 3331.0-2.0 mg/L and active carbon 0.5-1.0 g/L according to polarity, and culturing for 35-40 days under the conditions that the temperature is 23-27 ℃, the illumination intensity is 2000-3000 lux and the single-day illumination time is 12h, wherein the rooting rate is about 97%.
The invention has the beneficial effects that:
according to the tissue culture and rapid propagation method of the stem segments of the rhubarb vine, the combination of auxin and TDZ is adopted as a culture medium for explant induction, the induction rate of cluster buds is 70-80%, and meanwhile, due to the existence of TDZ, the induction of callus is basically prevented, and the variation risk caused by redifferentiation after induced callus in tissue culture is reduced;
compared with a comparison document, the cluster bud multiplication medium adopts the combination of NAA, 6-BA and KT, the theoretical multiplication coefficient can reach more than 6.0, but the combination of auxin and cytokinin is adopted, internodes are extremely short, generally 2-3 buds can be cut as an inoculation material in the actual inoculation process, and the actual multiplication coefficient is reduced, but the GA3 of 0.5-1.0 mg/L is used in the multiplication medium, so that on one hand, the internodes can be effectively lengthened, the inoculation operation link in the production process is facilitated, the production efficiency is improved, on the other hand, the multiplication coefficient is improved by about 1.5 times compared with that of the method without adding GA3 in the actual production, and the production cost is reduced; according to the rapid propagation method, the combination of NAA, IBA and PPP333 is adopted in the rooting culture medium, strong seedlings and rooting are synchronously carried out, the rooting rate can reach 97%, and the survival rate can reach 93% when the seedlings are strong, fresh and green and are acclimatized.
Detailed Description
The present invention will be further described with reference to examples. Embodiments of the present invention include, but are not limited to, the following examples.
Example 1: 1) selection of explants: selecting a robust male plant of the Fibraurea recisa pierre, and cutting the top 10-20 cm of a new shoot which is free from diseases and insect pests and is not lignified as an explant;
2) cleaning explants: removing leaves of the selected explant, cutting the explant into stem segments with buds (the terminal buds can also be used as the explant) with the size of about 2-3 cm, putting the stem segments into a 500ml beaker, rinsing the stem segments with a detergent solution for 20min, washing the stem segments with tap water for 4 times until no detergent remains, and then washing the stem segments with the tap water for 6 hours;
3) and (3) disinfection of explants: after the explants are cleaned, pouring out all tap water in a beaker, transferring the tap water into a clean bench, sucking surface moisture by using sterile paper, putting 20-30 explants in sterile bottles, adding 75% alcohol, shaking for 30s, rinsing with sterile water for 3 times, adding a saturated bleaching powder solution, shaking for 20min, rinsing with sterile water for 3 times, finally adding 0.05% mercuric chloride solution and 1 drop of Tween-80, shaking for 10min, rinsing with sterile water for 7 times, sucking off the surface moisture of the explants by using the sterile paper, wherein the 7-day pollution rate is 24.5%, and the 30-day pollution rate is 26.7%;
4) and (3) induction culture: cutting off wounds at two ends of a sterilized explant to form stem sections with single buds of 1.0-1.5 cm, obliquely inserting the stem sections into an induction culture medium of WPM, NAA 0.2mg/L, TDZ 1.0mg/L and active carbon 0.5g/L according to polarity, carrying out dark culture at the temperature of 23-27 ℃ for the first 20 days, and then carrying out culture for 70 days under the conditions of illumination intensity of 1000-2000 lux and single-day illumination time of 10h, wherein the induction rate of the cluster buds is 74.5%.
5) And (3) proliferation culture: after the stem section is induced to generate cluster buds, the cluster buds are cut into small sections with single buds according to internodes and are obliquely inserted into WPM + NAA 0.2mg/L +6-BA 0.2mg/L + KT 2.0mg/L + GA according to polarity30.5mg/L + 0.5g/L activated carbon in a growth medium at temperatureDark culture is carried out for 5 days at the temperature of 23-27 ℃, then culture is carried out for 45 days under the conditions that the illumination intensity is 1000-2000 lux and the single-day illumination time is 12h, and the multiplication coefficient is 6.2;
6) rooting culture: cutting cluster buds obtained by induction culture or propagation culture into stem sections with 2-3 buds, vertically inserting the cluster buds into a rooting culture medium of WPM + NAA 2.0mg/L + IBA 1.0mg/L + PP3331.0 mg/L + activated carbon 1.0g/L according to polarity, culturing for 40 days at the temperature of 23-27 ℃ under the conditions of illumination intensity of 2000-3000 lux and single-day illumination time of 12h, wherein the rooting rate is 97.5%, the seedlings are 5.8 cm high, and the basal stems are 0.09 cm thick.
