CN110432150B - Method for obtaining and rapidly proliferating aseptic seedlings of lamiophlomis rotata - Google Patents
Method for obtaining and rapidly proliferating aseptic seedlings of lamiophlomis rotata Download PDFInfo
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Abstract
The invention provides a method for obtaining a lamiophlomis rotata aseptic seedling, which comprises the following steps: (1) washing Lamiophlomis rotata seed with tap water, sterilizing in a super clean bench, sterilizing with 75% ethanol solution, washing with sterile water, soaking with 30% hydrogen peroxide solution, soaking with 10% hydrogen peroxide solution, washing with sterile water, and inoculating to germination culture medium for dark culture; (2) after the seeds germinate, transferring the seeds to illumination culture; after the cotyledon is stretched, shearing the cotyledon and hypocotyl of the aseptic seedling into small pieces/sections on an ultra-clean workbench, and horizontally placing the small pieces/sections on a cluster bud induction culture medium for inducing cluster buds; or cutting 4-5 true leaf seedlings or test-tube seedlings in subculture, cutting into small pieces, and inoculating to cluster bud induction culture medium for inducing cluster buds. The invention also provides a propagation method thereof. The invention improves the proliferation rate of lamiophlomis rotata tissue culture, and shortens the acquisition time of aseptic seedlings to 3-4 months.
Description
Technical Field
The invention belongs to the technical field of tissue culture and development and utilization of secondary metabolites, and particularly relates to a method for inducing lamiophlomis rotata cluster buds through tissue culture and rapidly propagating the lamiophlomis rotata cluster buds.
Background
At present, the lamiophlomis rotata medicinal material is mainly derived from wild resources, the medicine demand is increased along with the wide clinical application of lamiophlomis rotata, wild resources are excessively harvested in a predatory manner, so that the wild resources of lamiophlomis rotata are nearly exhausted and are listed as endangered Tibetan medicinal materials. On the other hand, the excessive excavation of wild resources also causes great damage to the local ecological environment, the desertification of the grassland is caused by the excavation of the high-cold grassland, and the deterioration of the grassland is accelerated. The lamiophlomis rotata is mainly propagated by rootstocks, the germination rate of seeds is lower, and the wild resources are greatly reduced because the lamiophlomis rotata cannot be regenerated after being dug. Excavation of underground parts is forbidden at present, but medicinal value of the underground parts recovered after excavation is generally 4 years, and the medicinal value of the underground parts is more than 5 years in high altitude areas, but in fact, a plurality of main production areas are excavated in a new round in less than 4 years, which is a common problem faced by a plurality of plateau medicinal plants. The artificial domestication and cultivation of the lamiophlomis rotata also has a plurality of limiting factors, and the lamiophlomis rotata can only be planted in high-cold and high-altitude areas due to the special ecological adaptability, which undoubtedly increases the cultivation difficulty. The plateau environment has short growing season, the time for cultivating medicinal materials to reach the medicinal standard needs 6 years, the long period limits the large-scale production, the artificial cultivation shows slow growth in both laboratories and fields, and the requirement of the current medicinal industry cannot be solved at all.
In order to prevent the damage of wild resources caused by excessive mining to the ecological environment and the extinction of the unique germplasm resources, a new way is necessary to be developed, and the problem is solved by a biotechnology means to become the most reliable source of the unique medicinal materials. The development of the tissue culture of the lamiophlomis rotata not only provides a theoretical basis for the large-scale industrial production of the lamiophlomis rotata, but also plays an important role in the development of national medicines, the protection of lamiophlomis rotata wild resources and the protection of the ecological environment of alpine grassland.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a method for obtaining aseptic seedlings of lamiophlomis rotata, and a large amount of aseptic seedlings can be obtained in a short time by quickly propagating in a cluster bud induction mode.
