CN116326479A - Culture method for obtaining madder tissue culture seedlings through callus induction - Google Patents
Culture method for obtaining madder tissue culture seedlings through callus induction Download PDFInfo
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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
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract
The invention provides a method for culturing gamene tissue culture seedlings by callus induction, belonging to the technical field of gamene culture. The invention solves the problem of poor tissue culture effect of the madder in the prior art by adopting the culture medium with specific components. The invention can directly establish sterile line by inducing and differentiating adventitious buds and gamene stem sections by using callus, and the proliferation coefficient of the gamene in-vitro regeneration system formed by the method is higher. The culture method is efficient and quick, and provides an effective way for breeding the excellent variety of the madder and preserving in-vitro germplasm resources; compared with the traditional madder propagation mode, the method has high propagation coefficient, can perform material selection and propagation in any growth stage, and provides convenient conditions for breeding of good madder varieties and industrialized seedling. The method can obtain a large number of madder tissue culture seedlings through simple steps, and provides technical support for industrial seedling and domestication cultivation in future.
Description
Technical Field
The invention relates to the technical field of cultivation of madder, in particular to a cultivation method for obtaining madder tissue culture seedlings through callus induction.
Background
Rubia cordifolia.L. is a perennial vine plant of Rubia genus of Rubiaceae family, which is up to 60 or more, distributed in Europe, asia, south Africa and America. The madder is a hemostatic drug which has the effects of cooling blood, removing blood stasis, stopping bleeding and dredging channels, so the madder is used as a traditional Chinese medicine. Modern pharmacological research shows that Chinese madder contains high-efficiency low-toxicity anticancer component cyclohexapeptide, but European madder R.tinctorum has no anticancer effect, and European madder also contains forbidden medicine-Lukidine. Not only does chinese madder contain doxycycline, but also 4 monomeric compounds having anticancer effects have been isolated, so that the sales of chinese madder in the world have proliferated. However, since madder belongs to perennial herbs, natural growth is very slow, and wild resources tend to be exhausted. In order to protect the wild resources of the Chinese madder, realize the modern cultivation of the madder, develop the research of the isolated rapid propagation and callus induction of the madder, it is more urgent and important. Wild resources of the madder are very limited, and main propagation modes of the madder are seed direct seeding, cutting, grafting and the like, but the propagation modes have low propagation coefficient and long growth period and are easily influenced by factors such as external environment conditions and the like. And the existing tissue culture schemes are not suitable for cultivation of the madder, so that the madder callus with good quality and poor proliferation effect are difficult to obtain.
Disclosure of Invention
The invention aims to provide a method for culturing gamene tissue culture seedlings obtained through callus induction, so as to solve the problem of poor gamene tissue culture effect in the prior art.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a culture method for obtaining gamene tissue culture seedlings through callus induction, which comprises a scheme S1 or a scheme S2;
the scheme S1 comprises the following steps:
s1.1, taking gamene leaves or gamene stem sections without buds as explants, and inducing callus growth in an induction culture medium;
s1.2, replacing the culture medium with a differentiation culture medium, and inducing the differentiation of adventitious buds;
s1.3, replacing the culture medium with a proliferation culture medium to induce bud proliferation;
s1.4, replacing the seedling culture medium with a rooting culture medium, and inducing rooting to obtain the madder tissue culture seedling;
the scheme S2 comprises the following steps:
s2.1, taking a bud stem section of madder as an explant, and inducing bud proliferation in a proliferation culture medium;
s2.2, replacing the seedling culture medium with a rooting culture medium, and inducing rooting to obtain the madder tissue culture seedlings;
the induction culture medium contains 1.8-2.2 mg/L of 2,4-D and 0.5-1.0 mg/L of NAA;
the proliferation culture medium contains 1.0-1.5 mg/L, IBA 0.3.0.3-0.7 mg/L thidiazuron and 120-180 g/L bermudagrass water extract.
Preferably, the induction medium further comprises MS basic culture medium, agar 7.0-7.5 g/L and sucrose 25-30 g/L.
Preferably, the proliferation medium further comprises MS basic culture medium, 7.2-7.8 g/L agar and 27-33 g/L sucrose.
