CN116602215B - Establishment method of high-efficiency induction Wu Lihu branch callus regeneration system - Google Patents
Establishment method of high-efficiency induction Wu Lihu branch callus regeneration system Download PDFInfo
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- 206010020649 Hyperkeratosis Diseases 0.000 title claims abstract description 63
- 230000006698 induction Effects 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 36
- 230000008929 regeneration Effects 0.000 title claims abstract description 22
- 238000011069 regeneration method Methods 0.000 title claims abstract description 22
- 230000035755 proliferation Effects 0.000 claims abstract description 21
- 230000004069 differentiation Effects 0.000 claims abstract description 20
- 230000012010 growth Effects 0.000 claims abstract description 20
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- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 claims description 27
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- CGIDKJRJBMFXKV-UHFFFAOYSA-N 6-n'-benzylpurine-6,6-diamine Chemical compound N1=CN=C2N=CN=C2C1(N)NCC1=CC=CC=C1 CGIDKJRJBMFXKV-UHFFFAOYSA-N 0.000 claims description 13
- 229920001817 Agar Polymers 0.000 claims description 13
- 229930006000 Sucrose Natural products 0.000 claims description 13
- 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 13
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- 238000011010 flushing procedure Methods 0.000 claims description 13
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- 238000002791 soaking Methods 0.000 claims description 11
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- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 claims description 10
- 239000011159 matrix material Substances 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 9
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 9
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- 230000002745 absorbent Effects 0.000 claims description 8
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 5
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- 235000020679 tap water Nutrition 0.000 claims description 5
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- 239000006870 ms-medium Substances 0.000 claims description 2
- OVSKIKFHRZPJSS-DOMIDYPGSA-N 2-(2,4-dichlorophenoxy)acetic acid Chemical compound OC(=O)[14CH2]OC1=CC=C(Cl)C=C1Cl OVSKIKFHRZPJSS-DOMIDYPGSA-N 0.000 claims 1
- 230000002068 genetic effect Effects 0.000 abstract description 5
- 239000000463 material Substances 0.000 abstract description 3
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- HXKWSTRRCHTUEC-UHFFFAOYSA-N 2,4-Dichlorophenoxyaceticacid Chemical compound OC(=O)C(Cl)OC1=CC=C(Cl)C=C1 HXKWSTRRCHTUEC-UHFFFAOYSA-N 0.000 description 5
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- 244000025254 Cannabis sativa Species 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000004459 forage Substances 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
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- 239000007640 basal medium Substances 0.000 description 1
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- 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
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- 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
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- 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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Abstract
The invention discloses a method for establishing a high-efficiency induction Wu Lihu branch callus regeneration system, and belongs to the technical field of tissue culture. Comprises the steps of explant disinfection, primary callus induction, adventitious bud differentiation, adventitious bud proliferation and elongation growth, rooting induction, seedling hardening and transplanting. The invention selects wild up to Wu Lihu branch seeds in the current year as an explant material, and establishes a set of stable and mature up to Wu Lihu branch tissue culture regeneration system by optimizing the components and the content of a callus induction medium and a rooting medium. Breaks the seasonal limitation, the primary callus is easier to obtain, the success rate of the callus can reach 100 percent, and the adventitious bud is successfully induced, so that the survival rate of the obtained plant is high. Not only provides good technical support for high-efficiency industrial seedling raising of branches reaching Wu Lihu, but also lays a foundation for subsequent genetic transformation and directional genetic improvement research and variety cultivation of branches reaching Wu Lihu.
Description
Technical Field
The invention belongs to the technical field of tissue culture, and particularly relates to a method for establishing a high-efficiency induction Wu Lihu dendritic callus regeneration system.
Background
Up to Wu Lihu branches (lespeezadarica (laxm.) schindl.) are a perennial herb-like half bush-like plant of the genus lespedeza (lespeezamichx.) of the subfamily of the family Leguminosae (eguinosae), widely distributed in northeast, north-China, northwest, china to the Yunnan region of China. The branches of Wu Lihu are one of the important dominant species of loess plateau, have the characteristics of cold resistance, drought resistance, salt and alkali resistance, barren resistance and the like, have developed root systems, and are important pioneer plants for improving local degraded lands, repairing mining areas, planting artificial grasslands and recovering natural grassland vegetation. In addition, the forage grass is used as high-quality forage grass, has complete protein types, and has quite large nutritional value and feeding value. At present, the propagation of the branches up to Wu Lihu mainly adopts seed propagation, but the seeds are smaller, the nutrition components are lower, the seeds are greatly influenced by the environment in the germination period, the propagation efficiency is low, the period is long, the seedlings cannot be produced in a large-scale and efficient manner, the market demand cannot be met, and the popularization and the application of the branches up to Wu Lihu are severely limited.
