CN113207695A - Formula for tissue culture of ulmus parvifolia - Google Patents
Formula for tissue culture of ulmus parvifolia Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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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
- A01H4/001—Culture apparatus for tissue culture
Abstract
The invention relates to a formula for tissue culture of Ulmus parvifolia, wherein a Ulmus parvifolia stem section is used as an explant, and a Ulmus parvifolia tissue culture efficient regeneration system is successfully established by using a special culture medium formula for induction culture, proliferation culture and rooting. The method saves occupied space, has no limitation on propagation time by seasons, short subculture period and no trouble of diseases and insect pests, is more suitable for storing and quickly and efficiently propagating wild ulmus parvifolia varieties, has high propagation efficiency and good nursery stock quality, provides a referable technology and method for industrial seedling culture of ulmus parvifolia, and has important application value in ulmus parvifolia seedling production and scientific research.
Description
Technical Field
The invention relates to the field of propagation methods of Ulmus parvifolia, and in particular relates to a tissue culture formula of Ulmus parvifolia.
Background
Ulmus parvifolia Jacq, a deciduous arbor of Ulmaceae Ulmus, also known as mosquito hammer, peeled Ulmus pumila, Ulmus parvifolia, etc., with a height of 25m, a diameter at breast height of 1.2 m, a flowering period of 8-9 months, and mature winged fruit in 10 months. The adaptability is strong, and the fertilizer is mainly distributed from the central south of north China to the east China, the central south China and the southwest China, is suitable for the south China and the central China, and is also distributed in Japan and Korean. The most suitable habitat is neutral soil with warm climate, fertile soil and good drainage. The garden hammerhead prefers light, warm and humid climate, drought resistance and barrenness, has low requirement on the pH value of soil, and can grow normally under different soil textures. Ulmus parvifolia is a precious tree species, and has beautiful tree shape, rich leaf color, mottled bark and high ornamental value. Ulmus parvifolia is a precious hard and wide tree species, is listed as a main recommended tree species in the Chinese main cultivation precious tree species reference book by the national forestry bureau, and is also the most suitable native high-quality precious tree species for use in the country of Jiangsu.
At present, propagation methods of ulmus parvifolia mainly include seeding, cutting and grafting, and have low survival rate and long period. Tissue culture is an important means for rapid propagation of forest seedlings, wherein rooting of test-tube seedlings is a necessary process for tissue rapid propagation. But the test-tube seedling is rooted in the bottle, so that the production cost is high, the seedling period is long (60 days are needed for rooting and transplanting in the bottle), the transplanting survival rate is low (the transplanting survival rate of the seedling with the roots in the tissue culture bottle is lower than 70 percent), and the like. The tissue culture technology of the garden burnet is not reported at present, the project breaks through the technical bottleneck of tissue culture by means of tissue culture and container seedling culture, a large-scale breeding system of excellent clones is established, a large number of high-quality clone seedlings can be provided for production, and the requirement of precious color tree species afforestation is met.
Disclosure of Invention
The invention aims to provide a tissue culture formula of Ulmus parvifolia, wherein an Ulmus parvifolia tissue culture efficient regeneration system is successfully established by taking an Ulmus parvifolia stem section as an explant and utilizing a special induction culture, multiplication culture and rooting culture medium formula of the Ulmus parvifolia, and a referable technology and method are provided for industrial seedling culture of Ulmus parvifolia.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
a formula for tissue culture of Ulmus parvifolia is characterized in that:
(1) taking a semi-lignified stem section of the robust and disease and pest-free Ulmus parvifolia as an explant;
(2) washing the explants selected in the step (1) with neutral soap water for 2 times, then cutting the explants into 2-4 cm long stem sections with buds, disinfecting the stem sections with 70% alcohol for 8-12s, then disinfecting the stem sections with 5% sodium hypochlorite solution for 5-8 minutes, and finally washing the stem sections with sterile water for 5 times;
(3) cutting off the wound part of the explant subjected to disinfection treatment in the step (2) and contacting with a disinfectant, reserving 1-2 cm of stem with buds, inoculating the stem in a culture bottle containing an induction culture medium, culturing for 25 days, and growing into a sterile tissue culture seedling with the height of 4-6 cm;
(4) shearing the sterile tissue culture seedlings in the step (3) into 1.5-2.0 cm stem sections with 1-2 axillary buds, transferring the stem sections into a culture bottle containing a proliferation culture medium for proliferation culture for 40 days to form a subculture period, wherein the proliferation coefficient is 8-10 times;
(5) and (4) cutting the propagation cultured seedlings in the step (4) into tender seedlings with the length of 1.5-2.0 cm, transferring the tender seedlings into a culture bottle containing a rooting culture medium for rooting culture, wherein 14 days of culture is a subculture period, and the rooting rate is 100%.