Preferably, the differentiation medium composition comprises: MS basic culture medium, thidiazuron 1-3 mg/L, NAA, 0.08-0.12 mg/L, agar 7.2-7.8 g/L and sucrose 27-33 g/L.
Preferably, the rooting medium comprises the following components: 1/2MS basic culture medium, IBA 2-3 mg/L, NAA 0.2-0.5 mg/L, agar 7.0-7.5 g/L and sucrose 25-30 g/L.
Preferably, the temperature for inducing the growth of the callus and the proliferation of the buds is 24-26 ℃, the humidity is 75-85%, the illumination is 14-16 h/d, and the illumination intensity is 1500-2000 Lx.
Preferably, the time for inducing bud proliferation is 7-14 d.
Preferably, the time for inducing the adventitious bud differentiation is 14-20 d, and the whole-course dark culture is adopted.
Preferably, the time for inducing rooting is 25-30 d.
Preferably, the culture method further comprises hardening and transplanting the madder tissue culture seedlings, wherein seedling culture matrixes adopted by transplanting comprise vermiculite, nutrient soil and river sand, and the vermiculite comprises the following components: nutrient soil: the weight ratio of river sand is 0.5-1.5:0.5-1.5:1.5-2.5.
The invention has the technical effects and advantages that:
the invention provides a culture method for obtaining gamene tissue culture seedlings through callus induction, which can directly establish a sterile line by using adventitious buds and gamene stem segments induced and differentiated by callus, and the proliferation coefficient of a gamene in-vitro regeneration system formed by the method is higher. The culture method is efficient and quick, and provides an effective way for breeding the excellent variety of the madder and preserving in-vitro germplasm resources; compared with the traditional madder propagation mode, the tissue culture method has high propagation coefficient, can perform material selection and propagation at any growth stage, and provides convenient conditions for breeding of good madder varieties and industrialized seedling. According to the method, a large number of gamene tissue culture seedlings can be obtained through simple steps, a complete gamene in-vitro regeneration technology system is established, technical support is provided for industrial seedling culture and domestication cultivation in the future, and therefore the problems of high difficulty in gamene introduction and domestication and lack of wild resources are solved.
Drawings
FIG. 1 is a schematic diagram showing the results of callus induction induced by the stem segments of Rubia cordifolia;
FIG. 2 is a schematic diagram showing the result of differentiating adventitious buds from calli of Rubia cordifolia;
FIG. 3 is a schematic diagram showing the result of cluster bud cultivation of Rubia cordifolia.
Detailed Description
The invention provides a culture method for obtaining gamene tissue culture seedlings through callus induction, wherein the culture method comprises a scheme S1 or a scheme S2;
the scheme S1 comprises the following steps:
s1.1, taking gamene leaves or gamene stem sections without buds as explants, and inducing callus growth in an induction culture medium;
s1.2, replacing the culture medium with a differentiation culture medium, and inducing the differentiation of adventitious buds;
s1.3, replacing the culture medium with a proliferation culture medium to induce bud proliferation;
s1.4, replacing the culture medium with a rooting culture medium, and inducing rooting to obtain the madder tissue culture seedlings.
The scheme S2 comprises the following steps:
s2.1, taking a bud stem section of madder as an explant, and inducing bud proliferation in a proliferation culture medium;
s2.2, replacing the culture medium with a rooting culture medium, and inducing rooting to obtain the madder tissue culture seedlings.
In the invention, the explant comprises gamene leaves and gamene stem segments, the explant is preferably disinfected before inducing callus growth, the disinfection is preferably that the gamene leaves or gamene stem segments are washed by washing powder and distilled water in sequence, then the gamene leaves or gamene stem segments are disinfected by alcohol, the volume fraction of the alcohol is preferably 70-80%, the alcohol disinfection time is preferably 30-40 s, the mercuric solution concentration is preferably 0.05-0.15%, the mercuric solution disinfection time is preferably 5-7 min, then the gamene leaves or gamene stem segments are washed by flowing water for 30-40 min, preferably 33-37 min, the surface water is absorbed, then the gamene leaves or gamene stem segments washed by sterile water are inoculated in MS culture medium for 3-5 min, and inducing callus growth can be carried out.
