CN114946657B - Hispid fig tissue culture method - Google Patents
Hispid fig tissue culture method Download PDFInfo
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- CN114946657B CN114946657B CN202210590683.5A CN202210590683A CN114946657B CN 114946657 B CN114946657 B CN 114946657B CN 202210590683 A CN202210590683 A CN 202210590683A CN 114946657 B CN114946657 B CN 114946657B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G17/00—Cultivation of hops, vines, fruit trees, or like trees
- A01G17/005—Cultivation methods
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/40—Afforestation or reforestation
Abstract
The invention discloses a hispid fig tissue culture method, which comprises the following steps: selecting the top bud of the hispid fig as an explant, cleaning and disinfecting the explant, washing the explant, and sucking surface water; inoculating the explant into a cluster bud induction culture medium, transferring the induced cluster buds into the cluster bud induction culture medium, and continuously proliferating and growing the cluster buds to obtain a large number of cluster buds and plantlets for subculture proliferation and rooting culture in a proliferation culture medium; and transferring the robust plantlets to a rooting culture medium to induce rooting so as to obtain robust plants. According to the invention, by adjusting the composition of the culture medium at each culture stage, the inductivity, the multiplication coefficient, the rooting rate and the transplanting survival rate of the hispid fig tissue culture are improved, the pollution rate and the browning rate are reduced, and the rapid propagation efficiency of the hispid fig is improved. The method for culturing the hispid fig tissue is simple and easy to operate, has high emergence rate and robust plants, shortens the seedling culture time and reduces the seedling culture cost.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a hispid fig tissue culture method.
Background
Ficus hirta Vahl (Ficus hirta Vahl), also known as Ficus palmata, ficus benjamina, guangdong, also known as Wuzhualong, wuzhuaniu milk, tuhuangqi, nanqi, etc. The plant shape, shrub or arbour, the whole stem, pericarp and leaves contain emulsion, and the root and bark have fragrance. The hispid fig is distributed in the south and the southwest of China, is a common herb in Lingnan, is used as a medicine by using the root of ficus microcarpa which belongs to the ficus genus of Moraceae, has the advantages of mild nature, sweet and pungent taste, has the functions of strengthening spleen, tonifying lung, promoting diuresis and relaxing tendons, and is used for treating symptoms such as spleen deficiency and edema, anorexia and weakness, phthisis cough, night sweat, rheumatic arthralgia, anorexia and abdominal distension, edema, postpartum agalactia and the like. In addition, the hispid fig is a plant with homology of food and medicine and is used for cooking soup among people in Guangdong region. In recent years, the hispid fig, a precious plant resource, draws high attention of pharmaceutical workers, researches on chemical components, pharmacological activity and other aspects are deepened, and the psoralen is proved to be one of the main active components of the hispid fig and has the effects of resisting bacteria, viruses, blood coagulation, inhibiting tumors, regulating immunity and the like. Has the functions of invigorating spleen, nourishing lung, promoting qi circulation and eliminating dampness.
In recent years, with the development of south medicine industry, hispid fig is abused and dug, wild resources are exhausted, the natural reproduction speed is slow, and the application and popularization of economic interplanting of hispid fig under forest is one of the development directions of modern south medicine industry. Traditional five-finger wild peach seedling breeds through the seed seeding, and the time of growing seedlings is long, is difficult to satisfy the seedling supply demand, and the demand of seedling appears the supply and demand trend moreover. The rapid and stable propagation of the hispid fig is realized through a tissue culture way, and the ever-increasing market demand can be met.
Disclosure of Invention
Based on the above, the present invention aims to provide a hispid fig tissue culture method.
The method for culturing the hispid fig tissue comprises the following steps:
selecting the top bud of the hispid fig as an explant, cleaning and disinfecting the explant, washing the explant, and sucking surface water;
inoculating the explant into a cluster bud induction culture medium, transferring the induced cluster buds into the cluster bud induction culture medium, and continuously proliferating and growing the cluster buds to obtain a large number of cluster buds and plantlets for subculture proliferation and rooting culture in a proliferation culture medium;
and transferring the robust plantlets to a rooting culture medium to induce rooting, so as to obtain robust plants.
