CN114868650A - Tissue culture and rapid propagation culture medium and tissue culture and rapid propagation method for veneers - Google Patents
Tissue culture and rapid propagation culture medium and tissue culture and rapid propagation method for veneers Download PDFInfo
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- CN114868650A CN114868650A CN202210428615.9A CN202210428615A CN114868650A CN 114868650 A CN114868650 A CN 114868650A CN 202210428615 A CN202210428615 A CN 202210428615A CN 114868650 A CN114868650 A CN 114868650A
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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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- 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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- 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
Abstract
The invention belongs to the technical field of tissue culture and rapid propagation of veneers, in particular to a tissue culture and rapid propagation culture medium and a tissue culture and rapid propagation method thereof, wherein the tissue culture and rapid propagation culture medium comprises a start culture medium, a propagation culture medium and a rooting culture medium, the propagation culture medium takes the start culture medium as a basal culture medium, and 0.5-3.0mgZT is added into each liter of the basal culture medium; the rooting culture medium takes 1/2MS as a basic culture medium, 20-30g of sucrose, 5-7g of agar, 0.1-0.5mg of IBA and 0.05-0.1mg of NAA are added into 1/2MS per liter, the maximum proliferation coefficient can reach 6.03 by utilizing the culture medium provided by the invention, and the rooting rate can reach 100%.
Description
Technical Field
The invention relates to the technical field of tissue culture and rapid propagation of veneers, in particular to a tissue culture and rapid propagation culture medium and a tissue culture and rapid propagation method for veneers.
Background
The tissue-cultured rapid propagation system of the veneer is of great significance for developing landscaping plant resources, and has the advantages that the tissue-cultured rapid propagation system of the veneer is provided.
Disclosure of Invention
In order to solve the technical problems, the invention provides a tissue culture rapid propagation medium and a tissue culture rapid propagation method for bark veneers.
The tissue culture and rapid propagation culture medium for the veneer comprises a starting culture medium, a multiplication culture medium and a rooting culture medium, wherein the multiplication culture medium takes the starting culture medium as a basic culture medium, and 0.5-3.0mgZT is added into each liter of the basic culture medium;
the rooting culture medium takes 1/2MS as a basic culture medium, and 20-30g of sucrose, 5-7g of agar, 0.1-0.5mg of IBA and 0.05-0.1mg of NAA are added into 1/2MS per liter.
Preferably, the start culture medium takes MS as a basic culture medium, 20-30g of sucrose and 5-7g of agar are added in each liter of MS,
the tissue culture and rapid propagation method of the veneer by using the tissue culture and rapid propagation culture medium of the veneer comprises the following steps:
s1, taking the stem section with axillary buds of the veneer as a test material, cleaning and disinfecting;
s2, after disinfection, the test material is inoculated into a start culture medium for induction culture, and then is sequentially inoculated into the multiplication culture medium and the rooting culture medium;
and S3, domesticating and hardening the rooted test material, and transplanting the test material into a matrix for management.
Preferably, in S1, the method for obtaining axillary bud stem segments comprises: the leaves of the veneer are removed, and the tender branches are cut into small sections with 2-4 axillary buds.
Preferably, in S1, the sterilization method is: sterilizing with 75% alcohol for 10-30s, washing with sterile water, and sterilizing with 5% NaClO for 8-12 min.
Preferably, in S2, after performing the start culture in the start culture medium, the non-contaminated and well-grown test material is inoculated into the enrichment culture medium for enrichment culture, and when the sprouts grow to 2-3cm, the sprouts are inoculated into the rooting culture medium for rooting induction.
Preferably, in S2, the initiation culture is performed under the conditions of 25 + -2 deg.C, dark culture for 3d, and then under the conditions of 2000lx illumination intensity and 16h/d photoperiod.
Preferably, in S2, the conditions for the proliferation culture are: temperature: 25 +/-2 ℃, the illumination intensity is 2000lx, and the light period is 16 h/d.
Preferably, in S2, the rooting induction is: culturing at 25 + -2 deg.C for 3 days in the dark, and culturing under the conditions of illumination intensity of 2000lx and photoperiod of 16 h/d.