In the invention, when the explant is gamene leaf or gamene stem without bud, the explant is inoculated to a callus induction culture medium, wherein the callus induction culture medium contains 2, 4-D1.8-2.2 mg/L and NAA 0.5-1.0 mg/L, the concentration of 2,4-D in the induction culture medium is preferably 1.9-2.1 mg/L, and the concentration of NAA in the induction culture medium is preferably 0.7-0.9 mg/L; preferably, the induction medium further comprises: MS basic culture medium, agar 7.0-7.5 g/L and sucrose 25-30 g/L, wherein the concentration of agar in the induction culture medium is more preferably 7.2-7.4 g/L, the concentration of sucrose in the induction culture medium is more preferably 26-28 g/L, and the pH of the induction culture medium is preferably 5.8-6.0; after bud proliferation, the culture medium is replaced by a differentiation culture medium, and the differentiation culture medium is used for inducing the differentiation of adventitious buds, wherein the differentiation culture medium preferably comprises the following components: MS basic culture medium, thidiazuron 1-3 mg/L, NAA 0.08.08-0.12 mg/L, agar 7.2-7.8 g/L and sucrose 27-33 g/L, wherein the thidiazuron concentration in the differentiation culture medium is preferably 1.5-2.5 mg/L, the NAA concentration in the differentiation culture medium is preferably 0.09-0.11 mg/L, and the time for inducing adventitious bud differentiation is preferably 14-20 d, and the whole-course dark culture is adopted; changing the adventitious bud into a proliferation culture medium after the adventitious bud is differentiated, and inducing the bud proliferation; when the explant is a bud-bearing madder stem segment, the explant is directly inoculated to a proliferation medium, wherein the proliferation medium contains 1.0-1.5 mg/L, IBA 0.3.3-0.7 mg/L of thidiazuron and 120-180 g/L of a water extract of bermudagrass, the concentration of the thidiazuron in the proliferation medium is preferably 1.2-1.4 mg/L, the concentration of the IBA in the proliferation medium is preferably 0.4-0.6 mg/L, the concentration of the water extract of the bermudagrass in the proliferation medium is preferably 140-160 g/L, and the bermudagrass refers to a perennial herb Cynodon (L.) Pers. The preparation method of the bermuda grass water extract comprises the following steps: weighing 150g of whole plant of the bermuda grass, cleaning, placing in 1L of sterile water for boiling, and then fishing out, wherein the left 1L of water containing the bermuda grass juice is used as a subsequent culture medium for configuration, namely the bermuda grass water extract; preferably, the proliferation medium further comprises an MS basic medium, agar 7.2-7.8 g/L and sucrose 27-33 g/L, wherein the concentration of the agar in the proliferation medium is preferably 7.3-7.6 g/L, the concentration of the sucrose in the proliferation medium is preferably 29-30 g/L, the pH of the proliferation medium is preferably 5.8-6.0, and the bud proliferation induction time is preferably 7-14 d, and more preferably 9-12 d; changing the buds to rooting culture medium after proliferation, and inducing rooting, wherein the subsequent step is preferably carried out by using 3-5 cm long gamene seedlings, and the rooting induction time is preferably 25-30 d; the rooting medium components preferably comprise: 1/2MS basic culture medium, IBA 2-3 mg/L, NAA 0.2-0.5 mg/L, agar 7.0-7.5 g/L and sucrose 25-30 g/L, wherein the concentration of IBA in the rooting culture medium is preferably 2.3-2.7 mg/L, the concentration of NAA in the rooting culture medium is preferably 0.15-0.25 mg/L, the concentration of agar in the rooting culture medium is preferably 7.2-7.4 g/L, and the concentration of sucrose in the rooting culture medium is preferably 26-28 g/L.
In the present invention, the temperature during the culture is preferably 24 to 26 ℃, more preferably 24.5 to 25.5 ℃, the humidity is preferably 75 to 85%, more preferably 78 to 82%, the illumination is preferably 14 to 16h/d, more preferably 14.5 to 15.5h/d, and the illumination intensity is preferably 1500 to 2000Lx, more preferably 1600 to 1800Lx, except for the induced differentiation process.
