CN116458430A - Tissue culture medium for sauropus spatulifolius and application thereof - Google Patents
Tissue culture medium for sauropus spatulifolius and application thereof Download PDFInfo
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- 239000003104 tissue culture media Substances 0.000 title claims abstract description 10
- 241001331775 Sauropus spatulifolius Species 0.000 title description 2
- 241001555141 Sauropus Species 0.000 claims abstract description 129
- 239000001963 growth medium Substances 0.000 claims abstract description 48
- 230000006698 induction Effects 0.000 claims abstract description 47
- 230000035755 proliferation Effects 0.000 claims abstract description 39
- 239000012883 rooting culture medium Substances 0.000 claims abstract description 12
- 239000002609 medium Substances 0.000 claims description 47
- 239000012882 rooting medium Substances 0.000 claims description 16
- 238000005286 illumination Methods 0.000 claims description 11
- 239000008223 sterile water Substances 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 229920001817 Agar Polymers 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 239000008272 agar Substances 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 8
- 230000001954 sterilising effect Effects 0.000 claims description 8
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 7
- 229930006000 Sucrose Natural products 0.000 claims description 7
- 238000005520 cutting process Methods 0.000 claims description 7
- 239000002689 soil Substances 0.000 claims description 7
- 239000005720 sucrose Substances 0.000 claims description 7
- 238000011282 treatment Methods 0.000 claims description 7
- 238000004140 cleaning Methods 0.000 claims description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 241001145025 Saussurea involucrata Species 0.000 claims description 5
- 229960002523 mercuric chloride Drugs 0.000 claims description 4
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 4
- 239000004094 surface-active agent Substances 0.000 claims description 4
- 239000003415 peat Substances 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 230000001939 inductive effect Effects 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 21
- 238000011419 induction treatment Methods 0.000 abstract description 6
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- 108010051696 Growth Hormone Proteins 0.000 description 5
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- 241000196324 Embryophyta Species 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
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- 241001448411 Dracaena draco Species 0.000 description 2
- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 description 2
- 239000006013 carbendazim Substances 0.000 description 2
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- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000000644 propagated effect Effects 0.000 description 2
- 241001184073 Basilicum Species 0.000 description 1
- 241001164374 Calyx Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000221017 Euphorbiaceae Species 0.000 description 1
- 235000010582 Pisum sativum Nutrition 0.000 description 1
- 240000004713 Pisum sativum Species 0.000 description 1
- 241000534017 Saururus chinensis Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 229910052799 carbon Inorganic materials 0.000 description 1
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- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 1
- 239000004062 cytokinin Substances 0.000 description 1
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- 238000011081 inoculation Methods 0.000 description 1
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- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 230000004048 modification Effects 0.000 description 1
- 239000006870 ms-medium Substances 0.000 description 1
- 230000001151 other effect Effects 0.000 description 1
- 239000003375 plant hormone Substances 0.000 description 1
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- 230000001737 promoting effect Effects 0.000 description 1
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- 238000005507 spraying Methods 0.000 description 1
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- SWGJCIMEBVHMTA-UHFFFAOYSA-K trisodium;6-oxido-4-sulfo-5-[(4-sulfonatonaphthalen-1-yl)diazenyl]naphthalene-2-sulfonate Chemical compound [Na+].[Na+].[Na+].C1=CC=C2C(N=NC3=C4C(=CC(=CC4=CC=C3O)S([O-])(=O)=O)S([O-])(=O)=O)=CC=C(S([O-])(=O)=O)C2=C1 SWGJCIMEBVHMTA-UHFFFAOYSA-K 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/40—Afforestation or reforestation
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The application relates to a tissue culture medium of sauropus basilicus and application thereof, belonging to the technical field of plant tissue culture. The sauropus She Zupei culture medium comprises adventitious bud induction culture medium, cluster bud proliferation culture medium and rooting culture medium. The application also provides the application of the culture medium in the rapid propagation of sauropus, which comprises the steps of explant pretreatment, adventitious bud induction treatment, cluster bud proliferation culture and rooting induction treatment. According to the method, the adventitious bud induction culture medium, the cluster bud proliferation culture medium and the rooting culture medium are selected as the rapid propagation culture medium of the sauropus, so that the propagation time of the sauropus can be shortened, and the propagation efficiency of the sauropus can be improved.
