CN109362568B - Tissue culture rapid propagation method of clematis' cherry lips - Google Patents

Tissue culture rapid propagation method of clematis' cherry lips Download PDF

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CN109362568B
CN109362568B CN201811462522.8A CN201811462522A CN109362568B CN 109362568 B CN109362568 B CN 109362568B CN 201811462522 A CN201811462522 A CN 201811462522A CN 109362568 B CN109362568 B CN 109362568B
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callus
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CN109362568A (en
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王连翠
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Linyi University
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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
    • A01H4/001—Culture apparatus for tissue culture
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G22/00—Cultivation of specific crops or plants not otherwise provided for
    • A01G22/60—Flowers; Ornamental plants
    • 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
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • 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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  • Developmental Biology & Embryology (AREA)
  • Engineering & Computer Science (AREA)
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Abstract

The invention discloses a tissue culture and rapid propagation method of clematis 'cherry lips', belonging to the technical field of plant tissue culture. The method comprises the following steps: step 1: selecting materials; step 2: sterilizing; and step 3: inducing and culturing callus; and 4, step 4: multiplication culture of the callus; and 5: culturing callus differentiation induction buds; step 6: and (4) rooting culture of the rootless seedlings. The invention reduces the possibility that the callus finally applied to the induction of bud differentiation carries virus to the lowest through callus induction and proliferation, and increases the survival rate of clematis.

Description

Tissue culture rapid propagation method of clematis' cherry lips
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a tissue culture and rapid propagation method of clematis chinensis 'cherry lips'.
Background
Clematis florida Thunb belongs to a vine flower of Clematis of Ranunculaceae. Because of the changeable colors, the various shapes and the various colors, the utility model enjoys the reputation of the vine queen in the garden. And is deeply favored by flower enthusiasts all over the world due to the strong drought resistance, cold resistance and other performances. The clematis has various varieties, namely large flower type, single petal type and double petal type; both petal fully developed types and bell-like types. Wherein the clang cherry lip clematis has petal combination, and the top end looks like a girl cherry labial, and is ruddy and elastic. Thus obtaining the name of 'cherry lip'. The iron wire lotus is popular in this year. The traditional propagation mode of clematis is mainly cuttage and seed propagation. Clematis internodes are long, and semi-lignified branches are easy to be cut and survive. The survival rate of cuttage is generally low. The number of clematis seeds is relatively small and certain low-temperature treatment is needed for germination. Some varieties of clematis cannot form seeds at all. Therefore, the breeding rate of clematis is very low, the requirement of the market on clematis is far from being met, and the application of the clematis in garden and family cultivation and appreciation is limited.
At present, plant tissue culture techniques are relatively mature and have been successfully cultivated and rapidly propagated on a large number of plants. The tissue culture method is used for the rapid propagation of clematis, and is a feasible and effective method.
Patent CN104206281A discloses a tissue culture method of clematis, comprising the following steps:
(1) obtaining aseptic seedling material; (2) primary culture; (3) carrying out proliferation culture; (4) rooting and seedling strengthening culture; when the root length is 2-4cm, domestication and transplantation can be carried out. The clematis tissue culture method provided by the invention has the advantages of short propagation period, high propagation coefficient, easiness in rooting, stout plant root system, good new growth vigor, stout stem and easiness in survival after domestication.
Patent CN 104396750A discloses a tissue culture propagation method of clematis pilosa, which mainly achieves the excellent effects of high propagation speed, stable hereditary character, high propagation coefficient and vigorous growth of test-tube plantlets by the steps of primary culture, propagation culture, rooting culture and seedling hardening and transplanting, the germination rate of the callus of clematis pilosa reaches 98%, the multiplication times reach 5.7 times, the rooting rate reaches 100%, and the final survival rate reaches 96%.
Patent CN 103202229A provides a tissue culture and rapid propagation method of clematis chinensis with green flower and double petals, which comprises the following steps: taking the stem tip of the caulicle of the clematis tangutica Dutch, washing the stem tip with sterile water, soaking the stem tip with 75% alcohol for 20-30 seconds on a super clean bench, soaking and disinfecting the stem tip with 0.1-0.2% mercuric chloride for 6-10 minutes, washing the stem tip with the sterile water, and then inoculating the stem tip into a cluster bud induction culture medium for primary generation induction culture; 2) dividing cluster buds obtained by primary induction culture into single buds, and inoculating the buds to a cluster bud multiplication culture medium for subculture; 3) and when the bud seedlings grow to 2-3 cm, inoculating the bud seedlings into a strong seedling rooting culture medium for rooting culture. The method can rapidly propagate the green flower clematis filamentosa, not only can save the cost for cultivating the seedlings and improve the production efficiency, but also is not limited by external conditions, can be carried out all the year round, saves the occupied area for seedling cultivation, can form a large number of excellent test-tube seedlings in a short period, and carries out large-scale and industrial production.
