CN110896858B - Tissue culture method of andrographis paniculata - Google Patents
Tissue culture method of andrographis paniculata Download PDFInfo
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Abstract
The invention discloses a tissue culture method of andrographis paniculata, which comprises the following steps: s1 explant treatment: collecting complete axillary buds of Andrographis paniculata Nees as explants, and sterilizing for later use; s2 callus culture: putting axillary buds into callus culture medium, and performing dark cultureCulturing, and then culturing by light; s3 induced bud culture: culturing in light for 7-10 days; 4, propagation culture: transferring the obtained plumule into subculture multiplication culture medium for continuous light culture; s5 rooting culture: transferring to rooting culture medium, and culturing for 3-5 days; s6 seedling hardening: placing the rooted seedlings in a nutrient solution, and performing light culture for 15-20 days; wherein: the rooting medium comprises the following components in parts by weight: 1/3 MS: +0.1-0.12mg/L NAA, +0.08-0.12mg/L BR1+0.06-0.08mg/L penicillin, +20g/L sucrose and +6.5g/L agar, pH 5.8-6.0. The culture method of the invention has the advantages of high germination rate, rooting rate and survival rate of the tissue culture of the andrographis paniculata plant.
Description
Technical Field
The invention relates to the technical field of Chinese herbal medicine culture, in particular to a tissue culture method of andrographis paniculata.
Background
Andrographis paniculata (Burm.f.) Nees) is an annual herb plant, belongs to medicinal plants, and has effects of clearing away heat and toxic materials, relieving inflammation, and relieving swelling and pain. Andrographis paniculata Nees prefer high temperature and humid climate, and prefer sunlight and fertilizer. The proper temperature of the seed germination and seedling growth period is 25-30 ℃, the growth is slow when the air temperature is reduced to 15-20 ℃, the air temperature is reduced to about 8 ℃, and the growth is stopped; when the plants are withered at low temperature of about 0 ℃ or frost. The cultivation is preferably carried out by using fertile, loose and well-drained acidic and neutral sandy loam soil, and the alkaline soil with the pH value of 8.0 can still grow normally. The traditional common andrographis herb grows for 5-6 months, the seedling culture time is long, and the growth period is long.
The traditional breeding method of the common andrographis herb is to sow and breed seeds, and the seeds are hard in skin, so that the germination time is long and the seedlings emerge irregularly. The plant tissue culture technology is used for propagation, so that the defects of the traditional propagation can be overcome. Currently, in plant tissue culture, an MS culture medium is often used as a basic culture medium and is added with corresponding plant hormones to perform seed germination induction, callus culture, bud induction culture, rooting culture and the like, so that higher germination rate, rooting rate and survival rate are obtained.
As for the selection of phytohormones, 6-benzyladenine (6-BA), naphthylacetic acid (1-naphthylacetic acid, NAA), indolebutyric acid (IBA), Kinetin (KT), 2,4-Dichlorophenoxyacetic acid (2,4-Dichlorophenoxyacetic acid, 2,4-D) are currently the most used in tissue culture of Andrographis paniculata. However, when the phytohormone is used for the plant tissue culture of the andrographis paniculata, the final induced germination rate is 60-85%, and the survival rate after transplanting is only 45-86%, which is relatively low.
Therefore, it is necessary to develop cultivation methods with high germination rate, rooting rate, survival rate and short period, and the method is also an urgent need of the market.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a tissue culture method of andrographis paniculata, which has the advantages of high germination rate, rooting rate and survival rate of andrographis paniculata plant tissue culture.