In one embodiment, the washing step of washing the explant after washing and sterilizing, and the blotting of surface moisture comprises: cleaning explant with clear water, placing into sterilized culture container, placing on super clean bench, sterilizing with 75% ethanol surface for 1-2min, and adding 0.1% (w/v) HgCl 2 Soaking for 8-12min, washing with sterile water for 3-5 times, and drying with sterile filter paper.
In one embodiment, the culture medium for inducing the multiple shoots is MS +6-BA2.0mg/L + sucrose 30g/L.
In one embodiment, the cluster buds continuously proliferate and grow, and the average propagation coefficient is 5-8.
In one embodiment of the above technical scheme, the proliferation medium is MS +6-BA1.5mg/L + NAA0.1mg/L + sucrose 30g/L.
In one embodiment of the above technical means, the multiplication factor is 6 times or more by repeating subculture in a multiplication medium.
In one implementation mode of the technical scheme, the rooting culture medium is 1/2MS + NAA0.5mg/L + IBA0.1mg/L + sucrose 40g/L + alginic acid 0.5mg/L +0.25% active carbon +0.1% common rhodamine foliar fertilizer.
In one embodiment of the technical scheme, robust seedlings growing to 3-4cm are transferred to a rooting culture medium to induce rooting, and after 20-30 days, the seedlings grow into plants with the plant height of 6-8cm, 2-3 canopy leaves and 2-3 roots, the root systems are strong and the branches are multiple, so that robust plants are obtained.
In one embodiment, the culture conditions of the method for culturing the hispid fig tissue are as follows: the temperature is 27 +/-1 ℃, the light source is a fluorescent lamp, the illumination intensity is 2000-3000lx, and the illumination time is 12 h.d < -1 >.
In one embodiment of the above technical solution, the method for culturing hispid fig tissue further comprises: culturing the robust plant with the plant height of 6-8cm continuously, obtaining the bottle seedling of the hispid fig when the total culture time on a rooting culture medium is 30-50d, moving the bottle seedling into a greenhouse, hardening the seedling under natural light, then opening a cover to harden the seedling in a shady and ventilated environment, spraying water at proper time, then moving the rooting seedling out of the bottle, cleaning the root culture medium, soaking and sterilizing the root, cleaning with clear water, transplanting the root into a substrate which is sterilized in advance, watering and shading properly, managing conventionally, and then culturing and transplanting the tissue culture seedling.
In one implementation mode of the technical scheme, the seedlings are trained for 2-5 days under natural light, then the seedlings are placed in a cool and ventilated environment, the cover is opened and the seedlings are trained for 2-5 days, water is sprayed at proper time, then the rooted seedlings are moved out of a bottle, a root culture medium is cleaned, the roots are soaked and disinfected for 5-10min by using 0.1% carbendazim solution, the roots are transplanted into a substrate which is disinfected in advance after being cleaned by using clear water, watering and drenching are carried out, proper moisture preservation and shading are carried out, nutrient solution is sprayed once when the roots are transplanted into the substrate for 12-18 days, conventional management is carried out, and the seedlings are transplanted into the substrate after being transplanted for 25-35 days.
Compared with the prior art, the method has the advantages that the induction rate, the multiplication coefficient, the rooting rate and the transplanting survival rate of the hispid fig tissue culture are improved by adjusting the composition of the culture medium in each culture stage, the pollution rate and the browning rate are reduced, and the rapid propagation efficiency of the hispid fig is improved. The method for culturing the hispid fig tissue is simple and easy to operate, has high emergence rate and robust plants, shortens the seedling culture time and reduces the seedling culture cost.
For a better understanding and practice, the invention is described in detail below with reference to the accompanying drawings.
Drawings
FIG. 1 is a schematic representation of explant inoculation.
FIG. 2 is a schematic diagram of the acquisition of a proliferating shoot.
FIG. 3 is a schematic of the proliferation process.
FIG. 4 is a schematic diagram showing the obtaining of multiple shoots.
FIG. 5 is a schematic diagram of robust seedling body acquisition.
FIG. 6 is a schematic representation of induced rooting.
FIG. 7 is a schematic diagram of a transplanted seedling.
FIG. 8 is a schematic diagram comparing a transplanted seedling with a normal seedling.
Fig. 9 is a real beat diagram of the field after the transplantation of the hispid fig.
FIG. 10 is a diagram of a hispid fig fruit cultured by the present invention.
