CN111034615B - Tissue culture method for rapid propagation of alum root "tiramisu - Google Patents
Tissue culture method for rapid propagation of alum root "tiramisu Download PDFInfo
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Abstract
The invention relates to a tissue culture method for the rapid propagation of alum root "tiramisu", and relates to the technical field of plant tissue culture. The invention takes vitriol root petioles as explants, relates to the steps of sterile system establishment, callus induction, callus formation adventitious bud induction, adventitious bud proliferation, rooting induction, hardening seedling transplantation and the like, and establishes a set of complete vitriol root tiramisu tissue culture rapid propagation method.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a tissue culture method for the rapid propagation of alum root "tiramisu".
Background
The alum root "tiramisu" (Heuchera micrantha), a plant of the genus alum of the family Saxifragaceae, a perennial root evergreen herb, is cold resistant, can tolerate low temperature of about-20 ℃, is fond of a half-shady environment, is not resistant to direct irradiation of strong light, has strong adaptability to various soils, and is preferably fertile and loose weakly acidic soil. The leaves of the variety are in a broad-heart shape, the color of the leaves is light green in spring and summer and is provided with unobvious white stripes, the color of the leaves sequentially turns red from inside to outside along the veins in autumn and winter until the whole leaves become wine red, the appearance of the plants changes in rich hue along with seasons, the ornamental value is high, and the leaves are deeply loved by gardening enthusiasts. In recent years, a large number of greening projects in northeast China begin to use alum roots as good shade-resistant color-leaf perennial root herbaceous plants, the alum roots are commonly used in scenes such as flower bed color blocks, natural flower beds and shade ground covers, and the like, and the market prospect is wide.
The conventional alum root propagation mode mainly adopts a plant division propagation mode and a leaf cutting propagation mode. The plant division breeding is that after the pot is filled with alum root, the pot is changed by dividing the alum root, after the plant division, the obtained alum root small plants are arranged and respectively planted in the pot. The leaf cutting propagation is that the leaves taken from the alum root mother plant are inserted on the perlite to ensure the wettability of the perlite, and the leaves are placed in a shade place for maintenance, the temperature is controlled between 10 ℃ and 30 ℃, and the roots can be rooted in about 1 month. Meanwhile, there is a report in the literature that a stem segment with a bud is used as an explant, and tissue culture is performed to propagate the shoot by a direct seedling path of the bud.
The alum root plant obtained by the conventional alum root propagation method has low propagation efficiency and long period, and is not beneficial to mass propagation of seedlings. The stem segment with the bud is used as an explant for propagating plants, the explant has high pollution rate, the stem segment with the bud is continuously taken, great damage is easily caused to a mother plant, and the multiplication rate of the adventitious bud obtained by the propagation method is low. Therefore, the aforementioned method of propagating alum roots has problems of slow propagation speed, low proliferation rate and high contamination rate.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a tissue culture method for the rapid propagation of alum root "tiramisu", which has the advantages of high propagation speed, high propagation rate and low pollution rate, can rapidly produce high-quality seedlings in the production process, and has important practical application value for the commercial development of alum root.
The above object of the present invention is achieved by the following technical solutions: a tissue culture method for the quick reproduction of alum root "tiramisu" includes such steps as creating aseptic system, inducing callus to become adventitious bud, proliferating adventitious bud, inducing root, hardening seedling and transplanting,
(1) establishing an aseptic system: selecting a stock plant which grows strongly and has no plant diseases and insect pests, cutting a petiole of a new leaf from the stock plant, and sterilizing the petiole to obtain a sterilized explant;
(2) inducing callus: inoculating the explant obtained in the step (1) to a culture medium for inducing callus, and culturing to obtain callus integrated with the explant;
(3) inducing callus to form adventitious buds: cutting the callus obtained in the step (2) from the explant, transferring the cut callus to a culture medium for inducing differentiation to form adventitious buds, and culturing to obtain the adventitious buds integrated with the callus;
(4) adventitious bud proliferation: cutting the healthy adventitious bud obtained in the step (3) from the callus, transferring the cut healthy adventitious bud to a culture medium for adventitious bud proliferation, and culturing to obtain a proliferated plantlet;
(5) inducing and rooting: transferring the plantlets of 3-4cm obtained in the step (4) to a rooting induction culture medium to obtain rooted plantlets;
(6) hardening and transplanting seedlings: hardening the seedlings obtained in the step (5) and transplanting the seedlings to a planting substrate.