Description
Technical Field
The application relates to the technical field of plant tissue culture, in particular to a tissue culture medium of saururus chinensis leaves and application thereof.
Background
Sauropus palustris (L.) pers of Euphorbiaceae. The shrubs are 30-40 cm high, and the roots belong to the root systems. Branches are more and less distorted. The single leaves are mutually generated, the meat is slightly fleshy, and the needle shape or the long round spoon shape is inverted. Flowers are open in summer, very small, purple red and axillary. The capsule is as large as peas, and is covered with the calyx. The sauropus leaves are used as medicines, the quality of the sauropus leaves is uneven, and the large-scale planting of the sauropus leaves is not carried out in China. In addition, sauropus has slow growth, mainly depends on the propagation of the branches, has low efficiency and is difficult to meet the current market demand. Therefore, the large-scale standard planting of the sauropus is a development trend, but the number of the current seedlings is small, the large-scale production is difficult to form, and a large number of seedling problems are urgently needed to be solved in production. If large-scale planting is desired, and the yield and economic benefit are improved in production, it is quite important to promote the asexual propagation speed of sauropus. However, few reports on tissue culture of sauropus, and industrial production of sauropus are not available, so that a rapid propagation method capable of providing a large amount of sauropus seedlings in a short period is needed rapidly to ensure market demands.
Disclosure of Invention
The purpose of the application is to overcome the defects of the prior art and provide the sauropus rapid propagation culture medium capable of shortening the sauropus propagation period and improving the propagation coefficient and the application thereof.
In order to achieve the above purpose, the technical scheme adopted by the application is as follows:
in a first aspect, the present application provides a rapid propagation medium of sauropus, comprising adventitious bud induction medium, cluster bud propagation medium and rooting medium;
the adventitious bud induction medium comprises the following components: MS culture medium, NAA 0.1-0.2mg/L, 6-BA 0.5-1.0mg/L and pH 5.8-6.0;
the cluster bud proliferation medium comprises the following components: MS culture medium, NAA 0.1-0.15mg/L, 6-BA 1.5-2.0mg/L and pH 5.8-6.0;
the rooting medium comprises the following components: 1/2MS culture medium, 0.1-0.5mg/L NAA, pH value is 5.8-6.0.
According to the method, the adventitious bud induction culture medium, the cluster bud proliferation culture medium and the rooting culture medium are selected as the rapid propagation culture medium of the sauropus, so that the propagation time of the sauropus can be shortened, and the propagation efficiency of the sauropus can be improved. According to the adventitious bud induction culture medium and the cluster bud proliferation culture medium, growth hormones NAA and 6-BA are added on the basis of an MS culture medium, so that the induction rate of the adventitious buds and the proliferation coefficient of the cluster buds can be effectively improved; the rooting culture medium is added with the plant growth hormone NAA on the basis of a 1/2MS culture medium, so that the induction rate of adventitious roots can be effectively improved. NAA is a plant hormone with auxin-like activity, and has effect of promoting plant adventitious root formation; 6-BA is an artificially synthesized cytokinin, and can promote plant cell division, non-meristematic tissue differentiation, bud differentiation induction and other effects. NAA and 6-BA are artificially synthesized plant growth hormone, are easy to obtain and low in cost, and can reduce the cost of rapidly propagating sauropus.
As a preferred embodiment of the culture medium described herein, the adventitious bud induction medium comprises the following components: MS culture medium, NAA 0.1mg/L, 6-BA 1.0mg/L and pH 5.8;
the cluster bud proliferation medium comprises the following components: MS culture medium, NAA 0.1mg/L, 6-BA 2.0mg/L and pH 5.8;
the rooting medium comprises the following components: 1/2MS medium, 0.1mg/L NAA, pH 5.8.