In the invention, the culture method further comprises transplanting the madder tissue culture seedlings after rooting culture, the madder tissue culture seedlings are preferably transferred to an outdoor shade place before transplanting, the cover is opened for domestication culture for 5-7 d, then the tissue culture seedlings are taken out, agar adhered to the base is washed off by clear water, and then the tissue culture seedlings are transplanted into seedling culture matrixes, wherein the seedling culture matrixes adopted by the transplanting preferably comprise vermiculite, nutrient soil and river sand, and the vermiculite comprises the following components: nutrient soil: the weight ratio of river sand is preferably 0.5-1.5:0.5-1.5:1.5-2.5, more preferably 0.8-1.1:0.8-1.1:1.8-2.2, the culture temperature after transplanting is preferably 24-26 ℃, more preferably 24.5-25.5 ℃, and the illumination is preferably 6-10 h/d, more preferably 7-9 h/d.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Step 1: establishment of sterile line
Taking a wild madder plant collected in a Beijing traditional Chinese medicine university campus as an explant source, and planting the wild madder plant in a climatic chamber 35d for material taking; selecting tender madder leaves, sequentially washing the tender madder leaves with washing powder and distilled water, sterilizing the tender madder leaves with 75% alcohol for 35s, sterilizing the tender madder leaves with 0.1% mercuric chloride for 6min, washing the tender madder leaves with running water for 30min, sucking water on the surfaces of the tender madder leaves, taking out the tender madder leaves, washing the tender madder leaves with sterile water, and inoculating the tender madder leaves to a buffer culture medium to obtain the aseptic madder leaf explants.
Step 2: callus induction
Inoculating the sterilized madder leaves to a callus induction culture medium, wherein the callus induction culture medium comprises the following components: MS+2, 4-D2.0 mg/L+NAA 0.8 mg/L+agar 7.3 g/L+sucrose 27g/L, pH 6.0;
culture conditions: the temperature is 25 ℃, the humidity is 80 percent, the illumination is 14h/d, and the illumination intensity is 1500Lx.
Step 3: callus differentiation
Taking induced radix Rubiae callus (shown in figure 1), and performing cluster bud differentiation culture on the induced radix Rubiae callus, wherein the used radix Rubiae callus differentiation culture medium is as follows: MS+2.0mg/LTDZ+0.1mg/LNAA+30g/L sucrose+7.6 g/L agar, pH=5.86, and the whole process of dark culture, after the leaves are cultured for 12 days in a callus induction culture medium, callus is generated, the callus is formed at the shearing part, and the calli of the madder are light yellow and compact. Culturing the callus in differentiation medium for 16d can produce a large number of adventitious buds, as shown in FIG. 2.
Culture conditions: the temperature is 25 ℃, the humidity is 80 percent, and the whole process is dark culture.
Step 4: seedling multiplication culture
Inoculating the differentiated seedlings obtained in the step 3 into a proliferation culture medium, wherein the formula comprises the following components: MS+1.3mg/L thidiazuron+0.5 mg/LIBA+150 g/L of aqueous extract of bermuda grass (150 g of whole plant of bermuda grass is weighed, washed and placed in 1L of sterile water to be boiled, and fished out to obtain the aqueous extract of bermuda grass) +30g/L sucrose+7.6 g/L agar, and the pH value is 5.86.
Culture conditions: the temperature is 25 ℃, the humidity is 80 percent, the illumination is 14h/d, and the illumination intensity is 1500Lx.
Step 5: rooting culture of seedling
Transplanting the 4cm long gamene seedlings (shown in figure 3) obtained by culturing in the step 4 into rooting culture medium for rooting culture for 25d, wherein the rooting culture medium is as follows: 1/2MS+2.5mg/L IBA+NAA 0.4mg/L+23g/L sucrose+7.2 g/L agar, pH6.0, and culturing for 27d to obtain radix Rubiae rooting tissue culture seedling.
Culture conditions: the temperature is 25 ℃, the humidity is 80 percent, the illumination is 14h/d, and the illumination intensity is 1500Lx.