By adopting the technical scheme, the stock plant to be taken is disinfected before the material is taken, so that the material used as the explant can be prevented from carrying pathogens on the stock plant, and the pollution rate of plant tissue culture is reduced from the root source; the explant taken from the stock plant is induced to form callus, plant cells have totipotency, so the plant cells of the explant are induced to differentiate to form the callus under the combined action of a culture medium, cytokinin and auxin added into the culture medium, the formed callus cells are free from the constraint of original organ tissues, and the totipotency of the cells can be expressed under the proper culture condition; on the basis of the callus formed by induction, under the combined action of a culture medium, cytokinin and auxin, the callus is induced to differentiate to form adventitious buds, and the callus cells of the plant have high totipotency and can differentiate to form the adventitious buds; the adventitious bud formed by the differentiation of the induced callus is cut off from the callus, the adventitious bud is transferred to an adventitious bud culture medium, the adventitious bud inoculated into the adventitious bud culture medium has higher totipotency, and the adventitious bud proliferates and differentiates to form cluster buds under the combined action of cytokinin and auxin; and transferring the cluster buds obtained in the last step into a rooting culture medium, further culturing the cluster buds into rooted plantlets, transplanting the rooted plantlets to a planting substrate, and further growing the plantlets. Compared with the existing culture method, the tissue culture method for the alum root 'tiramisu' rapid propagation has the advantages of high propagation speed, high propagation rate and low pollution rate, and can rapidly produce high-quality seedlings in the production process.
Further, the petiole of the newly grown leaf in the step (1) means a petiole of a leaf grown on the mother plant for 30 to 40 days.
According to the technical scheme, the petioles in the growth period of 30-40 days are used as materials, the leaf opening degree of the leaf in the growth period is good and the petioles are thick, compared with the petioles in the growth period of 30 days, the petioles in the growth period of 30-40 days have stronger tolerance to a disinfectant, and the survival rate of the disinfected petioles is greatly improved; in addition, the plant cells in the growth period have vigorous metabolic activity and strong division capability, the concentration of the hormone in the body is higher, the explant can be stimulated to dedifferentiate by only using the hormone with lower concentration, and spherical embryogenic callus with higher quality is induced, while old petioles in the growth period, such as petioles of two-year or multi-year growth, have higher fibrosis degree due to weaker cell metabolic activity and higher concentration of the hormone used for inducing the callus, and the problems of vitrification and the like of the callus and adventitious buds are easily caused in the later period, wherein the vitrification refers to a specific physiological disorder or pathological change in the tissue culture process, so that the test-tube plantlet is in a semitransparent appearance and abnormal shape. Therefore, compared with the adoption of the old petiole with the growth period within 30 days or longer, the induction rate of the explant and the quality of the formed callus are obviously improved by adopting the petiole with the growth period of 30-40 days as a material for induction.
Further, in the step (1), the first 7-10 days of cutting the petiole of the newly grown leaf from the stock plant, and a sterilizing agent is sprayed to the stock plant.
By adopting the technical scheme, the sterilizing agent is sprayed to the mother plant in advance 7-10 days before the material is obtained, so that the interference of a pollution source can be effectively reduced; compared with the method that the stem section with the terminal bud and the axillary bud is used as the explant, the method that the petiole is used as the explant eliminates the interference of a pollution source hidden in the stem section, and meanwhile, the sterilizing agent can fully sterilize the explant, so that the pollution rate is obviously reduced.
Furthermore, the sterilizing agent is carbendazim, and the dosage of the sterilizing agent is 1000 times of the volume of the thalli to be sterilized.
By adopting the technical scheme, the carbendazim is a widely-used bactericide, is also a metabolite of benomyl, is a high-efficiency low-toxicity systemic bactericide, has systemic treatment and protection effects, is sprayed to the mother plant in advance before material drawing, can effectively reduce the interference of a pollution source, has better systemic treatment and protection effects on the mother plant and leaves to be drawn, and obviously reduces the pollution rate of explants.
Further, the step (1) of sterilizing the petioles comprises sterilizing the petioles by using 75% alcohol and then sterilizing the petioles by using a disinfectant.
By adopting the technical scheme, the mode of disinfecting by alcohol is to lightly wipe the petioles by alcohol cotton, so that a large amount of surface skin hair can be cleaned, and dust and mixed bacteria attached to the surface can be reduced; compared with the method that the leaf stalks are taken as explants and the disinfection treatment is not carried out, the method that the leaf stalks are disinfected in advance can effectively eliminate the interference of the epidermal hair with a pollution source, and further reduce the pollution rate of the explants.
Further, the disinfectant is prepared by soaking the petiole to be treated in 0.1% mercuric chloride solution for 6-8 min.
By adopting the technical scheme, the mercuric chloride, namely the mercuric chloride can be used as a disinfectant, has a disinfection effect, can kill propagules, lipophilic viruses and the like, is a high-efficiency disinfectant when the explants for plant tissue culture are disinfected, has a strong killing effect on pathogens and the like carried by the explants, and further reduces the pollution rate of the explants.
Further, the formula of the culture medium for inducing the callus in the step (2) is as follows: MS culture medium, 6-benzylamino adenine (6-BA)0.1-0.2mg/l,2, 4-dichlorophenoxyacetic acid (2,4-D)0.8-1.0mg/l, sucrose 25-30g/l, agar 4-4.5g/l, pH value at 5.8-6.0; the culture temperature is 23-27 ℃, the illumination intensity is 1500-.