Under the preferable proportion, the induction rate of adventitious buds of the saussurea involucrata, the proliferation coefficient of cluster buds and the rooting rate are highest, the induction rate is 114%, the proliferation coefficient is 3.6 times and the rooting rate is 88.9%.
As a preferred embodiment of the medium described herein, the adventitious bud induction medium, the multiple bud proliferation medium, and the rooting medium further comprise 6-8g/L agar and 28-32g/L sucrose.
According to the preparation method, agar is added into a tissue culture medium, the final concentration is 6-8wt% to form a solid culture medium, and as the content of the agar is in the range of 6-8wt%, the prepared culture medium can enable sauropus to treat the surface of the culture medium, has moderate hardness, is suitable for root growth of sauropus seedling, and plays a role in moisture preservation in the subsequent seedling hardening step; and a carbon source sucrose is added into the tissue culture medium, so that the growth of sauropus basilicum leaves is facilitated.
As a preferred embodiment of the medium described herein, the adventitious bud induction medium, the multiple bud proliferation medium, and the rooting medium further comprise 7g/L agar and 30g/L sucrose.
In a second aspect, the present application provides the use of the above medium in the rapid propagation of sauropus.
As a preferred embodiment of the application described herein, the method comprises the steps of:
pretreatment of explants: selecting a single young sauropus plant with side bud stem as an explant, and cleaning and surface sterilizing the young sauropus plant with side bud stem to obtain a sterile explant;
induction of adventitious bud treatment: cutting the aseptic explant into small stem segments with an axillary bud, and culturing in the adventitious bud induction culture medium for 20-30 days to obtain adventitious buds of sauropus;
proliferation culture of cluster buds: transferring adventitious buds of sauropus into the cluster bud multiplication medium, and culturing for 25-30 days to obtain cluster buds with sauropus;
and (3) induction rooting treatment: cutting the cluster buds of the sauropus, transferring to the rooting culture medium, and culturing for 25-30 days to obtain sauropus She Youmiao with root systems.
According to the method, healthy sauropus leaves are selected as explants, and after the steps of cleaning and surface disinfection, the sauropus leaves are transferred into an adventitious bud induction culture medium to induce meristem adventitious buds, transferred into a cluster bud proliferation culture medium to induce meristem cluster buds, transferred into a rooting culture medium to induce rooting, so that the effect of rapidly propagating sauropus She Zupei seedlings in a short time is achieved.
As a preferred embodiment of the application described herein, in the pretreatment of explants, the sauropus plant is sprayed with 0.125wt% carbendazim, and then cleaned and surface sterilized.
As a preferred embodiment of the application described herein, in the explant pretreatment, the cleaning and surface disinfection of the saussurea involucrata stem segment is specifically as follows:
soaking the stem segments in surfactant for 5min, and washing for 15min to obtain cleaned saussurea involucrata leaf stem segments;
and (3) transferring the cleaned sauropus leaf stem into 75wt% ethanol for sterilization for 30s, rinsing with sterile water for 2 times, sterilizing with 0.1wt% mercuric chloride for 10min, rinsing with sterile water for 3-5 times, and rinsing with sterile water for 4-5min each time to obtain the sterile explant.
According to the method, the sauropus leaf stem sections are soaked in the surfactant, so that pollutants such as soil on the surface of the stem sections can be effectively removed, and lipid on the surface of the stem sections can be removed; after cleaning, the stem segments are disinfected by adopting 75wt% of ethanol and 0.1wt% of mercuric chloride, so that microorganisms on the surfaces of the stem segments can be effectively eliminated, the stem segments of the sauropus are prevented from being polluted by microorganisms in the nature in a tissue culture stage, a large amount of microorganisms are propagated in an MS culture medium, and the growth of the sauropus She Zupei seedlings is limited or even dead.