Further, the disinfection and inoculation work in the steps (2) to (4) is operated in a clean bench, the culture of the test-tube plantlet is carried out in a tissue culture chamber, and the culture conditions are as follows: the quantity of light was controlled to 40. mu. mol. m-2·s-1Setting the illumination time to 12 h.d-1At a temperature of 25 ℃. + -. 1 ℃.
The induction culture medium in the step (3) is MS +6 BA1.0mg.L-1+IBA0.002mg·L-1+ sucrose 30 g.L-1+8g·L-1Agar, pH 5.8.
The enrichment culture medium in the step (4) is MS +6BA 0.5 mg.L-1+NAA0.1mg·L-1+TDZ0.01mg·L-1+30g·L-1Sucrose +8 g.L-1Agar, pH 5.8.
The rooting culture medium in the step (5) is WPM basic culture medium and NAA 0.5 mg.L-1+30g·L-1Sucrose +8 g.L-1Agar, pH 5.8.
The rhizomes with buds in the step (1) are short sections with axillary buds, the lower ends of the short sections are provided with raw fleshy roots, and the stem sections are provided with 1-2 leaves.
Furthermore, the WPM minimal medium, namely the woody plant tissue culture medium, consists of macroelements, trace elements, iron salts and organic components.
Preferably, in the WPM minimal medium, the macroelements are: NH (NH)4NO3 400mg/L、Ca(NO3)2·4H2O 556mg/L、MgSO4·7H2O 370mg/L、KH2PO4 170mg/L、K2SO4 990mg/L；
The trace elements are as follows: MnSO4·H2O 22.3mg/L、ZnSO4·7H2O 8.6mg/L、H3BO3 6.2mg/L、Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O 0.025mg/L；
The iron salt is as follows: na (Na)2·EDTA 37.3mg/L、FeSO4·7H2O 27.8mg/L；
The organic components are as follows: 100mg/L inositol, 2mg/L glycine, 1mg/L thiamine hydrochloride, 0.5mg/L pyridoxine hydrochloride, and 0.5mg/L nicotinic acid.
The stem section of the ulmus parvifolia is used as an explant, a plant sterile system is established, and suitable conditions for induction, proliferation and rooting tissue culture and rapid propagation of the ulmus parvifolia are researched, so that the method has important technical significance for large-scale propagation of an ulmus parvifolia tissue culture and rapid propagation system. Specifically, the invention has the following advantages:
1. convenient for stock preservation and development of genetic improvement research: compared with cutting propagation, the method saves occupied space, has no limitation on propagation time by seasons, has short subculture period and no trouble of diseases and insect pests, and is more suitable for storing and rapidly and efficiently propagating the wild ulmus parvifolia variety.
2. The breeding efficiency is high: by adopting the tissue culture method, the multiplication factor of ulmus parvifolia reaches 8-10 times in 40 days, and is improved by about 3-5 times compared with the conventional tissue culture method.
3. The nursery stock has good quality: the method has simple formula components and robust seedling growth. Can obtain a large amount of seedlings in a short time, and maintain the excellent characters of the mother plant, and the descendant is inherited orderly.
In conclusion, the tissue culture method of Ulmus parvifolia has the advantages of high propagation coefficient, energy conservation, good seedling quality and the like, and has important application value in Ulmus parvifolia seedling production and scientific research.
Drawings
FIG. 1 explant.
FIG. 2 is a graph of detoxification of different concentrations of treatment.