The invention provides a method for obtaining a lamiophlomis rotata aseptic seedling, which comprises the following steps:
(1) culturing explants: washing the lamiophlomis rotata seeds with tap water, then sterilizing the lamiophlomis rotata seeds on a super clean bench, sterilizing the lamiophlomis rotata seeds for 90 to 120s by using a 75 percent ethanol solution, washing the lamiophlomis rotata seeds for 2 to 3 times by using sterile water, soaking the lamiophlomis rotata seeds in a 30 percent hydrogen peroxide solution for 15 to 20min, then soaking the lamiophlomis rotata seeds in a 10 percent hydrogen peroxide solution for 30 to 60min, washing the lamiophlomis rotata seeds for 2 to 3 times by using the;
(2) inducing cluster buds: after the seeds germinate, transferring the seeds to illumination culture; after the cotyledon is stretched, shearing the cotyledon and hypocotyl of the aseptic seedling into 1.8-2.2mm small blocks/sections on an ultra-clean workbench, and horizontally placing the small blocks/sections on a cluster bud induction culture medium for inducing cluster buds; or cutting 4-5 true leaf seedlings or test-tube seedlings in subculture, cutting into small pieces of 2-5mm, inoculating to cluster bud induction culture medium, and inducing cluster buds.
Preferably, in the step (1), the lamiophlomis rotata seeds are firstly washed by running water and then disinfected on a super clean bench, and are firstly sterilized by 75% ethanol solution for 2min, washed by sterile water for 3 times, soaked by 30% hydrogen peroxide solution for 20min, then soaked by 10% hydrogen peroxide solution for 30min, washed by sterile water for 3 times and then inoculated to a germination culture medium for germination culture.
Preferably, in step (1), the medium for germination culture is 1/2MSB medium, the culture conditions are dark culture, and the culture temperature is 25 + -2 deg.C.
Preferably, in step (2), the light culture conditions are: 2000LuX culturing under light for 14h and 10h in dark at 25 + -2 deg.C.
Preferably, in step (2), the multiple shoot induction medium is MSB medium supplemented with 800 mg. L of 300--1CH, 2.2g Gelrite, pH 5.8, and 1.0-2.0 mg. L-1KT+0.1-0.5mg·L-1IBA；
Preferably, in step (2), the multiple shoot induction medium is MSB medium supplemented with 500 mg.L- 1CH, 2.2g Gelrite, pH 5.8, and an addition of 1.0 mg. L-1KT+0.1mg·L-1IBA。
The invention provides a rapid proliferation method of a lamiophlomis rotata aseptic seedling, which is characterized in that the lamiophlomis rotata aseptic seedling is obtained by applying the method of any one of claims 1 to 5, and then the lamiophlomis rotata aseptic seedling is transferred into a subculture proliferation culture medium for subculture proliferation culture under the condition of illumination culture; the formula of the subculture multiplication medium is as follows: adding 300-800 mg.L into MSB culture medium-1CH, 2.2g Gelrite, and 0.2-1.0 mg. L-1KT+0.05-0.2mg·L-1IBA, after 2 subcultures, the hormone concentration in the subculture multiplication medium was halved and the culture was continued.
Preferably, the formulation of the subculture multiplication medium is as follows: adding 500 mg.L into MSB culture medium-1CH, and 0.2 mg. L-1KT+0.05mg·L-1IBA。
Preferably, the light culture conditions are: 2000LuX culturing under light for 14h and 10h in dark at 25 + -2 deg.C.
The invention provides a rooting method of lamiophlomis rotata aseptic seedlings, which is characterized in that lamiophlomis rotata aseptic seedlings obtained by the method of claim 6 or 7 are selected, strong buds with vigorous growth are selected, cut off from a base part, and inoculated into a rooting culture medium under the illumination culture condition for inductionRooting in 1/2MSB culture medium and adding 0.05-0.2 mg/L-1NAA+0.5-1.0mg·L-1IBA。
Preferably, the light culture conditions are: 2000LuX culturing under light for 14h and 10h in dark at 25 + -2 deg.C.
The invention successfully overcomes the problems of incomplete sterilization and low germination rate of the lamiophlomis rotata seeds, and improves the proliferation rate of lamiophlomis rotata tissue culture. The invention researches out an obtaining and rapid propagation system of aseptic seedlings suitable for lamiophlomis rotata, so that the obtaining time of the aseptic seedlings is shortened to 3-4 months, and the multiplication times of subculture are 10-25 times through the induction of cluster buds.
