Tissue culture method of semiliquidambar cathayensis

Abstract

The invention discloses a tissue culture method of semiliquidambar cathayensis and belongs to the technical field of tissue culture of semiliquidambar cathayensis, the method comprises the steps of explant collection and treatment, explant disinfection, induction culture, proliferation-rooting integrated culture, seedling hardening, transplanting and the like, by improving a culture medium, proliferation-rooting integrated culture of semiliquidambar cathayensis is achieved, and the tissue culture rate of semiliquidambar cathayensis is increased. The tissue culture time is shortened, and the operation cost is reduced; the tissue culture method has the advantages that the tissue culture seedlings of the semiliquidambar cathayensis can be rapidly cultured, dry matter accumulation of the tissue culture seedlings of the semiliquidambar cathayensis is promoted, hormone level, illumination time, illumination intensity, temperature and the like are controlled, vitrification is effectively inhibited, the problem of propagation coefficient reduction caused by vitrification of the tissue culture seedlings in the tissue culture process of the semiliquidambar cathayensis is solved, and efficient tissue culture of the semiliquidambar cathayensis is realized; the method has important significance on promoting the development of the semiliquidambar cathayensis industry.

Description

Technical Field

[0001] The invention relates to the technical field of tissue culture of Hemiptera: Hemiptera, in particular to a tissue culture method of Hemiptera: Hemiptera. Background Art

[0002] Semiliquidambar cathayensis belongs to the genus Semiliquidambar of the family Hamamelidaceae. It is a newly discovered species endemic to my country in 1962. Semiliquidambar cathayensis is an excellent ornamental tree species for landscaping, and its wood is of high quality and has good wrapping properties, making it an excellent material for making spiral products. At the same time, the rhizome, branches, leaves, bark and nectar of Semiliquidambar cathayensis have good medicinal value and can treat symptoms such as rheumatoid arthritis, lumbar muscle strain, and congestion. It is an effective folk local specialty medicine. However, due to the low natural reproduction rate of Semiliquidambar cathayensis and its frequent felling and mining, its wild resources are becoming increasingly scarce and its population is becoming endangered. How to protect and expand the resources of Semiliquidambar cathayensis has become a key issue that needs to be solved urgently.

[0003] At present, the main propagation methods of Helianthus annuus are seed propagation and cutting propagation, but traditional seed seedling cultivation cannot obtain a large number of high-quality seedlings in a short time, and the offspring cannot stably maintain the excellent shape of the parents. In addition, Helianthus annuus is a tree species that is difficult to root, and the survival rate of cuttings is low. Therefore, seed propagation and cutting propagation cannot meet the large-scale industrialization needs of Helianthus annuus excellent strains, which limits the development of Helianthus annuus industry. Tissue culture has the characteristics of short cycle, fast speed and stable maintenance of the excellent traits of the parents compared to seed propagation and cutting propagation. Therefore, establishing the tissue culture technology of Helianthus annuus is an effective way to solve the shortage of Helianthus annuus resources and maintain its excellent varieties. However, the traditional tissue culture method needs to go through multiple stages such as induction culture, proliferation culture, rooting culture and seedling hardening and transplanting. The procedure is cumbersome, the cycle is long, and the cost is high. In addition, vitrification is prone to occur during the proliferation culture of Helianthus annuus, resulting in a low propagation coefficient of Helianthus annuus and poor quality of tissue culture seedlings.

[0004] Therefore, there is an urgent need to find a tissue culture method for Hemiptera: Acer truncatum to solve the problems of long tissue culture time and complicated procedures under conventional tissue culture methods, as well as the easy occurrence of vitrification leading to low reproduction coefficient and poor seedling quality. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a tissue culture method for Hemiptera: Hemiptera: Sophora flavescens, so as to solve the problems of long tissue culture time and complicated procedures of Hemiptera: Sophora flavescens under conventional tissue culture methods, as well as easy vitrification leading to low propagation coefficient and poor seedling quality.

[0006] The present invention solves the above technical problems by the following technical means:

[0007] A tissue culture method for Hemiptera: the tissue culture method is as follows:

[0008] (1) Explant collection and processing: In spring, healthy new shoots are collected from excellent mother trees, and semi-lignified stem segments are cut and the leaves are removed. The segments are placed in water to wash away surface stains, and then soaked in soapy water for 10 to 20 minutes, and then rinsed with running water for 30 minutes to obtain explants;

[0009] (2) Explant disinfection: On a clean bench, cut the explant into stem segments with 1 to 2 axillary buds and a length of 2 to 3 cm, wash them once in sterile water, and then soak them in a 75% ethanol solution for disinfection for 15 to 30 seconds. Then, wash them 3 to 5 times in sterile water, and then transfer them to a 0.1% mercuric chloride solution for disinfection for 5 to 8 minutes. After disinfection, wash them again with sterile water 5 to 7 times to obtain the disinfected explants.