FIG. 3 Induction Medium screening Panels.
FIG. 4 shows a diagram of a medium for proliferating multiple shoots.
FIG. 5 shows a diagram of shoot elongation medium.
FIG. 6 rooting medium diagram.
Detailed Description
The above-mentioned contents of the present invention are further described in detail by way of examples below, but it should not be understood that the scope of the above-mentioned subject matter of the present invention is limited to the following examples, and any technique realized based on the above-mentioned contents of the present invention falls within the scope of the present invention.
The experimental procedures used in the examples below are conventional procedures unless otherwise specified, and the reagents, methods and equipment used therein are conventional in the art unless otherwise specified.
Example 1
1. Experimental materials and methods
1.1 Experimental materials
Selecting new variety seedling of robust Ulmus parvifolia, adopting stem with bud of annual growth without lignification or semi-lignification as explant, performing detoxification, inducing cluster seedling, further performing enrichment culture, and finally performing strong seedling and rooting culture. The test is carried out in plant tissue culture laboratories and greenhouses of the scientific research institute of forestry, Jiangsu province. The test comprises the following steps: a bud induction culture medium, a proliferation culture medium and a rooting culture medium. The temperature of the culture room is 25-2 ℃, the illumination intensity is 2000-3000 Lux, and the illumination culture is carried out for 24 hours.
1.2 Primary Medium
The starting culture medium is MS culture medium, and the WPM is improved, and the specific improvement is as follows: with Ca (NO)3)2·4H2O 684mg·L-1、KNO3 190mg·L-1、C10H3FeN2NaO873.4 mg.L-1 and thiamine hydrochloride 0.1 mg.L-1 substituted K in the original WPM medium2SO4、CaCl2、FeSO4And Na2EDTA，pH5.8。
1.3 explant detoxification
Placing the triangular flask, the empty triangular flask, the scissors, the tweezers, the scalpel, the culture dish and the filter paper filled with the distilled water into a high-pressure steam sterilization pot for 30 min: the special electronic sterilizer is arranged on one side of the superclean workbench. Surface sterilization of explants: all test materials are young branches cut from plants, washed for 10min by tap water, rinsed for 5-10 min by a washing powder solution, soaked in 70% alcohol for 30s, then soaked for 2min, 5-8min and 8-10min by sodium hypochlorite solutions with different concentrations of 2% and 5%, and finally washed for 5 times by sterile water, wherein each time lasts for 10 min. After washing, the sprouts were placed on sterile filter paper, the surface moisture was blotted, and the sprouts were inoculated on sterilized primary medium after the hard wound parts at the base were excised with forceps and scalpel, and 10 flasks of 3 sprouts per flask were inoculated.
1.4 Induction culture
Cutting off two ends of the stem segment, inoculating the stem segment with buds into an induction culture medium, inoculating one explant (avoiding cross contamination) in each bottle, placing in a tissue culture rack, and performing dark culture for 7.0d to prevent the explant from browning. The induction medium used is as follows (table 1). 30g/L of sucrose and 8g/L of agar are added, and an induction formula is obtained according to the growth condition of axillary buds.
TABLE 1 Induction Medium formulation
1.5 multiplication culture
Phytohormones were added to a suitable minimal medium (sucrose 4%, agar 0.6%, pH5.8) and the formulation is given in Table 1. 10 flasks were inoculated per treatment, 3 stem segments per flask. After inoculation, the seeds are irradiated for 24 hours at the temperature of 25 +/-2 ℃ and the illumination intensity of 1800 Lux. After 20 days of culture, the number of new shoots of seedlings cultured on different medium compositions was investigated. The explant inoculated in the induction culture medium starts to germinate after l weeks, and the bud can grow into a seedling of about 4-5 cm after 6-8 weeks. The seedlings were cut into about 2cm stem sections and transferred to different proliferation media for culture (sucrose 30g/L, agar 8g/L, pH5.8) with the formulation shown in Table 2.
TABLE 2 phytohormone formulation of the multiplication Medium
1.6 in-flask rooting culture
And (4) putting the induced plantlets into a rooting culture medium for culturing, and counting the number of roots and the growth condition after the roots grow. The sucrose is sucrose 30g/L, agar 8g/L, and pH5.8.