In order to achieve the purpose, the invention provides the following technical scheme: a tissue culture method of herba Andrographitis comprises the following steps:
s1 explant treatment: collecting sterile complete axillary buds of Andrographis paniculata Nees as explants, washing with sterile water after disinfection and sterilization, and naturally air-drying for later use;
s2 callus culture: under the aseptic environment, putting axillary buds into a callus culture medium for culture, firstly culturing in a dark place, and then culturing in a light place to obtain a callus;
s3 induced bud culture: culturing in callus culture medium at 23-25 deg.C under 3200-;
s4 proliferation culture: transferring the buds obtained in the bud induction culture in the step S3 into a subculture multiplication culture medium for continuous culture, wherein the culture environment is as follows: the temperature is 23-25 ℃, the illumination intensity is 3200-;
s5 rooting culture: transferring to a rooting culture medium for rooting culture, wherein the culture conditions are as follows: culturing at 21-25 deg.C under 4500-;
s6 seedling hardening: placing the rooted seedlings together with the paper ring on planting cotton, placing the planting cotton in a seedling tray, simultaneously placing 5-10mm of nutrient solution in the seedling tray, periodically supplementing water, performing dark culture at 25 ℃ for 3-5 days, and performing light culture at 22-25 ℃ and light intensity of 8000-15000lux for 15-20 days;
wherein: the rooting medium comprises the following components in parts by weight: 1/3 MS: +0.1-0.12mg/L NAA, +0.08-0.12mg/L BR1+0.06-0.08mg/L penicillin, +20g/L of sucrose and +6.5g/L of agar, and the pH value is 5.8-6.0.
By adopting the technical scheme, the condition parameters of the processes of explant treatment, callus culture, bud induction culture, proliferation culture, rooting culture and the like in the process of the andrographis paniculata plant tissue culture are gradually optimized, the adopted culture mode is gradually confirmed, the formula of the corresponding culture medium is optimized and confirmed, and the rooting of the andrographis paniculata is promoted jointly by using the penicillin and the auxin in a rooting culture medium in a matching way, so that the later-stage higher survival rate can be obtained.
Further, the formula of the callus culture medium is as follows: MS: +0.12-0.16mg/L BR5+1.8-2.0mg/L IBA, +0.08-0.10mg/L penicillin, +16g/L sucrose, +6g/L agar, pH 5.8-6.0.
By adopting the technical scheme, IBA and BR are added into the culture medium of the callus5And penicillin is added into the culture medium in a certain dosage, and then the three are matched with the external conditions of callus culture, so that the forming rate of the callus is higher; meanwhile, the callus culture medium is matched with a bud inducing condition, so that the germination rate of the plant is higher, and the later-stage proliferation and rooting process is further facilitated.
Further, the formula of the subculture multiplication medium is as follows: MS: +0.12-0.16mg/L BR5+0.1-0.14mg/L NAA, +0.08-0.10mg/L penicillin, +16g/L sucrose and +6g/L agar, pH 5.8-6.0.
By adopting the technical scheme, NAA and BR are added into the subculture multiplication medium5And penicillin is added into the culture medium in a certain dosage, and the three are matched with the external conditions of subculture proliferation, so that the performance of the proliferated plant is stable, the rooting process in the later period is facilitated, and the final survival rate of the andrographis paniculata is higher.
Further, the formula of the nutrient solution during seedling hardening is as follows: calcium nitrate 200-250mg/L, potassium nitrate 250-300mg/L, potassium fulvate 200-250mg/L, magnesium sulfate 160-200mg/L, zinc sulfate 30-60mg/L, manganese sulfate 20-30mg/L, potassium dihydrogen phosphate 80-90mg/L, and copper sulfate 1-3mg/L, NaFeEDTA 3-5 mg/L.
By adopting the technical scheme, the growth of the andrographis paniculata seedlings is facilitated, and the survival rate of the andrographis paniculata seedlings is higher.
Further, the specific operation of step S2 is: under sterile environment, putting axillary buds into a callus culture medium, culturing at 25 +/-1 ℃ for 3-5 days in a dark environment, and then culturing at 22-25 ℃ under the light intensity of 800 plus 1200lux for 7-10 days in a light environment.
By adopting the technical scheme, the callus differentiation rate of the callus of the andrographis paniculata is higher in the process of performing callus culture on the bud embryo of the andrographis paniculata by adopting the combined dark culture and light culture mode for culture.
Further, the humidity in the culture environment is ensured to be 65-85% in the processes of bud induction culture and propagation culture.
Through adopting above-mentioned technical scheme, avoid comparatively moist at the root of cultivateing the in-process plant, and then guarantee the better growth of plant.
Further, the step of sterilizing in step S1 is: the collected explants are sterilized by 75% alcohol for 30s, then soaked in 30ppm hypochlorous acid for 8min, and then washed with sterile water for 3 times.