However, at present, no report about a callus regeneration system of up to Wu Lihu branches exists in China, and no culture medium suitable for generating callus of up to Wu Lihu branches exists. Therefore, the establishment of the efficient induction culture medium and the regeneration system for the callus of the lespedeza with the length of Wu Lihu is of great significance for accelerating the asexual regeneration and propagation process of the lespedeza and establishing a lespedeza genetic transformation system and new variety cultivation.
Disclosure of Invention
Aiming at the problems and defects in the prior art, the invention aims to provide a method for establishing a high-efficiency induction up to Wu Lihu branch callus regeneration system, which can effectively improve the induction success rate of up to Wu Lihu branch callus.
The technical scheme adopted for achieving the aim of the invention is as follows: a method for establishing a high-efficiency induction up to Wu Lihu branch callus regeneration system comprises the following steps:
1) Explant sterilization:
selecting full seeds of branches reaching Wu Lihu in the current year as explants, sterilizing, and airing to obtain detoxified seeds;
2) Primary callus induction:
inoculating the detoxified seed in the step 1) into a callus induction culture medium to induce primary callus;
3) Differentiation of adventitious bud:
transferring the primary callus of the step 2) to a differentiation medium after multiple subcultures to promote induction of adventitious buds;
4) Proliferation and elongation growth of adventitious buds:
transferring the adventitious buds induced in the step 3) to a proliferation culture medium to promote the proliferation of the adventitious buds, and further continuing to extend the adventitious buds to obtain robust adventitious buds;
5) Rooting induction:
after the length of the robust adventitious bud obtained in the step 4) is about 3-5cm high, transferring the separated plant to a rooting culture medium for culture until 3-4 young roots are formed;
6) Hardening and transplanting:
and 5) taking out the tissue culture seedlings after the root systems of the tissue culture seedlings in the step 5) are developed, cleaning a culture medium, transplanting a matrix, watering a proper amount, culturing under an indoor greenhouse, transplanting the tissue culture seedlings into soil after the new growth She Fuzhuang is generated, and culturing in the outdoor greenhouse.
Further, in the step 1), the selected seeds are soaked for 30min with tap water, then the seeds are washed once with ultra-pure water, the ultra-pure water is filtered off, then the seeds are put into a sterilizing bottle and placed into an ultra-clean bench, the seeds are soaked for 30sec with 75% alcohol, washed for 3-4 times with sterile water, then soaked for 10min with 1% NaClO, washed for 3-4 times with sterile water continuously, and finally the seeds are spread on a culture dish filled with sterilizing water-absorbing filter paper and dried to obtain detoxified seeds; during the process, each soaking or flushing process needs to be oscillated to make the treatment of the seeds full, and each flushing process needs to be spread on sterilized absorbent filter paper after the sterile water flushing process is finished, so that dust and the like carried by the seeds can be attached to the absorbent filter paper.
Further, the callus induction culture medium is a basal culture medium taking an MS culture medium, and further comprises the following components in percentage by weight: naphthalene Acetic Acid (NAA) 0.25-0.5 mg/L, 6-benzylaminoadenine (6-BA) 0.5mg/L,2, 4-dichlorophenoxyacetic acid (2, 4-D) 0.5-1 mg/L, sucrose 30g/L and agar 8g/L.
Furthermore, the adventitious bud differentiation medium and the adventitious bud proliferation medium have the same formula, take an MS culture medium as a basic culture medium, and further comprise the following components in parts by weight: naphthalene Acetic Acid (NAA) 0.2mg/L, 6-benzylaminoadenine (6-BA) 1mg/L, sucrose 30g/L, agar 8g/L.
Further, the rooting culture medium takes 1/2MS as a basic culture medium and also comprises the following components in percentage by weight: 0-0.5 mg/L indolebutyric acid (IBA), 30g/L sucrose and 8g/L agar.
All the above media were pH adjusted to 5.81-5.83 with 1% HCl or 1mol/LNaOH prior to sterilization.
Further, the matrix is a Pink matrix.