TABLE 3 rooting Medium formulation
2 test results and analysis
2.1 obtaining of sterile seedlings
The treatment in different sodium hypochlorite solutions shows that the detoxified stem segments can be obtained by detoxifying non-lignified tender stems in a 2% NaCLO solution for 2-5min, and the detoxified stem segments can be obtained by detoxifying semi-lignified tender stems in a 5% sodium hypochlorite solution for 8-10 min.
2.2 Induction Medium
Cutting tender single-bud stem segments with the length of about 3-5cm, and inoculating the cut tender single-bud stem segments to a prepared culture medium to induce tender buds. In FIG. 3, it can be seen that S14, S16, S9, S3 and S11 can all induce shoots, S14 induces seedlings to grow faster and can be subcultured in 20 days, and S16 induces seedlings to grow slower and weak and have no significant elongation growth. S3 gave more callus and S11 gave smaller shoots. Healthy seedlings were not induced in the remaining medium. Therefore, the induction culture medium of Ulmus parvifoliaS14：MS+6BA 1.0mg·L-1+IBA 0.002mg·L-130 g.L of sucrose-1，Ph 5.8。
2.3 enrichment culture
In order to increase the propagation coefficient of ulmus parvifolia seedlings, a propagation method of ulmus parvifolia cluster buds is explored. The explant inoculated in the primary culture medium starts to germinate after 1 week, the bud can grow into a new shoot of about 4-5 cm after 2 weeks, phytohormone is added on a proper basic culture medium (sucrose 30, agar 0.6%, pH5.8), and the induced tender seedling is placed on the culture medium of the table 2 so as to induce the cluster bud of the Ulmus parvifolia. As can be seen in FIG. 4, the lateral bud of the culture medium 1 starts to sprout in 3 days, seedlings are generated in about 7 days, 2-5 can generate cluster buds, the culture media 3 and 4 generate more calluses, the buds are light green, the calluses are yellow and become top, the browning is easy, the multiplication coefficient of No. 5 is higher, 8-10 buds can be grown, the bud green color is strong, so that the No. 5 is a multiplication culture medium, and the formula is MS +6BA 0.5 mg.L-1+NAA0.1mg·L-1+TDZ0.01mg·L-1。
2.4 shoot elongation culture
After the cluster buds are induced, the buds need to grow into plants through elongation. As can be seen from fig. 5, the S1, S4 and 5 induced elongation growth of shoots, and the S5 and S6 shoots were strong but did not elongate. S1 and S4 are tender shoot elongation, the No. 5 culture medium is a proliferation culture medium for continuous growth, the step of changing the culture medium can be omitted, but lignification is easy, and tender buds are easy to brown. Therefore, S4 can be selected as the elongation medium, and the formula is S4: MS +6BA 0.5 mg.L-1+NAA 0.05mg·L-1+TDZ 0.002mg·L-1。
2.5 rooting culture
The survival rate of transplanting can be improved by rooting in the bottle to form a complete plant. In the formulation of the rooting medium table 3, R3 has the fastest rooting speed, can root in 7-10 days and has stronger roots (figure 6). And can induce lateral buds to continue growing into seedlings. Therefore, R3: WPM + NAA 0.5 mg.L-1Is the most suitable rooting culture medium.
3 conclusion
The tissue culture seedling of Ulmus parvifolia obtains a tissue culture rapid propagation system of aged Ulmus parvifolia after detoxification, induction, proliferation and rooting, and the propagation coefficient is more than 5. The specific formula is as follows:
(1) detoxification: non-lignified tender stem: 2% sodium hypochlorite solution for 2-5 min; semi-lignified tender stem: dissolving 5% sodium hypochlorite for 8-10 min.