In the prior art, the rapid propagation of clematis is realized by different treatment methods and different culture media.
Disclosure of Invention
The invention aims to provide a tissue culture rapid propagation method of clematis 'cherry lips', which reduces the possibility that callus finally applied to induced bud differentiation carries viruses to the lowest extent through callus induction and proliferation, and increases the survival rate of clematis.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a tissue culture and rapid propagation method of clematis 'cherry lips', which comprises the following steps:
step 1: selection of materials
Selecting strong and disease and insect pest-free tender branches, and shearing stem sections with nodes;
step 2: sterilizing and disinfecting
Cutting the stem segments into sections with the length of 0.8-1cm on a superclean workbench; washing with sterile water for 1 time, soaking in 70% alcohol for 30 s, soaking with 10% sodium hypochlorite for 10-15 min, soaking in 0.1% mercuric chloride for 7-8 min, shaking to sterilize, and washing with sterile water for 4-5 times;
and step 3: callus induction culture
Taking the stem segments, transversely placing the stem segments in a culture medium for inducing callus to perform induction culture, and generating a large amount of light green callus on the base parts of the stem segments after 15 days;
induction callus culture medium: 1/4 modified MS + NAA 1.0mg/L +2,4-D0.5mg/L +6-BA 0.5mg/L + 10% banana mud + active carbon 1g/L, the modified MS culture medium is: mixing MS culture medium with major element KNO3The dosage of (A) is changed to 2500mg/L by (NH)4)2SO4Replace NH4NO3The dosage is 200mg/L, other basic components are the same as MS, 25g/L of sucrose and 7.5g/L of agar are also added into the improved MS culture medium, and the pH value is 5.8;
and 4, step 4: multiplication culture of callus
Taking the induced callus, dividing the callus into small blocks of 0.4-0.6cm, and inoculating the small blocks into a liquid shallow culture medium to perform proliferation culture on the callus;
the proliferation culture medium is: 3/4 modified MS + NAA 1.0mg/L +2,4-D0.5mg/L +6-BA 0.5mg/L + active carbon 1 g/L;
and 5: culture of callus differentiation-induced buds
Taking callus, cutting the callus into small blocks of 1cm, and inoculating the small blocks into a differentiation culture medium for bud induction;
the culture medium is a liquid shallow culture medium and has the formula: 3/4 modified MS +6-BA 1.5mg/L + TDZ 0.5mg/L + NAA 0.01mg/L + active carbon 1.5 g/L;
step 6: rooting culture of rootless seedlings
When the cluster buds grow to 3-5cm, cutting branches, and inoculating the branches to a rooting culture medium to induce rooting;
the rooting culture medium comprises: 1/2 modified MS + NAA 0.5mg/L + IBA 0.1mg/L + activated carbon 0.5 g/L.
Further, the method also comprises the step 7: domestication and transplantation of test-tube plantlets: firstly, transplanting the test-tube plantlet to a greenhouse, then taking out the plantlet, cleaning the culture medium attached to the root, transplanting the plantlet to a substrate with the mixture ratio of: 1 part of sandy soil and 2 parts of vermiculite: 2 parts of perlite: culturing in 5 parts of mixed matrix of garden soil under natural conditions.
Further, in the step 1, every three nodes are taken as one section when the stem sections are cut, the cut stem sections are firstly washed under running water for 3-4 hours and then washed once by sterile water.
Furthermore, in the step 4, the increment rate in the proliferation culture of the callus is 2 to 3 times.
Furthermore, VC is added into the mixed matrix, and 800 times of carbendazim is sprayed and evenly stirred.
Further, the addition amount of VC is 2.5 g/L.
Further, in the step 5 and the step 6, the temperature is 22-26 ℃, the illumination intensity is 1500-.