By adopting the technical scheme, the 6-BA is a common cytokinin, has the main function of promoting the formation of buds and can also induce the differentiation of callus; 2,4-D is a common auxin for inducing callus, the induction effect of the auxin on the callus is better than that of other auxins, and the lack of 2,4-D is the reason for direct budding. In the stage of callus induction, 6-BA with the concentration of 0.1-0.2mg/l is added to mainly play a role in callus induction differentiation, and meanwhile, under the 6-BA with low concentration, the budding of the explant can be inhibited; meanwhile, 2,4-D is added into the MS culture medium, so that the formation of callus can be effectively induced, and 0.8-1.0mg/l of 2,4-D has an inhibiting effect on the budding of the explant. Therefore, the concentration of the 6-BA in the step of 0.1-0.2mg/l and the concentration of the 2,4-D in the step of 0.8-1.0 are more than that of the 6-BA, so that the addition of the 6-BA and the 2,4-D in the concentrations can play a better role in inducing the callus.
Further, the formula of the culture medium for inducing differentiation to form adventitious buds in the step (3) is as follows: MS culture medium, 6-BA 1.5-2.0mg/l,2, 4-D0.1-0.2 mg/l, sucrose 25-30g/l, agar 4-4.5g/l, pH value at 5.8-6.0; the culture temperature is 23-27 ℃, the illumination intensity is 1500-.
By adopting the technical scheme, on the basis of obtaining the callus through induction, the callus is used as the basis to induce the differentiation adventitious bud, at the moment, the 6-BA used as the cytokinin and the 2,4-D used as the auxin are added to induce the differentiation adventitious bud of the callus, and because the callus used as the basis is not the explant but the callus formed by the explant differentiation, the 6-BA of 1.5-2.0mg/l and the 2,4-D of 0.1-0.2mg/l are added, and the concentration of the 6-BA added is relatively far greater than that of the 2,4-D added, so that the 6-BA and the 2,4-D are matched to better promote the adventitious bud formed by the callus differentiation.
Further, the formula of the culture medium for adventitious bud proliferation in the step (4) is as follows: MS culture medium, 6-BA 0.05-0.1mg/l, naphthylacetic acid (NAA)0.05-0.1mg/l, sucrose 25-30g/l, agar 4-4.5g/l, pH value at 5.8-6.0; the culture temperature is 23-27 ℃, the illumination intensity is 1500-.
By adopting the technical scheme, the NAA is a broad-spectrum plant growth regulator and can promote cell division and expansion, the adventitious bud obtained through differentiation is cut from callus and is placed in an adventitious bud multiplication culture medium, the adventitious bud is multiplied on the basis of the adventitious bud formed through differentiation, the low-concentration 6-BA with cell division number is added to promote division and multiplication of plant cells, and the NAA is added as the plant growth regulator and has the effect of promoting cell multiplication and expansion, so that the 6-BA with 0.05-0.1mg/l and the NAA with 0.05-0.1mg/l are added into the culture medium for adventitious bud multiplication to promote multiplication of the adventitious bud.
Further, the formula of the culture medium for inducing rooting in the step (5) is as follows: 1/2MS culture medium, sucrose 25-30g/l, agar 4-4.5g/l, pH 5.8-6.0; the culture temperature is 23-27 ℃, the illumination intensity is 1500-.
By adopting the technical scheme, the proliferated adventitious buds are transplanted into a culture medium for inducing rooting, and the proliferated adventitious buds are promoted to grow roots and form seedlings under the combined action of hormones accumulated in each culture stage and various self-synthesized hormones.
Compared with the existing tissue culture method, the alum root 'tiramisu' tissue culture rapid propagation method provided by the invention has the following advantages:
the culture method has the advantages of high propagation speed, high propagation rate and low pollution rate, can quickly produce high-quality seedlings in the production process, and has important practical application value for the commercial development of alum roots.
1. The contamination rate of the explants is obviously reduced: the mother plant is cultured in a greenhouse and is sprayed with the carbendazim in advance before material drawing, so that the interference of indoor and outdoor pollution sources can be effectively reduced, the petioles are used as explants, compared with the method that stem sections with terminal buds and axillary buds are used as explants, the interference of the pollution sources hidden in the stem sections is eliminated, the explants can be fully disinfected by a disinfectant, and the pollution rate is obviously reduced; the petiole is lightly wiped by alcohol cotton, so that a large amount of epidermal hair can be cleaned, dust and infectious microbes attached to the petiole can be reduced, and compared with a method that the petiole is taken as an explant and is not subjected to pre-disinfection treatment, the method can effectively eliminate the interference of an epidermal hair attached with a pollution source and further reduce the disinfection pollution rate of the explant;
2. the survival rate and the induction rate of the explants are high: the petioles in the growth period of 30-40 days are used as materials, the leaf opening degree is good, the petioles are thick, the tolerance to a disinfectant is higher than that of the petioles in the growth period of 30 days, and the survival rate of young and tender petioles after disinfection is greatly improved; the plant cells in the growth period have vigorous metabolic activity and strong division capability, the concentration of hormones in the body is higher, the explants can be excited to dedifferentiate by using the hormones with lower concentration, spherical embryogenic callus with higher quality is induced, the metabolic activity of biennial (perennial) old petiole cells is weaker, the fibrosis degree of the petioles is higher, the hormones with higher concentration are often needed to induce the callus, and the problems that the callus and adventitious buds are easy to vitrify and the like are easily caused in the later period;
3. short induction bud time and high multiplication rate: in the process of inducing the callus by the method, dark culture is not needed, the two ends of the petioles can obviously expand in about 7 days under the illumination condition, green and healthy embryonic callus can be induced and formed in about 15-25 days, a large number of healthy adventitious buds can be induced in about 30-40 days after the callus is transferred to the induction bud-separating culture medium, the overall induction rate can be up to 20 times and more, the adventitious buds have good growth vigor, grow robustly and have no problems of vitrification, variation and the like, the adventitious buds can be induced in 45 days at the fastest speed, the induction bud-emergence time is greatly shortened, a large number of tissue culture materials can be provided in a short time, and the rapid production requirement is met; after cutting buds of the callus for inducing germination, the culture medium for inducing germination is adopted for at least two times of culture, and the germination can be continuously induced, and the germination quality is not obviously reduced.