(2) Induction medium: MS +6BA1.0 mg.L-1+IBA 0.002mg·L-130 g.L of sucrose-1，PH 5.8。
(3) Proliferation culture medium: MS +6BA 0.5 mg.L-1+NAA0.1mg·L-1+IBA 0.002mg·L-130 g.L of sucrose-1，PH 5.8
(4) Shoot elongation medium: MS +6BA 0.5 mg.L-1+NAA 0.05mg·L-1+TDZ 0.002mg·L-1。
(5) Rooting culture medium: WPM + NAA 0.5 mg.L-1。
Example 2
Preparing a basic culture medium:
ammonium Nitrate (NH)4NO3)1650mg/L potassium nitrate (KNO)3)1900mg/L potassium dihydrogen phosphate (KH)2PO4)170mg/L magnesium sulfate (MgSO)4·7H2O)370mg/L calcium chloride (CaCl)2·2H2O)440mg/L；
The composition of the trace elements and their corresponding use concentrations are as follows: potassium iodide (KI)0.83mg/L, manganese sulfate (MnSO)4·H2O)22.3mg/L, zinc sulfate (ZnSO)4·7H2O)8.6mg/L, boric acid (H)3BO3)6.2mg/L of sodium molybdate (Na)2MoO4·2H2O)0.25mg/L, copper sulfate (CuSO)4·5H2O)0.025mg/L, cobalt chloride (CoCl)2.6H2O)0.025mg/L；
The components of the iron salts and their corresponding use concentrations are as follows: disodium ethylene diamine tetraacetate (Na)2EDTA)37.3mg/L, ferrous sulfate (FeSO)4·7H2O)27.8mg/L；
The components of the organic components and their corresponding concentrations are as follows: inositol 100mg/L, glycine (Gly)2mg/L, thiamine hydrochloride (VB)1)1mg/L of pyridoxine hydrochloride (VB)6)0.5mg/L and 0.5mg/L of nicotinic acid (VPP).
The induction culture medium and the proliferation (rooting) culture medium are prepared by using the basic culture medium.
Wherein, the induction culture medium: per liter of basic culture medium, 1.0mg/L NAA and 0.05 mg/L6-BA 30 g.L-1Sucrose +8 g.L-1Agar, pH 5.8.
Proliferation culture medium: per liter of basic culture medium, 0.05mg/L NAA, 0.005mg/L IBA and 30 g.L-1Sucrose +8 g.L-1Agar, pH 5.8.
Injecting the prepared culture medium into a glass bottle, and sterilizing at high temperature and high pressure for 20 minutes for later use, wherein the temperature is 120-125 ℃, and the pressure is 1.1KG/CM2。
The tissue culture and rapid propagation method of Ulmus parvifolia comprises the following steps:
(1) material selection and disinfection treatment: selecting healthy and strong Ulmus parvifolia plants without diseases and insect pests, transplanting the Ulmus parvifolia plants to a sunlight greenhouse for curing for 1-2 months, selecting rhizomes with buds, washing the rhizomes in running water for 40min, washing the surfaces of the rhizomes with neutral soap water, shearing the selected explants into stem sections with buds of 2-4 cm in length, disinfecting the stem sections with buds for 10s by 70% of alcohol, disinfecting the stem sections with 0.1% of sodium hypochlorite for 8-10min, and finally washing the stem sections with sterile water for 5 times;
(2) and (3) sterile seedling culture: cutting off the wound part contacting with the disinfectant from the explant subjected to the sterilization treatment in the step (1) on a clean bench under an aseptic condition, reserving 1-2 cm of stem segments with buds, inoculating the stem segments into a culture bottle containing an induction culture medium, placing the culture bottle in an environment with a white fluorescent lamp as a light source, and controlling the light quantity to be 40 mu mol.m-2·s-1Setting the illumination time to 12 h.d-1Culturing at 25 +/-1 ℃ for 30 days to grow aseptic seedlings of 3-4 cm;
(3) proliferation and rooting culture: and (3) cutting the tissue culture seedlings grown in the step (2) into stem sections with the length of 1.5-2.0 cm and 1-2 axillary buds, transferring the stem sections into a multiplication culture medium for multiplication culture, wherein clustered buds cannot be obtained in the culture medium, and the multiplication multiple of 40 days in one period is 3 times.
