CN117837497A - Two-step seedling method for rapid propagation of cinnamon buds - Google Patents
Two-step seedling method for rapid propagation of cinnamon buds Download PDFInfo
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- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 claims description 12
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Abstract
The invention discloses a two-step seedling method for rapid propagation of cinnamon buds, which comprises the following steps: collecting tender buds of cinnamon as explants, sterilizing the explants, performing anti-browning treatment, inoculating the explants into an induction culture medium, performing induction culture to obtain cluster buds, and continuously culturing until the proliferation coefficient of the cluster buds reaches more than 5; cutting off robust single buds with bud height more than 2cm, transferring the single buds into a rooting induction culture medium, continuously culturing to obtain rooting tissue culture seedlings with bud height of 3-6 cm and root length of 1-2 cm, and then hardening off and transplanting. The cluster buds produced by the invention have excellent quality and large quantity, the hereditary character is stable, and the proliferation coefficient reaches more than 5; the invention has simple operation process, short culture period, low production cost and good economic, ecological and social benefits.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture methods in agricultural biotechnology, relates to a tissue culture technology of perennial woody plants, and particularly relates to a two-step seedling method for rapid propagation of cinnamon buds.
Background
At present, the propagation mode of cinnamon is mainly seed propagation, and because cinnamon seeds belong to recalcitrant seeds, seedling raising is generally carried out in a mode of picking and sowing at the same time in production. The traditional seed propagation has the defects of high seedling raising cost, uneven seedlings, insufficient fine variety potential and the like, thereby leading to the lack of good cinnamon varieties in the current industry. For perennial tree species, tissue culture techniques can provide an effective way to develop a large number of elite clones in a short period of time. Zhang Shi in 2018, a tissue culture and rapid propagation method of cinnamon (CN 109392717A) takes a twig as an explant, 3 steps of induction, proliferation and rooting are needed, the time consumption is long, the cost is high, and the proliferation coefficient is not detailed; recently, the agricultural university of south China discloses a method for establishing a sterile culture system by taking seeds as explants in the same genus of the plant of Vietnam cortex Cinnamomi (CN 115707378A), but the induction of cluster buds is not realized. Therefore, the current research on the technique of tissue culture and rapid propagation of cinnamon is less, and particularly, the tissue culture and rapid propagation technique system which uses tender buds as explants severely restricts the breeding process of excellent clone of cinnamon. The efficient tissue culture and rapid propagation technology system of cinnamon is established, and the method has very important significance for breeding of excellent varieties of cinnamon.
Disclosure of Invention
It is an object of the present invention to solve at least the above problems and to provide at least the advantages to be described later.
The invention aims to provide a two-step seedling method for rapid propagation of cinnamon buds, which can realize simultaneous induction and propagation of cluster buds in an induction stage, and can omit links of secondary propagation and strong seedling culture so as to realize two-step seedling, thereby shortening the culture period, improving the propagation coefficient and reducing the production cost.
To achieve these objects and other advantages and in accordance with the purpose of the invention, there is provided a two-step seedling method for rapid propagation of cinnamon buds, comprising the steps of:
collecting tender buds of cinnamon as explants, sterilizing the explants, performing anti-browning treatment, inoculating the explants into an induction culture medium, performing induction culture to obtain cluster buds, and continuously culturing until the proliferation coefficient of the cluster buds reaches more than 5;
cutting off robust single buds with bud height more than 2cm, transferring the single buds into a rooting culture medium, continuously culturing to obtain rooting tissue culture seedlings with bud height of 3-6 cm and root length of 1-2 cm, and then hardening off and transplanting.
Preferably, the induction medium is prepared by adding the following raw material components into a modified WPM medium: 4.0-6.0 mg/L, KT 0.5.5-1.0 mg/L, GA of 6-BA 3 0.2-0.6 mg/L, NAA 0.2-0.5 mg/L Active Carbon (AC)
0.1 to 0.2 percent, 0.8 to 1.5mg/L of riboflavin, 5.0 to 10.0mg/L of Vitamin C (VC), 25 to 30g/L of sucrose and 5.0 to 7.0g/L, pH of agar are 6.0.