By adopting the technical scheme, the explant is effectively prevented from being polluted by bacteria, so that the normal operation of later steps is ensured, and the callus culture, proliferation culture, rooting culture and the like in the later period are not influenced.
In conclusion, the invention has the following beneficial effects:
firstly, because the invention adopts the plant tissue culture mode to culture the andrographis paniculata seedlings, optimizes the components of the rooting culture medium in the culture process, and adds penicillin and plant growth regulator, the obtained plants have high rooting rate, and the higher rooting rate ensures that the andrographis paniculata has more efficient absorption and material exchange to nutrient substances in the culture medium, in the planted cotton and in the transplanted soil, thereby promoting the final plant seedlings to have high survival rate after transplantation.
Secondly, penicillin is preferably added into a callus culture medium and a subculture multiplication culture medium, the dosage between penicillin and plant growth regulator is optimized and confirmed, so that the callus culture medium is suitable for forming callus, and the subculture multiplication culture medium is beneficial to plant multiplication.
Detailed Description
The present invention will be described in further detail with reference to examples.
Wherein NAA (1-naphthylacetic acid) is naphthylacetic acid, IBA is indolebutyric acid, BR1Is Brassinolide (BL), BR5Is 6-deoxybrassinosteroid (6-deoxyocastasterone), BR8Is Theasterone (TE).
Examples
Example 1
A tissue culture method of herba Andrographitis comprises the following steps:
s1 explant treatment: collecting sterile complete axillary buds of herba Andrographitis as explant, sterilizing with 75% alcohol for 15s, soaking with 30ppm hypochlorous acid for 5min, washing with sterile water for 2 times, and naturally air drying;
s2 callus culture: under the aseptic environment, putting axillary buds into a callus culture medium for culturing, performing dark culture at the temperature of 25 +/-1 ℃ for 3 days, and performing light culture at the temperature of 22 ℃ and the light intensity of 800lux for 7 days to obtain callus;
s3 induced bud culture: culturing in callus culture medium at 23 deg.C under 3200lux illumination intensity for 7 days in sterile environment while ensuring indoor humidity to be 65%;
s4 proliferation culture: transferring the buds obtained in the bud induction culture in the step S3 into a subculture multiplication culture medium for continuous culture, wherein the culture environment is as follows: the temperature is 23 ℃, the illumination intensity is 3200lux, the illumination time is 10 h/day, the culture time is 10 days, the indoor humidity is ensured to be 65% in the period, and the process is finished when the color of the leaves of the rootless seedlings begins to change;
s5 rooting culture: transferring to a rooting culture medium for rooting culture, wherein the culture conditions are as follows: the temperature is 23 +/-2 ℃, and the illumination intensity is 4500 lux; culturing for 3 days until 98-100% of the seedling root length is more than 2 mm;
s6 seedling hardening: placing the rooted seedlings together with the paper rings on planting cotton, placing the planting cotton in a seedling tray, simultaneously placing 5mm of nutrient solution in the seedling tray, periodically supplementing water, performing dark culture at 25 +/-1 ℃ for 3-5 days, and performing light culture at 22 ℃ and 8000lux of light intensity for 15 days;
wherein:
the formula of the callus culture medium is as follows: MS: +0.12mg/L BR5+1.8mg/L IBA, +0.08mg/L penicillin, +16g/L sucrose, +6g/L agar, pH 5.8-6.0. Mixing BR according to the above formula5Adding IBA, penicillin and sucrose into deionized water respectively, adding agar, stirring, sterilizing, and cooling.
The formula of the subculture multiplication medium is as follows: MS: +0.12mg/L BR5+0.1mg/L NAA, +0.08mg/L penicillin, +16g/L sucrose and +6g/L agar, pH 5.8-6.0. Mixing BR according to the above formula5Adding NAA, penicillin and sucrose into deionized water, adding agar, stirring, sterilizing, and cooling.