Compared with the prior art, the invention has the following beneficial effects:
the tissue culture and rapid propagation method of clematis 'cherry lips' provided by the invention can rapidly propagate plants through tissue culture, and meets the market requirements. At the same time, the possibility of virus-carrying of the callus finally applied to the induction of shoot differentiation is minimized by callus induction and proliferation. The test-tube plantlet cultured by the bud induced and differentiated by the callus greatly reduces the possibility of virus. Therefore, after the test-tube plantlets are acclimatized and transplanted, careful management is carried out, and the probability of later-stage virus infection is greatly reduced. This brings high benefit for production and can enhance ecological environment protection.
Meanwhile, the invention also adjusts the nutrient components of the culture medium, so that the invention is more suitable for the rapid propagation of clematis, reduces the possibility of virus treatment of callus, and increases the survival rate.
Detailed Description
In order to make the technical problems, solutions and advantages of the present invention more apparent, specific embodiments will be described in detail below, but the present invention is by no means limited to these examples. The following description is only a preferred embodiment of the present invention, and is only for the purpose of explaining the present invention, and should not be construed as limiting the scope of the present invention. It should be understood that any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
The components used in the present invention are all commercially available products unless otherwise specified.
The invention provides a tissue culture and rapid propagation method of clematis 'cherry lips', and a specific embodiment is as follows.
Example 1
A tissue culture and rapid propagation method of clematis 'cherry lip' comprises the following steps:
step 1: selection of materials
Selecting strong and disease and insect pest-free tender branches, shearing stem sections with nodes, wherein every three nodes are one section, flushing the sheared stem sections under running water for 4 hours, and then flushing the stem sections with sterile water for one time;
step 2: and (3) disinfection and sterilization: cutting the stem segments into 1cm long with one segment on a superclean bench; washing with sterile water for 1 time, soaking in 70% alcohol for 30 s, soaking in 10% sodium hypochlorite for 10 min, soaking in 0.1% mercuric chloride for 8 min, shaking to sterilize, and washing with sterile water for 5 times;
and step 3: callus induction culture: taking the stem segments, transversely placing the stem segments in a culture medium for inducing callus to perform induction culture, and generating a large amount of light green callus on the base parts of the stem segments after 15 days;
induction callus culture medium: 1/4 modified MS + NAA 1.0mg/L +2,4-D0.5mg/L +6-BA 0.5mg/L + 10% banana mud + active carbon 1 g/L;
and 4, step 4: proliferation culture of callus: taking the induced callus, dividing the callus into small blocks of 0.4-0.6cm, inoculating the small blocks into a liquid shallow culture medium to perform callus proliferation culture, wherein the proliferation rate in the callus proliferation culture is 2 times, and performing the next culture; the proliferation culture medium is: 3/4 modified MS + NAA 1.0mg/L +2,4-D0.5mg/L +6-BA 0.5mg/L + active carbon 1 g/L;
and 5: culturing callus differentiation induction buds: taking callus, cutting the callus into small blocks of 1cm, and inoculating the small blocks into a differentiation culture medium for bud induction; culturing at 22 deg.C under illumination intensity of 2000Lx and relative humidity of 70%; the culture medium is a liquid shallow culture medium and has the formula: 3/4 modified MS +6-BA 1.5mg/L + TDZ 0.5mg/L + NAA 0.01mg/L + active carbon 1.5 g/L.
Step 6: rooting culture of rootless seedlings: when the cluster buds grow to 3cm, cutting branches, and inoculating the branches to a rooting culture medium to induce rooting; the rooting culture medium comprises: 1/2 modified MS + NAA 0.5mg/L + IBA 0.1mg/L + activated carbon 0.5 g/L.
And 7: domestication and transplantation of test-tube plantlets: firstly, transplanting the test-tube plantlet to a greenhouse, then taking out the plantlet, cleaning the culture medium attached to the root, transplanting the plantlet to a substrate with the mixture ratio of: 1 part of sandy soil and 2 parts of vermiculite: 2 parts of perlite: culturing in 5 parts of garden soil mixed matrix under natural conditions, adding VC in the mixed matrix, spraying 800 times of carbendazim, and stirring uniformly, wherein the addition amount of VC is 2.5 g/L.
The improved MS culture medium is as follows: mixing MS culture medium with major element KNO3The dosage of (A) is changed to 2500mg/L by (NH)4)2SO4Replace NH4NO3The dosage is 200mg/L, and other basic components are the same as MS. The improved MS culture medium is also added with 25g/L of sucrose and 7.5g/L of agar, and the pH value is 5.8.