[0010] (3) Induction culture: inoculating the sterilized explants into an induction culture medium for induction culture to obtain adventitious buds;

[0011] (4) Proliferation-rooting integrated culture: single buds are cut from the adventitious buds obtained by the induced culture, and then inoculated into a proliferation-rooting integrated culture medium for proliferation-rooting integrated culture to obtain rooted seedlings;

[0012] (5) Hardening and transplanting: Harden the rooted seedlings with root length ≥1 cm and number of roots ≥3 obtained by integrated proliferation-rooting culture. After hardening, take the rooted seedlings out of the tissue culture bottle, wash off the root culture medium, soak them in 800-1000 times carbendazim solution for 20 minutes, and then transplant them into the substrate tray.

[0013] Under the traditional tissue culture method, steps such as induction culture, proliferation culture, rooting culture, and seedling hardening and transplanting are required, which are complicated, costly, and time-consuming. Therefore, the present invention prepares a proliferation-rooting integrated culture medium for proliferation-rooting integrated culture, which can effectively shorten the tissue culture time of Hemiptera: Scutellaria baicalensis and reduce the cost of transfer operations, thereby improving the tissue culture propagation efficiency of Hemiptera: Scutellaria baicalensis.

[0014] Furthermore, the induction medium components are: MS+1.0-1.2 mg / L 6-BA+0.2-0.5 mg / L NAA+30 g / L sucrose+6.8 g / L agar, pH=5.8.

[0015] Furthermore, the proliferation-rooting integrated culture medium in step (4) is a solid-liquid double-layer culture medium, wherein the upper layer is a liquid proliferation culture medium and the lower layer is a solid rooting culture medium, and the proliferation-rooting integrated culture medium comprises the following raw materials:

[0016] Upper liquid proliferation medium: MS + 0.2-0.3 mg / L 6-BA + 0.05-0.15 mg / L NAA + 30 g / L sucrose + 2-3 g / L cyclohexylamine + 2-3 g / L isopropyl acetate + 3-4 g / L polyethylene glycol + 8-15 g / L stabilizer, pH = 5.8;

[0017] Lower layer solid rooting medium: MS + 0.6-0.8 mg / L IBA + 0.4 mg / L NAA + 0.6 mg / LABT + 30 g / L sucrose + 6.8 g / L agar, pH = 5.8.

[0018] Further, the preparation method of the proliferation-rooting integrated culture medium is as follows:

[0019] According to the component ratio of the lower solid rooting medium, IBA, NAA and ABT are added to the MS medium, mixed evenly, then agar is added, and then heated until the agar is dissolved, sucrose is added, stirred to dissolve and mixed evenly, the pH is adjusted to 5.8, and then put into a culture bottle, and sterilized at high temperature at 121°C for 20 minutes; according to the component ratio of the upper liquid proliferation medium, 6-BA, NAA, sucrose, cyclohexylamine, isopropyl acetate and polyethylene glycol are added to the MS medium, stirred and mixed evenly, and then a stabilizer is added to mix evenly, sterilized at 121°C for 20 minutes, and added after the lower solid rooting medium in the culture bottle is solidified, to obtain a proliferation-rooting integrated medium.

[0020] Further, the stabilizer comprises the following raw materials:

[0021] Sodium alginate, 6-BA, NAA, 2wt% calcium chloride solution, rice bran wax, triethylamine, thiourea, 2,4,6-tris(dimethylaminomethyl)phenol, epoxy resin.

[0022] Further, the stabilizer preparation method is as follows:

[0023] A: Dissolve sodium alginate in water to prepare a 5wt% sodium alginate solution, then add 6-BA and NAA and mix well, then slowly drop into a 2wt% calcium chloride solution to gel to obtain sodium alginate coated spheres, wash with sterile water for 3 times, and dry at 45°C for 20-30 min to obtain semi-dried sodium alginate coated spheres for standby use;

[0024] B: Grind rice bran wax through a 300-mesh sieve to obtain rice bran wax microparticles; put triethylamine, thiourea, and 2,4,6-tris(dimethylaminomethyl)phenol into a reaction kettle and mix them evenly, heat to 110-130° C. and react for 0.5-1h, then cool to 55° C., add epoxy resin, mix well, and then add rice bran wax microparticles and mix evenly to obtain a coating solution;

[0025] C: Spray the coating liquid onto the surface of the dried sodium alginate coated ball, place it in a cool and ventilated place for 12 to 24 hours, then place it at 60 to 70°C for 2 to 3 hours to allow the rice bran wax particles on the surface of the coated ball to melt and flow out, and then wash it with deionized water 3 to 5 times to obtain a stabilizer.