The rooting medium comprises the following components: 1/3 MS: +0.1mg/L NAA, +0.08mg/L BR1+0.06mg/L penicillin, +20g/L sucrose and +6.5g/L agar, pH 5.8-6.0. Mixing BR according to the above formula1Adding NAA, penicillin and sucrose into deionized water, adding agar, stirring, sterilizing, and cooling.
The formula of the nutrient solution during seedling exercising is as follows: 200mg/L of calcium nitrate, 250mg/L of potassium nitrate, 200mg/L of potassium fulvate, 160mg/L of magnesium sulfate, 30mg/L of zinc sulfate, 20mg/L of manganese sulfate, 80mg/L of monopotassium phosphate and 1mg/L, NaFeEDTA 3mg/L of copper sulfate.
The above reagents are all commercially available, wherein the potassium fulvate is purchased from kimberl, and the model is 01; the CAS number for penicillin is 61-33-6, and is purchased from Shanghai Nuotai chemical Co., Ltd; MS culture medium is purchased from Beijing Kulyeba science and technology Limited and has the model of PM1013-1 KG; 1/3MS culture medium is purchased from Ku Laibobu technology of Beijing, and has model number of PM 1181-500G; NAA (Naphthylacetic acid) has CAS number of 86-87-3, all purchased from Saibei science and technology Limited, Yuancheng Saisha, Hubei; CAS number of IBA (indolebutyric acid) 133-32-4, BR1The CAS number of (brassinolide) is 72962-43-7, all from Shanghai leaf Biotech Co., Ltd; CAS number for 24-epioleolactone (Epibarassinolide) 78821-43-9, purchased from Zhongkoita; BR (BR)5CAS number of (6-deoxybrassinosteroid) is 690627-49-7; the CAS number of high typasterol (28-Homoteasterone) is 90524-90-6; BR (BR)8CAS number for Theasterone (TE) is 92751-21-8.
Example 2
A tissue culture method of herba Andrographitis comprises the following steps:
s1 explant treatment: collecting sterile complete axillary buds of herba Andrographitis as explant, sterilizing with 75% alcohol for 45s, soaking with 30ppm hypochlorous acid for 10min, washing with sterile water for 3 times, and naturally air drying;
s2 callus culture: under the aseptic environment, putting axillary buds into a callus culture medium for culturing, carrying out dark culture at the temperature of 25 +/-1 ℃ for 5 days, and then carrying out light culture at the temperature of 25 ℃ and under the light intensity of 1200lux for 10 days to obtain callus;
s3 induced bud culture: culturing in callus culture medium at 25 deg.C under 3400lux for 10 days in sterile environment while ensuring the humidity in the culture environment to be 85%;
s4 proliferation culture: transferring the buds obtained in the bud induction culture in the step S3 into a subculture multiplication culture medium for continuous culture, wherein the culture environment is as follows: the temperature is 25 ℃, the illumination intensity is 3500lux, the illumination time is 12 h/day, the culture time is 15 days, the humidity of the external environment is ensured to be 85% in the period, and the process is finished when the color of the leaves without the root seedlings begins to change;
s5 rooting culture: transferring to a rooting culture medium for rooting culture, wherein the culture conditions are as follows: the temperature is 23 +/-2 ℃, and the illumination intensity is 6000 lux; culturing for 5 days until 98-100% of the seedling root length is more than 2 mm;
s6 seedling hardening: placing the rooted seedlings together with the paper ring on planting cotton, placing the planting cotton in a seedling tray, simultaneously placing 10mm of nutrient solution in the seedling tray, periodically supplementing water, performing dark culture at 25 +/-1 ℃ for 5 days, and performing light culture at 25 ℃ and 15000lux light intensity for 20 days;
wherein:
the formula of the callus culture medium is as follows: MS: +0.16mg/L BR5, +2.0mg/L IBA, +0.10mg/L penicillin, +16g/L sucrose, +6g/L agar, pH 5.8-6.0.
The formula of the subculture multiplication medium is as follows: MS: +0.16mg/L BR5, +0.14mg/L NAA, +0.10mg/L penicillin, +16g/L sucrose and +6g/L agar, pH 5.8-6.0.
The rooting medium comprises the following components: 1/3 MS: +0.12mg/L NAA, +0.12mg/L BR1+0.08mg/L penicillin, +20g/L sucrose and +6.5g/L agar, pH 5.8-6.0.