Drawings
FIG. 1 is a schematic diagram of callus formation induced by explants according to the invention.
FIG. 2 is a schematic diagram showing the dedifferentiation of callus to form adventitious buds in the present invention.
FIG. 3 is a schematic view showing the formation of a large number of adventitious buds from an adventitious bud mass in the present invention.
FIG. 4 is a schematic view showing the formation of a seedling by the proliferation of an adventitious bud in the present invention.
FIG. 5 is a schematic diagram showing the rooting of the plantlets in the present invention.
Detailed Description
The carbendazim used in the invention is Guoguang carbendazim which is purchased from Sichuan Guoguang agro-chemical company Limited; the alcohol is 500ml of 75% disinfection solution of Lierkang, and is purchased from Shandong Lierkang medical science and technology GmbH; the mercuric chloride is a mercuric chloride standard solution purchased from the institute of optometry and fine chemistry in Tianjin. The alum root "tiramisu" parent strain used in the invention is selected from Beijing Huaxiang flower science and technology research institute, Inc.
Referring to fig. 1-5, the invention provides a tissue culture method for the rapid propagation of alum root "tiramisu", which mainly relates to the steps of sterile system establishment, callus induction, callus formation adventitious bud induction, adventitious bud proliferation, rooting induction, hardening seedling transplantation and the like, and comprises the following specific steps,
1. establishing an aseptic system:
1) selecting a strong parent plant without diseases and insect pests, placing the parent plant in a greenhouse for culturing, and spraying 1000 times of 800-fold carbendazim 7-10 days before the material is taken;
2) selecting and cutting 30-40 days of petioles of the new leaves when the materials are taken, washing with running water for 10 minutes, and airing;
3) wiping the petiole with 75% alcohol-containing cotton, wiping off epidermal hair on the petiole, cleaning the petiole with sterile water for 1-2 times;
4) sterilizing the material in 0.1% mercuric chloride solution for 6-8min, washing with sterile water for 5-8 times, and shaking for 3-5min each time;
5) cutting petiole into 1-1.5cm segments to obtain sterilized explant.
2. Inducing callus:
the petiole disinfected in the step (1) is spread and inoculated on a culture medium for inducing callus, and the formula of the culture medium is as follows: MS minimal medium, 6-BA 0.1-0.2mg/l,2, 4-D0.8-1.0 mg/l, sucrose 25-30g/l, agar 4-4.5g/l, pH value 5.8-6.0; the culture temperature of the induced callus is 23-27 ℃, the illumination intensity is 1500-1800lux, the illumination time is 9-10h/d, and the callus integrated with the explant is obtained after culture.
3. Inducing callus to form adventitious buds:
cutting the callus obtained in the step (2) from an explant, and transferring the cut callus to a culture medium for inducing differentiation to form adventitious buds, wherein the formula of the culture medium is as follows: MS minimal medium, 6-BA 1.5-2.0mg/l,2, 4-D0.1-0.2 mg/l, sucrose 25-30g/l, agar 4-4.5g/l, pH value 5.8-6.0; the culture temperature for inducing differentiation to form adventitious buds is 23-27 ℃, the illumination intensity is 1500-1800lux, the illumination time is 9-10h/d, and the adventitious buds integrated with the callus tissues are obtained after culture.
4. Adventitious bud proliferation:
cutting the healthy adventitious buds obtained in the step (3) from the callus, and transferring the healthy adventitious buds to a culture medium for adventitious bud proliferation, wherein the formula of the culture medium is as follows: MS minimal medium, 6-BA 0.05-0.1mg/l, NAA 0.05-0.1mg/l, sucrose 25-30g/l, agar 4-4.5g/l, pH value 5.8-6.0; the culture temperature for adventitious bud multiplication is 23-27 ℃, the illumination intensity is 1500-1800lux, and the illumination duration is 9-10h/d, so as to obtain the multiplied plantlet.
5. Inducing and rooting:
transferring the plantlets with the plant height of 3-4cm obtained in the step (4) to a rooting induction culture medium, wherein the culture medium comprises the following components in parts by weight: 1/2MS minimal medium, sucrose 25-30g/l, agar 4.0g/l, pH 5.8-6.0; the culture temperature for inducing rooting is 23-27 ℃, the illumination intensity is 1500-1800lux, the illumination duration is 9-10h/d, and the rooted plantlet is obtained.