The method for obtaining and rapidly proliferating the aseptic seedlings of the lamiophlomis rotata provided by the invention comprises the steps of sterilizing wild seeds to germinate the seeds, inducing cluster buds by using cotyledon, hypocotyl slices (segments) and leaves, optimizing culture conditions in the inducing and proliferating processes of the cluster buds, and finally establishing an aseptic seedling culture system. The method for obtaining the aseptic seedlings of the lamiophlomis rotata can obtain a large amount of aseptic seedlings within 3-4 months, the cluster bud induction rate can reach 80%, and the rooting rate can reach 50% -70% by further applying a rooting method. The seed disinfection is the first step of tissue culture, the 30% hydrogen peroxide solution is soaked for 20min, which is beneficial to the softening and surface disinfection of the seed coat, and the hydrogen peroxide solution with the concentration of 10% is replaced, which can completely eliminate endophytes in the softened seed coat. The hydrogen peroxide solution is selected as the disinfectant, and the mercuric chloride solution is not used, so that the environment is protected. Mercuric chloride solution is widely used as plant explant disinfectant, and the waste liquid of the mercuric chloride solution enters the environment to cause pollution.
During the induction of the cluster buds, the addition of CH is beneficial to the formation of the cluster buds, and compared with the treatment without CH, the number of the cluster buds can be increased by about 5 percent. The formation of secondary metabolites in the culture process of medicinal plants enables browning phenomena to be ubiquitous in the culture process, agar culture medium is added, the culture medium which is in contact with leaf sections of about 20d of the lamiophlomis rotata leaf cut blocks is inoculated to begin browning and then spread to the periphery, and the induction rate of cluster buds is low, only about 40%, and the cluster buds grow slowly; gelrite is added into the culture medium to replace the traditional agar as a coagulator, so that browning of the base of the culture material can be obviously reduced, the nutrient components can be absorbed by leaves, the induction rate of the cluster buds is high and can reach 80%, and the growth is vigorous. Because the germination rate of the lamiophlomis rotata wild seeds is low and the endophyte is relatively complex, in the culture process, the leaves of the test-tube plantlet are taken and are cut into pieces to induce cluster buds, so that the proliferation rate can be increased, and the culture period can be effectively shortened. Reducing the concentration of hormones and adjusting the type of hormones during the subculture process is beneficial to the healthy growth of cluster buds. In the rooting induction stage, two auxin matching modes are used, the rooting rate can be improved, and after the test-tube plantlet roots, the nutrition is fully absorbed, which obviously shows that the leaf area is increased, the thickness is increased, the plant grows vigorously, and the later-stage transplanting or other researches are facilitated.
In conclusion, the invention fully considers the influence of disinfectant selection in the seed disinfection process, auxin and mitogen in cluster bud induction and culture, and finally obtains the lamiophlomis rotata aseptic seedlings by screening the optimal culture medium, adding CH, replacing the traditional agar by Gelrite and adjusting the concentration and the type of exogenous hormone in the culture process, and establishes the culture method for rapid proliferation. The method has the advantages of short time consumption, high induction rate, and strong growth of aseptic seedlings. The method is suitable for wild seed of Lamiophlomis rotata Benth, and has positive effects on development and utilization of Lamiophlomis rotata Benth and molecular biology research.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 shows the germination of Lamiophlomis rotate kudo seeds.
FIG. 2 shows the induction of the clumped buds of the leaf of Lamiophlomis rotate.
FIG. 3 shows the cluster buds of Lamiophlomis rotate kudo.
FIG. 4 shows the proliferation culture of aseptic Lamiophlomis rotata seedlings.
FIG. 5 shows the whole plant of Lamiophlomis rotate kudo.
FIG. 6 shows the culture of the aseptic test-tube plantlet of Lamiophlomis rotate (Benth.) kudo in the culture shelf.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are commercially available unless otherwise specified.
In the present invention:
6-BA-6-benzylaminopurine, KT-N6-isopentenyladenine; NAA-naphthylacetic acid; IAA-indoleacetic acid, IBA-indolebutyric acid, Gelrite-vegetable gel, CH-hydrolysed casein; all the above were analytical reagents purchased from Sigma.
The method for obtaining the aseptic seedlings of the lamiophlomis rotata comprises the following steps:
(1) test materials: wild lamiophlomis rotata seeds collected from Gansu Maqu Gansu grassland.
(2) Culturing explants: washing the lamiophlomis rotata seeds collected in the field with running water for 2 hours, then sterilizing on a super clean workbench, sterilizing for 90-120s with 75% ethanol solution, washing with sterile water for 2-3 times, soaking with 30% hydrogen peroxide solution for 15-20min, changing into 10% hydrogen peroxide solution for soaking for 30-60min, washing with sterile water for 2-3 times, inoculating into a germination culture medium which is 1/2MSB culture medium, and culturing in dark at 25 +/-2 ℃.