The formula of the nutrient solution during seedling exercising is as follows: 250mg/L of calcium nitrate, 300mg/L of potassium nitrate, 250mg/L of potassium fulvate, 200mg/L of magnesium sulfate, 60mg/L of zinc sulfate, 30mg/L of manganese sulfate, 90mg/L of monopotassium phosphate and 3mg/L, NaFeEDTA 5mg/L of copper sulfate.
Example 3
A tissue culture method of herba Andrographitis comprises the following steps:
s1 explant treatment: collecting sterile complete axillary buds of herba Andrographitis as explant, sterilizing with 75% alcohol for 30s, soaking with 30ppm hypochlorous acid for 8min, washing with sterile water for 3 times, and naturally air drying;
s2 callus culture: under the aseptic environment, putting axillary buds into a callus culture medium for culturing, performing dark culture at the temperature of 25 +/-1 ℃ for 4 days, and performing light culture at the temperature of 23 ℃ and the light intensity of 1000lux for 9 days to obtain callus;
s3 induced bud culture: culturing in callus culture medium at 24 deg.C under 3300lux illumination intensity for 9 days under sterile environment to ensure humidity of external environment to be 75%, generating more axillary buds and growing young and tender leaf;
s4 proliferation culture: transferring the buds obtained in the bud induction culture in the step S3 into a subculture multiplication culture medium for continuous culture, wherein the culture environment is as follows: the temperature is 24 ℃, the illumination intensity is 3300lux, the illumination time is 11 h/day, the culture time is 12 days, the humidity of the external environment is ensured to be 75% in the period, and the process is finished when the color of the leaves without the root seedlings begins to change;
s5 rooting culture: transferring to a rooting culture medium for rooting culture, wherein the culture conditions are as follows: the temperature is 23 +/-2 ℃, and the illumination intensity is 5200 lux; culturing for 4 days until 98-100% of the seedling root length is more than 2 mm;
s6 seedling hardening: placing the rooted seedlings together with the paper ring on planting cotton, placing the planting cotton in a seedling tray, simultaneously placing 8mm of nutrient solution in the seedling tray, periodically supplementing water, performing dark culture at 25 +/-1 ℃ for 3-5 days, and performing light culture at 24 ℃ under 12000lux light intensity for 18 days;
wherein:
the formula of the callus culture medium is as follows: MS: +0.14mg/L BR5+1.9mg/L IBA, +0.09mg/L penicillin, +16g/L sucrose, +6g/L agar, pH 5.8-6.0.
The formula of the subculture multiplication medium is as follows: MS: +0.14mg/L BR5+0.12mg/L NAA, +0.09mg/L penicillin, +16g/L sucrose and +6g/L agar, pH 5.8-6.0.
The rooting medium comprises the following components: 1/3 MS: +0.11mg/L NAA, +0.10mg/L BR1+0.07mg/L penicillin, +20g/L sucrose and +6.5g/L agar, pH 5.8-6.0.
The formula of the nutrient solution during seedling exercising is as follows: 230mg/L of calcium nitrate, 230mg/L of potassium fulvate, 180mg/L of magnesium sulfate, 45mg/L of zinc sulfate, 25mg/L of manganese sulfate, 85mg/L of monopotassium phosphate and 2mg/L, NaFeEDTA 4mg/L of copper sulfate.
Examples 4 to 6
Examples 4-6 differ from example 3 in the light culture time for callus culture, and are otherwise the same as example 3, as detailed in Table 1.
Examples 7 to 8
Examples 7 to 8 differ from example 5 in the light intensity of callus culture, and are otherwise the same as example 5, as shown in Table 1.
Examples 9 to 12
Examples 9 to 12 are different from example 5 in the light intensity or the cultivation time of the bud-inducing cultivation, and are otherwise the same as example 5, specifically shown in Table 1.
Examples 13 to 14
Examples 13 to 14 differ from example 11 in the culture time for rooting culture, and are otherwise the same as example 11, as shown in Table 1.
Examples 15 to 19
Examples 15 to 19 are different from example 11 in the culture time or light intensity of the seedling culture, and are otherwise the same as example 11, as shown in Table 1.