In the method for establishing the high-efficiency induction up to Wu Lihu branch callus regeneration system, wild up to Wu Lihu branch seeds in the current year are selected as the explant materials, and a large amount of the explant materials can be obtained only by collecting the seeds in a concentrated manner within 9-10 months and used in subsequent experiments.
In the method for establishing the high-efficiency induction Wu Lihu branch callus regeneration system, the concentration of plant hormone is optimized, and the result shows that when the concentration of Naphthalene Acetic Acid (NAA) is 0.5mg/L and the concentration of 2, 4-dichlorophenoxyacetic acid (2, 4-D) is 1mg/L, the callus induction rate is about 87%; the concentration of Naphthalene Acetic Acid (NAA) is 0.25mg/L, the concentration of 2, 4-dichlorophenoxyacetic acid (2, 4-D) is 0.5mg/L, which is the optimal formula for inducing the callus, the callus induction rate can reach 100%, the callus is yellow and loose, the growth state is good, and a foundation is laid for the subsequent construction of a regeneration system reaching Wu Lihu branches.
The invention provides a method for establishing a regeneration system of up to Wu Lihu branches, which is stable and mature, takes wild up to Wu Lihu branches seeds in the current year as explants to induce callus, and has high regeneration rate. The invention not only provides good technical support for high-efficiency industrial seedling raising of up to Wu Lihu branches, but also lays a foundation for genetic transformation and directional genetic improvement of up to Wu Lihu branches.
Drawings
FIG. 1 is a flow chart of a process for constructing up to Wu Lihu shoot callus and regeneration system.
FIG. 2 is a plot of growth at stage a during induction of callus formation following inoculation into the medium.
FIG. 3 is a plot of growth at stage b in the induction of callus formation following inoculation into the medium.
FIG. 4 is a plot of growth at stage c during induction of callus formation following inoculation into the medium.
FIG. 5 is a plot of growth at stage d during induction of callus formation following inoculation into the medium.
FIG. 6 is a plot of the growth of callus proliferation.
FIG. 7 is a graph showing the differentiation and growth vigor of adventitious buds.
FIG. 8 shows a graph of robust adventitious bud growth.
FIG. 9 rooting induction growth vigor diagram.
Detailed Description
The following describes in detail the method for establishing the callus induction culture medium of up to Wu Lihu branch seeds and the regeneration system of up to Wu Lihu branches according to the embodiments.
Example 1
The invention provides a method for establishing a regeneration system of up to Wu Lihu branches, which comprises the following steps:
1) Explant sterilization:
selecting full seeds of branches reaching Wu Lihu in the current year as explants, sterilizing, and airing to obtain detoxified seeds;
2) Primary callus induction:
inoculating the detoxified seed in the step 1) into a callus induction culture medium to induce primary callus;
3) Differentiation of adventitious bud
Transferring the primary callus of the step 2) to a differentiation medium after multiple subcultures to promote induction of adventitious buds;
4) Proliferation and elongation growth of adventitious buds:
transferring the adventitious buds induced in the step 3) to a proliferation culture medium to promote the proliferation of the adventitious buds, and further continuing to extend the adventitious buds to obtain robust adventitious buds;
5) Rooting induction:
transferring the separated plants to a rooting culture medium to be cultured until 4 young roots are formed after the robust adventitious buds obtained in the step 4) are about 5cm in height;
6) Hardening and transplanting:
and 5) taking out the tissue culture seedlings after the root systems of the tissue culture seedlings in the step 5) are developed, cleaning a culture medium, transplanting a matrix, watering a proper amount, culturing under an indoor greenhouse, transplanting the tissue culture seedlings into soil after the new growth She Fuzhuang is generated, and culturing in the outdoor greenhouse.
In the step 1), the selected seeds are soaked in tap water for 30min, then are washed once by ultrapure water, are put into a sterilizing bottle to be placed in an ultra-clean bench after being filtered off, are soaked in 75% alcohol for 30sec, are washed with sterile water for 4 times, are soaked in 1% NaClO for 10min, are continuously washed with sterile water for 3 times, and are spread on a culture dish filled with sterilizing water-absorbing filter paper for airing to obtain detoxified seeds; during the process, each soaking or flushing process needs to be oscillated to make the treatment of the seeds full, and each flushing process needs to be spread on sterilized absorbent filter paper after the sterile water flushing process is finished, so that dust and the like carried by the seeds can be attached to the absorbent filter paper.