By applying the sterilization method, the lamiophlomis rotata seeds are sterilized thoroughly, and the sterilization rate is between 70 and 80 percent; by applying the germination method, the germination rate of the lamiophlomis rotata seeds is high and is between 20 and 30 percent.
(3) Inducing cluster buds: after 35-45 days, germinating the seeds, transferring the seeds to illumination culture, cutting cotyledons and hypocotyls of the aseptic seedlings into small blocks/sections of 1.8-2.2mm on an ultra-clean workbench after the cotyledons are stretched, and horizontally placing the small blocks/sections on a cluster bud induction culture medium; or cutting 4-5 true leaf seedlings or late subcultured test-tube seedlings, cutting into small pieces of 2-5mm, inoculating to cluster bud induction culture medium, and inducing cluster buds. After 20-30 days, cluster buds are successfully induced to obtain the second generation of aseptic seedlings of the lamiophlomis rotata.
Wherein the illumination culture conditions are as follows: 2000LuX culturing under light for 14h and 10h in dark at 25 + -2 deg.C. (the same light culture conditions are used for proliferation culture and rooting culture).
The cluster bud induction medium is prepared by adding 300-800 mg.L to MSB medium-1CH, 2.2g Gelrite, pH 5.8, and 1.0-2.0 mg. L-1KT+0.1-0.5mg·L-1IBA。
By applying the induction method, the induction rate of the cluster buds is 80-85%.
The rapid propagation method of the lamiophlomis rotata aseptic seedlings comprises the following steps:
transferring the cluster buds into a subculture multiplication medium for subculture multiplication culture to continue the growth of the cluster buds, and growing new cluster buds from the bases of the cluster buds. The subculture multiplication medium is prepared by adding 300-800 mg.L to the MSB medium-1CH, 2.2g Gelrite, and 0.2-1.0 mg. L-1KT+0.05-0.2mg·L-1IBA, the reduction of the hormone concentration is beneficial to the healthy growth of cluster buds; after 2 subcultures, the hormone concentration in the subculture multiplication medium was halved and the culture was continued. The proliferation culture method is applied, the proliferation culture time is 35-50 days, and the proliferation multiple is 10-25 times, so that the proliferated lamiophlomis rotata aseptic seedlings are obtained. Repeatedly carrying out the steps of inducing cluster buds and quickly proliferating the aseptic seedlings of the lamiophlomis rotata to obtain a large batch of aseptic seedlings of the lamiophlomis rotata.
When transplanting is needed, the proliferation-cultured lamiophlomis rotata aseptic seedlings are subjected to rooting culture, and the rooting culture can adopt the following steps:
and (3) selecting vigorous and robust buds, shearing the buds from the base part, inoculating the buds to a rooting culture medium for inducing rooting, culturing for 30-45 days, allowing adventitious roots to grow at the lower end, allowing the roots to grow for 3-5cm, and allowing the rooting rate to be 50% -70%, so as to obtain a complete plant. The radix Lamiophlomidis Rotatae culture medium is 1/2MSB culture medium, and 0.05-0.2 mg.L-1NAA+0.5-1.0mg·L-1IBA. The two growth factors are used in a composite way, which is beneficial to improving the rooting rate.
FIGS. 1-6 show the growth process of aseptic Lamiophlomis rotata seedlings. Wherein, figure 1 shows the germination of lamiophlomis rotata seeds; FIG. 2 shows the induction of the cutting of lamiophlomis rotata leaves to form cluster buds; FIG. 3 shows cluster buds of Lamiophlomis rotate kudo; FIG. 4 shows the proliferation culture of aseptic seedlings of Lamiophlomis rotate (Benth.) kudo; FIG. 5 shows a Lamiophlomis rotate whole plant; FIG. 6 shows the culture of the aseptic test-tube plantlet of Lamiophlomis rotate (Benth.) kudo in the culture shelf.
The MSB medium used in the present invention is a medium using a combination of the inorganic elements of the MS medium and the organic elements of the B5 medium. The main components and the amounts thereof are shown in table 1.
1/2MSB medium is MSB medium with half of all components.
TABLE 1 MSB Medium formulation
The reagents in Table 1 are all analytical reagents, and are purchased from chemical reagents of national drug group, Inc.