Example 3
Preparing a basic culture medium:
basicThe culture medium is MS culture medium, wherein, the components of macroelements and the corresponding use concentrations are as follows: ammonium Nitrate (NH)4NO3)1650mg/L potassium nitrate (KNO)3)1900mg/L potassium dihydrogen phosphate (KH)2PO4)170mg/L magnesium sulfate (MgSO)4·7H2O)370mg/L calcium chloride (CaCl)2·2H2O)440mg/L；
The composition of the trace elements and their corresponding use concentrations are as follows: potassium iodide (KI)0.83mg/L, manganese sulfate (MnSO)4·H2O)22.3mg/L, zinc sulfate (ZnSO)4·7H2O)8.6mg/L, boric acid (H)3BO3)6.2mg/L of sodium molybdate (Na)2MoO4·2H2O)0.25mg/L, copper sulfate (CuSO)4·5H2O)0.025mg/L, cobalt chloride (CoCl)2.6H2O)0.025mg/L；
The components of the iron salts and their corresponding use concentrations are as follows: disodium ethylene diamine tetraacetate (Na)2EDTA)37.3mg/L, ferrous sulfate (FeSO)4·7H2O)27.8mg/L；
The components of the organic components and their corresponding concentrations are as follows: inositol 100mg/L, glycine (Gly)2mg/L, thiamine hydrochloride (VB)1)1mg/L of pyridoxine hydrochloride (VB)6)0.5mg/L and 0.5mg/L of nicotinic acid (VPP).
The induction culture medium and the proliferation (rooting) culture medium are prepared by using the basic culture medium.
Wherein, the induction culture medium: per liter of basic culture medium, 0.1mg/L of 6BA, 0.005mg/L of IBA and 30 g.L-1Sucrose +8 g.L-1Agar, pH 5.8.
Proliferation culture medium: per liter of basic culture medium, 0.1mg/L NAA, 0.5 mg/L6-BA, 0.01mg/L TDZ and 30 g.L-1Sucrose +8 g.L-1Agar, pH 5.8.
Injecting the prepared culture medium into a glass bottle, and sterilizing at high temperature and high pressure for 20 minutes for later use, wherein the temperature is 120-125 ℃, and the pressure is 1.1KG/CM2。
The tissue culture and rapid propagation method of Ulmus parvifolia comprises the following steps:
(1) material selection and disinfection treatment: selecting a new Ulmus parvifolia variety plant which grows strongly and has no disease or insect pest, moving the plant to a sunlight greenhouse for potting and maintaining for 1-2 months, selecting a rhizome with buds, placing the rhizome in running water for washing for 40min, washing the surface of the stem with neutral soap water, shearing the selected explant into stem sections with buds with the length of 2-4 cm, sterilizing the stem sections with 70% of alcohol for 10s, sterilizing the stem sections with 5% of sodium hypochlorite for 8-10min, and finally washing the stem sections with sterile water for 5 times;
(2) and (3) sterile seedling culture: cutting off the wound part contacting with the disinfectant from the explant subjected to the sterilization treatment in the step (1) on a clean bench under an aseptic condition, reserving 1-2 cm of stem segments with buds, inoculating the stem segments into a culture bottle containing an induction culture medium, placing the culture bottle in an environment with a white fluorescent lamp as a light source, and controlling the light quantity to be 40 mu mol.m-2·s-1Setting the illumination time to 12 h.d-1Culturing at 25 +/-1 ℃ for 25 days to grow aseptic seedlings of 4-6 cm;
(3) proliferation and rooting culture: and (3) shearing the tissue culture seedling grown in the step (2) into a stem section with the length of 1.5-2.0 cm and 1-2 axillary buds, transferring the stem section into a multiplication culture medium for multiplication culture, and obtaining robust tender seedlings in the culture medium at the same time, wherein the multiplication times of 35 days in one period are 3 times, and the rooting rate is 100%.
The above description is only a preferred embodiment of the present invention, and should not be taken as limiting the invention in any way, and any person skilled in the art can make any simple modification, equivalent replacement, and improvement on the above embodiment without departing from the technical spirit of the present invention, and still fall within the protection scope of the technical solution of the present invention.
