CN113383707B - Method for establishing high-efficiency in-vitro regeneration system of Qishu mature embryos - Google Patents
Method for establishing high-efficiency in-vitro regeneration system of Qishu mature embryos Download PDFInfo
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Abstract
The invention discloses a method for establishing an efficient in-vitro regeneration system of an Qishu mature embryo, which comprises the following steps: selecting and pretreating seeds, and applying low-temperature plasma activated water to a pretreatment process; inoculating the pretreated mature embryo into an adventitious bud induction culture medium, and performing adventitious bud induction culture; transferring the mature embryo with the adventitious bud induced to a multiplication culture medium for multiplication culture of the adventitious bud; cutting the proliferated adventitious bud into pieces with proper size, and transferring the pieces into an elongation culture medium for elongation culture of adventitious buds; and when the adventitious bud grows to 2-3 cm and is accompanied with complete leaves, transferring the adventitious bud into a rooting culture medium for rooting culture, and finally obtaining a complete keemun regenerated plant. The invention applies low-temperature plasma activated water to the pretreatment process, and then realizes the rapid propagation of the keemun germchit by directly inducing the regeneration of the cluster buds, thereby providing powerful technical guarantee for the efficient propagation and the large-scale production of the keemun germchit.
Description
Technical Field
The invention belongs to the technical field of plant biology, and particularly relates to a method for establishing an efficient in-vitro regeneration system of an Qishu mature embryo.
Background
The keemun refers to a perennial medicinal plant of atractylodes of Compositae, which is named after being produced in Qimen, and the rhizome of the keemun can be used as a medicine, is one of the most representative traditional Chinese medicinal materials of Qimen, and is also a famous and precious medicinal material of Anhui-dao. A large number of clinical studies of traditional Chinese medicine show that the keemun skill is warm in nature, sweet in taste and slightly bitter, has the effects of strengthening spleen and tonifying qi, eliminating dampness and diuresis, tranquilizing mind, preventing miscarriage, eliminating fatigue and the like of common bighead atractylodes rhizome, and has remarkable treatment effects on jaundice, heart diseases, gastric ulcer and ascites. Besides the properties of rare medicinal materials, Qishu can be directly eaten as a tonic, and can be made into Qishu cake after being mixed with fried sesame or soybeans, glutinous rice and the like and ground into powder, so that the Qishu cake can be quickly recovered after being eaten by a long-term patient, and can prolong the life of a patient without the Qishu. Based on the remarkable curative effect, lasting medicinal effect and no greasiness, the keemun skilled person is used as a precious medicinal material tonic. In addition, the keemun technology is widely applied to industries such as beauty treatment and skin care, and has wide market application prospect.
Because the qishu has higher requirement on the growth environment, the qishu is pleasantly cool in nature, high temperature and water accumulation are avoided, and the growth area is only limited to shady slopes, valley woodlands and shrubs which are 1000m above the height of the yellow mountain and fertile in soil. In addition, because the keemun art grows slowly, flowers and seeds are few, part of the seeds have no vitality, and the ecological environment of the keemun art is seriously damaged along with the daily increase of the civil excavation amount, so that the wild keemun art is extremely sparsely distributed in the natural ecological environment. At present, keemun operas are usually bred in a traditional sowing mode in production, but have the problems of low seed germination rate, weak seedling resistance, susceptibility to the damage of various plant diseases and insect pests such as damping-off, root rot and the like. In addition, propagation of the keemun is feasible by utilizing rhizome, but propagation in such a way has the problems of high cost, low propagation speed, material limitation on propagation, easy introduction of diseases and the like because the rhizome is used as a main part for medicinal keemun. At present, the research on the propagation of Qishu by utilizing a tissue culture technology has been reported, but the problems of incomplete disinfection, low propagation coefficient and the like exist in the propagation process, and the large-scale production, popularization and application of Qishu seedlings are seriously limited. Aiming at a plurality of problems in the prior art of keemun breeding, a method for establishing a keemun mature embryo high-efficiency in-vitro regeneration system is urgently needed to meet the requirements of rapid propagation and variety improvement of keemun seedlings.
Disclosure of Invention
The invention aims to solve the technical problem of meeting the requirements of rapid propagation and variety improvement of keemun seedlings.