Preferably, the formulation of the modified WPM medium comprises: KNO (KNO) 3 200mg/L、Ca(NO 3 ) 2 .4H 2 O 250mg/L、(NH 4 ) 2 SO 4 750mg/L、MgSO 4 ·7H 2 O 400mg/L、CaCl 2 ·2H 2 O 150mg/L、KH 2 PO 4 300mg/L、Na 2 MoO 4 ·2H 2 O 0.25mg/L、MnSO 4 ·H 2 O 22.4mg/L、ZnSO 4 ·7H 2 O 15.0mg/L、CuSO 4 ·5H 2 O 0.25mg/L、FeSO 4 ·7H 2 O 27.8mg/L、Na 2 EDTA37.3mg/L, inositol 100mg/L, vitamin B 1 1.0mg/L, nicotinic acid 0.5mg/L, vitamin B 6 0.5mg/L, glycine 2.0mg/L.
Preferably, the rooting medium is prepared by adding the following raw material components into a 1/2MS medium: IBA 0.8-1.5 mg/L, NAA 0.3-0.8 mg/L, AC 0.1.1-0.15%, sucrose 25-30 g/L, agar 5.0-7.0 g/L, pH is 6.0.
Preferably, the conditions for induction culture are: the temperature is 24-28 ℃, the illumination intensity is 2000Lx, and the illumination time is 10 h/day; the rooting culture conditions are as follows: the temperature is 24-28 ℃, the illumination intensity is 1 000Lx, and the illumination time is 10 h/day.
Preferably, the sterilization and browning prevention treatment of the explant specifically comprises the following steps:
s1, immediately placing cinnamon buds after collection in a refrigerator at 4 ℃ for cold treatment for 24 hours;
s2, taking out the cinnamon buds refrigerated in the step S1, soaking the cinnamon buds in detergent water with the volume concentration of 0.05% for 15min, taking out the cinnamon buds, flushing the cinnamon buds with running water for 30min, and then soaking the cinnamon buds in NaClO solution with the volume concentration of 2% on an ultra-clean workbench for 10min;
s3, taking out the cinnamon buds treated in the step S2, soaking in the anti-browning combination liquid subjected to filtration sterilization for 5min, and then inoculating into an induction culture medium.
Preferably, the formula of the anti-browning combination liquid comprises: 50-200 mg/L of cysteine salt, 3-5 g/L of polyvinylpyrrolidone, 50-100 ml/L of cinnamon water extract and 1.0-3.0 g/L of agar.
Preferably, the cinnamon water extract is obtained by crushing cinnamon, sieving with a 50-mesh sieve, mixing with purified water according to the mass ratio of 1:5-10, soaking for 3 hours, heating and boiling for 30 minutes, and filtering.
Preferably, the specific seedling hardening and transplanting method comprises the following steps: the rooting tissue culture seedlings are subjected to cover hardening in the room for 3-5 days, and roots of the rooting tissue culture seedlings are cleaned to obtain transplanting seedlings; soaking the transplanted seedlings in a disinfectant for 30 seconds, then transplanting the transplanted seedlings to a seedling bed, keeping the humidity of the seedling bed to be 80-90% 10 days before transplanting, keeping the humidity of the seedling bed to be 60-80% 10 days after transplanting, controlling the temperature in the transplanting process to be 22-30 ℃, growing new leaves of the transplanted seedlings, and spraying compound fertilizer liquid once every 10-15 days.
Preferably, the formula of the disinfectant comprises 1-1.5 g/L of potassium permanganate and 0.5-1 g/L of carbendazim;
the matrix on the seedling bed is obtained by mixing perlite and seedling soil according to the volume ratio of 1:2, and the pH value is 4.5-6.5;
sterilizing the substrate by using a potassium permanganate solution with mass fraction of 0.2% -0.3% 12-24 hours before transplanting;
the mass fraction of the compound fertilizer liquid is 0.1-0.3%, and the mass ratio of N to P to K in the compound fertilizer liquid is 1:1:1.