Step 6: seedling hardening, transplanting and culturing
Selecting the madder tissue culture seedlings with developed root systems and strong plant growth in the step 5, performing seedling hardening and transplanting, transferring the madder tissue culture seedlings into outdoor shade places, opening the cover, performing domestication and cultivation for 6d, taking out, washing off agar adhered to the base parts of the tissue culture seedlings with clean water, transplanting the madder tissue culture seedlings into sterilized and watered seedling culture matrixes (vermiculite: nutrient soil: river sand=1:1:2), and performing cultivation in an illumination incubator at 25 ℃ under an illumination condition of 8 h/d.
Example 2
Step 1: establishment of sterile line
Taking a wild madder plant collected in a Beijing traditional Chinese medicine university campus as an explant source, and planting the wild madder plant in a climatic chamber for 30d for material taking; selecting tender madder bud stem segments, sequentially washing the tender madder bud stem segments with washing powder and distilled water, sterilizing the tender madder bud stem segments with 75% alcohol for 30s, sterilizing the tender madder bud stem segments with 0.1% mercuric chloride for 5min, washing the tender madder bud stem segments with running water for 30min, sucking water on the surface of the terminal buds, taking out the tender madder bud stem segments, washing the tender madder bud stem segments with sterile water, and inoculating the madder bud stem segments with sterile culture medium to obtain the madder bud stem segment sterile explants.
Step 2: bud multiplication culture
Directly inoculating the sterilized bud-bearing stem to a proliferation culture medium, wherein the formula comprises the following steps: MS+1.3mg/L Thidiazuron+0.5 mg/L IBA+Cynodon dactylon aqueous extract 150g/L+30g/L sucrose+7.6 g/L agar, pH 5.86.
The culture condition is that the temperature is 25 ℃, the humidity is 80%, the illumination is 14h/d, and the illumination intensity is 1500Lx.
Step 3: rooting culture of seedling
And (2) transferring the 5cm long gamene seedlings obtained in the step (2) into a rooting medium for rooting culture for 22d, wherein the rooting medium is as follows: 1/2MS+2.0mg/L IBA+NAA0.2mg/L+20g/L sucrose+7.0 g/L agar, pH5.8.
Culture conditions: the temperature is 25 ℃, the humidity is 80 percent, the illumination is 14h/d, and the illumination intensity is 1500Lx.
Step 4: seedling hardening, transplanting and culturing
Selecting a tissue culture seedling of madder with developed root system and strong plant growth after rooting culture, performing seedling hardening and transplanting, transferring the tissue culture seedling into an outdoor shade place, opening a cover, performing domestication and culture for 5 days, taking out, washing agar adhered to the base of the tissue culture seedling with clear water, transplanting the tissue culture seedling into a sterilized and watered seedling culture medium (vermiculite: nutrient soil: river sand=0.8:0.8:2), and culturing in an illumination incubator at 24 ℃ under the illumination condition of 7 h/d.
Example 3
Step 1: establishment of sterile line
Taking a wild madder plant collected in a Beijing traditional Chinese medicine university campus as an explant source, and planting the wild madder plant in a climatic chamber 35d for material taking; washing the stem segments of the madder without buds with washing powder and distilled water in sequence, sterilizing the stem segments with 75% alcohol for 40s, sterilizing the stem segments with 0.1% mercuric chloride for 7min, washing the stem segments with running water for 32min, sucking the surface water, taking out the stem segments, washing the stem segments with sterile water, and inoculating the stem segments to a buffer culture medium to obtain the aseptic stem segment explants of the madder.
Step 2: callus induction
Inoculating the sterilized madder stem to a callus induction culture medium, wherein the callus induction culture medium comprises the following components: MS+2, 4-D2.2 mg/L+NAA 0.5 mg/L+agar 7.5 g/L+sucrose 30g/L, pH 6.0;
culture conditions: the temperature is 26 ℃, the humidity is 85 percent, the illumination is 16h/d, and the illumination intensity is 2000Lx.
Step 3: callus differentiation
Performing cluster bud differentiation culture on the induced madder callus, wherein the used madder callus differentiation culture medium is as follows: MS+2.0mg/LTDZ+0.1mg/LNAA+30g/L sucrose+7.6 g/L agar, pH=5.86,
culture conditions: dark culture is carried out at the temperature of 25 ℃ and the humidity of 80 percent in the whole process, the leaves are cultured in a callus induction culture medium for 10 days to generate callus, the callus is formed at the shearing part, and the callus of the madder is light yellow and compact. Culturing the callus 17d in a differentiation medium can produce a large number of adventitious buds.