The invention solves the technical problems through the following technical means: a method for establishing an efficient in-vitro regeneration system of an Qishu mature embryo comprises the following steps:
(1) selecting and pretreating seeds, soaking the selected seeds in low-temperature plasma activated water subjected to filtration sterilization for 2-4 min, and then, absorbing water on the surfaces of the seeds by using sterile filter paper for later use;
(2) inoculating the pretreated mature embryo into an adventitious bud induction culture medium, and performing adventitious bud induction culture;
(3) transferring the mature embryo with the adventitious bud induced to a multiplication culture medium for multiplication culture of the adventitious bud;
(4) cutting the proliferated adventitious bud into pieces with proper size, and transferring the pieces into an elongation culture medium for elongation culture of adventitious buds;
(5) and when the adventitious bud grows to 2-3 cm and is accompanied with complete leaves, transferring the adventitious bud into a rooting culture medium for rooting culture, and finally obtaining a complete keemun regenerated plant.
The invention applies low-temperature plasma activated water to the pretreatment process of the keemun seeds, accelerates the start of mature embryo regeneration while reducing the pollution rate, and realizes the rapid propagation of keemun seedlings by directly inducing the regeneration of cluster buds.
Preferably, the selection and pretreatment of the seeds in the step (1) are that viable mature and full Keemu seeds are selected and placed in a 250ml plastic culture bottle and washed under running water for 10-20 min. Then placing the seeds in an aseptic operation table, washing the seeds with aseptic water for 3-4 times, then carrying out surface disinfection for 2 times by using 75% absolute ethyl alcohol, wherein the disinfection is carried out for 15-20 s each time, and the seeds are washed with the aseptic water for 2-3 times between 2 times; then sterilizing the mixture for 3-5 min by using 0.1% (w/v) mercuric chloride solution, and then washing the mixture for 6-8 times by using sterile water.
Preferably, in the low-temperature plasma activated water, Ar is an ion source, and double distilled water is treated for 2min through a dielectric barrier discharge device.
Preferably, the adventitious bud induction medium in the step (2) is: DKW + 0.5-2.0 mg/L ZT + 2.0-5.0 mg/L AgNO3+ 3.0% (w/v) sucrose + 0.7% (w/v) agar, pH 5.8.
Preferably, the adventitious bud induction medium in the step (2) is: DKW +1.0mg/L ZT +3.0mg/LAgNO3+ 3.0% (w/v) sucrose + 0.7% (w/v) agar, pH 5.8.
Preferably, the proliferation medium of the adventitious bud in the step (3) is: DKW + 0.2-1.5 mg/L ZT + 0.05-0.5 mg/L TDZ + 3.0% (w/v) sucrose + 0.7% (w/v) agar, pH 5.8.
Preferably, the proliferation medium of the adventitious bud in the step (3) is: the adventitious bud multiplication culture medium comprises: DKW +0.75mg/L ZT +0.25mg/L TDZ + 3.0% (w/v) sucrose + 0.7% (w/v) agar, pH 5.8.
Preferably, the elongation medium of the adventitious bud in the step (4) is: DKW + 0.05-0.5 mg/L GA3+0.2～10mg/L KT + 3.0% (w/v) sucrose + 0.7% (w/v) agar, pH 5.8.
Preferably, the elongation medium of the adventitious bud in the step (4) is: the adventitious bud elongation culture medium comprises: DKW +0.25mg/L GA3+0.5mg/L KT + 3.0% (w/v) sucrose + 0.7% (w/v) agar, pH 5.8.
Preferably, the rooting medium for the adventitious bud in the step (5) is: 1/8DKW + 10-50 μ M melatonin + 0.1-2.0 mg/L KIBA + 1.0-2.0% (w/v) sucrose + 0.7% (w/v) agar, pH 5.8.
Preferably, the rooting medium for the adventitious bud in the step (5) is: 1/8DKW +25 μ M melatonin +1.0mg/L KIBA + 1.0-2.0% (w/v) sucrose + 0.7% (w/v) agar, pH 5.8.