Claims (8)
1. A formula for tissue culture of Ulmus parvifolia is characterized in that:
(1) taking a semi-lignified stem section of the robust and disease and pest-free Ulmus parvifolia as an explant;
(2) washing the explants selected in the step (1) with neutral soap water for 2 times, then cutting the explants into 2-4 cm long stem sections with buds, disinfecting the stem sections with 70% alcohol for 8-12s, then disinfecting the stem sections with 5% sodium hypochlorite solution for 5-8 minutes, and finally washing the stem sections with sterile water for 5-6 times;
(3) cutting off the wound part of the explant subjected to disinfection treatment in the step (2) and contacting with a disinfectant, reserving 1-2 cm of stem with buds, inoculating the stem in a culture bottle containing an induction culture medium, culturing for 25 days, and growing into a sterile tissue culture seedling with the height of 4-6 cm;
(4) shearing the sterile tissue culture seedlings in the step (3) into 1.5-2.0 cm stem sections with 1-2 axillary buds, transferring the stem sections into a culture bottle containing a proliferation culture medium for proliferation culture for 40 days to form a subculture period, wherein the proliferation coefficient is 8-10 times;
(5) and (4) cutting the propagation cultured seedlings in the step (4) into tender seedlings with the length of 1.5-2.0 cm, transferring the tender seedlings into a culture bottle containing a rooting culture medium for rooting culture, wherein 14 days of culture is a subculture period, and the rooting rate is 100%.
2. The tissue culture formula of Ulmus parvifolia as claimed in claim 1, wherein: the disinfection and inoculation work in the steps (2) to (4) is operated in a super clean workbench, the culture of the test-tube plantlet is carried out in a tissue culture chamber, and the culture conditions are as follows: the quantity of light was controlled to 40. mu. mol. m-2·s-1Setting the illumination time to 12 h.d-1At a temperature of 25 ℃. + -. 1 ℃.
3. The tissue culture formula of Ulmus parvifolia as claimed in claim 1, wherein: the induction culture medium in the step (3) is MS +6 BA1.0mg.L-1+IBA0.002mg·L-1+ sucrose 30 g.L-1+8g·L-1Agar, pH 5.8.
4. The tissue culture formula of Ulmus parvifolia as claimed in claim 1, wherein: the enrichment culture medium in the step (4) is MS +6BA 0.5 mg.L-1+NAA0.1mg·L-1+TDZ0.01mg·L-1+30g·L-1Sucrose +8 g.L-1Agar, pH 5.8.
5. The tissue culture formula of Ulmus parvifolia as claimed in claim 1, wherein: the rooting culture medium in the step (5) is WPM basic culture medium and NAA 0.5 mg.L-1+30g·L-1Sucrose +8 g.L-1Agar, pH 5.8.
6. The tissue culture formula of Ulmus parvifolia as claimed in claim 1, wherein: the rhizomes with buds in the step (1) are short sections with axillary buds, the lower ends of the short sections are provided with raw fleshy roots, and the stem sections are provided with 1-2 leaves.
7. The tissue culture formula of Ulmus parvifolia as claimed in claim 5, wherein: the WPM basic culture medium, namely woody plant tissue culture medium, consists of macroelements, microelements, iron salts and organic components.
8. The tissue culture formula of Ulmus parvifolia as claimed in claim 7, wherein: in the WPM basic culture medium, the macroelements are as follows: NH (NH)4NO3 400mg/L、Ca(NO3)2·4H2O 556mg/L、MgSO4·7H2O 370mg/L、KH2PO4170mg/L、K2SO4 990mg/L；
The trace elements are as follows: MnSO4·H2O 22.3mg/L、ZnSO4·7H2O 8.6mg/L、H3BO3 6.2mg/L、Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O 0.025mg/L；
The iron salt is as follows: na (Na)2·EDTA 37.3mg/L、FeSO4·7H2O 27.8mg/L；
The organic components are as follows: 100mg/L inositol, 2mg/L glycine, 1mg/L thiamine hydrochloride, 0.5mg/L pyridoxine hydrochloride, and 0.5mg/L nicotinic acid.
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