Preferably, in S3, the matrix is peat: vermiculite: perlite is 3:1: 1.
Compared with the prior art, the invention has the beneficial effects that:
1. the tissue culture and rapid propagation culture medium for the veneer provided by the invention has the advantages that the multiplication rate of the veneer is as high as 0.92-6.03, and is obviously higher than that of a culture medium comprising 6-BA and NAA, when the multiplication culture medium is MS + sucrose 30g/L +6g/L agar + ZT1.0mg/L, the multiplication rate is the highest and is 6.03, and the rooting culture medium is as follows: 1/2MS, 30g/L of cane sugar, 6g/L of agar, 0.1 mg/L of IBA and 0.05 mg/L of NAA, the rooting rate of the veneer can reach 100 percent, and the transplanting survival rate after culture by the culture medium reaches more than 90 percent.
2. The germination rate of the tissue culture and rapid propagation method of the veneer provided by the invention is 42.59-90.74% of the disinfection combination of alcohol and NaClO, which is obviously higher than that of alcohol and HgCl 2 Disinfection combining germination Rate, alcohol and HgCl 2 The germination rate of the disinfection combination is between 27.70 and 90.74 percent, and mercury bichloride has residue in disinfection, has toxic action on plant materials and pollutes the environment.
Drawings
FIG. 1 is a graph showing the growth of roots of tissue culture seedlings according to example 1;
FIG. 2 is a diagram showing the growth of the tissue-cultured seedlings in example 1 after transplantation;
FIG. 3 is a graph showing the growth of roots of a tissue culture seedling of comparative example 4;
FIG. 4 is a diagram showing the growth of the tissue-cultured seedlings in comparative example 4 after transplantation.
Detailed Description
The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The experimental methods described in the examples of the present invention are all conventional methods unless otherwise specified.
Example 1
The tissue culture and rapid propagation method of the veneer comprises the following steps:
s1, collecting well-grown stem sections with axillary buds of the veneer, removing leaves, and shearing tender branches into small sections with 2-4 axillary buds to obtain a test material;
s2, putting the test material into a beaker, adding 3 drops of liquid detergent, covering a layer of gauze on the beaker, flushing the beaker for 30min by running water from a tap water pipe, then flushing the beaker for 3 times by using sterile water, and airing the beaker;
s3, sterilizing the dried test material in 75% alcohol for 30S, washing with sterile water for 3 times, and sterilizing in 5% NaClO solution for 10 min;
s4, washing the disinfected test material with sterile water for 5 times, cutting off two ends of the test material, inoculating the test material into a start culture medium for 30 bottles, inoculating 1 test material into each bottle, and counting the survival rate, the pollution rate and the death rate after 20 days, wherein the start culture medium is MS + sucrose 30g/L + agar 6 g/L; when the culture is started, firstly culturing in the dark for 3d under the condition of 25 ℃ after inoculation, and then culturing under the conditions that the illumination intensity is 2000lx and the photoperiod is 16 h/d;
s5, when the bud on the starting culture medium grows to 1-2cm, cutting axillary buds, inoculating the axillary buds on the enrichment culture medium for enrichment culture, inoculating 30 bottles, and inoculating 1 axillary bud in each bottle; the proliferation culture medium is: MS + sucrose 30g/L +6g/L agar + ZT2.0mg/L, and the conditions of proliferation culture are as follows: the temperature is 25 ℃, the illumination intensity is 2000lx, and the light period is 16 h/d;
s6, when the sprouts in the enrichment culture grow to 2cm, cutting the sprouts into a rooting culture medium for induction culture to obtain wild tissue culture seedlings for rooting of the veneers, and inoculating 15 bottles of sprouts, wherein each bottle is inoculated with 2 sprouts; the rooting culture medium comprises: 1/2MS + sucrose 30g/L + agar 6g/L + IBA0.1 mg/L + NAA0.05 mg/L, during induction culture, dark culture is carried out for 3d under the condition of 25 ℃, and then culture is carried out under the conditions of illumination intensity of 2000lx and photoperiod of 16 h/d;
s7, moving the wild bark tree rooting tissue culture seedlings to room temperature, domesticating the seedlings for 5 days under natural light, taking out the seedlings, ensuring that the roots are not damaged when the seedlings are taken out, cleaning a root culture medium, and transplanting the seedlings to turf: vermiculite: and (3) putting the perlite in the sterilized substrate with the ratio of 3:1:1 in a shading environment after planting, and performing normal environmental condition management after seedling rejuvenation.