The invention has the advantages that:
the invention provides a method for establishing an efficient in-vitro regeneration system of an Qishu mature embryo, and particularly relates to a method for realizing rapid propagation of Qishu seedlings by directly inducing regeneration of cluster buds by taking Qishu mature seeds as explants. Has the following outstanding advantages: firstly, the invention optimizes the operation method of each stage of Keemun tissue culture, effectively reduces the pollution rate, improves the regeneration rate and obtains a large number of strong adventitious buds. Meanwhile, low-temperature plasma activated water is applied to the pretreatment process of Qishu seeds, so that the pollution rate is reduced, and the regeneration start of mature embryos is accelerated; secondly, the pretreatment method, the type of the basic culture medium and the combination of plant growth regulators of explants at different growth stages can ensure the efficient regeneration of mature embryos of the keemun process; thirdly, the use of melatonin in the rooting culture medium overcomes the problems of low survival rate and poor growth vigor of keemun transplantation in the prior art, and further remarkably improves the growth capacity of keemun seedlings. The conventional keemun seedling raising technology has no report related to direct induction of cluster bud regeneration by taking mature embryos as explants, and the method can greatly improve the propagation coefficient of keemun and provides powerful technical guarantee for efficient breeding and large-scale production of keemun seedlings.
Drawings
FIG. 1 mature embryos of Qishu inoculated on DKW medium for 2 weeks;
FIG. 2 shows an immature embryo inoculated on an adventitious bud induction medium and cultured for 2 weeks;
FIG. 3 proliferation of adventitious buds of mature embryos;
FIG. 4 elongation of adventitious bud of mature embryo;
FIG. 5 induction of adventitious roots;
FIG. 6 regenerated plant of an Qishu after 6 weeks of rooting culture.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The present invention will be described in further detail with reference to examples.
Example 1
A method for establishing an efficient in-vitro regeneration system of an Qishu mature embryo comprises the following specific operations:
(1) mature and plump seeds with active vigor are selected as explants and are placed in a 250ml plastic culture bottle to be washed for 10min under running water. Then placing the seeds in a sterile operation table, washing the seeds for 3 times by using sterile water, then carrying out surface disinfection for 2 times by using 75% absolute ethyl alcohol, carrying out disinfection for 15s each time, washing the seeds for 2 times by using the sterile water between 2 times, then disinfecting the seeds for 3min by using 0.1% (w/v) mercuric chloride solution, and then washing the seeds for 6 times by using the sterile water; further soaking the above seeds in low temperature plasma activated water (Ar is ion source, double distilled water is treated by dielectric barrier discharge device for 2min) for 2min, and then drying the surface water with sterile filter paper.
(2) Inoculating the seeds pretreated in the step (1) into an induction culture medium, and carrying out induction culture on adventitious buds in a constant-temperature culture chamber at the temperature of 22 ℃, the illumination intensity of 2200lx and the illumination time of 16 h. After 4 weeks of illumination culture, the seeds induced adventitious buds, the induction rate of the adventitious buds was 59.2%, and 2.3 adventitious buds were generated per explant on average. Wherein the adventitious bud induction culture medium comprises DKW, 0.5mg/L ZT, 2.0mg/L AgNO3, 3.0% (w/v) sucrose and 0.7% (w/v) agar, and has pH of 5.8.
(3) Transferring the mature embryo with the adventitious bud induced in the step (2) into a multiplication culture medium, and performing multiplication culture of the adventitious bud in the constant-temperature culture chamber. After 4 weeks of light culture, an average of 3.6 adventitious shoots were produced per explant. Wherein the proliferation culture medium of the adventitious bud is as follows: DKW +0.2mg/L ZT +0.05mg/L TDZ + 3.0% (w/v) sucrose + 0.7% (w/v) agar, pH 5.8.
(4) And (4) cutting the adventitious bud cluster in the step (3) into blocks with proper sizes, and inoculating the blocks into an elongation culture medium for elongation culture of adventitious buds. After 4 weeks of light culture, Qishu sprouts, having an average height of 2.1cm and accompanied by 2-3 intact leaves, were obtained. Wherein the elongation culture medium of the adventitious bud is as follows: DKW +0.05mg/L GA3+0.2mg/L KT + 3.0% (w/v) sucrose + 0.7% (w/v) agar, pH 5.8.