In the method, a sterilization method and a culture medium formula optimization experiment:
sterilization of lamiophlomis rotata seeds
Collecting Lamiophlomis rotata seeds in the field, taking the seeds back to a laboratory, cleaning the seeds for 2 hours by running water, performing sterilization treatment on a super clean workbench, sterilizing the seeds for 2 minutes by using 75% alcohol solution, washing the seeds for 3 times by using sterile water, and respectively sterilizing by using 3 methods, wherein the method comprises the following steps of: sterilizing with 30% hydrogen peroxide solution for 20min, soaking in 10% hydrogen peroxide solution for 60min, and washing with sterile water for 3 times; the method 2 comprises the following steps: sterilizing 0.1% mercuric chloride solution for 15min, and washing with sterile water for 5 times; the method 3 comprises the following steps: 0.1% mercuric chloride solution was sterilized for 15min, rinsed 5 times with sterile water, then soaked with 10% hydrogen peroxide for 60min, rinsed 3 times with sterile water, and the results were as follows:
TABLE 2 Effect of different sterilization methods on seed Germination
The mercury bichloride and hydrogen peroxide compound sterilization pollution rate is lowest, but the chemical injury is easy to generate, and the damage is caused to seeds; the sterilizing effect of the mercuric chloride on the surfaces of the seeds is better than that of hydrogen peroxide, but the long sterilizing time can cause phytotoxicity, damage the embryo of the seeds and reduce germination, the short sterilizing time cannot achieve good sterilizing effect on the condition that endophytes exist in the seed skins, even the seeds are killed, but the mould still exists; the pollution can be effectively reduced by soaking the seeds in the hydrogen peroxide with high and low concentrations, the seeds can be soaked in the hydrogen peroxide with low concentration for a long time, the seed coats can be softened, meanwhile, the permeability is good, endophytes can be killed without damaging the seeds, the pollution rate is low, and the germination rate can reach 21.7%. The low germination rate of the lamiophlomis rotata is also caused by the non-uniform maturity of wild seeds, poor germination capacity of the seeds, no pretreatment and the like.
Differentiation and proliferation culture of cluster buds of lamiophlomis rotata and lamiophlomis rotata
1. Cutting leaves or leaves of aseptic seedlings of Lamiophlomis rotata into small pieces of 1.8-2.2mm, inoculating to cluster bud induction culture Medium (MSB), adding CH 500mg, 2.2g Gelrite, pH 5.8, wherein the added hormones are as follows:
TABLE 3 Cluster bud Induction Medium composition (mg. L)-1)
The influence of different culture media on cluster bud differentiation is counted, and the results are as follows:
TABLE 4 Effect of different media on Cluster bud differentiation
2. Cutting leaves or leaves of aseptic Lamiophlomis rotata (Benth.) kudo seedlings into small pieces of 1.8-2.2mm, inoculating to cluster buds for inductionIn the culture medium, MSB is used as cluster bud inducing culture medium, and CH 500mg, 5g agar, 1.0 mg. L are added-1KT+0.1mg·L-1IBA, pH 5.8. The induced differentiation rate is 40%, and the culture medium around the culture material is yellow brown.
Example 1
The method for obtaining the aseptic seedlings of the lamiophlomis rotata comprises the following steps:
(1) test materials: wild lamiophlomis rotata seeds collected from Gansu Maqu Gansu grassland.
(2) Culturing explants: washing wild collected lamiophlomis rotata seeds with running water for 2h, sterilizing on a super clean workbench, sterilizing for 2min with 75% ethanol solution, washing with sterile water for 3 times, soaking with 30% hydrogen peroxide solution for 20min, changing to 10% hydrogen peroxide solution for 30min, washing with sterile water for 3 times, inoculating to a germination culture medium which is 1/2MSB culture medium, and culturing in dark at 25 ℃. By applying the sterilization method, the lamiophlomis rotata seeds are thoroughly sterilized, and the sterilization rate is 80%; by applying the germination method, the germination rate of the lamiophlomis rotata seeds is high and is 30 percent.
(3) Inducing cluster buds: after 35 days, the seeds germinate, and are transferred to illumination culture, the test-tube plantlets subcultured at the later stage are cut into small pieces with the length of 4mm, and the small pieces are horizontally placed on a cluster bud induction culture medium for inducing cluster buds. After 20 days, cluster buds are successfully induced to obtain the lamiophlomis rotata aseptic seedlings.
Wherein the illumination culture conditions are as follows: 2000LuX culturing under light for 14h and dark for 10h at 25 deg.C. (the same light culture conditions are used for proliferation culture and rooting culture).