[0026] Furthermore, in step A, the mass ratio of sodium alginate solution, 6-BA and NAA is (160-300): (0.03-0.05): (0.01-0.02).

[0027] Furthermore, in step B, the mass ratio of triethylamine, thiourea, 2,4,6-tris(dimethylaminomethyl)phenol, epoxy resin and rice bran wax particles is (8-16):(10-20):(10-20):(50-70):(10-20).

[0028] Furthermore, the induction culture temperature is 25-27°C, the illumination time is 8-12h / d, and the illumination intensity is 2000-3000lx; the proliferation-rooting integrated culture temperature is 25-27°C, the illumination time is 8-12h / d, and the illumination intensity is 4500-5000lx.

[0029] Furthermore, the particle size of the stabilizer is 0.5-1 mm.

[0030] During tissue culture of the half-maple, vitrification is easy to occur in the proliferation stage. The vitrified seedlings show the conditions of high water content, low dry matter and cellulose accumulation, poor growth, and difficulty in further rooting. It is also difficult to survive after transplanting, which greatly reduces the reproduction coefficient. Therefore, in the preparation of the proliferation-rooting integrated culture medium, the present invention adds isopropyl acetate, cyclohexylamine and polyethylene glycol to the upper liquid proliferation culture medium. Cyclohexylamine enhances the ion exchange between nutrients in the culture medium and the root system of the tissue culture seedlings by adjusting the charge properties of the culture medium. Further, isopropyl acetate acts on the cell membrane, changes the arrangement of the cell membrane structure to increase the permeability of the cell membrane, and then the nutrients in the culture medium enter the tissue culture seedlings through the cell membrane with enhanced permeability under stronger ion exchange, so as to accelerate the dry matter accumulation of the tissue culture seedlings, promote the lignification of the tissue culture seedlings, and improve the quality of the seedlings; at the same time, the added polyethylene glycol can adjust the osmotic potential of the culture medium to regulate the water content in the tissue culture seedlings, and then the components in the culture medium work together to reduce the vitrification of the half-maple tissue culture seedlings, so that it can better root and obtain high-quality rooted seedlings.

[0031] In order to shorten the tissue culture time and improve the breeding efficiency, the present invention prepares a double-layer culture medium for integrated proliferation-rooting culture. However, the components in the upper liquid proliferation culture medium tend to settle downward and penetrate into the lower solid rooting culture medium after a long time. Among them, 6-BA and NAA are small amounts of added hormone costs. After penetrating into the lower rooting culture medium, it will lead to insufficient hormones required for proliferation. Adding more hormones to the upper liquid proliferation culture medium will further lead to the occurrence of vitrification.

[0032] Based on the above problems, the present invention adopts sodium alginate gel to embed 6-BA and NAA in a specific ratio to prepare a stabilizer and add it to the upper liquid proliferation medium. The 6-BA and NAA embedded in the sodium alginate gel are slowly released into the upper liquid proliferation medium for tissue culture seedlings to use, and will not enter the lower rooting proliferation medium due to long-term deposition, thereby ensuring the appropriate amount of hormones in the culture medium. However, after the sodium alginate coated ball enters the culture medium, it is easy to quickly absorb water and swell, causing the sudden release of 6-BA and NAA. Therefore, the present invention sprays the epoxy resin coating liquid containing rice bran wax after semi-drying the sodium alginate coated ball. After the epoxy resin is cured, a hard shell layer is formed on the surface of the sodium alginate coated ball, thereby inhibiting the swelling of the sodium alginate coated ball, and better solves the problem of sudden swelling release. The hole structure formed by the melting of the rice bran wax allows the hormone components released inside to smoothly enter the culture medium for tissue culture seedlings to use.

[0033] Beneficial effects:

[0034] 1. The present invention effectively controls the culture medium components, culture temperature, culture light time and intensity during the tissue culture process of Schizonepeta tenuifolia, inhibits the production of Schizonepeta tenuifolia vitrified seedlings, improves the tissue culture propagation coefficient of Schizonepeta tenuifolia, and improves the quality of tissue culture seedlings, which is of great significance to promoting the development of the Schizonepeta tenuifolia industry.

[0035] 2. The present invention prepares a proliferation-rooting integrated culture medium for integrated proliferation and rooting culture, which can effectively shorten the tissue culture time and reduce the cost of transfer operations; and further improves the upper liquid proliferation culture medium in the proliferation-rooting integrated culture medium to maintain an appropriate concentration of hormones in the culture medium, promote the accumulation of dry matter in tissue culture seedlings, inhibit the excessive increase of water content in the seedlings, further inhibit the vitrification of tissue culture seedlings, and better improve the quality of tissue culture seedlings, thereby effectively improving the rooting rate and transplant survival rate, and improving the reproduction coefficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 : It is the picture of tissue culture seedling status of embodiment 4 of the present invention. DETAILED DESCRIPTION