The rooting rate of the rootless seedlings in the embodiment is 98.4%, and the survival rate of domesticated 25-day clematis is 94.6%.
Example 2
A tissue culture and rapid propagation method of clematis 'cherry lip' comprises the following steps:
step 1: selection of materials
Selecting strong and disease and insect pest-free tender branches, shearing stem sections with nodes, wherein every three nodes are one section, flushing the sheared stem sections under running water for 3 hours, and then flushing the stem sections with sterile water for one time;
step 2: and (3) disinfection and sterilization: cutting the stem segments into sections with the length of 0.8cm on a superclean bench; washing with sterile water for 1 time, soaking in 70% alcohol for 30 s, soaking in 10% sodium hypochlorite for 10 min, soaking in 0.1% mercuric chloride for 8 min, shaking to sterilize, and washing with sterile water for 5 times;
and step 3: callus induction culture: taking the stem segments, transversely placing the stem segments in a culture medium for inducing callus to perform induction culture, and generating a large amount of light green callus on the base parts of the stem segments after 15 days; induction callus culture medium: 1/4 modified MS + NAA 1.0mg/L +2,4-D0.5mg/L +6-BA 0.5mg/L + 10% banana mud + active carbon 1 g/L;
and 4, step 4: proliferation culture of callus: taking the induced callus, dividing the callus into small blocks of 0.4cm, inoculating the small blocks into a liquid shallow culture medium to perform callus proliferation culture, wherein the proliferation rate in the callus proliferation culture is 3 times, and performing the next culture; the proliferation culture medium is: 3/4 modified MS + NAA 1.0mg/L +2,4-D0.5mg/L +6-BA 0.5mg/L + active carbon 1 g/L;
and 5: culturing callus differentiation induction buds: taking callus, cutting the callus into small blocks of 1cm, and inoculating the small blocks into a differentiation culture medium for bud induction; culturing at 22 deg.C under illumination intensity of 2000Lx and relative humidity of 70%; the culture medium is a liquid shallow culture medium and has the formula: 3/4 modified MS +6-BA 1.5mg/L + TDZ 0.5mg/L + NAA 0.01mg/L + active carbon 1.5 g/L;
step 6: rooting culture of rootless seedlings: when the cluster buds grow to 5cm, cutting branches, and inoculating the branches to a rooting culture medium to induce rooting; the rooting culture medium comprises: 1/2 modified MS + NAA 0.5mg/L + IBA 0.1mg/L + active carbon 0.5 g/L;
and 7: domestication and transplantation of test-tube plantlets: firstly, transplanting the test-tube plantlet to a greenhouse, then taking out the plantlet, cleaning the culture medium attached to the root, transplanting the plantlet to a substrate with the mixture ratio of: 1 part of sandy soil and 2 parts of vermiculite: 2 parts of perlite: culturing in 5 parts of garden soil mixed matrix under natural conditions, adding VC in the mixed matrix, spraying 800 times of carbendazim, and stirring uniformly, wherein the addition amount of VC is 2.5 g/L.
The improved MS culture medium is as follows: mixing MS culture medium with major element KNO3The dosage of (A) is changed to 2500mg/L by (NH)4)2SO4Replace NH4NO3The dosage is 200mg/L, and other basic components are the same as MS. The improved MS culture medium is also added with 25g/L of sucrose and 7.5g/L of agar, and the pH value is 5.8.
The rooting rate of the rootless seedlings in the embodiment is 99.2%, and the survival rate of domesticated 25-day clematis is 96.8%.