The cluster bud induction medium is prepared by adding 500 mg.L to MSB medium-1CH, 2.2g Gelrite, pH 5.8, and add.
By applying the induction method, the induction rate of the cluster buds is 85 percent.
The rapid propagation method of the lamiophlomis rotata aseptic seedlings comprises the following steps:
transferring the cluster buds into a subculture multiplication medium for subculture multiplication culture to continue the growth of the cluster buds, and growing new cluster buds from the bases of the cluster buds. Subculture multiplicationThe culture medium is MSB culture medium added with 500 mg.L-1CH, 2.2g Gelrite, and 0.2 mg. L-1KT+0.05mg·L-1IBA, the reduction of the hormone concentration is beneficial to the growth of the cluster buds; after 2 subcultures, the hormone concentration in the subculture multiplication medium was halved and the culture was continued. The proliferation culture method is applied, the proliferation culture time is 35 days, and the proliferation multiple is 25 times, so that the proliferated lamiophlomis rotata aseptic seedlings are obtained. Repeatedly carrying out the steps of inducing cluster buds and quickly proliferating the aseptic seedlings of the lamiophlomis rotata to obtain a large batch of aseptic seedlings of the lamiophlomis rotata.
When transplanting is needed, the proliferation-cultured lamiophlomis rotata aseptic seedlings are subjected to rooting culture, and the rooting culture can adopt the following steps:
selecting vigorous and strong buds, cutting off the buds from the base, inoculating the buds to a rooting culture medium to induce rooting, culturing for 30 days, allowing adventitious roots to grow at the lower end, allowing the roots to grow for 5cm, and allowing the rooting rate to be 70% to obtain complete plants. The radix Lamiophlomidis Rotatae culture medium is 1/2MSB culture medium, and 0.1 mg.L is added-1NAA+0.5mg·L-1IBA. The two growth factors are used in a composite way, which is beneficial to improving the rooting rate.
Example 2
The method for obtaining the aseptic seedlings of the lamiophlomis rotata comprises the following steps:
(1) test materials: wild lamiophlomis rotata seeds collected from Gansu Maqu Gansu grassland.
(2) Culturing explants: washing wild collected lamiophlomis rotata seeds with running water for 2h, sterilizing on a super clean workbench, sterilizing with 75% ethanol solution for 2min, washing with sterile water for 2 times, soaking with 30% hydrogen peroxide solution for 20min, changing to 10% hydrogen peroxide solution for 30min, washing with sterile water for 2 times, inoculating to germination medium which is 1/2MSB medium, and dark culturing at 25 deg.C. By applying the sterilization method, the lamiophlomis rotata seeds are thoroughly sterilized, and the sterilization rate is 75 percent; by applying the germination method, the germination rate of the lamiophlomis rotata seeds is high and is 26 percent.
(3) Inducing cluster buds: after 40 days, the seeds germinate, are transferred to illumination culture, the seedlings are grown into complete plants or test-tube seedlings subcultured at the later stage, the leaves are cut, the small pieces with the diameter of 5mm are cut, and the small pieces are inoculated in a cluster bud induction culture medium for inducing cluster buds. After 25 days, cluster buds are successfully induced to obtain the lamiophlomis rotata aseptic seedlings.
Wherein the illumination culture conditions are as follows: 2000LuX culturing under light for 14h and 10h in dark at 25 + -2 deg.C. (the same light culture conditions are used for proliferation culture and rooting culture).
The culture medium for inducing cluster buds is MSB culture medium supplemented with 300 mg.L-1CH, 2.2g Gelrite, pH 5.8, and an addition of 1.5 mg. L-1KT+0.3mg·L-1IBA。
By applying the induction method, the induction rate of the cluster buds is 82 percent.
The rapid propagation method of the lamiophlomis rotata aseptic seedlings comprises the following steps:
transferring the cluster buds into a subculture multiplication medium for subculture multiplication culture to continue the growth of the cluster buds, and growing new cluster buds from the bases of the cluster buds. The subculture growth medium is prepared by adding 300 mg. L to MSB medium-1CH, 2.2g Gelrite, and 0.5 mg. L-1KT+0.15mg·L-1IBA, the reduction of the hormone concentration is beneficial to the growth of the cluster buds; after 2 subcultures, the hormone concentration in the subculture multiplication medium was halved and the culture was continued. The proliferation culture method is applied, the proliferation culture time is 42 days, and the proliferation multiple is 20 times, so that the proliferated lamiophlomis rotata aseptic seedlings are obtained. Repeatedly carrying out the steps of inducing cluster buds and quickly proliferating the aseptic seedlings of the lamiophlomis rotata to obtain a large batch of aseptic seedlings of the lamiophlomis rotata.