[0037] The present invention will be described in detail below with reference to specific embodiments and accompanying drawings:

[0038] Example 1: Stabilizer Preparation

[0039] A: 230g sodium alginate was dissolved in water to prepare a 5wt% sodium alginate solution, and then 0.04g 6-BA and 0.015g NAA were added and mixed evenly, and then slowly dripped into a 2wt% calcium chloride solution to gel to prepare sodium alginate coated spheres with a particle size of about 1mm. After washing with sterile water for 3 times, the spheres were dried at 45°C for 25min to obtain semi-dried sodium alginate coated spheres for standby use;

[0040] B: Grind rice bran wax through a 300-mesh sieve to obtain rice bran wax microparticles; put 12 g of triethylamine, 15 g of thiourea, and 15 g of 2,4,6-tris(dimethylaminomethyl)phenol into a reaction kettle and mix them evenly, heat to 120° C. and react for 0.7 h, then cool to 55° C. and add 60 g of epoxy resin and mix well, then add 15 g of rice bran wax microparticles and mix evenly to obtain a coating solution;

[0041] C: Spray the coating liquid onto the surface of the dried sodium alginate coated ball with a spray thickness of about 0.02 mm, place it in a cool and ventilated place for 18 hours, then place it at 65°C for 2.5 hours to allow the rice bran wax particles on the surface of the coated ball to melt and flow out, then wash it with deionized water 4 times to obtain a stabilizer with a particle size of about 0.8 cm.

[0042] Example 2: Preparation of stabilizer

[0043] A: 160g sodium alginate was dissolved in water to prepare a 5wt% sodium alginate solution, and then 0.03g 6-BA and 0.01g NAA were added and mixed evenly, and then slowly dripped into a 2wt% calcium chloride solution to gel to prepare sodium alginate coated spheres with a particle size of about 1mm. After washing with sterile water for 3 times, the spheres were dried at 45°C for 20min to obtain semi-dried sodium alginate coated spheres for standby use;

[0044] B: Grind rice bran wax through a 300-mesh sieve to obtain rice bran wax microparticles; put 8 g of triethylamine, 10 g of thiourea, and 10 g of 2,4,6-tris(dimethylaminomethyl)phenol into a reaction kettle and mix them evenly, heat to 110° C. and react for 0.5 h, then cool to 55° C. and add 50 g of epoxy resin and mix well, then add 10 g of rice bran wax microparticles and mix evenly to obtain a coating solution;

[0045] C: Spray the coating liquid onto the surface of the dried sodium alginate coated ball with a spraying thickness of about 0.02 mm, place it in a cool and ventilated place for 12 hours, then place it at 60°C for 3 hours to allow the rice bran wax particles on the surface of the coated ball to melt and flow out, and then wash it with deionized water 4 times to obtain a stabilizer with a particle size of about 0.8 cm.

[0046] Example 3: Preparation of stabilizer

[0047] A: 300g sodium alginate was dissolved in water to prepare a 5wt% sodium alginate solution, and then 0.05g 6-BA and 0.02g NAA were added and mixed evenly, and then slowly dripped into a 2wt% calcium chloride solution to gel to prepare sodium alginate coated spheres with a particle size of about 1mm. After washing with sterile water for 3 times, the spheres were dried at 45°C for 30min to obtain semi-dried sodium alginate coated spheres for standby use;

[0048] B: Grind rice bran wax through a 300-mesh sieve to obtain rice bran wax microparticles; put 16 g of triethylamine, 20 g of thiourea, and 20 g of 2,4,6-tris(dimethylaminomethyl)phenol into a reaction kettle and mix them evenly, heat to 130° C. and react for 1 h, then cool to 55° C. and add 70 g of epoxy resin and mix them evenly, then add 20 g of rice bran wax microparticles and mix them evenly to obtain a coating solution;

[0049] C: Spray the coating liquid onto the surface of the dried sodium alginate coated ball with a spraying thickness of about 0.02 mm, place it in a cool and ventilated place for 24 hours, then place it at 70°C for 2 hours to allow the rice bran wax particles on the surface of the coated ball to melt and flow out, and then wash it with deionized water 5 times to obtain a stabilizer with a particle size of about 0.8 cm.

[0050] After the stabilizer was prepared, tissue culture of the half-maple was further carried out, as shown below.