Step 4: seedling multiplication culture
Inoculating the differentiated seedlings obtained in the step 3 into a proliferation culture medium, wherein the formula comprises the following components: MS+1.3mg/L Thidiazuron+0.6 mg/LIBA+Cynodon dactylon aqueous extract 150g/L+32g/L sucrose+7.6 g/L agar, pH 5.86.
Culture conditions: the temperature is 25 ℃, the humidity is 80 percent, the illumination is 14h/d, and the illumination intensity is 1500Lx.
Step 5: rooting culture of seedling
Transferring the 4cm long gamene seedling obtained by culturing in the step 4 into rooting culture medium for rooting culture for 25d, wherein the rooting culture medium is as follows: 1/2MS+2.7mg/L IBA+0.5mg/L NAA+23g/L sucrose+7.2 g/L agar, pH5.9, and culturing 29d to obtain radix Rubiae rooting tissue culture seedling.
Culture conditions: the temperature is 25 ℃, the humidity is 80 percent, the illumination is 14h/d, and the illumination intensity is 1500Lx.
Step 6: seedling hardening, transplanting and culturing
Selecting the madder tissue culture seedlings with developed root systems and strong plant growth in the step 5, performing seedling hardening and transplanting, transferring the madder tissue culture seedlings into outdoor shade places, opening the cover, performing domestication and cultivation for 6d, taking out, washing agar adhered to the base parts of the tissue culture seedlings with clean water, transplanting the madder tissue culture seedlings into sterilized and watered seedling culture matrixes (vermiculite: nutrient soil: river sand=1.1:1.1:2), and performing cultivation in an illumination incubator at 25 ℃ under an illumination condition of 8 h/d.
Comparative example 1
The difference from example 1 was only that the concentration of 2,4-D in the callus induction medium was 1.0mg/L, NAA and that of 0.5mg/L.
Comparative example 2
The difference from example 1 was only that the concentration of 2,4-D in the callus induction medium was 1.0mg/L, NAA and that of 0.8mg/L.
Comparative example 3
The difference from example 1 was only that the concentration of 2,4-D in the callus induction medium was 1.0mg/L, NAA and 1.0mg/L.
Comparative example 4
The difference from example 1 was only that the concentration of 2,4-D in the callus induction medium was 2.0mg/L, NAA and that of 0.5mg/L.
Comparative example 5
The difference from example 1 was only that the concentration of 2,4-D in the callus induction medium was 2.0mg/L, NAA and 1.0mg/L.
Comparative example 6
The difference from example 1 was only that the concentration of 2,4-D in the callus induction medium was 2.5mg/L, NAA and that of 0.5mg/L.
Comparative example 7
The difference from example 1 was only that the concentration of 2,4-D in the callus induction medium was 2.5mg/L, NAA and that of 0.8mg/L.
Comparative example 8
The difference from example 1 was only that the concentration of 2,4-D in the callus induction medium was 2.5mg/L, NAA and 1.0mg/L.
Comparative example 9
The only difference from example 1 is that the concentration of thidiazuron in the propagation medium is 0.5mg/L, IBA and 0.1mg/L.
Comparative example 10
The only difference from example 1 is that the concentration of thidiazuron in the propagation medium is 0.5mg/L, IBA and 0.2mg/L.
Comparative example 11
The only difference from example 1 is that the concentration of thidiazuron in the propagation medium is 0.5mg/L, IBA and 1.5mg/L.
Comparative example 12
The only difference from example 1 is that the concentration of thidiazuron in the propagation medium is 0.5mg/L, IBA and 0.1mg/L.
Comparative example 13
The only difference from example 1 is that the concentration of thidiazuron in the propagation medium is 2.0mg/L, IBA and 1.5mg/L.
Comparative example 14
The only difference from example 1 is that the concentration of thidiazuron in the propagation medium is 3.0mg/L, IBA and 2.0mg/L.
Comparative example 15
The only difference from example 1 is that the concentration of thidiazuron in the propagation medium is 0.1mg/L, IBA and 0.1mg/L.