As a preferred embodiment of the application described herein, in the adventitious bud induction treatment, the small stem section with one axillary bud has a length of 1 to 1.5cm. The aseptic explant is cut into small stem segments with an axillary bud in the adventitious bud induction treatment, and the length is 1-1.5cm, and the retention of the axillary bud combined with growth hormone can promote the generation of the adventitious bud of the explant. Cutting to a length of 1-1.5cm can make maximum use of the explant, if the length is too long, the growth of the explant can be inhibited, if the length is too short, the growth is too slow.
As a preferred embodiment of the method described herein, in the adventitious bud induction treatment or the cluster bud proliferation culture or the rooting induction treatment, the culture conditions are as follows: the illumination time is 14 h/day, the illumination intensity is 2000lx, and the culture temperature is 25+/-1 ℃.
As a preferred embodiment of the application, the application further comprises a seedling hardening and transplanting step, and the specific operation is as follows:
covering sauropus She Youmiao with root system with natural light, standing for 2-3 days, removing the bottle cap, adding sterile water to keep the culture medium moist, and standing for 2-3 days to obtain sauropus She Youmiao after seedling hardening;
taking out the seedling-hardening sauropus She Youmiao from the rooting culture medium, washing the root culture medium, transplanting the seedling-hardening sauropus She Youmiao into peat soil and planting the seedling-hardening sauropus She Youmiao in the field.
Experiments for multiple times find that the survival rate of the sauropus She Youmiao obtained through rapid propagation after seedling hardening treatment can be improved. If the sauropus seedling is transplanted into the soil directly without hardening off, the sauropus seedling is suddenly transferred from a single-structure sterile culture medium to a complex soil environment, so that the stress response of sauropus She Youmiao is easily caused, and the transplanting survival rate of the sauropus seedling is reduced. Transplanting the sauropus seedling into soil after hardening seedlings are found through multiple transplanting experiments, and combining field management (keeping humidity to 90%, shading properly, keeping ventilation and increasing illumination time length) can greatly improve the transplanting survival rate of sauropus She Youmiao and enable the sauropus leaf quality to be more stable.
Compared with the prior art, the beneficial effects of this application are:
(1) According to the method, the adventitious bud induction culture medium, the cluster bud proliferation culture medium and the rooting culture medium are selected as the tissue culture medium of the sauropus, so that the propagation time of sauropus can be shortened, the induction rate of the adventitious buds, the proliferation coefficient of the cluster buds and the rooting rate of the sauropus in the rapid propagation process of the sauropus can be effectively improved, and a large amount of sauropus She Youmiao can be obtained in a short time.
(2) The sauropus leaf rapid propagation technology has the advantages of simple and feasible process route and easy operation, the sauropus leaf stem section is used as an explant, the sauropus leaf in-vitro regenerated plant is successfully obtained through the processes of adventitious bud induction, proliferation, rooting, hardening seedling transplanting and the like, a sauropus leaf tissue culture rapid propagation technology system is established, the seed amount in cultivation production is saved, and a foundation is laid for providing high-quality sauropus leaf seedlings for the market in a large number. Meanwhile, NAA and 6-BA are selected as plant growth hormones to be added into the tissue culture medium, so that the cost of the tissue culture medium can be greatly reduced, and the economic benefit of sauropus is improved.
Drawings
FIG. 1 shows a rapidly propagated sauropus patent She Youmiao according to example 1 of the present application;
FIG. 2 shows the growth of adventitious buds of sauropus for rapid propagation of sauropus for examples 1-3 of the present application, wherein A is example 1, B is example 2, and C is example 3;
FIG. 3 shows the growth of the cluster buds of sauropus for rapid propagation of sauropus for example 1, examples 4-5, wherein A is example 4, B is example 5, and C is example 1;
FIG. 4 shows growth of sauropus She Youmiao obtained by rapid propagation of sauropus for example 1, example 6, and comparative example 7 of the present application, wherein A is example 1, B is example 86, and C is comparative example 7.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present application, the present application will be further described with reference to specific examples.