Example 2: 1) selection of explants: selecting a robust male plant of the Fibraurea recisa pierre, and cutting the top 10-20 cm of a new shoot which is free from diseases and insect pests and is not lignified as an explant;
2) cleaning explants: removing leaves of the selected explant, cutting the explant into stem segments with buds (the terminal buds can also be used as the explant) with the size of about 2-3 cm, putting the stem segments into a 500ml beaker, rinsing the stem segments with a detergent solution for 10min, washing the stem segments with tap water for 4 times until no detergent remains, and then washing the stem segments with the tap water for 12 hours;
3) and (3) disinfection of explants: after the explants are cleaned, pouring out all tap water in a beaker, transferring the tap water into a clean bench, sucking surface water by using sterile paper, putting 20-30 explants in sterile bottles, adding 75% alcohol, shaking for 20s, rinsing with sterile water for 3 times, adding a saturated bleaching powder solution, shaking for 30min, rinsing with sterile water for 4 times, adding 0.05% mercuric chloride solution and 1 drop of Tween-80, shaking for 12min, rinsing with sterile water for 8 times, sucking the surface water of the explants by using the sterile paper, wherein the 7-day pollution rate is 20.4%, and the 30-day pollution rate is 21.2%;
4) and (3) induction culture: cutting off wounds at two ends of a sterilized explant to form stem sections with single buds of 1.0-1.5 cm, obliquely inserting the stem sections into an induction culture medium of WPM + NAA0.5mg/L + TDZ 2.0mg/L + activated carbon 1.0g/L according to polarity, carrying out dark culture at the temperature of 23-27 ℃ for the first 20 days, and then carrying out culture for 70 days under the conditions of illumination intensity of 1000-2000 lux and single-day illumination time of 10h, wherein the induction rate of the cluster buds is 77.8%;
5) and (3) proliferation culture: after the stem section is induced to produce cluster bud, the cluster bud is cut into small sections with single bud according to internode, and pressed into polarObliquely inserted into WPM + NAA0.5mg/L +6-BA 0.5mg/L + KT 4.0mg/L + GA3Culturing in a proliferation culture medium of 1.0mg/L + activated carbon of 1.0g/L in dark at the temperature of 23-27 ℃ for the first 5 days, and then culturing for 45 days under the conditions of illumination intensity of 1000-2000 lux and single-day illumination time of 12h, wherein the proliferation coefficient is 6.7;
6) rooting culture: cutting cluster buds obtained by induction culture or propagation culture into stem sections with 2-3 buds, vertically inserting the cluster buds into a rooting culture medium of WPM + NAA 1.0mg/L + IBA 0.5mg/L + PP3332.0 mg/L + activated carbon 1.0g/L according to polarity, culturing for 40 days at the temperature of 23-27 ℃ under the conditions of illumination intensity of 2000-3000 lux and single-day illumination time of 12h, wherein the rooting rate is more than 96.4%, the seedlings are 5.2 cm high, and the basal stems are 0.10 cm thick.
Claims (5)
1. A tissue culture and rapid propagation method of a caulis Fibraureae stem segment is characterized by comprising the following steps:
1) selection and treatment of explants: cutting 20cm rattan at the top end of the non-lignified rhubarb vine, removing leaves, cutting into small sections with single buds and the length of about 2-3 cm, rinsing with detergent water for 10-20 min, and showering for 6-12 hours;
2) and (3) disinfection of explants: disinfecting the cleaned explant in a super clean bench by using alcohol, bleaching powder solution and mercuric chloride solution in sequence;
3) and (3) induction culture: inoculating the sterilized stem segments into an induction culture medium which takes WPM as a basic culture medium and is added with NAA, TDZ and active carbon, and culturing under the environment of certain temperature, illumination intensity and photoperiod;
4) and (3) proliferation culture: inoculating the induced cluster buds into a multiplication culture medium which takes WPM as a basic culture medium and is added with NAA, 6-BA, KT, GA3 and active carbon, and culturing under the environment of certain temperature, illumination intensity and photoperiod;
5) strong seedling and rooting culture: inducing or proliferating to obtain cluster buds, inoculating to rooting culture medium containing WPM as basic culture medium and NAA, IBA, paclobutrazol and activated carbon, and culturing at certain temperature, illumination intensity and photoperiod.
2. The tissue culture and rapid propagation method of the stem segments of the fibraurea recisa pierre as claimed in claim 1, wherein the optimal disinfection method of the explant in step 2) is 75% alcohol disinfection for 20-30 s, saturated bleaching powder disinfection for 20-30 min, and 0.05% mercuric chloride disinfection for 10-12 min.
3. The tissue culture and rapid propagation method of the stem segments of the rhubarb vine according to claim 1, characterized in that the induction culture medium in the step 3) is WPM + NAA 0.2-0.5 mg/L + TDZ 1.0-2.0 mg/L + activated carbon 0.5-1.0 g/L, the culture environment is 23-27 ℃, dark culture is carried out in the first 20 days, and then culture is carried out under the conditions that the illumination intensity is 1000-2000 lux and the single-day illumination time is 10 h.
4. The tissue culture and rapid propagation method of the stem segments of the rhubarb vine according to claim 1, characterized in that the propagation medium in the step 4) is WPM + NAA 0.2-0.5 mg/L +6-BA 0.2-0.5 mg/L + KT 2.0-4.0 mg/L + GA30.5-1.0 mg/L of activated carbon, wherein the culture environment is 23-27 ℃, dark culture is carried out for the first 5 days, and then culture is carried out under the conditions that the illumination intensity is 1000-2000 lux, and the single-day illumination time is 12 hours.
5. The tissue culture and rapid propagation method of the stem segments of the rhubarb vine according to claim 1, characterized in that the strong seedling rooting culture medium in the step 5) is WPM + NAA 1.0-2.0 mg/L + IBA 0.5-1.0 mg/L + PP 3331.0-2.0 mg/L + activated carbon, the culture environment is 23-27 ℃, and the culture is carried out under the conditions of the illumination intensity of 2000-3000 lux and the single-day illumination time of 12 h.
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