When transplanting is needed, the proliferation-cultured lamiophlomis rotata aseptic seedlings are subjected to rooting culture, and the rooting culture can adopt the following steps:
selecting vigorous and strong buds, cutting off the buds from the base, inoculating the buds to a rooting culture medium to induce rooting, culturing for 40 days, allowing adventitious roots to grow at the lower end, allowing the roots to grow for 4cm, and allowing the rooting rate to be 60% to obtain complete plants. The radix Lamiophlomidis Rotatae culture medium is 1/2MSB culture medium, and 0.05 mg.L-1NAA+0.8mg·L-1IBA. The two growth factors are used in a composite way, which is beneficial to improving the rooting rate.
Example 3
The method for obtaining the aseptic seedlings of the lamiophlomis rotata comprises the following steps:
(1) test materials: wild lamiophlomis rotata seeds collected from Gansu Maqu Gansu grassland.
(2) Culturing explants: washing wild collected lamiophlomis rotata seeds with running water for 2h, sterilizing on a clean bench, sterilizing with 75% ethanol solution for 2min, washing with sterile water for 3 times, soaking with 30% hydrogen peroxide solution for 20min, changing to 10% hydrogen peroxide solution, soaking for 50min, washing with sterile water for 2 times, inoculating to germination medium, dark culturing at 23 deg.C, and culturing with 1/2 MSB. By applying the sterilization method, the lamiophlomis rotata seeds are thoroughly sterilized, and the sterilization rate is 70%; by applying the germination method, the germination rate of the lamiophlomis rotata seeds is high and is 20 percent.
(3) Inducing cluster buds: after 45 days, the seeds germinate, are transferred to illumination culture, are directly cultured to grow into seedlings, and then cotyledons and hypocotyls of the aseptic seedlings are cut into 1.8mm small blocks/sections on an ultra-clean workbench after the cotyledons are stretched, and are horizontally placed on a cluster bud induction culture medium for inducing cluster buds. After 30 days, cluster buds are successfully induced to obtain the lamiophlomis rotata aseptic seedlings.
Wherein the illumination culture conditions are as follows: 2000LuX culturing under light for 14h and 10h in dark at 25 + -2 deg.C. (the same light culture conditions are used for proliferation culture and rooting culture).
The cluster bud induction medium is prepared by adding 800 mg.L to MSB medium-1CH, 2.2g Gelrite, pH 5.8, and an addition of 2.0 mg. L-1KT+0.5mg·L-1IBA。
By applying the induction method, the induction rate of the cluster buds is 80 percent.
The rapid propagation method of the lamiophlomis rotata aseptic seedlings comprises the following steps:
transferring the cluster buds into a subculture multiplication medium for subculture multiplication culture to continue the growth of the cluster buds, and growing new cluster buds from the bases of the cluster buds. The subculture growth medium is prepared by adding 800 mg.L to MSB medium-1CH, 2.2g Gelrite, and 1.0 mg. L-1KT+0.2mg·L-1IBA, reducing hormone concentration is beneficial to cluster budsThe growth and growth of (2); after 2 subcultures, the hormone concentration in the subculture multiplication medium was halved and the culture was continued. By applying the proliferation culture method, the proliferation culture time is 50 days, and the proliferation multiple is 10 times, so that the proliferated lamiophlomis rotata aseptic seedlings are obtained. Repeatedly carrying out the steps of inducing cluster buds and quickly proliferating the aseptic seedlings of the lamiophlomis rotata to obtain a large batch of aseptic seedlings of the lamiophlomis rotata.