6. Hardening and transplanting seedlings:
selecting small seedlings with plant height of 4-5cm, at least 4 roots and root length of more than 2cm obtained in the step (5), and opening a bottle cap to harden the seedlings for 2 days; taking out and cleaning the plantlets, planting the plantlets in a 105-hole plug tray, wherein the matrix is peat soil: perlite is 4:1, water is poured and 1000 times of carbendazim is sprayed, direct sunlight is avoided, and good ventilation, heat preservation and moisture preservation are realized.
The cultivation method of the alum root "tiramisu" provided by the invention has the advantages of high propagation speed, high multiplication rate and low pollution rate, and can quickly produce high-quality seedlings in the production process.
The present invention will be described in further detail with reference to examples 1 to 3 and comparative examples 1 to 4, and the results of the tests of the respective test items in examples 1 to 3 and comparative examples 1 to 4 are shown in Table 1.
Examples
Example 1
The culture method provided by the invention is used for rapid propagation, and the specific steps are as follows:
(1) establishing an aseptic system: selecting a strong parent plant without diseases and insect pests, placing the parent plant in a greenhouse for culturing, and spraying 1000 times of carbendazim 7 days before material selection; selecting and cutting the petiole of a new leaf growing for 30 days when the material is taken, washing for 10 minutes with running water, and airing; wiping the petiole with 75% alcohol-containing cotton, wiping off epidermal hair on the petiole, and cleaning with sterile water for 2 times without folding the petiole; sterilizing the material in 0.1% mercuric chloride solution for 6min, washing with sterile water for 5 times, shaking for 3min each time, and cutting petiole into 1cm segments to obtain sterilized explant.
(2) Inducing callus: inoculating, flatly paving and inoculating the explants sterilized in the step (1) to a culture medium for inducing callus, wherein the culture temperature is 25 ℃, the illumination intensity is 1500lux, the illumination time is 9h/d, and the formula of the culture medium is as follows: MS basic culture medium, 6-BA 0.1mg/l, 2, 4-D1.0 mg/l, sucrose 30g/l, agar 4.5g/l, pH value 5.8, and after culturing for 7 days, counting the callus induction rate.
(3) Inducing callus to form adventitious buds: cutting the callus obtained in the step (2) from an explant, transferring the callus to a culture medium for inducing differentiation to form adventitious buds, wherein the culture temperature is 25 ℃, the illumination intensity is 1500lux, the illumination time is 9h/d, and the formula of the culture medium is as follows: MS minimal medium, 6-BA 1.5mg/l, 2, 4-D0.1 mg/l, sucrose 30g/l, agar 4.5g/l, pH value 5.8, forming adventitious buds after culturing for 30 days, counting the number of each adventitious bud and calculating the adventitious bud induction rate.
(4) And (3) adventitious bud multiplication culture: cutting the healthy adventitious bud group obtained in the step (3) from the callus, transferring the bud group to a culture medium for propagation culture, wherein the culture temperature is 25 ℃, the illumination intensity is 1500lux, the illumination time is 9h/d, and the formula of the culture medium is as follows: MS minimal medium, 6-BA 0.05mg/l, NAA 0.1mg/l, sucrose 30g/l, agar 4.5g/l, pH value 5.8, after 25 days of culture, proliferation multiple and proliferation rate of each bud are calculated.
(5) Inducing and rooting: transferring the plantlets with the plant height of 3-4cm obtained in the step (4) to a rooting induction culture medium, wherein the culture temperature is 25 ℃, the illumination intensity is 1500lux, and the illumination time is 9h/d, and the culture medium comprises the following components in parts by weight: 1/2MS minimal medium, 30g/l sucrose, 4.0g/l agar, pH 5.8, after 20 days of culture, counting the rooting number of each seedling, and calculating the induced rooting rate.
(6) Hardening and transplanting seedlings: selecting small seedlings with plant height of 4-5cm, at least 4 roots and root length of more than 2cm obtained in the step (5), and opening a bottle cap to harden the seedlings for 2 days; taking out and cleaning the plantlets, planting the plantlets in a 105-hole plug tray, wherein the matrix is peat soil: and (3) pouring water and spraying 800 times of carbendazim on the perlite which is 4:1, paying attention to ventilation, heat preservation and moisture preservation and preventing direct sunlight, and calculating the survival rate after planting for 15 days.
Example 2
The present embodiment is different from embodiment 1 in many ways, and the specific steps of the cultivation method of alum root "tiramisu" in the present embodiment are as follows:
(1) establishing an aseptic system: selecting a strong parent plant without diseases and insect pests, placing the parent plant in a greenhouse for culturing, and spraying 800 times of carbendazim 7 days before material selection; selecting and cutting 35 days of newborn petioles when the materials are taken, washing with running water for 10 minutes, and airing; wiping the petiole with 75% alcohol-containing cotton, wiping off epidermal hair on the petiole, and cleaning with sterile water for 2 times without folding the petiole; sterilizing the material in 0.1% mercuric chloride solution for 7min, washing with sterile water for 5 times, shaking for 3min each time, and cutting petiole into 1.5cm segments to obtain sterilized explant.