Examples 20 to 23
Examples 20-23 differ from example 16 in the penicillin or BR content of the callus culture medium5The amount of the compound (D) was varied, and the same as in example 16, specifically shown in Table 1.
Examples 24 to 27
Examples 24 to 27 differ from example 18 in the penicillin or BR of the subculture multiplication medium5The amount of the compound (D) was varied, and the same as in example 16, specifically shown in Table 1.
Examples 28 to 31
Examples 28-31 differ from example 16 in the penicillin or BR of the rooting medium1The amount of the compound (D) was varied, and the same as in example 16, specifically shown in Table 1.
TABLE 1 table of different implementation conditions for different examples
Comparative example
Comparative example 1
Comparative example 1 differs from example 16 in that:
the culture process of the callus tissue comprises the following steps: axillary buds were cultured in a callus culture medium under aseptic conditions, and cultured at 23 ℃ under light of 1000lux for 13 days to obtain callus, as in example 18.
Comparative examples 2 to 5
Comparative examples 2 to 5 differ from example 16 in the light culture time or intensity of callus culture, and are otherwise the same as in example 18, see Table 2.
Comparative examples 6 to 9
Comparative examples 6 to 9 are different from example 16 in the cultivation time or light intensity of the induced-shoot culture, and are otherwise the same as example 18, as shown in Table 2.
Comparative examples 10 to 11
Comparative examples 10 to 11 differ from example 16 in the culture time for rooting culture, and are otherwise the same as example 18, as shown in Table 2.
Comparative examples 12 to 15
Comparative examples 12 to 15 differ from example 16 in the culture time or light intensity of the acclimatization culture, and the others are the same as example 18, as shown in Table 2.
Comparative example 16
Comparative example 16 differs from example 16 in that penicillin was replaced with an equal amount of streptomycin in the callus culture medium, and the other example 18.
Comparative example 17
Comparative example 17 differs from example 16 in that penicillin was not added to the callus culture medium, and the other example is the same as example 18.
Comparative example 18
Comparative example 18 differs from example 16 in that 1.9mg/L of IBA was replaced with 0.11mg/L of NAA in the callus medium, otherwise the same as example 18.
Comparative example 19
Comparative example 19 differs from example 16 in that no BR was added to the callus culture medium5Otherwise, the same procedure as in example 18 was repeated.
Comparative example 20
Comparative example 20 differs from example 16 in the amount of BR in the callus culture medium1Alternative BR5Otherwise, the same procedure as in example 18 was repeated.
Comparative example 21
Comparative example 21 differs from example 16 in that penicillin was replaced with an equal amount of streptomycin in the subculture multiplication medium, and the other example is the same as example 18.
Comparative example 22
Comparative example 22 is different from example 16 in that penicillin was not added to the subculture multiplication medium, and the other example is the same as example 18.
Comparative example 23
Comparative example 23 differs from example 16 in that no BR was added to the subculture multiplication medium5Otherwise, the same procedure as in example 18 was repeated.
Comparative example 24
Comparative example 24 differs from example 16 in that the same amount of BR was used in the subculture multiplication medium1Alternative BR5Otherwise, the same procedure as in example 18 was repeated.
Comparative example 25
Comparative example 25 differs from example 16 in that an equivalent amount of 24-epioleolactone was substituted for BR in the subculture multiplication medium5Otherwise, the same procedure as in example 18 was repeated.
Comparative example 26
Comparative example 26 differs from example 16 in that an equal amount of high typosterol was substituted for BR in the subculture multiplication medium5Otherwise, the same procedure as in example 18 was repeated.
Comparative example 27
Comparative example 27 differs from example 16 in that the same amount of BR was used in the subculture multiplication medium8Alternative BR5Otherwise, the same procedure as in example 18 was repeated.
Comparative example 28
Comparative example 28 differs from example 16 in that the penicillin was replaced with an equal amount of streptomycin in rooting medium, otherwise the same as example 18.
Comparative example 29
Comparative example 29 differs from example 16 in that penicillin was not added to the rooting medium, and it is otherwise the same as example 18.