(5) And (4) separating the adventitious buds elongated in the step (4), and inoculating the adventitious buds into a rooting culture medium for adventitious root induction culture. After 4 weeks of light culture, the induction rate of adventitious roots was 74.2%, on average 3.2 adventitious roots were produced per explant, and the average length of the adventitious roots was 3.6 cm. Wherein the rooting culture medium is 1/8DKW, 10 μ M melatonin, 0.1mg/L KIBA, 1.0% (w/v) sucrose and 0.7% agar, and the pH value is 5.8.
Example 2
A method for establishing an efficient in-vitro regeneration system of an Qishu mature embryo comprises the following specific operations:
(1) mature and plump seeds with active vigor are selected as explants and placed in 250ml plastic culture bottles to be washed for 20min under running water. Then placing the seeds in a sterile operation table, washing the seeds with sterile water for 4 times, then carrying out surface disinfection for 2 times by using 75% absolute ethyl alcohol, carrying out disinfection for 20s each time, washing the seeds with sterile water for 3 times between 2 times, then disinfecting the seeds with 0.1% (w/v) mercuric chloride solution for 4min, and then washing the seeds with sterile water for 8 times; soaking the seeds in low-temperature plasma activated water (Ar is ion source, double distilled water is treated by a dielectric barrier discharge device for 2min) for 3min, and finally, absorbing the water on the surfaces of the seeds by using sterile filter paper for later use.
(2) Inoculating the seeds pretreated in the step (1) into an induction culture medium, and carrying out induction culture on adventitious buds in a constant-temperature culture chamber at the temperature of 22 ℃, the illumination intensity of 2200lx and the illumination time of 16 h. After 2 weeks of light culture, the keemun seeds inoculated in DKW medium without plant growth regulator showed only germination and no adventitious bud formation (fig. 1). The seeds inoculated in the adventitious bud induction medium induced an adventitious bud cluster, and the mature embryos induced adventitious buds (FIG. 2). After 2 weeks of continued culture, the rate of induction of adventitious buds was 95.8%, and an average of 3.7 adventitious buds per explant was produced. Wherein the adventitious bud induction culture medium comprises DKW +1.0mg/L ZT +3.0mg/L AgNO3+ 3.0% (w/v) sucrose + 0.7% (w/v) agar, and has pH of 5.8.
(3) Transferring the mature embryo with the adventitious bud induced in the step (2) into a multiplication culture medium, and performing multiplication culture of the adventitious bud in the constant-temperature culture chamber. After 4 weeks of light culture, an average of 8.9 adventitious shoots were produced per explant (FIG. 3). Wherein the proliferation culture medium of the adventitious bud is as follows: DKW +0.75mg/L ZT +0.25mg/L TDZ + 3.0% (w/v) sucrose + 0.7% (w/v) agar, pH 5.8.
(4) And (4) cutting the adventitious bud cluster in the step (3) into blocks with proper sizes, inoculating the blocks into an elongation culture medium, and performing elongation culture on the adventitious buds in a constant-temperature culture chamber. After 4 weeks of light culture, an average height of 2.9cm was obtained with 2-3 full leaf keels (FIG. 4). Wherein the elongation culture medium of the adventitious bud is as follows: DKW +0.25mg/L GA3+0.5mg/L KT + 3.0% (w/v) sucrose + 0.7% (w/v) agar, pH 5.8.
(5) And (4) separating the adventitious buds elongated in the step (4), inoculating the adventitious buds into a rooting culture medium, and performing induction culture of adventitious roots in a constant-temperature culture chamber. After 4 weeks of light culture, the induction rate of the adventitious roots is 97.5%, each explant averagely produces 8.3 adventitious roots, the average length of the adventitious roots is 3.1cm (figure 5), and after 2 weeks of continuous culture, robust keemun fully-regenerated plants are obtained (figure 6). Wherein the rooting culture medium is 1/8DKW, 25 μ M melatonin, 1.0mg/L KIBA, 1.5% (w/v) sucrose and 0.7% agar, and the pH value is 5.8.