(2) Inducing callus: inoculating, flatly paving and inoculating the explants sterilized in the step (1) to a culture medium for inducing callus, wherein the culture temperature is 25 ℃, the illumination intensity is 1500lux, the illumination time is 9h/d, and the formula of the culture medium is as follows: MS basic culture medium, 6-BA 0.1mg/l, 2, 4-D0.8 mg/l, sucrose 30g/l, agar 4.5g/l, pH value 5.8, and after culturing for 7 days, counting the callus induction rate.
(3) Inducing callus to form adventitious buds: cutting the callus obtained in the step (2) from an explant, transferring the callus to a culture medium for inducing differentiation to form adventitious buds, wherein the culture temperature is 25 ℃, the illumination intensity is 1500lux, the illumination time is 9h/d, and the formula of the culture medium is as follows: MS minimal medium, 6-BA 1.5mg/l, 2, 4-D0.2 mg/l, sucrose 30g/l, agar 4.5g/l, pH value 5.8, forming adventitious buds after culturing for 30 days, counting the number of each adventitious bud and calculating the adventitious bud induction rate.
(4) And (3) adventitious bud multiplication culture: cutting the healthy adventitious bud group obtained in the step (3) from the callus, transferring the bud group to a culture medium for propagation culture, wherein the culture temperature is 25 ℃, the illumination intensity is 1500lux, the illumination time is 9h/d, and the formula of the culture medium is as follows: MS minimal medium, 6-BA 0.05mg/l, NAA 0.05mg/l, sucrose 30g/l, agar 4.5g/l, pH value 5.8, after 25 days of culture, proliferation multiple and proliferation rate of each bud are calculated.
(5) Inducing and rooting: transferring the plantlets with the plant height of 3-4cm obtained in the step (4) to a rooting induction culture medium, wherein the culture temperature is 25 ℃, the illumination intensity is 1500-: 1/2MS minimal medium, 30g/l sucrose, 4.0g/l agar, pH 5.8, after 20 days of culture, counting the rooting number of each seedling, and calculating the induced rooting rate.
(6) Hardening and transplanting seedlings: selecting small seedlings with plant height of 4-5cm, at least 4 roots and root length of more than 2cm obtained in the step (5), and opening a bottle cap to harden the seedlings for 2 days; taking out and cleaning the plantlets, planting the plantlets in a 105-hole plug tray, wherein the matrix is peat soil: and (3) pouring water and spraying 800 times of carbendazim on the perlite which is 4:1, paying attention to ventilation, heat preservation and moisture preservation and preventing direct sunlight, and calculating the survival rate after planting for 15 days.
Example 3
The present embodiment is different from both embodiment 1 and embodiment 2 in many ways, and the specific steps of the cultivation method of alum root "tiramisu" in the present embodiment are as follows:
(1) establishing an aseptic system: selecting a strong parent plant without diseases and insect pests, placing the parent plant in a greenhouse for culturing, and spraying 1000 times of carbendazim 10 days before the material is taken; selecting and cutting 40 days of newborn petioles when the materials are taken, washing with running water for 10 minutes, and airing; wiping the petiole with 75% alcohol-containing cotton, wiping off epidermal hair on the petiole, and cleaning with sterile water for 2 times without folding the petiole; sterilizing the material in 0.1% mercuric chloride solution for 8min, washing with sterile water for 6 times, shaking for 4min each time, and cutting petiole into 1cm segments to obtain sterilized explant.
(2) Inducing callus: inoculating, flatly paving and inoculating the explants sterilized in the step (1) to a culture medium for inducing callus, wherein the culture temperature is 27 ℃, the illumination intensity is 1500lux, the illumination time is 10h/d, and the formula of the culture medium is as follows: MS basic culture medium, 6-BA 0.2mg/l,2, 4-D1.0 mg/l, sucrose 30g/l, agar 4.5g/l, pH 6.0, and after culturing for 7 days, counting the callus induction rate.
(3) Inducing callus to form adventitious buds: cutting the callus obtained in the step (2) from an explant, transferring the callus to a culture medium for inducing differentiation to form adventitious buds, wherein the culture temperature is 27 ℃, the illumination intensity is 1500lux, the illumination time is 10h/d, and the formula of the culture medium is as follows: MS minimal medium, 6-BA 2.0mg/l,2, 4-D0.1 mg/l, sucrose 30g/l, agar 4.5g/l, pH value 6.0, forming adventitious buds after culturing for 30 days, counting the number of each adventitious bud and calculating the adventitious bud induction rate.
(4) And (3) adventitious bud multiplication culture: cutting the healthy adventitious bud group obtained in the step (3) from the callus, transferring the bud group to a culture medium for propagation culture, wherein the culture temperature is 25 ℃, the illumination intensity is 1500lux, and the illumination time is 10h/d, and the formula of the culture medium is as follows: MS minimal medium, 6-BA 0.1mg/l, NAA 0.05mg/l, sucrose 30g/l, agar 4.5g/l, pH value 6.0, after 25 days of culture, proliferation multiple and proliferation rate of each bud are calculated.