The conditions of the callus induction culture are preferably as follows:
the whole callus induction process is under dark condition, and the temperature is 22 ℃; the time of the callus induction culture is preferably 42d, more preferably 39d.
The proliferation culture is preferably carried out every two weeks for 2 times continuously. The differentiation and proliferation culture is preferably performed on the callus differentiation and proliferation medium according to the above-described technical scheme. The conditions for the differentiation and proliferation culture are preferably as follows: the photoperiod is 16h illumination/8 h darkness, the illumination intensity is 2000Lux, and the temperature under the illumination condition is 22 ℃; the time of the induction culture is preferably 23d, more preferably 21d.
The rooting induction is preferably carried out by inoculating plants with the height of 3cm to a rooting culture medium. The rooting culture is a 1/2MS culture medium. The time for rooting induction is preferably 4 weeks. The conditions for rooting induction are preferably as follows: the photoperiod is 16h illumination/8 h darkness, the illumination intensity is 2000Lux, and the temperature is 22 ℃. More preferably, the illumination intensity is 2500Lux and the temperature is 22 ℃. The time of the induction culture is preferably 28d, more preferably 23d. And obtaining regenerated seedlings after the plants grow 4 roots.
The high-efficiency induction culture medium is a basal culture medium taking an MS culture medium and further comprises the following components in percentage by weight: naphthalene Acetic Acid (NAA) 0.25mg/L, 6-benzylaminoadenine (6-BA) 0.5mg/L,2, 4-dichlorophenoxyacetic acid (2, 4-D) 1mg/L, sucrose 30g/L, and agar 8g/L.
The adventitious bud differentiation culture medium and the adventitious bud proliferation culture medium are based on MS, and further comprise the following components in percentage by weight: 0.2mg/L Naphthalene Acetic Acid (NAA), 1 mg/L6-benzylaminoadenine (6-BA), 30g/L sucrose, 8g/L agar powder.
The adventitious bud differentiation culture medium is subjected to bud differentiation culture for about one week, the differentiation rate can reach 85.7143%, and the adventitious buds are elongated and grow well; by adopting the adventitious bud proliferation culture medium, a large number of adventitious buds can be obtained, and the leaves of the adventitious buds are dark green and grow healthily after continuous culture for about 9 days.
The rooting culture medium is based on 1/2MS, and further comprises the following components in percentage by weight: the concentration of the indolebutyric acid (IBA) is 0.5mg/L, the concentration of the preferred indolebutyric acid (IBA) is 0mg/L,30g/L sucrose and 8g/L agar powder.
All of the above media were pH adjusted to 5.81 with 1% HCl or 1mol/LNaOH prior to sterilization.
Example 2
A method for establishing a high-efficiency induction up to Wu Lihu branch callus regeneration system comprises the following steps:
1) Selecting full seeds of branches reaching Wu Lihu in the current year as explants, soaking the explants for 30min with tap water, washing the explants with ultrapure water once, filtering out ultrapure water, placing the ultrapure water into a sterilizing bottle, placing the sterilizing bottle on an ultra-clean bench, soaking the sterilized bottle with 75% alcohol for 30sec, washing the sterilized bottle with sterile water for 3 times, soaking the sterilized bottle with 1% NaClO for 10min, continuously washing the sterilized bottle with sterile water for 4 times, spreading the sterilized bottle on a culture dish filled with sterilizing water-absorbing filter paper, and airing the sterilized bottle to obtain sterilized seeds; during the period, each soaking or flushing process needs to be oscillated to make the treatment of the seeds full, and each flushing process with sterile water needs to be spread on sterilized absorbent filter paper, so that dust and the like carried by the seeds can be attached to the absorbent filter paper;
2) Inoculating the detoxified seed into a callus induction culture medium (naphthalene acetic acid (NAA) with the concentration of 0.25mg/L and 2, 4-dichlorophenoxyacetic acid (2, 4-D) with the concentration of 0.5 mg/L) for primary callus induction;
3) Calli were subcultured every 2 weeks, 3 times later, and transferred to differentiation medium (basal medium with MS, further comprising the following content components: 1mg/L naphthylacetic acid, 1 mg/L6-benzylaminoadenine, 30g/L sucrose, 8g/L agar powder), promoting induction of adventitious bud;
4) The adventitious buds induced in the step (based on MS medium, further comprising the following components: 0.2mg/L naphthylacetic acid, 1 mg/L6-benzylaminoadenine, 30g/L sucrose, 8g/L agar powder), promote the propagation of adventitious buds, and further continue to elongate the adventitious buds to obtain robust adventitious buds;
5) After the robust adventitious bud is about 5cm in height, the separated plants are transferred to a rooting medium (based on a 1/2MS medium, and the content components are: 30g/L sucrose, 8g/L agar powder) until 4 young roots are formed;
6) After the root system of the tissue culture seedling develops, the tissue culture seedling is taken out, the culture medium is washed, and matrix soil (Pinshi matrix: vermiculite: perlite = 8:3: 1) After a proper amount of watering, culturing in an indoor greenhouse, transplanting the newly born She Fuzhuang into soil and culturing in an outdoor greenhouse.