Example 3
A method for establishing an efficient in-vitro regeneration system of an Qishu mature embryo comprises the following specific operations:
(1) mature and plump seeds with active vigor are selected as explants and placed in 250ml plastic culture bottles to be washed for 15min under running water. Then placing the seeds in an aseptic operation platform, washing the seeds for 3 times by using aseptic water, then carrying out 2 times of surface disinfection by using 75% absolute ethyl alcohol, washing the seeds for 2 times by using the aseptic water between 17s and 2 times of disinfection each time, then disinfecting the seeds for 5min by using 0.1% (w/v) mercuric chloride solution, and then washing the seeds for 7 times by using the aseptic water; further soaking the above seeds in low temperature plasma activated water (Ar is ion source, double distilled water is treated by dielectric barrier discharge device for 2min) for 4min, and then drying the surface water with sterile filter paper.
(2) Inoculating the seeds pretreated in the step (1) into an induction culture medium, and carrying out induction culture on adventitious buds in a constant-temperature culture chamber at the temperature of 22 ℃, the illumination intensity of 2200lx and the illumination time of 16 h. After 4 weeks of illumination culture, the seed induces adventitious bud clumps, the induction rate of the adventitious buds is 80.8%, and each explant averagely generates 3.1 adventitious buds. Wherein the adventitious bud induction culture medium comprises DKW +2.0mg/L ZT +5.0mg/L AgNO3+ 3.0% (w/v) sucrose + 0.7% (w/v) agar, and has pH of 5.8.
(3) Transferring the mature embryo with the adventitious bud induced in the step (2) into a multiplication culture medium, and performing multiplication culture of the adventitious bud in the constant-temperature culture chamber. After 4 weeks of light culture, an average of 6.4 adventitious shoots were produced per explant. Wherein the proliferation culture medium of the adventitious bud is as follows: DKW +1.5mg/L ZT +0.5mg/L TDZ + 3.0% (w/v) sucrose + 0.7% (w/v) agar, pH 5.8.
(4) And (4) cutting the adventitious bud cluster in the step (3) into blocks with proper sizes, and inoculating the blocks into an elongation culture medium for elongation culture of adventitious buds. After 4 weeks of light culture, Qishu sprouts, having an average height of 2.4cm and accompanied by 2-3 intact leaves, were obtained. Wherein the elongation culture medium of the adventitious bud is as follows: DKW +0.5mg/L GA3+1.0mg/L KT + 3.0% (w/v) sucrose + 0.7% (w/v) agar, pH 5.8.
(5) And (4) separating the adventitious buds elongated in the step (4), and inoculating the adventitious buds into a rooting culture medium for adventitious root induction culture. After 4 weeks of light culture, the induction rate of adventitious roots was 75.8%, and on average 4.2 adventitious roots were produced per explant, with an average adventitious root length of 1.6 cm. Wherein the rooting culture medium is 1/8DKW, 50 μ M melatonin, 2.0mg/L KIBA, 1.0% (w/v) sucrose and 0.7% agar, and the pH value is 5.8;
example 4:
this example tests the effect of low temperature plasma activated water on the contamination rate and germination time of Keemun seeds. The inventors selected viable mature and full Keemun seeds as explants and placed them in 250ml plastic culture flasks and washed under running water for 20 min. Then placing the seeds in a sterile operation table, washing the seeds with sterile water for 4 times, then carrying out surface disinfection for 2 times by using 75% absolute ethyl alcohol, carrying out disinfection for 20s each time, washing the seeds with sterile water for 3 times between 2 times, then disinfecting the seeds with 0.1% (w/v) mercuric chloride solution for 4min, and then washing the seeds with sterile water for 8 times; on the basis, the seeds were soaked in filter-sterilized low-temperature plasma activated water (Ar is an ion source, double distilled water was treated by a dielectric barrier discharge device for 2min) for various times (0, 1, 2, 3, 4 and 5min), then surface moisture of the seeds was absorbed by sterile filter paper, and the seeds were inoculated into the adventitious bud induction medium of example 2, and induction culture of adventitious buds was carried out in a constant-temperature culture chamber at a temperature of 22 ℃, an illumination intensity of 2200lx and an illumination time of 16 h. And (5) counting the seed pollution rate and the germination condition after 2 weeks of illumination culture. The research results (table 1) show that the use of low-temperature plasma activated water in the pretreatment process of the keemun mature embryos can greatly reduce the contamination rate of the keemun mature embryos, and the contamination rate is gradually reduced along with the prolonging of the treatment time. Meanwhile, the low-temperature plasma activated water can promote the germination of the mature embryos to a certain extent, the effect is the best when the mature embryos are placed in the plasma activated water for treatment for 3-4 min, the pollution rate is 0%, the germination rate is 100%, and the low-temperature plasma activated water has a certain inhibiting effect on the germination of the mature embryos along with the prolongation of the treatment period.