The invention at least comprises the following beneficial effects:
the whole set of method provided by the invention is simple and convenient to operate, low in production cost, high in proliferation coefficient and high in transplanting survival rate, is convenient for large-scale industrial production and application, can rapidly and continuously obtain high-quality transplanting seedlings in a large quantity, and has strong practicability;
secondly, the invention realizes the simultaneous carrying out of the induction and proliferation culture of the cinnamon cluster buds in the cluster bud induction stage, and can omit the link of subculture proliferation and strong seedling, thereby realizing the two-step seedling formation of the rapid propagation of the cinnamon aseptic seedlings, greatly shortening the period and obviously reducing the production cost;
thirdly, the formula of the induction culture medium can reach a proliferation coefficient of more than 5, and can realize better economic benefit.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a culture flow chart of the present invention;
FIG. 2 is the result of the induction effect of anti-browning treatment on cluster buds, wherein A is treated with the soaking anti-browning combination liquor; b is the treatment of the non-soaked anti-browning combined liquid;
FIG. 3 is the result of the influence of the culture medium on the induction of the cluster buds, wherein A is the modified WPM minimal medium; b is WPM minimal medium.
Detailed Description
The present invention is described in further detail below with reference to the drawings to enable those skilled in the art to practice the invention by referring to the description.
It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
The experimental methods described in the following embodiments are conventional methods unless otherwise indicated, and the reagents and materials are commercially available.
As shown in fig. 1, the invention provides a two-step seedling method for rapid propagation of cinnamon buds, which comprises the following steps:
collecting tender buds of cinnamon as explants, sterilizing the explants, performing anti-browning treatment, inoculating the explants into an induction culture medium, performing induction culture to obtain cluster buds, and continuously culturing until the proliferation coefficient of the cluster buds reaches more than 5;
cutting off robust single buds with bud height more than 2cm, transferring the single buds into a rooting culture medium, continuously culturing to obtain rooting tissue culture seedlings with bud height of 3-6 cm and root length of 1-2 cm, and then hardening off and transplanting.
In another technical scheme, the induction culture medium is prepared by adding the following raw material components into an improved WPM culture medium: 4.0-6.0 mg/L, KT 0.5.5-1.0 mg/L, GA of 6-BA 3 0.2 to 0.6mg/L, NAA 0.2.2 to 0.5mg/L, 0.1 to 0.2 percent of Activated Carbon (AC), 0.8 to 1.5mg/L of riboflavin, 5.0 to 10.0mg/L of Vitamin C (VC), 25 to 30g/L of sucrose and 5.0 to 7.0g/L, pH of agar are 6.0.
In another embodiment, the formulation of the modified WPM medium comprises: KNO (KNO) 3 200mg/L、Ca(NO 3 ) 2 .4H 2 O 250mg/L、(NH 4 ) 2 SO 4 750mg/L、MgSO 4 ·7H 2 O 400mg/L、CaCl 2 ·2H 2 O 150mg/L、KH 2 PO 4 300mg/L、Na 2 MoO 4 ·2H 2 O 0.25mg/L、MnSO 4 ·H 2 O 22.4mg/L、ZnSO 4 ·7H 2 O15.0 mg/L、CuSO 4 ·5H 2 O 0.25mg/L、FeSO 4 ·7H 2 O 27.8mg/L、Na 2 EDTA37.3mg/L, inositol 100mg/L, vitamin B 1 1.0mg/L, nicotinic acid 0.5mg/L, vitamin B 6 0.5mg/L, glycine 2.0mg/L.
In another technical scheme, the rooting medium is prepared by adding the following raw material components into a 1/2MS medium: IBA 0.8-1.5 mg/L, NAA 0.3-0.8 mg/L, AC 0.1.1-0.15%, sucrose 25-30 g/L, agar 5.0-7.0 g/L, pH is 6.0.
In another embodiment, the conditions for induction culture are: the temperature is 24-8 ℃, the illumination intensity is 2000Lx, and the illumination time is 10 h/day; the rooting culture conditions are as follows: the temperature is 24-28 ℃, the illumination intensity is 1 000Lx, and the illumination time is 10 h/day.
In another technical scheme, the sterilization and browning prevention treatment of the explant specifically comprises the following steps:
s1, immediately placing cinnamon buds after collection in a refrigerator at 4 ℃ for cold treatment for 24 hours;
s2, taking out the cinnamon buds refrigerated in the step S1, soaking the cinnamon buds in detergent water with the volume concentration of 0.05% for 15min, taking out the cinnamon buds, flushing the cinnamon buds with running water for 30min, and then soaking the cinnamon buds in NaClO solution with the volume concentration of 2% on an ultra-clean workbench for 10min;
s3, taking out the cinnamon buds treated in the step S2, soaking in the anti-browning combination liquid subjected to filtration sterilization for 5min, and then inoculating into an induction culture medium.