When transplanting is needed, the proliferation-cultured lamiophlomis rotata aseptic seedlings are subjected to rooting culture, and the rooting culture can adopt the following steps:
selecting vigorous and strong buds, cutting off the buds from the base, inoculating the buds to a rooting culture medium to induce rooting, culturing for 45 days, allowing adventitious roots to grow at the lower end, allowing the roots to grow for 3cm, and allowing the rooting rate to be 50% to obtain complete plants. The radix Lamiophlomidis Rotatae culture medium is 1/2MSB culture medium, and 0.2 mg.L-1NAA+1.0mg·L-1IBA. The two growth factors are used in a composite way, which is beneficial to improving the rooting rate.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. A method for obtaining aseptic seedlings of lamiophlomis rotata is characterized by comprising the following steps: the method comprises the following steps:
(1) culturing explants: washing the lamiophlomis rotata seeds with tap water, then sterilizing the lamiophlomis rotata seeds on a super clean bench, sterilizing the lamiophlomis rotata seeds for 90 to 120s by using a 75 percent ethanol solution, washing the lamiophlomis rotata seeds for 2 to 3 times by using sterile water, soaking the lamiophlomis rotata seeds in a 30 percent hydrogen peroxide solution for 15 to 20min, then soaking the lamiophlomis rotata seeds in a 10 percent hydrogen peroxide solution for 30 to 60min, washing the lamiophlomis rotata seeds for 2 to 3 times by using the; the culture medium for germination culture is 1/2MSB culture medium, and the culture temperature is 25 +/-2 ℃;
(2) clumpAnd (3) bud induction: after the seeds germinate, transferring the seeds to illumination culture; the illumination culture conditions are as follows: 2000LuX culturing under light for 14h and 10h in darkness at 25 + -2 deg.C; after the cotyledon is stretched, shearing the cotyledon and hypocotyl of the aseptic seedling into 1.8-2.2mm small blocks/sections on an ultra-clean workbench, and horizontally placing the small blocks/sections on a cluster bud induction culture medium for inducing cluster buds; or shearing 4-5 true leaf seedlings or test-tube seedlings in subculture, cutting into small pieces of 2-5mm, inoculating to cluster bud induction culture medium, and inducing cluster buds; the cluster bud induction medium is prepared by adding 300-800 mg.L to MSB medium-1 CH, 2.2g Gelrite, pH 5.8, and 1.0-2.0 mg. L-1 KT+0.1-0.5mg·L-1 IBA；
(3) And (3) rapid propagation culture: under the condition of illumination culture, the cluster buds are transferred to a subculture multiplication medium for subculture multiplication; the formula of the subculture multiplication medium is as follows: adding 300-800 mg.L into MSB culture medium-1 CH, and 0.2-1.0 mg. L-1 KT+0.05-0.2mg·L-1 IBA, after 2 subcultures, continuously culturing by reducing the hormone concentration in the subculture multiplication medium by half; the illumination culture conditions are as follows: 2000LuX culturing under light for 14h and 10h in darkness at 25 + -2 deg.C;
(4) rooting culture: selecting strong bud, cutting from the base, inoculating to rooting culture medium (1/2 MSB culture medium) with 0.05-0.2 mg.L for inducing rooting-1 NAA+0.5-1.0mg·L-1IBA; the illumination culture conditions are as follows: 2000LuX culturing under light for 14h and 10h in dark at 25 + -2 deg.C.
2. The method for obtaining aseptic seedlings of lamiophlomis rotata according to claim 1, wherein the method comprises the following steps: in the step (1), the lamiophlomis rotata seeds are firstly washed by running water, then disinfected on a super clean workbench, sterilized by 75% ethanol solution for 2min, washed by sterile water for 3 times, soaked by 30% hydrogen peroxide solution for 20min, soaked by 10% hydrogen peroxide solution for 30min, washed by sterile water for 3 times and then inoculated to a germination culture medium for germination culture.
3. The method for obtaining aseptic seedlings of lamiophlomis rotata according to claim 1 or 2, wherein the method comprises the following steps: in the step (2), the clumpy bud induction medium is the MSB medium added with 500 mg.L-1 CH, 2.2g Gelrite, pH 5.8, and an addition of 1.0 mg. L-1 KT+0.1mg·L-1 IBA。
4. The method for obtaining aseptic seedlings of lamiophlomis rotata according to claim 1, wherein the method comprises the following steps: the formula of the subculture multiplication medium is as follows: adding 500 mg.L into MSB culture medium-1 CH, 2.2g Gelrite, and 0.2 mg. L-1 KT+0.05mg·L-1 IBA。
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| CN105875405A (en) * | 2014-10-29 | 2016-08-24 | 四川深达生物科技有限公司 | Culture medium inducing proliferation of lamiophlomis rotata and preparation method thereof |
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| CN105875405A (en) * | 2014-10-29 | 2016-08-24 | 四川深达生物科技有限公司 | Culture medium inducing proliferation of lamiophlomis rotata and preparation method thereof |
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