Comparative example 16
The only difference from example 1 is that the concentration of the aqueous extract of bermuda grass in the propagation medium is 100g/L.
Comparative example 17
The only difference from example 1 is that the seedling substrate ratio used in the seedling hardening, transplanting and culturing is vermiculite: nutrient soil = 1:1.
Comparative example 18
The only difference from example 1 is that the seedling substrate ratio used in the seedling hardening, transplanting and culturing is vermiculite: perlite=2:1.
Comparative example 19
The difference from example 1 is that the seedling substrate ratio used in the seedling hardening, transplanting and culturing is river sand: vermiculite = 1:1.
Comparative example 20
The only difference from example 1 is that the seedling substrate ratio used in the seedling hardening, transplanting and culturing is vermiculite: nutrient soil: perlite=1:1:1.
Comparative example 21
The only difference from example 1 is that the proliferation medium formulation is: MS+6-BA2.0mg/L+NAA 0.2 mg/L+agar 7.3 g/L+sucrose 27g/L, pH5.9.
Comparative example 22
The only difference from example 1 is that the calli differentiation medium of madder is: MS+6-BA 0.2mg/L+2, 4-D0.03 mg/L, and light 14h/D.
Experimental example 1
The callus induction conditions of example 1 and comparative examples 1 to 8 were counted, the callus induction rate (callus induction rate=amount of callus grown/total inoculum size) was calculated, and the callus growth conditions were recorded, and the results are shown in table 1 below:
TABLE 1 callus induction effects of different experimental groups
Experimental example 2
The proliferation of the gamene seedlings of example 1 and comparative examples 9 to 16 was counted, the proliferation coefficient of adventitious buds (proliferation coefficient=total number of adventitious buds/number of explants to be inoculated×100%) was calculated, and the callus growth, i.e. the total number of days from the time of inoculation of leaf explants to the time of callus growth was recorded, and the results are shown in table 2 below:
TABLE 2 proliferation results of madder seedlings from different experimental groups
| Treatment group | Multiplication factor of adventitious bud | Proliferation conditions |
| Comparative example 9 | 2.3 | Slower (20 d) |
| Comparative example 10 | 1.7 | Slow (24 d) |
| Comparative example 11 | 1.1 | Very slow (28 d) |
| Comparative example 12 | 3.6 | Faster (16 d) |
| Comparative example 13 | 3.4 | Faster (16 d) |
| Comparative example 14 | 2.8 | Slower (20 d) |
| Comparative example 15 | 4.9 | Faster (16 d) |
| Example 1 | 5.5 | Fast (12 d) |
| Comparative example 16 | 4.3 | General (14 d) |
Experimental example 3
The transplanting conditions of example 1 and comparative examples 17 to 22 were counted, and the transplanting survival rate=transplanting survival plants/total number of plants×100% was calculated, and the transplanting number of each group was 30 plants, and the results are shown in table 3:
TABLE 3 transplanting survival results for different experimental groups
| Treatment group | Survival rate/% |
| Comparative example 17 | 76.67 |
| Comparative example 18 | 80.00 |
| Example 1 | 93.33 |
| Comparative example 19 | 83.33 |
| Comparative example 20 | 70.00 |
| Comparative example 21 | 0.00 |
| Comparative example 22 | 0.00 |
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A method for culturing gamene tissue culture seedlings obtained by callus induction, characterized in that the method comprises a scheme S1 or a scheme S2;
the scheme S1 comprises the following steps:
s1.1, taking gamene leaves or gamene stem sections without buds as explants, and inducing callus growth in an induction culture medium;
s1.2, replacing the culture medium with a differentiation culture medium, and inducing the differentiation of adventitious buds;
s1.3, replacing the culture medium with a proliferation culture medium to induce bud proliferation;
s1.4, replacing the seedling culture medium with a rooting culture medium, and inducing rooting to obtain the madder tissue culture seedling;
the scheme S2 comprises the following steps:
s2.1, taking a bud stem section of madder as an explant, and inducing bud proliferation in a proliferation culture medium;
s2.2, replacing the seedling culture medium with a rooting culture medium, and inducing rooting to obtain the madder tissue culture seedlings;
the induction culture medium contains 1.8-2.2 mg/L of 2,4-D and 0.5-1.0 mg/L of NAA;
the proliferation culture medium contains 1.0-1.5 mg/L, IBA 0.3.0.3-0.7 mg/L thidiazuron and 120-180 g/L bermudagrass water extract.