TABLE 1 Effect of Low temperature plasma activation Water treatment time on the contamination and germination rates of mature embryos of keemun surgery
| Treatment time (min) | Contamination ratio (%) | Percentage of sprout (%) |
| 0 | 24.2 | 87.5 |
| 1 | 11.7 | 95.0 |
| 2 | 3.3 | 98.3 |
| 3 | 0.0 | 100 |
| 4 | 0.0 | 100 |
| 5 | 0.0 | 67.7 |
Note: data are mean, 120 explants per treatment, triplicate per treatment
Example 5:
this example tests the effect of medium type and ZT and AgNO3 concentration on induction of adventitious buds in an Qioperative mature embryo. The inventors inoculated the keemun seeds pretreated in example 2 to different types of minimal medium supplemented with different concentrations of ZT and AgNO3 for induction culture of adventitious buds. The induction rate of adventitious buds was counted after 4 weeks of light culture. The results of the study (Table 2) show that the type of medium and the type and concentration of plant growth regulator have an important effect on induction of adventitious buds of an expert mature embryo. Among the three types of media tested, DKW media induced better than MS and WPM media. When ZT is added into the culture medium, the adventitious bud induction rate and the number of adventitious buds gradually increase with the increase of ZT concentration in the culture medium, and the induction effect of the adventitious buds is optimal at 1.0 mg/L. On the basis, further research results show that the use of AgNO3 and ZT play a synergistic and promoting role, the induction effect of adding 1.0mg/L ZT and 3.0mg/L AgNO3 adventitious buds in the DKW basic culture medium is optimal, the induction rate of the adventitious buds is as high as 95.8%, and each explant generates 3.7 adventitious buds on average.
TABLE 2 Effect of different concentrations of ZT and AgNO3 on induction of adventitious buds in Qishu
Note: data are mean, 120 explants per treatment, triplicate per treatment
Example 6:
this example tests the effect of ZT and TDZ concentrations on adventitious bud proliferation. The inventors inoculated the pretreatment of the Qishu seeds in example 2 into the adventitious bud induction medium in example 2 to induce adventitious buds. On the basis of adventitious bud acquisition, the influence of different ZT and TDZ concentrations on adventitious bud proliferation is further researched. This was counted for adventitious shoots after 4 weeks of light culture. The research results (Table 3) show that the use of low concentration ZT and low concentration TDZ has synergistic effect on the proliferation of adventitious buds, the proliferation effect of the culture medium added with 0.75mg/L ZT and 0.25mg/L TDZ is the best, the proliferation coefficient is 2.4, and each explant generates 8.9 adventitious buds on average.
TABLE 3 Effect of ZT and TDZ concentration in culture Medium on adventitious bud proliferation
| ZT(mg/L) | TDZ(mg/L) | Coefficient of proliferation | Adventitious bud number/explant |
| 0.75 | 0.0 | 1.1 | 3.9 |
| 1.0 | 0.0 | 1.2 | 4.3 |
| 1.5 | 0.0 | 1.4 | 5.1 |
| 0.2 | 0.05 | 1.6 | 5.8 |
| 0.75 | 0.25 | 2.4 | 8.9 |
| 1.5 | 0.5 | 1.7 | 6.4 |
Note: data are mean, 120 explants per treatment, triplicate per treatment
Example 7:
this example tests the effect of GA3 and KT concentrations in the medium on the elongation of the adventitious buds from Keemum. The present inventors cut the adventitious buds proliferated in example 2 into appropriate sized pieces and transferred them to a medium supplemented with GA3 and KT at different concentrations. The length of adventitious buds was counted after 4 weeks of light culture. The results of the study (Table 4) show that the addition of GA3 and KT to the medium promotes the elongation of the adventitious bud of Keemun, and that both have a dose effect. The combined use of GA3 and KT can obviously promote the elongation of adventitious buds, and the elongation effect of the adventitious buds is best when 0.25mg/L GA3 and 0.5mg/L KT are added into the culture medium.