Detailed Description
The terms of orientation of up, down, left, right, front, back, top, bottom, and the like, referred to or may be referred to in this specification, are defined relative to their configuration, and are relative concepts. Therefore, it may be changed according to different positions and different use states. Therefore, these and other directional terms should not be construed as limiting terms.
The implementations described in the exemplary embodiments below are not intended to represent all implementations consistent with the present disclosure. Rather, they are merely examples of implementations consistent with certain aspects of the present disclosure.
The terminology used in the disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. As used in this disclosure, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.
The invention takes the top bud of the hispid fig as an explant to induce cluster buds, finally realizes plant regeneration and establishes a tissue culture technical system.
The invention relates to a hispid fig tissue culture method, which comprises the following steps:
Selecting a hispid fig (Ficus hirta Vahl) terminal bud as an explant, cleaning and disinfecting the explant, washing, and sucking off surface water.
Preferably, the washing, disinfecting, rinsing, and blotting surface moisture of the explant comprises: cleaning explant with clear water, placing into sterilized culture container, placing on super clean bench, sterilizing with 75% ethanol surface for 1-2min, and adding 0.1% (w/v) HgCl 2 Soaking for 8 times12min, preferably 10min, and finally rinsing with sterile water 3-5 times, and blotting the surface water with sterile filter paper. Thus, a high induction rate and a low contamination rate can be maintained.
Description of the culture conditions:
the basic culture medium is MS or 1/2MS culture medium, 30mg/L or 40mg/L of cane sugar (rooting culture), 0.5mg/L of NAA0.1mg/L of IBA0.1mg/L of 6-BA (6-benzyladenine) with different types and concentrations, 0.5mg/L of alginic acid and 0.1% of common rhodamine foliar fertilizer are added, the pH value is 5.8, and after preparation and split charging, sterilization is carried out for 20min under the conditions of 1.05kg/cm pressure and 121 ℃. The culture conditions can lead the inductivity of the hispid fig to reach more than 70 percent and the average bud height to reach more than 3 cm.
The following steps 102-103, and the culturing conditions of the culturing phase continued on the rooting medium in step 104 are: the temperature is 27 +/-1 ℃, the light source is a fluorescent lamp, specifically an LED lamp is selected, the illumination intensity is 2000-3000lx, and the illumination time is 12 h.d < -1 >. Can obviously improve the multiplication coefficient of the hispid fig tissue culture.
102, inducing cluster buds, and carrying out subculture proliferation.
Please refer to fig. 1-5. Inoculating the explant to a cluster bud induction culture medium, transferring the induced cluster buds to the cluster bud induction culture medium, growing the buds into seedlings after 1 week, wherein the stems are thick and short, the leaves are green, each cluster bud continuously proliferates and grows, the average propagation coefficient is 5-8, and a large number of cluster buds and seedlings are obtained and used for subculture proliferation and rooting in the proliferation culture medium.
Preferably, the cluster bud induction medium is MS +6-BA2.0mg/L + sucrose 30g/L. Inducing to differentiate large amount of cluster buds, inducing root and culturing to form normal plant. Can obviously improve the inductivity of the hispid fig tissue culture.
The culture medium suitable for explant induction of multiple shoots is MS +6-BA2.0mg/L + sucrose 30g/L, the terminal shoots can be induced to differentiate a large number of multiple shoots, and after rooting culture, normal plants can be formed, and a rapid in vitro propagation system of hispid fig is established. Therefore, it is generally recommended to select the acromion as the explant.
Preferably, the proliferation medium is MS +6-BA1.5mg/L + NAA0.1mg/L + sucrose 30g/L. Can obviously improve the multiplication coefficient of the hispid fig tissue culture.
The selection of the type, level and combination of plant growth regulators is important for the formation and differentiation of tissues. The propagation speed of the cluster buds is greatly accelerated by adjusting the hormone content in the propagation medium, the propagation coefficient reaches 6-8 times, MS +6-BA1.5mg/L + NAA0.1mg/L + sucrose 30g/L is selected as the propagation medium, and a large amount of required cluster buds can be quickly and stably obtained.
And 103, rooting culture.
Referring to FIG. 6, the robust plantlets were transferred to rooting medium for root induction to obtain robust plants.