In another technical scheme, the formula of the anti-browning combination liquid comprises the following components: 50-200 mg/L of cysteine salt, 3-5 g/L of polyvinylpyrrolidone, 50-100 ml/L of cinnamon water extract and 1.0-3.0 g/L of agar.
In another technical scheme, the cinnamon water extract is obtained by crushing cinnamon materials, sieving with a 50-mesh sieve, mixing with purified water according to a mass ratio of 1:5-10, soaking for 3 hours, heating and boiling for 30 minutes, and filtering.
In another technical scheme, the seedling hardening and transplanting specific method comprises the following steps: the rooting tissue culture seedlings are subjected to cover hardening in the room for 3-5 days, and roots of the rooting tissue culture seedlings are cleaned to obtain transplanting seedlings; soaking the transplanted seedlings in a disinfectant for 30 seconds, then transplanting the transplanted seedlings to a seedling bed, keeping the humidity of the seedling bed to be 80-90% 10 days before transplanting, keeping the humidity of the seedling bed to be 60-80% 10 days after transplanting, controlling the temperature in the transplanting process to be 22-30 ℃, growing new leaves of the transplanted seedlings, and spraying compound fertilizer liquid once every 10-15 days.
In another technical scheme, the formula of the disinfectant comprises 1-1.5 g/L of potassium permanganate and 0.5-1 g/L of carbendazim;
the matrix on the seedling bed is obtained by mixing perlite and seedling soil according to the volume ratio of 1:2, and the pH value is 4.5-6.5;
sterilizing the substrate by using a potassium permanganate solution with mass fraction of 0.2% -0.3% 12-24 hours before transplanting;
the mass fraction of the compound fertilizer liquid is 0.1-0.3%, and the mass ratio of N to P to K in the compound fertilizer liquid is 1:1:1.
The induction rate, the multiplication factor, the rooting rate and the transplanting survival rate have the following calculation formulas:
inductivity (%) = (number of shoots induced into clump of shoots/number of shoots inoculated) ×100%;
multiplication times = number of effective shoots/number of inoculated shoots greater than 1.0cm in height;
rooting rate (%) = (number of rooted shoots/number of inoculated shoots) ×100%;
transplanting survival rate (%) = (number of surviving tissue culture seedlings/number of transplanted tissue culture seedlings) ×100%.
Example 1 ]
A two-step seedling method for rapid propagation of cinnamon buds comprises the following steps:
collecting cinnamon buds as explants, sterilizing the explants, performing anti-browning treatment, inoculating the explants into an induction culture medium, performing induction culture for 25 days to obtain cluster buds, and continuously culturing for 30 days until the proliferation coefficient of the cluster buds reaches more than 5;
cutting off robust single buds with bud height more than 2cm, transferring the single buds into a rooting culture medium, continuously culturing to obtain rooting tissue culture seedlings with bud height of 3-6 cm and root length of 1-2 cm, and then hardening off and transplanting the seedlings;
the induction culture medium is prepared by adding the following raw material components into an improved WPM culture medium: 6-BA 5.0mg/L, KT 1.0.0 mg/L, GA 3 0.5mg/L, NAA 0.2.2 mg/L, 0.2mg/L Active Carbon (AC), 1.0mg/L riboflavin, 8.0mg/L Vitamin C (VC), 25g/L sucrose, 5.5g/L, pH agar, 6.0;
the formula of the improved WPM culture medium comprises the following components: KNO (KNO) 3 200mg/L、Ca(NO 3 ) 2 .4H 2 O 250mg/L、(NH 4 ) 2 SO 4 750mg/L、MgSO 4 ·7H 2 O 400mg/L、CaCl 2 ·2H 2 O 150mg/L、KH 2 PO 4 300mg/L、Na 2 MoO 4 ·2H 2 O 0.25mg/L、MnSO 4 ·H 2 O 22.4mg/L、ZnSO 4 ·7H 2 O15.0 mg/L、CuSO 4 ·5H 2 O0.25mg/L、FeSO 4 ·7H 2 O 27.8mg/L、Na 2 EDTA37.3mg/L, inositol 100mg/L, vitamin B 1
1.0mg/L, nicotinic acid 0.5mg/L, vitamin B 6 0.5mg/L, glycine 2.0mg/L;
the rooting culture medium is prepared by adding the following raw material components into a 1/2MS culture medium: IBA1.0mg/L, NAA0.5mg/L, AC 0.15.15%, sucrose 25g/L, agar 6.0g/L, pH as 6.0;
the sterilization and browning prevention treatment of the explant specifically comprises the following steps:
s1, immediately placing cinnamon buds after collection in a refrigerator at 4 ℃ for cold treatment for 24 hours;
s2, taking out the cinnamon buds refrigerated in the step S1, soaking the cinnamon buds in detergent water with the volume concentration of 0.05% for 15min, taking out the cinnamon buds, flushing the cinnamon buds with running water for 30min, and then soaking the cinnamon buds in NaClO solution with the volume concentration of 2% on an ultra-clean workbench for 10min;
s3, taking out the cinnamon buds treated in the step S2, soaking in the anti-browning combination liquid subjected to filtration sterilization for 5min, and then inoculating into an induction culture medium;
the seedling hardening and transplanting concrete method comprises the following steps: the rooting tissue culture seedlings are subjected to cover hardening in an indoor environment for 3 days, and roots of the rooting tissue culture seedlings are cleaned to obtain transplanting seedlings; soaking the transplanted seedlings in a disinfectant for 30 seconds, then transplanting the transplanted seedlings to a seedling bed, keeping the humidity of the seedling bed to be 80% -90% 10 days before transplanting, keeping the humidity of the seedling bed to be 60% -80% 10 days after transplanting, controlling the temperature to be 25 ℃ in the transplanting process, growing new leaves of the transplanted seedlings, and spraying compound fertilizer liquid once every 10 days;
the formula of the anti-browning combination liquid comprises the following components: cysteine 100mg/L, polyvinylpyrrolidone 4g/L, cinnamon aqueous extract 80ml/L, agar 2.0g/L;
the formula of the disinfectant comprises 1.0g/L potassium permanganate and 1.0g/L carbendazim;
the matrix on the seedling bed is obtained by mixing perlite and seedling soil according to the volume ratio of 1:2, and the pH value is 6.5;
sterilizing the substrate with a potassium permanganate solution with the mass fraction of 0.2% for 12 hours before transplanting;
the mass fraction of the compound fertilizer liquid is 0.1%, and the mass ratio of N to P to K in the compound fertilizer liquid is 1:1:1.
The method provided by the embodiment can realize the induction and proliferation of the cinnamon cluster buds, the induction rate of the cluster buds is 100%, the proliferation multiple is 5.86 times, and simultaneously, the two-step seedling formation can be realized, the rooting rate reaches 100%, and the transplanting survival rate reaches 88.2%.
Example 2 ]
A two-step seedling method for rapid propagation of cortex Cinnamomi tender bud is the same as that provided in example 1, except for the formulation of induction medium and rooting medium, in this example, the following raw materials are added into modified WPM medium to prepare 6-BA4.0mg/L, KT 0.5.5 mg/L, GA 3 0.2mg/L, NAA 0.2.2 mg/L, 0.1% Active Carbon (AC), 1.5mg/L, VC 5.0.0 mg/L riboflavin, 25g/L sucrose, 5.0g/L, pH agar, 6.0. The rooting culture medium is prepared by adding the following raw material components into a 1/2MS culture medium to prepare IBA0.8mg/L, NAA 0.3.3 mg/L, AC 0.1.1%, sucrose 25g/L and agar 5.0g/L, pH of 6.0.
The method of the embodiment can realize the induction and proliferation of the cinnamon cluster buds, the induction rate of the cluster buds is 100%, the proliferation multiple is 5.63 times, and simultaneously can realize two-step seedling, the rooting rate reaches 100%, and the transplanting survival rate reaches 87.1%.
Example 3 ]
A two-step seedling method for rapid propagation of cortex Cinnamomi tender bud is the same as that provided in example 1, except for the formulation of induction medium and rooting medium, in this example, the following raw materials are added into modified WPM medium to prepare 6-BA6.0mg/L, KT 1.0mg/L, GA 3 0.6mg/L NAA0.5mg/L, AC 0.2.2%, riboflavin 1.5mg/L, VC 10.0.0 mg/L sucrose30g/L, agar 7.0g/L, pH is 6.0. The rooting culture medium is prepared by adding the following concentration component raw materials into a 1/2MS culture medium to prepare IBA1.5mg/L, NAA 0.8mg/L, AC 0.15.15%, sucrose 30g/L and agar 7.0g/L, pH of 6.0.