(5) Inducing and rooting: transferring the plantlets with the plant height of 3-4cm obtained in the step (4) to a rooting induction culture medium, wherein the culture temperature is 25 ℃, the illumination intensity is 1500lux, and the illumination time is 10h/d, and the culture medium comprises the following components in parts by weight: 1/2MS minimal medium, 30g/l sucrose, 4.0g/l agar, pH 6.0, after 20 days of culture, counting the rooting number of each seedling, and calculating the induced rooting rate.
(6) Hardening and transplanting seedlings: selecting small seedlings with plant height of 4-5cm, at least 4 roots and root length of more than 2cm obtained in the step (5), and opening a bottle cap to harden the seedlings for 2 days; taking out and cleaning the plantlets, planting the plantlets in a 105-hole plug tray, wherein the matrix is peat soil: and (3) pouring water and spraying 800 times of carbendazim on the perlite which is 4:1, paying attention to ventilation, heat preservation and moisture preservation and preventing direct sunlight, and calculating the survival rate after planting for 15 days.
Comparative example
Comparative example 1
Comparative example 1 is essentially the same as example 1 in the main steps, with some details differing, mainly in that:
(1) the explant material selected in comparative example 1 was alum leaf.
(2) The disinfection mode of the explant is simplified in the establishment process of a sterile system, and during material taking, a new leaf which is cut for 30 days is selected, washed by running water for 10 minutes and dried; sterilizing the material in 0.1% mercuric chloride solution for 6min, washing with sterile water for 5 times, shaking for 3min each time, and cutting into 1cm pieces2To obtain sterilized explants.
(3) Culturing for 20 days in the process of inducing to form the callus, and counting the callus induction rate after 20 days.
(4) Culturing for 60 days when adventitious buds are formed by callus induction, forming adventitious bud masses after 60 days, counting the number of each adventitious bud mass, and calculating the adventitious bud induction rate.
Comparative example 2
Comparative example 2 is essentially the same as example 2 in the main steps, with some details different, mainly in that:
(1) the explant material selected in comparative example 2 was alum leaf.
(2) The disinfection mode of the explant is simplified in the establishment process of a sterile system, and when materials are taken, fresh leaves which are cut for 35 days are selected, washed by running water for 10 minutes and dried; sterilizing the material in 0.1% mercuric chloride solution for 7min, washing with sterile water for 5 times, shaking for 3min each time, and cutting into 1cm pieces2To obtain sterilized explants.
(3) Culturing for 20 days in the process of inducing to form the callus, and counting the callus induction rate after 20 days.
(4) Culturing for 60 days when adventitious buds are formed by callus induction, forming adventitious bud masses after 60 days, counting the number of each adventitious bud mass, and calculating the adventitious bud induction rate.
Comparative example 3
Comparative example 3 is essentially the same as example 1 in the main steps, with some details different, mainly in that:
(1) the disinfection mode of the explant is simplified in the establishment process of a sterile system, and when the material is taken, a new petiole which is cut for 30 days is selected, washed by running water for 10 minutes and dried; sterilizing the material in 0.1% mercuric chloride solution for 6min, washing with sterile water for 5 times, shaking for 3min each time, and cutting the leaf into 1cm segments to obtain sterilized explant.
(2) In the process of inducing callus, the formula of the culture medium is as follows: MS minimal medium, 6-BA 1.0mg/l, NAA 0.5mg/l, sucrose 30g/l, agar 4.5g/l, pH value 5.8, after culturing for 40 days, counting the callus induction rate.
(3) In the process of inducing the callus to form adventitious buds, the formula of the culture medium is as follows: MS minimal medium, 6-BA 1.0mg/l, 2, 4-D0.1 mg/l, sucrose 30g/l, agar 4.5g/l, pH value 5.8, forming adventitious buds after culturing for 60 days, counting the number of each adventitious bud, and calculating the adventitious bud induction rate.
Comparative example 4
Comparative example 4 is essentially the same as example 2 in the main steps, with some details differing, mainly in that:
(1) the disinfection mode of the explant is simplified in the establishment process of a sterile system, and when materials are taken, a new petiole which is cut for 35 days is selected, washed by running water for 10 minutes and dried; sterilizing the material in 0.1% mercuric chloride solution for 7min, washing with sterile water for 5 times, shaking for 3min each time, and cutting the leaf into 1cm segments to obtain sterilized explant.
(2) In the process of inducing callus, the formula of the culture medium is as follows: MS minimal medium, 6-BA 1.0mg/l, NAA 1.0mg/l, sucrose 30g/l, agar 4.5g/l, pH value 5.8, after culturing for 30 days, counting the callus induction rate.
(3) In the process of inducing the callus to form adventitious buds, the formula of the culture medium is as follows: MS basic culture medium, 6-BA 1.0mg/l, 2, 4-D0.1 mg/l, cane sugar 25-30g/l, agar 4-4.5g/l, pH value 5.8, forming adventitious bud mass after culturing for 25 days, counting the number of each adventitious bud mass and calculating the adventitious bud induction rate.