[0051] Example 4: Method 1 for tissue culture of Hemiptera:

[0052] Before conducting tissue culture of P. serrata, the induction medium and proliferation and rooting medium were prepared as follows:

[0053] Induction medium: MS + 1.1 mg / L 6-BA + 0.3 mg / L NAA + 30 g / L sucrose + 6.8 g / L agar, pH = 5.8;

[0054] Proliferation-rooting integrated culture medium:

[0055] Add IBA, NAA, and ABT at a final concentration of 0.7 mg / L to the MS medium, mix well, add agar at a final concentration of 6.8 g / L, heat until the agar is dissolved, add sucrose at a final concentration of 30 g / L, stir to dissolve and mix well, adjust the pH to 5.8, put it into a culture bottle, and sterilize it at 121° C. for 20 min to obtain a lower solid rooting medium; add 6-BA, NAA, sucrose, cyclohexylamine, isopropyl acetate, and polyethylene glycol at final concentrations of 0.25 mg / L, 0.1 mg / L, 30 g / L, 2.5 g / L, and 3.5 g / L to the MS medium, stir to mix well, then add the stabilizer prepared in Example 1 at a final concentration of 11 g / L, mix well, sterilize at 121° C. for 20 min, and add it after the lower solid rooting medium in the culture bottle solidifies to obtain a proliferation-rooting integrated medium;

[0056] (1) Explant collection and processing: In spring, healthy new shoots were collected from excellent mother trees, and semi-lignified stem segments were cut and leaves were removed. The segments were washed in water to remove surface stains, and then soaked in soapy water for 15 min, and then rinsed in running water for 30 min to obtain explants.

[0057] (2) Explant disinfection: On a clean bench, cut the explant into stem segments with 1 to 2 axillary buds and a length of about 2 cm, wash them once in sterile water, and then soak them in a 75% ethanol solution for 25 seconds. Then, wash them four times in sterile water, and then transfer them to a 0.1% mercuric chloride solution for 6 minutes. After disinfection, wash them six times in sterile water to obtain the disinfected explants.

[0058] (3) Induction culture: The sterilized explants were inoculated into an induction medium and induced to obtain adventitious buds at a temperature of 26°C, a light duration of 10 h / d, and a light intensity of 2500 lx;

[0059] (4) Proliferation-rooting integrated culture: The adventitious buds obtained by the induced culture were cut into single buds, and then inoculated into a proliferation-rooting integrated culture medium, and the proliferation-rooting integrated culture was carried out under the conditions of a temperature of 26°C, a light duration of 10 h / d, and a light intensity of 4700 lx to obtain rooted seedlings;

[0060] (5) Hardening and transplanting: Harden the rooted seedlings with root length ≥1 cm and number of roots ≥3 obtained by integrated proliferation-rooting culture. After hardening, take the rooted seedlings out of the tissue culture bottle, wash off the root culture medium, soak them in 800 times carbendazim solution for 20 min, and then transplant them into the substrate tray.

[0061] Example 5: Method 2 for tissue culture of Hemiptera:

[0062] Before conducting tissue culture of P. serrata, the induction medium and proliferation and rooting medium were prepared as follows:

[0063] Induction medium: MS + 1.0 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 6.8 g / L agar, pH = 5.8;

[0064] Proliferation-rooting integrated culture medium:

[0065] Add IBA, NAA, and ABT at a final concentration of 0.6 mg / L to the MS medium, mix well, add agar at a final concentration of 6.8 g / L, heat until the agar is dissolved, add sucrose at a final concentration of 30 g / L, stir to dissolve and mix well, adjust the pH to 5.8 and put it into a culture bottle, sterilize at 121° C. for 20 min to obtain a lower solid rooting medium; add 6-BA, NAA, 30 g / L sucrose, 2 g / L cyclohexylamine, 2 g / L isopropyl acetate, and 3 g / L polyethylene glycol at final concentrations of 0.2 / L to the MS medium, stir to mix well, then add the stabilizer prepared in Example 2 at a final concentration of 8 g / L, mix well, sterilize at 121° C. for 20 min, and add it after the lower solid rooting medium in the culture bottle solidifies to obtain a proliferation-rooting integrated medium;

[0066] (1) Explant collection and processing: In spring, healthy new shoots were collected from excellent mother trees, and semi-lignified stem segments were cut and leaves were removed. The segments were washed in water to remove surface stains, and then soaked in soapy water for 10 min, and then rinsed in running water for 30 min to obtain explants.

[0067] (2) Explant disinfection: On a clean bench, cut the explant into stem segments with 1 to 2 axillary buds and a length of about 2 cm, wash them once in sterile water, and then soak them in a 75% ethanol solution for 15 seconds. Then, wash them three times in sterile water and then transfer them to a 0.1% mercuric chloride solution for 5 minutes. After disinfection, wash them five times in sterile water to obtain the disinfected explants.