The invention does not limit the up to Wu Lihu branch seeds, and can adopt the up to Wu Lihu branch seeds with different sources according to the test requirement. In the embodiment of the invention, up to Wu Lihu branch seeds are taken as explants. The explant is sterilized prior to inoculation, and the method of sterilization is not particularly limited in the present invention, and sterilization methods well known in the art may be used.
The preparation method of the culture medium composition is not particularly limited, and the preparation method of the culture medium composition known in the art may be adopted, for example, 4 kinds of culture mediums may be prepared respectively according to a conventional autoclaving method, and each culture medium may be separately packaged into a certain container.
Test example 1
FIG. 1 shows a flowchart of a method for establishing a tissue culture regeneration system of up to Wu Lihu branches, which comprises the following specific steps:
1) Explant sterilization:
selecting full seeds grown in the current year and reaching Wu Lihu branches as explants, soaking the explants for 30min with tap water, washing the explants with ultrapure water once, filtering out ultrapure water, placing the sterilized explants into a sterilizing bottle, placing the sterilizing bottle on an ultra-clean bench, soaking the sterilized bottle in 75% alcohol for 30sec, washing the sterilized bottle with sterile water for 4 times, soaking the sterilized bottle in 1% NaClO for 10min, washing the sterilized bottle with sterile water for 3 times, spreading the sterilized bottle on a culture dish filled with sterilizing water-absorbing filter paper, and airing the sterilized bottle to obtain the detoxified seeds. During the process, the culture dish is fully treated by oscillating for several times each time of soaking or flushing, and is paved on the culture dish filled with the sterilized water-absorbing filter paper after each time of flushing with sterile water.
2) Primary callus induction:
inoculating the detoxified seed in the step 1) into a callus induction culture medium, and performing primary callus induction under dark conditions in a tissue culture room at the temperature of 24 ℃.
9 kinds of callus induction culture mediums are prepared, and three concentration gradients of Naphthalene Acetic Acid (NAA) and 6-Benzylaminoadenine (6-BA) and 2,4-D as L 3 And (5) performing orthogonal test. The prepared explants were inoculated into 9 different callus induction media, the callus induction rate was counted after 5-6 weeks, and the best induction media were selected according to the callus color, texture judgment, as shown in fig. 2-5.
TABLE 1 screening of callus induction Medium
The research result shows that the hormone combination (MS+0.25 mg/L Naphthalene Acetic Acid (NAA) +0.5 mg/L6-benzylaminoadenine (6-BA) +0.5mg/L2, 4-dichlorophenoxyacetic acid 2, 4-D) of the culture medium No. 1 has the best performance in inducing the callus, the callus induction rate can reach 100%, and the callus is yellow, loose and has good growth state, as shown in figure 5.
3) Differentiation of adventitious bud:
transferring the primary callus of the step 2) to a differentiation medium (0.2 mg/L naphthylacetic acid (NAA) +1 mg/L6-benzylaminoadenine (6-BA)) after 3 times of subculture every 2 weeks under the illumination condition, so as to promote the induction of adventitious buds; the illumination intensity is 2500Lux, the illumination period is 16h/d, the temperature is 22 ℃, and the growth condition is shown in figure 7.
4) Proliferation and elongation growth of adventitious buds:
transferring the adventitious bud induced in the step 3) to a proliferation culture medium (0.2 mg/L Naphthalene Acetic Acid (NAA) +1 mg/L6-benzylaminoadenine (6-BA)), and promoting propagation of the adventitious bud, so that the adventitious bud is elongated, and a robust adventitious bud is obtained; the illumination intensity is 2500Lux, the illumination period is 16h/d, and the temperature is 22 ℃. The growth of which is shown in figure 8.