Example 3
A tissue culture and rapid propagation method of clematis 'cherry lip' comprises the following steps:
step 1: selection of materials
Selecting strong and disease and insect pest-free tender branches, shearing stem sections with nodes, wherein every three nodes are one section, flushing the sheared stem sections under running water for 4 hours, and then flushing the stem sections with sterile water for one time;
step 2: and (3) disinfection and sterilization: cutting the stem segments into 1cm long with one segment on a superclean bench; washing with sterile water for 1 time, soaking in 70% alcohol for 30 s, soaking in 10% sodium hypochlorite for 12 min, soaking in 0.1% mercuric chloride for 8 min, shaking to sterilize, and washing with sterile water for 5 times;
and step 3: callus induction culture: taking the stem segments, transversely placing the stem segments in a culture medium for inducing callus to perform induction culture, and generating a large amount of light green callus on the base parts of the stem segments after 15 days;
induction callus culture medium: 1/4 modified MS + NAA 1.0mg/L +2,4-D0.5mg/L +6-BA 0.5mg/L + 10% banana mud + active carbon 1 g/L;
and 4, step 4: proliferation culture of callus: taking the induced callus, dividing the callus into small blocks of 0.5cm, inoculating the small blocks into a liquid shallow culture medium to perform callus proliferation culture, wherein the proliferation rate in the callus proliferation culture is 3 times, and performing the next culture; the proliferation culture medium is: 3/4 modified MS + NAA 1.0mg/L +2,4-D0.5mg/L +6-BA 0.5mg/L + active carbon 1 g/L;
and 5: culturing callus differentiation induction buds: taking callus, cutting the callus into small blocks of 1cm, and inoculating the small blocks into a differentiation culture medium for bud induction; culturing at 26 deg.C under the conditions of illumination intensity of 1500Lx and relative humidity of 65%; the culture medium is a liquid shallow culture medium and has the formula: 3/4 modified MS +6-BA 1.5mg/L + TDZ 0.5mg/L + NAA 0.01mg/L + active carbon 1.5 g/L;
step 6: rooting culture of rootless seedlings: when the cluster buds grow to 4cm, cutting branches, and inoculating the branches to a rooting culture medium to induce rooting; the rooting culture medium comprises: 1/2 modified MS + NAA 0.5mg/L + IBA 0.1mg/L + active carbon 0.5 g/L;
and 7: domestication and transplantation of test-tube plantlets: firstly, transplanting the test-tube plantlet to a greenhouse, then taking out the plantlet, cleaning the culture medium attached to the root, transplanting the plantlet to a substrate with the mixture ratio of: 1 part of sandy soil and 2 parts of vermiculite: 2 parts of perlite: culturing in 5 parts of garden soil mixed matrix under natural conditions, adding VC in the mixed matrix, spraying 800 times of carbendazim, and stirring uniformly, wherein the addition amount of VC is 2.5 g/L.
The improved MS culture medium is as follows: mixing MS culture medium with major element KNO3The dosage of (A) is changed to 2500mg/L by (NH)4)2SO4Replace NH4NO3The dosage is 200mg/L, and other basic components are the same as MS. The improved MS culture medium is also added with 25g/L of sucrose and 7.5g/L of agar, and the pH value is 5.8.
The rooting rate of the rootless seedlings in the embodiment is 98.9%, and the survival rate of domesticated 25-day clematis is 95.7%.
To further illustrate the advantages of the tissue culture and rapid propagation method of clematis chinensis 'cherry lips', only example 2 is taken as an example to construct a comparative example as follows.
Comparative example 1
The modified MS medium of example 3 was replaced with MS medium, and the remaining conditions were the same as in example 2.
The clematis chinensis obtained by the method of the comparative example has the rooting rate of 81.2 percent and the survival rate of 75.8 percent after 25 days of acclimation.
Comparative example 2
In this comparative example, steps 1-2 are the same as in example 2;
and step 3: and (3) inducing and culturing cluster buds: inoculating the sterilized stem segments into a culture medium for inducing cluster buds to perform primary culture; induction medium of cluster buds: 3/4 modified MS + NAA 0.5mg/L + ZT 0.5mg/L +6-BA 2.0mg/L + active carbon 1 g/L;
and 4, step 4: and (3) propagation culture of the cluster buds: cutting the cluster buds induced in the step (3) into single buds, and inoculating the single buds to a subculture medium for multiplication and propagation culture of the cluster buds; subculture medium of cluster buds: modified MS + NAA 0.5mg/L +6-BA 2.0mg/L + GA 3.0mg/L activated carbon 1 g/L;
and 5: rooting culture of rootless seedlings: when the cluster buds grow to 3-5cm, cutting branches, and inoculating the branches to a rooting culture medium to induce rooting; the rooting culture medium comprises: 1/2 modified MS + NAA 0.5mg/L + IBA 0.1mg/L + active carbon 0.5 g/L;
culturing the above steps 3-5 at 22 deg.C under illumination intensity of 2000Lx and relative humidity of 70%;
the subsequent culture was the same as in example 2.
The rooting rate of the clematis in the comparative example is 90.1%, and the survival rate of the clematis after 25 days of acclimation is 81.4%.