[0068] (3) Induction culture: The sterilized explants were inoculated into an induction medium and induced to obtain adventitious buds at a temperature of 25°C, a light intensity of 8 h / d, and a light intensity of 2000 lx;

[0069] (4) Proliferation-rooting integrated culture: The adventitious buds obtained by the induced culture were cut into single buds, and then inoculated into a proliferation-rooting integrated culture medium, and the proliferation-rooting integrated culture was carried out under the conditions of a temperature of 25°C, a light duration of 8 h / d, and a light intensity of 5000 lx to obtain rooted seedlings;

[0070] (5) Hardening and transplanting: Harden the rooted seedlings with root length ≥1 cm and number of roots ≥3 obtained by integrated proliferation-rooting culture. After hardening, take the rooted seedlings out of the tissue culture bottle, wash off the root culture medium, soak them in 1000 times carbendazim solution for 20 min, and then transplant them into the substrate tray.

[0071] Example 6: Method 3 for tissue culture of Hemiptera:

[0072] Before conducting tissue culture of P. serrata, the induction medium and proliferation and rooting medium were prepared as follows:

[0073] Induction medium: MS + 1.2 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 6.8 g / L agar, pH = 5.8;

[0074] Proliferation-rooting integrated culture medium:

[0075] Add IBA, NAA, and ABT at a final concentration of 0.8 mg / L to the MS medium, mix well, add agar at a final concentration of 6.8 g / L, heat until the agar is dissolved, add sucrose at a final concentration of 30 g / L, stir to dissolve and mix well, adjust the pH to 5.8, put it into a culture bottle, and sterilize it at high temperature at 121° C. for 20 min to obtain a lower solid rooting medium; add 6-BA, NAA, sucrose, cyclohexylamine, isopropyl acetate, and polyethylene glycol at final concentrations of 0.3 mg / L, respectively, to the MS medium, stir and mix well, then add the stabilizer prepared in Example 1 at a final concentration of 15 g / L, mix well, sterilize at high temperature at 121° C. for 20 min, and add it after the lower solid rooting medium in the culture bottle solidifies to obtain a proliferation-rooting integrated medium;

[0076] (1) Explant collection and processing: In spring, healthy new shoots were collected from excellent mother trees, and semi-lignified stem segments were cut and leaves were removed. The segments were placed in water to wash away surface stains, and then soaked in soapy water for 20 minutes, and then rinsed with running water for 30 minutes to obtain explants;

[0077] (2) Explant disinfection: On a clean bench, cut the explant into stem segments with 1 to 2 axillary buds and about 3 cm in length, wash them once in sterile water, and then soak them in a 75% ethanol solution for disinfection for 30 seconds. Then, wash them five times in sterile water, and then transfer them to a 0.1% mercuric chloride solution for disinfection for 5 minutes. After disinfection, wash them five times in sterile water to obtain the disinfected explants.

[0078] (3) Induction culture: The sterilized explants were inoculated into an induction medium and induced to obtain adventitious buds at a temperature of 27°C, a light intensity of 12 h / d, and a light intensity of 3000 lx;

[0079] (4) Proliferation-rooting integrated culture: The adventitious buds obtained by the induced culture were cut into single buds, and then inoculated into the proliferation-rooting integrated culture medium, and the proliferation-rooting integrated culture was carried out under the conditions of a temperature of 27°C, a light duration of 12 h / d, and a light intensity of 4500 lx to obtain rooted seedlings;

[0080] (5) Hardening and transplanting: Harden the rooted seedlings with root length ≥1 cm and number of roots ≥3 obtained by integrated proliferation-rooting culture. After hardening, take the rooted seedlings out of the tissue culture bottle, wash off the root culture medium, soak them in 1000 times carbendazim solution for 20 min, and then transplant them into the substrate tray.

[0081] Comparative Example 1: Tissue Culture Method of Hemiptera:

[0082] In contrast to Example 4, the only difference is that cyclohexylamine is not added to the proliferation-rooting integrated culture medium during the tissue culture of Hemiptera: Acer truncatula in Comparative Example 1, and the remaining steps are the same as those in Example 4.

[0083] Comparative Example 2: Tissue Culture Method of Hemiptera:

[0084] In contrast to Example 4, the only difference is that in Comparative Example 2, isopropyl acetate is not added to the proliferation-rooting integrated culture medium during the tissue culture of Hemiptera: Scutellaria baicalensis, and the remaining steps are the same as those in Example 4.

[0085] Comparative Example 3: Tissue Culture Method of Hemiptera:

[0086] In contrast to Example 4, the only difference is that in Comparative Example 3, no stabilizer is added to the proliferation-rooting integrated culture medium during the tissue culture of Hemiptera: Schizonepeta tenuifolia, and the remaining steps are the same as those in Example 4.

[0087] Comparative Example 4: Tissue Culture Method of Hemiptera:

[0088] In contrast to Example 4, the only difference is that polyethylene glycol is not added to the proliferation-rooting integrated culture medium during the tissue culture of Hemiptera in Comparative Example 4, and the remaining steps are the same as those in Example 4.