Comparative example 30
Comparative example 30 differs from example 16 in that no BR was added to the rooting medium1Otherwise, the same procedure as in example 18 was repeated.
Comparative example 31
Comparative example 31 differs from example 16 in that equal amounts of BR were used in the rooting medium5Alternative BR1Otherwise, the same procedure as in example 18 was repeated.
Comparative example 32
Comparative example 32 differs from example 16 in that an equal amount of 24-epioleolactone was substituted for BR in the rooting medium1Otherwise, the same procedure as in example 18 was repeated.
Comparative example 33
Comparative example 33 differs from example 16 in that an equal amount of high typosterol was substituted for BR in the rooting medium1Otherwise, the same procedure as in example 18 was repeated.
Comparative example 34
Comparative example 34 differs from example 16 in that equal amounts of BR are used in the rooting medium8Alternative BR1Otherwise, the same procedure as in example 18 was repeated.
TABLE 2 table of different conditions for different comparative examples
Performance test
(I) callus formation Rate
The callus formation rates of examples 1 to 8, examples 20 to 23, comparative examples 1 to 5 and comparative examples 16 to 20 were measured and calculated, and the results are shown in Table 3.
The callus formation rate (%) — the number of axillary buds forming the callus/the number of initial axillary buds × 100%.
TABLE 3 statistical results of callus formation rates of various examples and comparative examples
By comparing the results of examples 1 to 8 and comparative examples 2 to 5 in Table 3, it is shown that the external conditions for callus culture of Andrographis paniculata Nees are: the light culture time and the light intensity have certain influence on the formation of the callus, and the optimal culture condition of the callus is that the light culture time is 8 days and the light intensity is 1000 lux. By comparing comparative example 1 and example 5, it is demonstrated that the culture of callus of Andrographis paniculata Nees favors the formation of callus of Andrographis paniculata Nees according to a culture mode of first dark culture followed by light culture.
As a result of comparing examples 20 to 23 and comparative examples 16 to 20, the addition of penicillin to the callus culture medium was advantageous for the tissue culture of Andrographis paniculata Nees, but the addition of penicillin to the callus culture medium was carried out by adding other substances: the streptomycin replaces or does not add penicillin, the formation rate of the callus of the andrographis paniculata is reduced, and the formula of the callus culture medium is more important for the callus culture of the andrographis paniculata; in all the embodiments carried out above, when +0.14mg/L BR is added to the MS medium5The best callus formation rate of Andrographis paniculata was found with +1.9mg/L IBA and +0.09mg/L penicillin.
(II) induced germination percentage
The induced germination rates of examples 5, 9-12, 20-23, 1-9 and 16-20 were measured and calculated, and the specific results are shown in Table 4.
The induced germination rate (%) was the number of axillary buds germinated/the number of axillary buds forming callus × 100%.
TABLE 4 statistical results of induced germination percentage for various examples and comparative examples
By comparing the results of example 5, examples 9 to 12 and comparative examples 6 to 9 in Table 3, it was revealed that the induced bud culture of Andrographis paniculata Nees was affected by the light culturing time and the light intensity, and the preferable culturing conditions for the induced bud culture were 9 days and 3300lux light intensity.
Comparing the results of examples 20 to 23 and comparative examples 16 to 20, the addition of penicillin to the germination-inducing medium facilitated the formation of axillary buds and the growth of young leaves of callus in the tissue culture of andrographis paniculata plants, but with other substances: callus of Andrographis paniculata (Burm. f.) Nees with or without penicillinThe rate is reduced, and the formula of the callus culture medium is more important for the induction bud culture of the andrographis paniculata; in all the embodiments carried out above, when +0.14mg/L BR is added to the MS medium5The germination rate of Andrographis paniculata is better when the plant contains IBA +1.9mg/L and penicillin +0.09 mg/L.
(III) inducing rooting rate
The induced rooting rates of the samples of example 11, examples 13-14, examples 24-31, comparative examples 10-11 and comparative examples 21-34 were measured and calculated, and the specific results are shown in Table 5.
The induced rooting rate (%) is the number of rooted seedlings/the number of initial non-rooted seedlings multiplied by 100%.