The method of the embodiment can realize the induction and proliferation of the cinnamon cluster buds, the induction rate of the cluster buds is 100%, the proliferation multiple is 5.02 times, and simultaneously, the two-step seedling formation can be realized, the rooting rate reaches 100%, and the transplanting survival rate reaches 85.7%.
Comparative example 1 ]
A two-step seedling method for rapid propagation of cinnamon buds is the same as that provided in example 1, except that the rooting medium is prepared by adding the following raw material components of IBA 0.5mg/L, AC 0.1.1%, sucrose 25g/L, agar 5.5g/L and pH 6.0 into a 1/2MS medium.
The method of the embodiment can realize the rooting of the aseptic buds of the cinnamon, but the rooting rate is 67.5%, the roots are slender, and the transplanting survival rate is 45.3%.
Comparative example 2 ]
A two-step seedling method for rapid propagation of cinnamon buds is the same as that provided in example 1, except that the rooting medium is prepared by adding the following raw material components into an MS medium to prepare 6-BA 5.0mg/L, NAA0.2 mg/L, AC 0.2.2%, sucrose 25g/L, agar 5.5g/L and pH 6.0.
The method of this comparative example did not induce cinnamon cluster buds.
Comparative example 3 ]
A two-step seedling method for rapid propagation of cortex Cinnamomi tender bud is the same as that provided in example 1, except for the formulation of rooting medium, and the induction medium in this example is prepared by adding the following raw materials into MS medium 3 0.5mg/L, NAA 0.2.2 mg/L, AC 0.2.2%, sucrose 25g/L, agar 5.5g/L, pH 6.0.
The method of this comparative example did not induce cinnamon cluster buds.
Comparative example 4 ]
A two-step seedling method for rapid propagation of cinnamon buds is the same as that provided in example 1, except that no anti-browning treatment is performed, namely, sterilization is performed after the cinnamon buds are collected, and then the cinnamon buds are placed in an induction medium for induction culture.
The results are shown in FIG. 2. The method of the comparative example cannot achieve a good browning prevention effect, all the buds are serious in browning after sterilization treatment, and cluster buds cannot be induced by culturing in an induction medium.
Comparative example 5 ]
A two-step seedling method for rapid propagation of cinnamon buds is the same as that provided in example 1, except that WPM minimal medium is used instead of modified WPM minimal medium.
The results are shown in FIG. 3. Although the method of the comparative example can realize the induction and proliferation of the cinnamon cluster buds, the induction rate of the cluster buds is reduced to 67.3% from 100%, the proliferation multiple is reduced to 2.37 from 5.86, and leaves of the cluster buds are easy to yellow and fall off, so that the rooting culture of the next step is seriously influenced.
Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.
Claims (10)
1. The two-step seedling method for rapid propagation of cinnamon buds is characterized by comprising the following steps:
collecting tender buds of cinnamon as explants, sterilizing the explants, performing anti-browning treatment, inoculating the explants into an induction culture medium, performing induction culture to obtain cluster buds, and continuously culturing until the proliferation coefficient of the cluster buds reaches more than 5;
cutting off robust single buds with bud height more than 2cm, transferring the single buds into a rooting culture medium, continuously culturing to obtain rooting tissue culture seedlings with bud height of 3-6 cm and root length of 1-2 cm, and then hardening off and transplanting.
2. The two-step seedling method for rapid propagation of cinnamon buds according to claim 1, wherein the induction medium is prepared by adding the following raw material components into an improved WPM medium: 4.0-6.0 mg/L, KT 0.5.5-1.0 mg/L, GA of 6-BA 3 0.2 to 0.6mg/L, NAA 0.2.2 to 0.5mg/L, 0.1 to 0.2 percent of activated carbon, 0.8 to 1.5mg/L of riboflavin, 5.0 to 10.0mg/L of vitamin c, 25 to 30g/L of sucrose and 5.0 to 7.0g/L, pH of agar of 6.0.