TABLE 4 Effect of GA3 and KT concentrations in Medium on adventitious bud elongation
| GA3(mg/L) | KT(mg/L) | Average indefinite bud length (cm) |
| 0.0 | 0.0 | 0.7 |
| 0.05 | 0.0 | 1.1 |
| 0.25 | 0.0 | 1.5 |
| 0.5 | 0.0 | 1.3 |
| 0.0 | 0.2 | 1.5 |
| 0.0 | 0.5 | 1.9 |
| 0.0 | 1.0 | 1.7 |
| 0.05 | 0.2 | 2.1 |
| 0.25 | 0.5 | 2.9 |
| 0.5 | 1.0 | 2.4 |
Note: data are mean, 120 explants per treatment, triplicate per treatment
Example 8:
this example tested the effect of melatonin and KIBA concentrations in the medium on induction of adventitious roots in keels, and the inventors changed the concentrations of exogenous melatonin and KIBA only in the rooting medium, and inoculated the elongated keels group culture sprouts obtained in step (4) of example 4 into rooting medium supplemented with melatonin and KIBA at different concentrations for induction culture of adventitious roots. After 4 weeks of light culture, the induction of adventitious roots was counted, and the results of the study are shown in Table 5. The results of the study showed that the concentrations of melatonin and KIBA in the medium had an important effect on induction of the adventitious roots of keemun. When the two are used independently, the induction rate of the adventitious roots, the number of the adventitious roots and the average length of the adventitious roots are gradually increased along with the increase of the concentrations of melatonin and KIBA in a certain range. When melatonin was used alone, 25 μ M melatonin had the best effect of induction of adventitious roots. When KIBA is used alone, the adventitious root induction effect is the best at 2.0mg/L, but the difference is not significant compared with 1.0 mg/L. Further research shows that the melatonin and KIBA have a synergistic promoting effect in the induction process of the adventitious roots of the keemun javelle bud grafting, the induction effect of the adventitious roots in a culture medium added with 25 mu M of melatonin and 1.0mg/L of KIBA is optimal, the induction rate of the adventitious roots is up to 97.5 percent, each explant averagely generates 8.3 adventitious roots, and the average root length is 3.1 cm.
TABLE 5 Effect of exogenous melatonin and KIBA concentrations on induction of adventitious roots in keemuny
| Melatonin (mu M) | KIBA(mg/L) | Rooting percentage (%) | Average number of roots | Average root length (cm) |
| 0 | 0 | 6.7 | 1.4 | 3.3 |
| 10 | 0.0 | 48.3 | 2.9 | 3.1 |
| 25 | 0.0 | 73.3 | 4.5 | 2.9 |
| 50 | 0.0 | 67.5 | 3.7 | 2.2 |
| 0.0 | 0.1 | 59.2 | 1.8 | 3.2 |
| 0.0 | 0.5 | 67.5 | 4.3 | 3.1 |
| 0.0 | 1.0 | 83.3 | 5.7 | 2.3 |
| 0.0 | 2.0 | 86.7 | 6.4 | 1.8 |
| 10 | 0.1 | 74.2 | 3.2 | 3.6 |
| 25 | 0.5 | 91.7 | 6.7 | 3.4 |
| 25 | 1.0 | 97.5 | 8.3 | 3.1 |
| 50 | 2.0 | 75.8 | 4.2 | 1.6 |
Note: data are mean, 120 explants per treatment, triplicate per treatment
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (5)
1. A method for establishing an efficient in-vitro regeneration system of an Qishu mature embryo is characterized by comprising the following steps: the method comprises the following steps: (1) selecting and pretreating seeds, soaking the selected seeds in low-temperature plasma activated water subjected to filtration sterilization for 2-4 min, and then, absorbing water on the surfaces of the seeds by using sterile filter paper for later use; (2) inoculating the pretreated mature embryo into an adventitious bud induction culture medium, and performing adventitious bud induction culture; (3) transferring the mature embryo with the adventitious bud induced to a multiplication culture medium for multiplication culture of the adventitious bud; (4) cutting the proliferated adventitious bud into pieces with proper size, and transferring the pieces into an elongation culture medium for elongation culture of adventitious buds; (5) when the adventitious bud grows to 2-3 cm and is accompanied by complete leaves, transferring the adventitious bud into a rooting culture medium for rooting culture, and finally obtaining a complete keemun regenerated plant;