5) Rooting induction:
and (3) transferring the separated plants to a rooting culture medium to be cultured until 4 young roots are formed after the robust adventitious buds obtained in the step (4) are about 4cm in height.
TABLE 2 screening of rooting Medium
| IBA concentration/mg/L | Root number/number of roots | Longest root length/cm |
| 0 | 4.3824 | 4.9709 |
| 0.5 | 2.7500 | 4.2094 |
| 1 | 3.7778 | 3.6967 |
The test results showed that when indolebutyric acid (IBA) was not added, the rooting number was the largest, and the rooting was the longest and strong, as shown in fig. 9.
(6) Hardening and transplanting:
after the root system of the tissue culture seedling in the step 5) is developed, taking out the tissue culture seedling, cleaning a culture medium, and transplanting a matrix, wherein matrix soil is used as a matrix: vermiculite: perlite = 8:3:1, a step of; after watering a proper amount, culturing under an indoor greenhouse, transplanting the newly born She Fuzhuang into soil and culturing in the outdoor greenhouse. The illumination intensity of the indoor greenhouse is 2500Lux, the illumination period is 16h/d, the temperature is 24 ℃, the first 3 days after 2 times of transplanting are all required to cover seedlings by using plastic cups with a plurality of small openings cut off, and then the plastic cups are gradually lifted according to the growth condition of the seedlings along with the time.
Claims (2)
1. The method for establishing the regeneration system of the callus of the branches of Wu Lihu by high-efficiency induction is characterized by comprising the following steps of:
1) Explant sterilization:
selecting full seeds of branches reaching Wu Lihu in the current year as explants, sterilizing, and airing to obtain detoxified seeds;
2) Primary callus induction:
inoculating the detoxified seed in the step 1) into a callus induction culture medium to induce primary callus;
3) Differentiation of adventitious bud:
transferring the primary callus of the step 2) to a differentiation medium after multiple subcultures to promote induction of adventitious buds;
4) Proliferation and elongation growth of adventitious buds:
transferring the adventitious buds induced in the step 3) to a proliferation culture medium to promote the proliferation of the adventitious buds, and further continuing to extend the adventitious buds to obtain robust adventitious buds;
5) Rooting induction:
transferring the separated plants to a rooting culture medium for culture until 3-4 young roots are formed after the length of the robust adventitious buds obtained in the step 4) is 3-5 cm;
6) Hardening and transplanting:
taking out the tissue culture seedlings after the root systems of the tissue culture seedlings in the step 5) develop, cleaning a culture medium, transplanting a matrix, watering a proper amount, culturing under an indoor greenhouse, transplanting the tissue culture seedlings into soil after the new growth She Fuzhuang is generated, and culturing in the outdoor greenhouse;
the callus induction culture medium is based on an MS culture medium and further comprises the following components in percentage by weight: 0.25-1 mg/L of naphthylacetic acid, 0.5mg/L of 6-benzylaminoadenine, 0.5-1 mg/L of 2, 4-dichlorophenoxyacetic acid, 30g/L of sucrose and 8g/L of agar powder;
the differentiation medium is based on an MS medium and further comprises the following components in percentage by weight: 0.2mg/L naphthalene acetic acid, 1 mg/L6-benzylaminoadenine, 30g/L sucrose, 8g/L agar powder;
the proliferation culture medium is based on an MS culture medium and further comprises the following components in percentage by weight: 0.2mg/L naphthalene acetic acid, 1 mg/L6-benzylaminoadenine, 30g/L sucrose, 8g/L agar powder;
the rooting culture medium is based on a 1/2MS culture medium, and comprises the following components: 0-0.5 mg/L indolebutyric acid, 30g/L sucrose and 8g/L agar powder.
2. The method for establishing a high-efficiency induction Wu Lihu branch callus regeneration system according to claim 1, wherein in the step 1), selected seeds are soaked in tap water for 30min, then washed once by ultra-pure water, filtered out, put into a sterilization bottle, placed into an ultra-clean bench, soaked in 75% alcohol for 30sec, washed 3-4 times with sterile water, soaked in 1% NaClO for 10min, washed 3-4 times with sterile water continuously, and finally spread on a culture dish filled with sterilization water-absorbing filter paper for airing to obtain detoxified seeds; during the process, each soaking or flushing process needs to be oscillated to make the treatment of the seeds full, and each flushing process needs to be spread on sterilized absorbent filter paper after the sterile water flushing process is finished, so that dust and the like carried by the seeds can be attached to the absorbent filter paper.
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