Other materials, reagents and the like used in examples, comparative examples and effect examples are commercially available unless otherwise specified.
Examples 1 to 6
The adventitious bud induction medium and the cluster bud proliferation medium in the sauropus leaf rapid propagation medium of examples 1-6 are added with 7g of agar, 30g of sucrose, NAA and 6-BA on the basis of MS medium, and the dosage of NAA and 6-BA is shown in Table 1; rooting medium 7g agar, 30g sucrose and NAA were added on the basis of 1/2MS medium, and the amount of NAA was shown in Table 1.
The method for rapidly propagating the sauropus soleus leaves in examples 1-6 comprises the following steps:
(1) Pretreatment of explants: selecting a single young leaf stem section with lateral buds of sauropus as an explant, spraying 0.125wt% of carbendazim on the young leaf stem section of sauropus, soaking the young leaf stem section with lateral buds of sauropus in a surfactant for 5min, washing for 15min, then transferring into 75wt% of ethanol for sterilization for 30s, rinsing with sterile water for 2 times, sterilizing with 0.1wt% of mercuric chloride for 10min, rinsing with sterile water for 3-5 times, and rinsing for 4-5min each time to obtain the sterile explant;
(2) Induction of adventitious bud treatment: cutting sterilized sauropus leaf stem into 1-1.5cm small stem with an axillary bud under aseptic condition, and culturing in adventitious bud induction culture medium for 20-30 days to obtain adventitious bud of sauropus leaf; the culture condition is that the illumination time is 14 h/day, the illumination intensity is 2000lx, the culture temperature is 25+/-1 ℃, and the culture time is prolonged;
(3) Proliferation culture of cluster buds: transferring adventitious buds of sauropus leaves into a cluster bud proliferation culture medium, and culturing for 25-30 days to obtain cluster buds with sauropus leaves; the culture condition is that the illumination time is 14 h/day, the illumination intensity is 2000lx, and the culture temperature is 25+/-1 ℃;
(4) And (3) induction rooting treatment: cutting cluster buds of sauropus, transferring to rooting culture medium, and culturing for 25-30 days to obtain sauropus She Youmiao with root system; the culture condition is that the illumination time is 14 h/day, the illumination intensity is 2000lx, and the culture temperature is 25+/-1 ℃;
(5) Hardening and transplanting: covering sauropus She Youmiao with root system with natural light, standing for 2-3 days, removing the bottle cap, adding sterile water to keep the culture medium moist, and standing for 2-3 days to obtain sauropus She Youmiao after seedling hardening;
taking out the seedling-hardening sauropus She Youmiao from the rooting culture medium, washing the root culture medium, transplanting the seedling-hardening sauropus She Youmiao into peat soil and planting the seedling-hardening sauropus She Youmiao in the field.
Example 1 the young sauropus after seedling hardening and transplanting is shown in figure 1, the leaves of the young sauropus are bigger and the growth state is good.
TABLE 1 grouping of the bases of the rapid propagation cultures of examples 1-6
Comparative examples 1 to 2
The rapid propagation media of comparative examples 1-2 were similar to example 1 except that NAA was not added to the adventitious bud induction medium, and IBA and 6-BA were added in different amounts, the specific amounts being shown in Table 2; the method for rapid propagation of sauropus leaves was the same as in example 1.
TABLE 2 composition and amount of growth hormone (in mg/L) in adventitious bud induction Medium of comparative examples 1 to 2
Comparative examples 3 to 4
The rapid propagation media of comparative examples 3 to 4 were similar to example 1 except that NAA was not added to the propagation medium of the cluster buds, and the amounts of IBA and 6-BA added were varied, and specific amounts are shown in Table 3; the method for rapid propagation of sauropus leaves was the same as in example 1.