in the step (1), in the low-temperature plasma activated water, Ar is an ion source, and double distilled water is treated for 2min by a dielectric barrier discharge device;
in the step (2), the adventitious bud induction culture medium is: DKW + 0.5-2.0 mg/L ZT + 2.0-5.0 mg/L AgNO3+ 3.0% (w/v) sucrose + 0.7% (w/v) agar, pH = 5.8;
in the step (3), the adventitious bud propagation medium is: DKW + 0.2-1.5 mg/L ZT + 0.05-0.5 mg/L TDZ + 3.0% (w/v) sucrose + 0.7% (w/v) agar, pH = 5.8;
in the step (4), the adventitious bud elongation culture medium is: DKW + 0.05-0.5 mg/L GA3+ 0.2-1.0 mg/L KT + 3.0% (w/v) sucrose + 0.7% (w/v) agar, pH = 5.8;
in the step (5), the adventitious bud rooting medium is as follows: 1/8DKW + 10-50 mu M melatonin + 0.1-2.0 mg/L KIBA + 1.0-2.0% (w/v) sucrose + 0.7% (w/v) agar, and pH = 5.8.
2. The method for establishing an efficient in-vitro regeneration system of an keemun mature embryo according to claim 1, which is characterized in that: in the step (1), the selection and pretreatment of the seeds refers to selecting mature and full Keemu seeds with vitality, placing the Keemu seeds in a 250ml plastic culture bottle, washing the Keemu seeds for 10-20 min under running water, then placing the Keemu seeds in an aseptic operation table, washing the Keemu seeds for 3-4 times by using sterile water, then carrying out surface disinfection for 2 times by using 75% absolute ethyl alcohol, disinfecting for 15-20 s each time, and washing for 2-3 times by using the sterile water between 2 times; then sterilizing the mixture for 3-5 min by using 0.1% (w/v) mercuric chloride solution, and then washing the mixture for 6-8 times by using sterile water; further soaking the seeds in low-temperature plasma activated water for 2-4 min, which is subjected to filtration sterilization, and then sucking water on the surfaces of the seeds by using sterile filter paper for later use.
3. The method for establishing an efficient in-vitro regeneration system of an keemun mature embryo according to claim 1, which is characterized in that: in the step (2), the adventitious bud induction culture medium is: DKW +1.0mg/L ZT +3.0mg/L AgNO3+ 3.0% (w/v) sucrose + 0.7% (w/v) agar, pH = 5.8.
4. The method for establishing an efficient in-vitro regeneration system of an keemun mature embryo according to claim 1, which is characterized in that: in the step (3), the adventitious bud propagation medium is: DKW +0.75mg/L ZT +0.25mg/L TDZ + 3.0% (w/v) sucrose + 0.7% (w/v) agar, pH = 5.8.
5. The method for establishing an efficient in-vitro regeneration system of an keemun mature embryo according to claim 1, which is characterized in that: in the step (4), the adventitious bud elongationThe long medium is: DKW +0.25mg/L GA3+0.5mg/L KT + 3.0% (w/v) sucrose + 0.7% (w/v) agar, pH = 5.8.
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| US5312801A (en) * | 1987-04-29 | 1994-05-17 | Dna Plant Technology Corporation | Somatic embryogenesis and plant regeneration of cacao |
| CN106718934A (en) * | 2017-02-09 | 2017-05-31 | 河北大学 | A kind of utilization plumular axis and radicle directly break up the bighead atractylodes rhizome regenerating system of adventitious bud |
| CN113475395A (en) * | 2021-07-06 | 2021-10-08 | 中国科学院合肥物质科学研究院 | Method for direct regeneration and in-vitro rooting of hypocotyls in Qishu |
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| CN106718934A (en) * | 2017-02-09 | 2017-05-31 | 河北大学 | A kind of utilization plumular axis and radicle directly break up the bighead atractylodes rhizome regenerating system of adventitious bud |
| CN113475395A (en) * | 2021-07-06 | 2021-10-08 | 中国科学院合肥物质科学研究院 | Method for direct regeneration and in-vitro rooting of hypocotyls in Qishu |
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