TABLE 5 statistical results of induced rooting rates for various examples and comparative examples
For the rooting of the andrographis paniculata non-rooted seedlings, the rooting rate of each non-rooted seedling in the example is 89-97%, while the rooting rate of each non-rooted seedling in the comparative example is 70-81%, which is far lower than that of each non-rooted seedling in the example.
(IV) survival rate after transplantation
The survival rate of the transplanted seedlings of the plants of example 11, examples 15 to 19 and comparative examples 12 to 15 was statistically calculated, and the specific results are shown in Table 6.
Survival (%) — number of transplanted seedlings/number of surviving seedlings × 100%.
TABLE 6 statistical results of induced rooting rates for various examples and comparative examples
The survival rate of each transplanted andrographis paniculata seedling in the example is 90-96%, and the survival rate of each transplanted andrographis paniculata seedling in the comparative example is 79-87%, which is lower than the rooting rate of each rootless seedling in the example.
The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.
Claims (5)
1. A tissue culture method of andrographis paniculata, which is characterized by comprising the following steps:
s1 explant treatment: collecting sterile complete axillary buds of Andrographis paniculata Nees as explants, washing with sterile water after disinfection and sterilization, and naturally air-drying for later use;
s2 callus culture: under the aseptic environment, putting axillary buds into a callus culture medium for culture, firstly culturing in a dark place, and then culturing in a light place to obtain a callus;
s3 induced bud culture: culturing in callus culture medium at 23-25 deg.C under 3200-;
s4 proliferation culture: transferring the buds obtained in the bud induction culture in the step S3 into a subculture multiplication culture medium for continuous culture, wherein the culture environment is as follows: the temperature is 23-25 ℃, the illumination intensity is 3200-;
s5 rooting culture: transferring to a rooting culture medium for rooting culture, wherein the culture conditions are as follows: culturing at 21-25 deg.C under 4500-;
s6 seedling hardening: placing the rooted seedlings together with the paper ring on planting cotton, placing the planting cotton in a seedling tray, simultaneously placing 5-10mm of nutrient solution in the seedling tray, periodically supplementing water, performing dark culture at 25 ℃ for 3-5 days, and performing light culture at 22-25 ℃ and light intensity of 8000-15000lux for 15-20 days;
wherein: the rooting medium comprises the following components in parts by weight: 1/3MS +0.1-0.12mg/L NAA +0.08-0.12mg/L BR1+0.06-0.08mg/L penicillin, 20g/L sucrose and 6.5g/L agar, pH 5.8-6.0;
of the callus culture mediumThe formula is as follows: MS +0.12-0.16mg/L BR5+1.8-2.0mg/L of IBA +0.08-0.10mg/L of penicillin +16g/L of sucrose +6g/L of agar, pH 5.8-6.0; the formula of the subculture multiplication medium is as follows: MS +0.12-0.16mg/L BR5+0.1-0.14mg/L NAA +0.08-0.10mg/L penicillin +16g/L sucrose +6g/L agar, pH 5.8-6.0.
2. The tissue culture method of andrographis paniculata according to claim 1, wherein the formula of the nutrient solution during hardening-seedling is: calcium nitrate 200-250mg/L, potassium nitrate 250-300mg/L, potassium fulvate 200-250mg/L, magnesium sulfate 160-200mg/L, zinc sulfate 30-60mg/L, manganese sulfate 20-30mg/L, potassium dihydrogen phosphate 80-90mg/L, and copper sulfate 1-3mg/L, NaFeEDTA 3-5 mg/L.
3. The method of claim 1, wherein the step S2 comprises the following steps: under sterile environment, putting axillary buds into a callus culture medium, culturing at 25 +/-1 ℃ for 3-5 days in a dark environment, and then culturing at 22-25 ℃ under the light intensity of 800 plus 1200lux for 7-10 days in a light environment.
4. The method of claim 1, wherein the humidity in the culture environment is maintained at 65-85% during the bud induction culture and the proliferation culture.
5. The method of claim 1, wherein the step of sterilizing S1 comprises: the collected explants are sterilized by 75% alcohol for 30s, then soaked in 30ppm hypochlorous acid for 8min, and then washed with sterile water for 3 times.
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