TABLE 3 composition and amount of growth hormone (in mg/L) in the Cluster bud propagation medium of comparative examples 3 to 4
Comparative examples 5 to 7
The rapid propagation media of comparative examples 5 to 7 are similar to example 1, except that the composition of the rooting medium is different, and the specific amounts are shown in Table 4; the method for rapid propagation of sauropus leaves was the same as in example 1.
Table 4 rooting medium compositions of comparative examples 5 to 7
Effect example 1 Effect of adventitious bud Induction Medium of different formulations on the rapid propagation of sauropus
The total number of buds, the induction rate and the average bud height of the sauropus leaves of examples 1-3 and comparative examples 1-2 after being cultured by the adventitious bud induction medium were calculated according to the rapid propagation method of example 1, the results are shown in Table 5, and the growth conditions of the sauropus leaves of examples 1-3 are shown in FIG. 2. The induction rate was calculated as follows:
inductivity (%) = total number of buds (one)/total number of explant inoculations (one) ×100%
TABLE 5 influence of different adventitious bud induction Medium on sauropus leaves
As shown in Table 5 and FIG. 2, the induction rate of the adventitious bud induction medium of the present application to sauropus leaf is 100% or more, and the growth state of sauropus leaf is good, and the higher the concentration of 6-BA, the more the number of the explants bud, and overall, the best effect of example 1 is seen. Compared with the comparative example, the sauropus leaf induction rate is higher and the bud height is also improved, which indicates that the adventitious bud induction culture medium can improve the sauropus leaf induction rate and the bud height in the rapid propagation process.
Effect example 2 Effect of different formulations of Cluster bud proliferation Medium on the rapid propagation of sauropus
The total number of buds, proliferation coefficients and growth conditions of the dragon tree leaves of examples 1, 4-5 and comparative examples 3-4 after being cultured by the cluster bud proliferation medium were calculated according to the rapid propagation method of example 1, the results are shown in Table 6, and the growth conditions of the dragon tree leaves of examples 1 and 4-5 are shown in FIG. 3. The proliferation factor is calculated as follows:
multiplication factor (times) =total number of shoots (one)/seed number (strain)
TABLE 6 influence of different Cluster bud propagation Medium on sauropus
As shown in Table 6 and FIG. 3, the higher the concentration of 6-BA in the bud of sauropus in a certain concentration range, the higher the proliferation factor of sauropus in the bud, and the best proliferation factor and growth condition of example 1 are seen as a whole. The example has higher coefficient She Zengshi compared with the comparative example, and can promote the growth shape of tissue culture seedlings, and although the proliferation coefficient of example 5 is slightly lower than that of comparative example 4, the growth condition of example 5 is better than that of comparative example 4, which shows that the cluster bud proliferation medium can promote the proliferation coefficient and the growth shape of the spatula sinensis leaves in the process of rapid proliferation of the spatula sinensis leaves.
Effect example 3 Effect of rooting Medium of different formulations on the rapid propagation of sauropus
The rooting number, rooting rate and growth condition of the sauropus leaves of example 1, example 6 and comparative examples 5-7 after being cultured by rooting medium were calculated according to the rapid propagation method of example 1, the results are shown in Table 7, and the growth states of the sauropus leaves of example 1, example 6 and comparative example 7 are shown in FIG. 4.
TABLE 7 influence of different rooting media on sauropus
As shown in table 7 and fig. 4, example 1 has a higher rooting rate compared to comparative example 7, indicating that 1/2MS medium can effectively promote sauropus She Shenggen; compared with comparative examples 5 and 6, the root system of sauropus in example 1 is more developed, better in growth vigor and higher in rooting rate; the rooting rate of example 6 is slightly lower than that of comparative example 7, but the plant growth vigor of comparative example 5 is not as good as that of example 6, which shows that the rooting medium of the application can improve the rooting rate and plant growth vigor of sauropus in the process of rapid propagation of sauropus.
Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present application and not for limiting the scope of protection of the present application, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present application without departing from the spirit and scope of the technical solutions of the present application.