[0089] Comparative Example 5: Tissue Culture Method of Hemiptera:

[0090] In contrast to Example 4, the only difference is that in Comparative Example 5, the amount of isopropyl acetate added to the proliferation-rooting integrated culture medium during tissue culture of Hemiptera: Acer truncatula is 5 g / L, and the remaining steps are the same as in Example 4.

[0091] Comparative Example 6: Tissue Culture Method of Semi-Maple

[0092] In contrast to Example 4, the only difference is that the stabilizer added to the tissue culture and proliferation-rooting integrated culture medium of Hemiptera in Comparative Example 6 is not sprayed with a coating liquid, and the remaining steps are the same as those in Example 4. The specific preparation method of the stabilizer is as follows:

[0093] 230g of sodium alginate was dissolved in water to prepare a 5wt% sodium alginate solution, and then 0.04g of 6-BA and 0.015g of NAA were added and mixed evenly. Then, the solution was slowly dripped into a 2wt% calcium chloride solution for gelation to obtain sodium alginate-coated spheres with a particle size of about 1mm. After washing with sterile water for 3 times, the spheres were dried at 45°C for 25min to obtain semi-dried sodium alginate-coated spheres, which served as a stabilizer.

[0094] Experiment: Tissue culture experiment of Hemiptera:

[0095] The vitrification rate (when transferred for proliferation and rooting culture), rooting rate (38 days after proliferation-rooting integrated culture) and transplanting survival rate (30 days after transplanting) of the tissue culture seedlings of Examples 4 to 6 and Comparative Examples 1 to 6 were recorded. In order to ensure the accuracy of the experimental structure, the experiment was repeated twice according to the methods of Examples 4-6 and Comparative Examples 1-6, for a total of 3 experiments, and the final average data were shown in Table 1.

[0096] Table 1

[0097]

[0098] According to the data analysis in Table 1:

[0099] (1) According to the method of the present invention, the vitrification incidence of Hemiptera was low when tissue culture was carried out. The rooting rate reached more than 85% after 38 days of integrated proliferation-rooting culture, and the transplant survival rate was high. This shows that the vitrification incidence of Hemiptera can be effectively reduced according to the method of the present invention. Most of the roots can be taken root in a relatively short time through integrated proliferation-rooting culture, and the survival rate of the rooted seedlings after transplantation is high. This can better realize the efficient tissue culture of Hemiptera and promote the development of the Hemiptera industry.

[0100] (2) In Comparative Example 1, cyclohexylamine was not added to the proliferation-rooting integrated culture medium during tissue culture, and isopropyl acetate was not added to the proliferation-rooting integrated culture medium during tissue culture in Comparative Example 2. The tissue culture seedlings in Comparative Examples 1 and 2 had weak absorption capacity for substances in the culture medium, low dry matter accumulation, and low degree of lignification, so the proportion of vitrified seedlings obtained during transfer was high.

[0101] (3) In comparative example 3, no stabilizer was added during tissue culture of Hemiptera: Maple Leaf, and thus the cytokinin concentration in the culture medium was low, and the vitrification phenomenon was reduced, but the growth of the tissue culture seedlings was restricted, and the rooting rate after transfer was low; in comparative example 4, no polyethylene glycol was added during tissue culture of Hemiptera: Maple Leaf, and the water absorption of the tissue culture seedlings increased, which promoted the formation of vitrified seedlings; in comparative example 5, too much isopropyl acetate was added during tissue culture of Hemiptera: Maple Leaf, which excessively damaged the cell membrane of the tissue culture seedlings and affected their growth and seedling quality; in comparative example 6, no epoxy resin coating liquid was used for spraying during tissue culture of Hemiptera: Maple Leaf, and after adding the culture medium, the Hemiptera: Maple Leaf rapidly absorbed water and expanded, releasing most of the 6-BA. The tissue culture seedlings absorbed too much 6-BA in a short period of time, causing the vitrification phenomenon to worsen.

[0102] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention. The techniques, shapes, and structural parts not described in detail in the present invention are all known technologies.

Claims

1. A tissue culture method of Hemiptera: characterized in that: The method is as follows: (1) Explant collection and processing: Collect branches from mother trees, cut lignified stem segments and remove leaves, and wash to obtain explants; (2) Explant disinfection: The explants were cut into stem segments with 1 to 2 axillary buds and 2 to 3 cm in length, washed with sterile water, and then placed in an ethanol solution and a mercuric chloride solution for disinfection to obtain disinfected explants; (3) Induction culture: inoculating the sterilized explants into an induction culture medium for induction culture to obtain adventitious buds; (4) Proliferation-rooting integrated culture: single buds are cut from the adventitious buds obtained by the induced culture, and then inoculated into a proliferation-rooting integrated culture medium for proliferation-rooting integrated culture to obtain rooted seedlings; (5) Hardening and transplanting: Harden the rooted seedlings. After hardening, take them out of the tissue culture bottle, wash off the root culture medium, soak them in carbendazim solution for 20 minutes, and then transplant them into the substrate tray.