As can be seen from the above examples and comparative examples, the tissue culture rapid propagation method of clematis chinensis 'cherry lips' provided by the invention minimizes the possibility that the callus finally applied to the induction of bud differentiation carries viruses through callus induction and proliferation. The test-tube plantlet cultured by the bud induced and differentiated by the callus greatly reduces the possibility of virus. Therefore, after the test-tube plantlets are acclimatized and transplanted, careful management is carried out, the probability of later-stage virus infection is greatly reduced, and the survival rate is increased.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (7)

1. A tissue culture and rapid propagation method of clematis 'cherry lips' is characterized by comprising the following steps:
step 1: selection of materials
Selecting strong and disease and insect pest-free tender branches, and shearing stem sections with nodes;
step 2: sterilizing and disinfecting
Cutting the stem segments into sections with the length of 0.8-1cm on a superclean workbench; washing with sterile water for 1 time, soaking in 70% alcohol for 30 s, soaking with 10% sodium hypochlorite for 10-15 min, soaking in 0.1% mercuric chloride for 7-8 min, shaking to sterilize, and washing with sterile water for 4-5 times;
and step 3: callus induction culture
Taking the stem segments, transversely placing the stem segments in a culture medium for inducing callus to perform induction culture, and generating a large amount of light green callus on the base parts of the stem segments after 15 days;
induction callus culture medium: 1/4 modified MS + NAA 1.0mg/L +2,4-D0.5mg/L +6-BA 0.5mg/L + 10% banana mud + active carbon 1g/L, the modified MS culture medium is: mixing MS culture medium with major element KNO3The dosage of (A) is changed to 2500mg/L by (NH)4)2SO4Replace NH4NO3The dosage is 200mg/L, other basic components are the same as MS, 25g/L of sucrose and 7.5g/L of agar are also added into the improved MS culture medium, and the pH value is 5.8;
and 4, step 4: multiplication culture of callus
Taking the induced callus, dividing the callus into small blocks of 0.4-0.6cm, and inoculating the small blocks into a liquid shallow culture medium to perform proliferation culture on the callus;
the proliferation culture medium is: 3/4 modified MS + NAA 1.0mg/L +2,4-D0.5mg/L +6-BA 0.5mg/L + active carbon 1 g/L;
and 5: culture of callus differentiation-induced buds
Taking callus, cutting the callus into small blocks of 1cm, and inoculating the small blocks into a differentiation culture medium for bud induction;
the culture medium is a liquid shallow culture medium and has the formula: 3/4 modified MS +6-BA 1.5mg/L + TDZ 0.5mg/L + NAA 0.01mg/L + active carbon 1.5 g/L;
step 6: rooting culture of rootless seedlings
When the cluster buds grow to 3-5cm, cutting branches, and inoculating the branches to a rooting culture medium to induce rooting;
the rooting culture medium comprises: 1/2 modified MS + NAA 0.5mg/L + IBA 0.1mg/L + activated carbon 0.5 g/L.
2. The tissue culture and rapid propagation method of clematis 'cherry lips' according to claim 1, further comprising the step 7: domestication and transplantation of test-tube plantlets: firstly, transplanting the test-tube plantlet to a greenhouse, then taking out the plantlet, cleaning the culture medium attached to the root, transplanting the plantlet to a substrate with the mixture ratio of: 1 part of sandy soil and 2 parts of vermiculite: 2 parts of perlite: culturing in 5 parts of mixed matrix of garden soil under natural conditions.
3. The tissue culture and rapid propagation method of clematis 'cherry lips' according to claim 1, wherein in the step 1, every three nodes are cut into one segment, and the cut stem is washed under running water for 3-4 hours and then washed with sterile water once.
4. The tissue culture and rapid propagation method of clematis 'cherry lips' according to claim 1, wherein in the step 4, the proliferation rate in the proliferation culture of callus is 2-3 times.
5. The tissue culture and rapid propagation method of clematis 'cherry lips' as claimed in claim 2, wherein VC is added to the mixed matrix and uniformly sprayed with 800 times of carbendazim.
6. The tissue culture and rapid propagation method of clematis chinensis 'cherry lips' according to claim 5, wherein the addition amount of VC is 2.5 g/L.
7. The tissue culture rapid propagation method of clematis filamentosa 'cherry lips' as claimed in claims 1-6, wherein in the steps 5 and 6, the temperature is 22-26 ℃, the illumination intensity is 1500-.
CN201811462522.8A 2018-12-03 2018-12-03 Tissue culture rapid propagation method of clematis' cherry lips Active CN109362568B (en)

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