And (3) transferring the robust seedlings growing to 3-4cm to a rooting culture medium to induce rooting, and growing into plants with the plant height of 6-8cm, 2-3 canopy leaves and 2-3 roots after 20-30 days, wherein the rooting rate is 100%, the root system is robust and has multiple branches, so that robust plants are obtained.
Preferably, the rooting medium is 1/2MS + NAA0.5mg/L + IBA0.1mg/L + sucrose 40g/L + alginic acid 0.5mg/L +0.25% active carbon +0.1% common rhodamine foliar fertilizer. Can obviously improve the rooting rate, the root length and the root number of the hispid fig tissue culture seedlings.
1/2MS + NAA0.5mg/L + IBA0.1mg/L + sucrose 40g/L + alginic acid 0.5mg/L +0.25% active carbon +0.1% common rhodamine foliar fertilizer are selected as a rooting culture medium, so that the nutrient content and the sugar content in the rooting culture medium are reduced, rooting can be promoted, and in a test, 1/2MS with half of nutrients reduced as the rooting culture medium has the rooting rate of 100% and the root system grows robustly.
And 104, hardening and transplanting the seedlings.
Please refer to fig. 7-10. Continuously culturing a robust plant with the plant height of 6-8cm, obtaining a bottle seedling of hispid fig when the total culture time on a rooting culture medium is 30-50d, moving the bottle seedling into a greenhouse, exercising the seedling under natural light, then opening a cover to exercise the seedling in a shady and ventilated environment, spraying water at proper time, then moving the rooting seedling out of the bottle, cleaning the root culture medium, soaking and disinfecting the root, cleaning with clear water, transplanting the root into a substrate which is disinfected in advance, watering and thoroughly watering, appropriately preserving moisture and shading, carrying out conventional management, and then transplanting tissue culture seedlings.
In specific implementation, preferably, the seedlings are acclimatized under natural light for 2-5 days, then the seedlings are acclimatized in a cool and ventilated environment for 2-5 days, water is sprayed timely, then the rooted seedlings are moved out of a bottle, a root culture medium is cleaned, the roots are soaked and disinfected for 5-10min by using 0.1% carbendazim solution, the roots are transplanted into a substrate which is disinfected in advance after being cleaned by using clear water, watering and drenching are carried out, proper moisture preservation and shading are carried out, a nutrient solution is sprayed once when the roots are transplanted into the substrate for 12-18 days, conventional management is carried out, and the tissue culture seedlings are transplanted after being transplanted into the substrate for 25-35 days.
The transplanting survival rate of the tissue culture seedlings reaches more than 90 percent, which indicates that the hardening-off seedlings are favorable for the transplanting of test-tube seedlings.
The beneficial effects of the hispid fig tissue culture method are as follows:
the invention takes the acrocarpus fimbriatus as an explant and determines that 0.1% (w/v) HgCl is adopted 2 The most suitable time for sterilization is 10min; the clustered shoot induction culture medium suitable for explant terminal shoot induction is MS +6-BA2.0mg/L + sucrose 30g/L, a large number of clustered shoots are induced, and a normal plant can be formed after rooting culture, so that a rapid in-vitro hispid fig propagation system is established. Therefore, it is generally recommended to select the top bud of hispid fig as the explant.
In plant tissue culture, the selection of the type, level and combination of plant growth regulators is important for the formation and differentiation of tissues. By regulating the hormone content in the culture medium, the differentiation speed of the cluster buds is greatly accelerated, the propagation coefficient reaches 6-8 times, and the proliferation culture medium is selected
MS +6-BA1.5mg/L + NAA0.1mg/L + sucrose 30g/L can obtain a large amount of required cluster buds.
In the rooting stage of plant tissue culture, the rooting can be promoted by reducing the content of nutrient components and sugar in the culture medium, and the rooting culture medium is selected from 1/2MS + NAA0.5mg/L + IBA0.1mg/L + sucrose 40g/L + alginic acid 0.5mg/L +0.25% active carbon +0.1% common rhodamine foliar fertilizer. In the experiment, 1/2MS with half of nutrients is found to be used as a rooting culture medium, the rooting rate can reach 100 percent, and the growth vigor is good.
Through hardening off the seedling, the survival rate of transplanting the tissue culture seedling reaches more than 90 percent, and the experiment shows that the hardening off the seedling is beneficial to the transplanting of the test-tube seedling.