2. The method according to claim 1, wherein the induction medium composition further comprises MS minimal medium, agar 7.0-7.5 g/L and sucrose 25-30 g/L.
3. The culture method according to claim 2, wherein the proliferation medium composition further comprises MS minimal medium, agar 7.2-7.8 g/L and sucrose 27-33 g/L.
4. The culture method of claim 1, wherein the differentiation medium composition comprises: MS basic culture medium, thidiazuron 1-3 mg/L, NAA, 0.08-0.12 mg/L, agar 7.2-7.8 g/L and sucrose 27-33 g/L.
5. The method of claim 1, wherein the rooting medium comprises: 1/2MS basic culture medium, IBA 2-3 mg/L, NAA 0.2-0.5 mg/L, agar 7.0-7.5 g/L and sucrose 25-30 g/L.
6. The method according to any one of claims 1 to 5, wherein the temperature for inducing callus growth and bud proliferation is 24 to 26 ℃, the humidity is 75 to 85%, the light is 14 to 16h/d, and the light intensity is 1500 to 2000Lx.
7. The culture method according to claim 6, wherein the time for inducing proliferation of buds is 7 to 14d.
8. The method according to claim 7, wherein the time for inducing the differentiation of adventitious buds is 14 to 20d, and the whole-course dark culture is adopted.
9. The method according to claim 8, wherein the time for inducing rooting is 25 to 30 days.
10. The method according to claim 1, further comprising transplanting the gamene tissue culture seedlings, wherein the transplanting adopts seedling culture substrates including vermiculite, nutrient soil and river sand, and the vermiculite: nutrient soil: the weight ratio of river sand is 0.5-1.5:0.5-1.5:1.5-2.5.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014167464A1 (en) * | 2013-04-08 | 2014-10-16 | Sederma | Production method of meristematic cells of plantago lanceolata, composition comprising said cells or their cellular extract, and cosmetic, nutraceutical and dermatological uses |
| WO2019006470A1 (en) * | 2017-06-30 | 2019-01-03 | Booshoot Llc | Media for rapid and reliable tissue culturing of plants |
| CN109392719A (en) * | 2018-11-25 | 2019-03-01 | 钟天路 | A kind of method for building up of madder regenerating system |
| CN110100736A (en) * | 2019-06-20 | 2019-08-09 | 南京农业大学 | A kind of water planting enrichment procedure of Thesium chinese tissue-cultured seedling |
| CN114793905A (en) * | 2022-05-23 | 2022-07-29 | 浙江省亚热带作物研究所(浙南林业科学研究院) | Rapid propagation method and culture medium for gardenia tissue culture seedlings |
-
2023
- 2023-03-17 CN CN202310265230.XA patent/CN116326479A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014167464A1 (en) * | 2013-04-08 | 2014-10-16 | Sederma | Production method of meristematic cells of plantago lanceolata, composition comprising said cells or their cellular extract, and cosmetic, nutraceutical and dermatological uses |
| WO2019006470A1 (en) * | 2017-06-30 | 2019-01-03 | Booshoot Llc | Media for rapid and reliable tissue culturing of plants |
| CN109392719A (en) * | 2018-11-25 | 2019-03-01 | 钟天路 | A kind of method for building up of madder regenerating system |
| CN110100736A (en) * | 2019-06-20 | 2019-08-09 | 南京农业大学 | A kind of water planting enrichment procedure of Thesium chinese tissue-cultured seedling |
| CN114793905A (en) * | 2022-05-23 | 2022-07-29 | 浙江省亚热带作物研究所(浙南林业科学研究院) | Rapid propagation method and culture medium for gardenia tissue culture seedlings |
Non-Patent Citations (1)
| Title |
|---|
| 薛建平等: ""茜草愈伤组织诱导与快速繁殖的研究"", 《中国中药杂志》, vol. 27, no. 2, pages 101 - 103 * |
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