3. The two-step seedling method for rapid propagation of cinnamon buds as claimed in claim 2, wherein the formulation of the modified WPM medium comprises: KNO (KNO) 3 200mg/L、Ca(NO 3 ) 2 .4H 2 O 250mg/L、(NH 4 ) 2 SO 4 750mg/L、MgSO 4 ·7H 2 O 400mg/L、CaCl 2 ·2H 2 O 150mg/L、KH 2 PO 4 300mg/L、Na 2 MoO 4 ·2H 2 O0.25mg/L、MnSO 4 ·H 2 O 22.4mg/L、ZnSO 4 ·7H 2 O 15.0mg/L、CuSO 4 ·5H 2 O 0.25mg/L、FeSO 4 ·7H 2 O 27.8mg/L、Na 2 EDTA37.3mg/L, inositol 100mg/L, vitamin B 1 1.0mg/L, nicotinic acid 0.5mg/L, vitamin B 6 0.5mg/L, glycine 2.0mg/L.
4. The two-step seedling method for rapid propagation of cinnamon buds according to claim 1, wherein the rooting medium is prepared by adding the following raw material components in a 1/2MS medium: IBA 0.8-1.5 mg/L, NAA 0.3-0.8 mg/L, AC 0.1.1-0.15%, sucrose 25-30 g/L, agar 5.0-7.0 g/L, pH is 6.0.
5. The two-step seedling method for rapid propagation of cinnamon buds as claimed in claim 1, wherein the conditions of the induction culture are as follows: the temperature is 24-28 ℃, the illumination intensity is 2000Lx, and the illumination time is 10 h/day; the rooting culture conditions are as follows: the temperature is 24-28 ℃, the illumination intensity is 1 000Lx, and the illumination time is 10 h/day.
6. The two-step seedling method for rapid propagation of cinnamon buds according to claim 1, wherein the sterilization and browning prevention treatment of the explant comprises the following steps:
s1, immediately placing cinnamon buds after collection in a refrigerator at 4 ℃ for cold treatment for 24 hours;
s2, taking out the cinnamon buds refrigerated in the step S1, soaking the cinnamon buds in detergent water with the volume concentration of 0.05% for 15min, taking out the cinnamon buds, flushing the cinnamon buds with running water for 30min, and then soaking the cinnamon buds in NaClO solution with the volume concentration of 2% on an ultra-clean workbench for 10min;
s3, taking out the cinnamon buds treated in the step S2, soaking in the anti-browning combination liquid subjected to filtration sterilization for 5min, and then inoculating into an induction culture medium.
7. The two-step seedling method for rapid propagation of cinnamon buds as in claim 6, wherein the anti-browning composition comprises the following components: 50-200 mg/L of cysteine salt, 3-5 g/L of polyvinylpyrrolidone, 50-100 ml/L of cinnamon water extract and 1.0-3.0 g/L of agar.
8. The two-step seedling method for rapid propagation of cinnamon buds according to claim 7, wherein the cinnamon water extract is obtained by crushing cinnamon materials, sieving with a 50-mesh sieve, mixing with purified water according to a mass ratio of 1:5-10, soaking for 3h, heating and boiling for 30min, and filtering.
9. The two-step seedling method for rapid propagation of cinnamon buds according to claim 1, wherein the specific seedling hardening and transplanting method is as follows: the rooting tissue culture seedlings are subjected to cover hardening in the room for 3-5 days, and roots of the rooting tissue culture seedlings are cleaned to obtain transplanting seedlings; soaking the transplanted seedlings in a disinfectant for 30 seconds, then transplanting the transplanted seedlings to a seedling bed, keeping the humidity of the seedling bed to be 80-90% 10 days before transplanting, keeping the humidity of the seedling bed to be 60-80% 10 days after transplanting, controlling the temperature in the transplanting process to be 22-30 ℃, growing new leaves of the transplanted seedlings, and spraying compound fertilizer liquid once every 10-15 days.
10. The two-step seedling method for rapid propagation of cinnamon buds according to claim 9, wherein the disinfectant comprises 1-1.5 g/L potassium permanganate and 0.5-1 g/L carbendazim;
the matrix on the seedling bed is obtained by mixing perlite and seedling soil according to the volume ratio of 1:2, and the pH value is 4.5-6.5;
sterilizing the substrate by using a potassium permanganate solution with mass fraction of 0.2% -0.3% 12-24 hours before transplanting;
the mass fraction of the compound fertilizer liquid is 0.1-0.3%, and the mass ratio of N to P to K in the compound fertilizer liquid is 1:1:1.
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