Claims (10)
1. The tissue culture medium for sauropus us is characterized by comprising an adventitious bud induction medium, a cluster bud proliferation medium and a rooting medium;
the adventitious bud induction medium comprises the following components: MS culture medium, NAA 0.1-0.2mg/L, 6-BA 0.5-1.0mg/L and pH 5.8-6.0;
the cluster bud proliferation medium comprises the following components: MS culture medium, NAA 0.1-0.15mg/L, 6-BA 1.5-2.0mg/L and pH 5.8-6.0;
the rooting medium comprises the following components: 1/2MS culture medium, 0.1-0.5mg/L NAA, pH value is 5.8-6.0.
2. The culture medium of claim 1, wherein the adventitious bud induction medium comprises the following components: MS culture medium, NAA 0.1mg/L, 6-BA 1.0mg/L and pH 5.8;
the cluster bud proliferation medium comprises the following components: MS culture medium, NAA 0.1mg/L, 6-BA 2.0mg/L and pH 5.8;
the rooting medium comprises the following components: 1/2MS medium, 0.1mg/L NAA, pH 5.8.
3. The medium of claim 1 or 2, wherein the adventitious bud induction medium, the multiple bud proliferation medium, and the rooting medium further comprise 6-8g/L agar and 28-32g/L sucrose.
4. The medium of claim 3, wherein the adventitious bud induction medium, the multiple bud proliferation medium, and the rooting medium further comprise 7g/L agar and 30g/L sucrose.
5. The use of the medium according to any one of claims 1-4 in the rapid propagation of sauropus.
6. The use according to claim 5, comprising the steps of:
pretreatment of explants: selecting a single young sauropus plant with side bud stem as an explant, and cleaning and surface sterilizing the young sauropus plant with side bud stem to obtain a sterile explant;
induction of adventitious bud treatment: cutting the aseptic explant into small stem segments with an axillary bud, and placing into an adventitious bud induction culture medium to culture for 20-30 days to obtain adventitious buds of sauropus fomithe;
proliferation culture of cluster buds: transferring adventitious buds of sauropus leaves into a cluster bud proliferation culture medium, and culturing for 25-30 days to obtain cluster buds with sauropus leaves;
and (3) induction rooting treatment: cutting cluster buds of sauropus, transferring to rooting culture medium, and culturing for 25-30 days to obtain sauropus She Youmiao with root system.
7. The use of claim 6, wherein in the step of explant pretreatment, the steps of cleaning and surface disinfection of the saussurea involucrata stem segment are as follows:
soaking the stem segments in surfactant for 5min, and washing for 15min to obtain cleaned saussurea involucrata leaf stem segments;
and (3) transferring the cleaned sauropus leaf stem into 75wt% ethanol for sterilization for 30s, rinsing with sterile water for 2 times, sterilizing with 0.1wt% mercuric chloride for 10min, rinsing with sterile water for 3-5 times, and rinsing with sterile water for 4-5min each time to obtain the sterile explant.
8. The use according to claim 6, wherein in the step of inducing adventitious buds, the length of the small stem section with one axillary bud is 1-1.5cm.
9. The use according to claim 6, wherein in the induction of adventitious bud or clumped bud proliferation culture or induction of rooting, the culture conditions are as follows: the illumination time is 14 h/day, the illumination intensity is 2000lx, and the culture temperature is 25+/-1 ℃.
10. The use according to claim 6, further comprising a seedling transplanting step, comprising the following operations:
covering sauropus She Youmiao with root system with natural light, standing for 2-3 days, removing the bottle cap, adding sterile water to keep the culture medium moist, and standing for 2-3 days to obtain sauropus She Youmiao after seedling hardening;
taking out the seedling-hardening sauropus She Youmiao from the rooting culture medium, washing the root culture medium, transplanting the seedling-hardening sauropus She Youmiao into peat soil and planting the seedling-hardening sauropus She Youmiao in the field.
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