2. The tissue culture method of Hemiptera: Acacia according to claim 1, characterized in that: The induction medium components in step (3) are: MS+1.0-1.2 mg / L 6-BA+0.2-0.5 mg / L NAA+30 g / L sucrose+6.8 g / L agar, pH=5.

8.

3. The tissue culture method of Hemiptera: Acacia according to claim 2, characterized in that: The proliferation-rooting integrated culture medium in step (4) is a solid-liquid double-layer culture medium, wherein the upper layer is a liquid proliferation culture medium and the lower layer is a solid rooting culture medium. The proliferation-rooting integrated culture medium comprises the following raw materials: Upper liquid proliferation medium: MS + 0.2-0.3 mg / L 6-BA + 0.05-0.15 mg / L NAA + 30 g / L sucrose + 2-3 g / L cyclohexylamine + 2-3 g / L isopropyl acetate + 3-4 g / L polyethylene glycol + 8-15 g / L stabilizer, pH = 5.8; Lower layer solid rooting medium: MS + 0.6-0.8 mg / L IBA + 0.4 mg / L NAA + 0.6 mg / LABT + 30 g / L sucrose + 6.8 g / L agar, pH = 5.

8.

4. The tissue culture method of Hemiptera: Acacia according to claim 3, characterized in that: The method for preparing the proliferation-rooting integrated culture medium is as follows: According to the component ratio of the lower solid rooting medium, IBA, NAA and ABT are added to the MS medium, mixed evenly, then agar is added, and then heated until the agar is dissolved, sucrose is added, stirred to dissolve and mixed evenly, the pH is adjusted to 5.8, and then put into a culture bottle, and sterilized at high temperature at 121°C for 20 minutes; according to the component ratio of the upper liquid proliferation medium, 6-BA, NAA, sucrose, cyclohexylamine, isopropyl acetate and polyethylene glycol are added to the MS medium, stirred and mixed evenly, and then a stabilizer is added to mix evenly, sterilized at 121°C for 20 minutes, and added after the lower solid rooting medium in the culture bottle is solidified, to obtain a proliferation-rooting integrated medium.

5. The tissue culture method of Hemiptera: Acacia according to claim 4, characterized in that: The stabilizer comprises the following raw materials: Sodium alginate, 6-BA, NAA, 2wt% calcium chloride solution, rice bran wax, triethylamine, thiourea, 2,4,6-tris(dimethylaminomethyl)phenol, epoxy resin.

6. The tissue culture method of Hemiptera: Acacia according to claim 5, characterized in that: The stabilizer preparation method is as follows: A: Dissolve sodium alginate in water to prepare a 5wt% sodium alginate solution, then add 6-BA and NAA and mix well, then slowly drop into a 2wt% calcium chloride solution to gel to obtain sodium alginate coated spheres, wash with sterile water for 3 times, and dry at 45°C for 20-30 min to obtain semi-dried sodium alginate coated spheres for standby use; B: Grind rice bran wax through a 300-mesh sieve to obtain rice bran wax microparticles; put triethylamine, thiourea, and 2,4,6-tris(dimethylaminomethyl)phenol into a reaction kettle and mix them evenly, heat to 110-130° C. and react for 0.5-1h, then cool to 55° C., add epoxy resin, mix well, and then add rice bran wax microparticles and mix evenly to obtain a coating solution; C: Spray the coating liquid onto the surface of the semi-dried sodium alginate coated ball, place it in a cool and ventilated place for 12 to 24 hours, then place it at 60 to 70°C for 2 to 3 hours to allow the rice bran wax particles on the surface of the coated ball to melt and flow out, and then wash it with deionized water 3 to 5 times to obtain a stabilizer.

7. The tissue culture method of Hemiptera: Acacia according to claim 6, characterized in that: The mass ratio of the sodium alginate solution, 6-BA and NAA in step A is (160-300): (0.03-0.05): (0.01-0.02).

8. The tissue culture method of Hemiptera: Acacia according to claim 7, characterized in that: In the step B, the mass ratio of triethylamine, thiourea, 2,4,6-tris(dimethylaminomethyl)phenol, epoxy resin and rice bran wax particles is (8-16):(10-20):(10-20):(50-70):(10-20).

9. The tissue culture method of Hemiptera: Acacia according to claim 8, characterized in that: The induction culture temperature is 25-27°C, the illumination time is 8-12h / d, and the illumination intensity is 2000-3000lx; the proliferation-rooting integrated culture temperature is 25-27°C, the illumination time is 8-12h / d, and the illumination intensity is 4500-5000lx.

10. The tissue culture method of Hemiptera: Acacia according to claim 9, characterized in that: The particle size of the stabilizer is 0.5-1 mm.

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