According to the invention, by adjusting the composition of the culture medium at each culture stage, the inductivity, the multiplication coefficient, the rooting rate and the transplanting survival rate of the hispid fig tissue culture are improved, the pollution rate and the browning rate are reduced, and the rapid propagation efficiency of the hispid fig is improved. The method for culturing the hispid fig tissue is simple and easy to operate, has high emergence rate and robust plants, shortens the seedling culture time and reduces the seedling culture cost.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Claims (7)
1. A hispid fig tissue culture method is characterized by comprising the following steps:
selecting the terminal bud of hispid fig as an explant, cleaning, disinfecting and washing the explant, and sucking surface moisture, wherein the cleaning, disinfecting and washing the explant, and sucking surface moisture comprises the following steps: cleaning explant with clear water, placing into sterilized culture container, placing on super clean bench, sterilizing with 75% ethanol surface for 1-2min, and adding 0.1% (w/v) HgCl 2 Soaking for sterilization for 8-12min, washing with sterile water for 3-5 times, and drying with sterile filter paper;
inoculating an explant into a cluster bud induction culture medium, transferring the induced cluster buds into the cluster bud induction culture medium, continuously proliferating and growing the cluster buds to obtain a large number of cluster buds and plantlets for subculture proliferation and rooting culture in a proliferation culture medium, wherein the cluster bud induction culture medium is MS +6-BA2.0mg/L + 30g/L of sucrose; the multiplication culture medium is MS +6-BA1.5mg/L + NAA0.1mg/L + sucrose 30g/L; the rooting culture medium is 1/2MS + NAA0.5mg/L + IBA0.1mg/L + sucrose 40g/L + alginic acid 0.5mg/L +0.25% active carbon +0.1% common rhodamine foliar fertilizer;
and transferring the robust plantlets to a rooting culture medium to induce rooting so as to obtain robust plants.
2. The hispid fig tissue culture method according to claim 1, wherein: the cluster buds continuously proliferate and grow, and the average proliferation coefficient is 5-8.
3. The method for tissue culture of hispid fig. 1, characterized in that: repeating subculture in the proliferation culture medium to obtain a proliferation multiple of 6 times or more.
4. The hispid fig tissue culture method according to any one of claims 1 to 3, wherein: and (3) transferring the robust seedlings growing to 3-4cm to a rooting culture medium to induce rooting, and growing into plants with the plant height of 6-8cm, 2-3 canopy leaves and 2-3 roots after 20-30 days, wherein the roots are strong and have more branches, so that robust plants are obtained.
5. The hispid fig tissue culture method according to any one of claims 1 to 3, wherein: the culture conditions of the method for culturing the hispid fig tissue are as follows: the temperature is 27 +/-1 ℃, the light source is a fluorescent lamp, the illumination intensity is 2000-3000lx, and the illumination time is 12 h.d < -1 >.
6. The hispid fig tissue culture method according to claim 4, further comprising: continuously culturing a robust plant with the plant height of 6-8cm, obtaining a bottle seedling of hispid fig when the total culture time on a rooting culture medium is 30-50d, moving the bottle seedling into a greenhouse, exercising the seedling under natural light, then opening a cover to exercise the seedling in a shady and ventilated environment, spraying water at proper time, then moving the rooting seedling out of the bottle, cleaning the root culture medium, soaking and disinfecting the root, cleaning with clear water, transplanting the root into a substrate which is disinfected in advance, watering and thoroughly watering, appropriately preserving moisture and shading, carrying out conventional management, and then transplanting tissue culture seedlings.
7. The hispid fig tissue culture method according to claim 6, characterized in that, the seedlings are acclimatized under natural light for 2-5d, then the seedlings are acclimatized in a cool and ventilated environment for 2-5d by opening the cover, water is sprayed at a proper time, then the rooted seedlings are moved out of the bottle, the root culture medium is cleaned, the roots are soaked and sterilized with 0.1% carbendazim solution for 5-10min, the roots are cleaned with clear water and then transplanted into a substrate which is sterilized in advance, the watering and the drenching are carried out, the proper moisture and the shading are kept, the nutrient solution is sprayed once when the roots are transplanted into the substrate for 12-18d, the conventional management is carried out, and the tissue culture seedlings are transplanted into the substrate for 25-35 d.
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