TABLE 1 test results of respective test items in examples 1 to 3 and comparative examples 1 to 4
The petioles of the regenerated leaves were used as explants in examples 1 to 3, while those of comparative examples 1 and 2 used the regenerated leaves as explants, and it was found from the above-mentioned results that the time for inducing callus formation and inducing callus formation of adventitious buds was significantly shorter when the petioles were used as explants than when the leaves were used as explants; in addition, the callus formed by taking the petiole as the explant is mostly spherical embryonic callus, the quality of the callus is higher than that of the callus formed by taking the leaf as the explant, the spherical embryonic callus is dedifferentiated to form a large amount of adventitious buds at the later stage, and the adventitious bud induction effect is obvious. Examples 1-3 in comparison with comparative examples 3-4, 6-BA and NAA were added to the medium during callus induction in comparative examples 3-4. According to the detection results, compared with the addition of 6-BA and NAA, the addition of 6-BA and 2,4-D in the process of inducing callus formation has the advantages that 2,4-D has more obvious effect on inducing callus formation, spherical embryogenic callus with higher quality can be induced and formed in a short time, a large amount of adventitious buds are induced, and the time for inducing adventitious buds is shorter. In addition, the cytokinin used in the primary culture process has a remarkable influence on the proliferation rate of the adventitious bud subculture, and the use of the cytokinin 2,4-D is beneficial to improving the proliferation rate of the adventitious bud subculture. As can be seen from the comparison between examples 1-3 and comparative examples 1-4, the various auxins and cytokinins added to the culture medium have little influence on "tiramisu" of alum root, and a good rooting effect can be achieved without adding hormones.
Through comparison between the embodiment and the comparative example, the tissue culture method for inducing the alum root "tiramisu" to rapidly propagate provided by the invention has the advantages that the propagation speed of the alum root "tiramisu" is accelerated, the proliferation rate is improved, the pollution rate is greatly reduced, and the tissue culture method has important practical application value for the commercial development of the alum root.
The embodiments of the present invention are preferred embodiments of the present invention, and the scope of the present invention is not limited by these embodiments, so: all equivalent changes made according to the structure, shape and principle of the invention are covered by the protection scope of the invention.
Claims (2)
1. A tissue culture method for the quick reproduction of alum root "tiramisu" is characterized by comprising the steps of establishing a sterile system, inducing callus, inducing the callus to form adventitious buds, proliferating the adventitious buds, inducing rooting and hardening off and transplanting, and comprises the following steps,
(1) establishing an aseptic system: selecting a stock plant which grows strongly and has no plant diseases and insect pests, cutting a petiole of a new leaf from the stock plant, and sterilizing the petiole to obtain a sterilized explant;
(2) inducing callus: inoculating the explant obtained in the step (1) to a culture medium for inducing callus, and culturing to obtain callus integrated with the explant;
the formula of the culture medium for inducing the callus tissue comprises the following components: MS culture medium, 6-benzylamino adenine (6-BA)0.1-0.2mg/l,2, 4-dichlorophenoxyacetic acid (2,4-D)0.8-1.0mg/l, sucrose 25-30g/l, agar 4-4.5g/l, pH value at 5.8-6.0; the culture temperature is 23-27 ℃, the illumination intensity is 1500-;
(3) inducing callus to form adventitious buds: cutting the callus obtained in the step (2) from the explant, transferring the cut callus to a culture medium for inducing differentiation to form adventitious buds, and culturing to obtain the adventitious buds integrated with the callus;
the formula of the culture medium for inducing differentiation to form adventitious buds is as follows: MS culture medium, 6-BA 1.5-2.0mg/l,2, 4-D0.1-0.2 mg/l, sucrose 25-30g/l, agar 4-4.5g/l, pH value at 5.8-6.0; the culture temperature is 23-27 ℃, the illumination intensity is 1500-;
(4) adventitious bud proliferation: cutting the healthy adventitious bud obtained in the step (3) from the callus, transferring the cut healthy adventitious bud to a culture medium for adventitious bud proliferation, and culturing to obtain a proliferated plantlet;
the formula of the culture medium for adventitious bud proliferation is as follows: MS culture medium, 6-BA 0.05-0.1mg/l, naphthylacetic acid (NAA)0.05-0.1mg/l, sucrose 25-30g/l, agar 4-4.5g/l, pH value at 5.8-6.0; the culture temperature is 23-27 ℃, the illumination intensity is 1500-;
(5) inducing and rooting: transferring the plantlets of 3-4cm obtained in the step (4) to a rooting induction culture medium to obtain rooted plantlets;
the formula of the culture medium for inducing rooting is as follows: 1/2MS culture medium, sucrose 25-30g/l, agar 4-4.5g/l, pH 5.8-6.0; the culture temperature is 23-27 ℃, the illumination intensity is 1500-;
(6) hardening and transplanting seedlings: hardening the seedlings obtained in the step (5) and transplanting the seedlings to a planting substrate.
2. The tissue culture method for alum root "tiramisu" rapid propagation according to claim 1, wherein the mother plant is sprayed with 800-1000 times of carbendazim 7-10 days before the material is obtained in step (1); selecting and cutting 30-40 days of petiole of new leaf when taking material; the disinfection of the petiole is to disinfect the petiole by 75% alcohol and then soak the petiole in 0.1% mercuric chloride solution for 6-8 min.
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