Example 2
The tissue culture and rapid propagation method of the veneer comprises the following steps:
s1, collecting well-grown stem sections with axillary buds of the veneer, removing leaves, and shearing tender branches into small sections with 2-4 axillary buds to obtain a test material;
s2, putting the test material into a beaker, adding 3 drops of liquid detergent, covering a layer of gauze on the beaker, flushing the beaker for 30min by running water from a tap water pipe, then flushing the beaker for 3 times by using sterile water, and airing the beaker;
s3, sterilizing the dried test material in 75% alcohol for 10S, washing with sterile water for 2 times, and sterilizing in 5% NaClO solution for 12 min;
s4, washing the sterilized test material with sterile water for 5 times, cutting off two ends of the test material, inoculating the test material to a starting culture medium, inoculating 30 bottles, inoculating 1 test material in each bottle, and counting the survival rate, the pollution rate and the death rate after 20 days, wherein the starting culture medium is MS + sucrose 30g/L + agar 6 g/L; when the culture is started, firstly culturing in the dark for 3d at the temperature of 25 ℃ after inoculation, and then culturing under the conditions that the illumination intensity is 2000lx and the light cycle is 16 h/d;
s5, when the bud on the starting culture medium grows to 1-2cm, cutting axillary buds, inoculating the axillary buds on the enrichment culture medium for enrichment culture, inoculating 30 bottles, and inoculating 1 axillary bud in each bottle; the proliferation culture medium is: MS + sucrose 30g/L +6g/L agar + ZT0.5mg/L, and the conditions of proliferation culture are as follows: the temperature is 25 ℃, the illumination intensity is 2000lx, and the light period is 16 h/d;
s6, when the sprouts in the enrichment culture grow to 2cm, cutting the sprouts to a rooting culture medium for induction culture to obtain wild tissue culture seedlings with thin bark wood roots, and inoculating 15 bottles of 2 sprouts in each bottle; the rooting culture medium comprises: 1/2MS + sucrose 30g/L + agar 6g/L + IBA0.1 mg/L + NAA0.05 mg/L, during induction culture, dark culture is carried out for 3d under the condition of 25 ℃, and then culture is carried out under the conditions of illumination intensity of 2000lx and photoperiod of 16 h/d;
s7, moving the wild bark tree rooted tissue culture seedlings to room temperature, domesticating the seedlings for 5 days under natural light, taking out the seedlings, ensuring that the roots are not damaged when the seedlings are taken out, cleaning a root culture medium, and transplanting the seedlings to turf: vermiculite: and (3) putting the perlite in the sterilized substrate with the ratio of 3:1:1 in a shading environment after planting, and performing normal environmental condition management after seedling rejuvenation.
Example 3
The tissue culture and rapid propagation method of the veneer comprises the following steps:
s1, collecting well-grown stem sections with axillary buds of the veneer, removing leaves, and shearing tender branches into small sections with 2-4 axillary buds to obtain a test material;
s2, putting the test material into a beaker, adding 3 drops of liquid detergent, covering a layer of gauze on the beaker, flushing the beaker for 30min by running water from a tap water pipe, then flushing the beaker for 3 times by using sterile water, and airing the beaker;
s3, sterilizing the dried test material in 75% alcohol for 20S, washing with sterile water for 2 times, and sterilizing in 5% NaClO solution for 10 min;
s4, washing the sterilized test material with sterile water for 5 times, cutting off two ends of the test material, inoculating the test material to a starting culture medium, inoculating 30 bottles, inoculating 1 test material in each bottle, and counting the survival rate, the pollution rate and the death rate after 20 days, wherein the starting culture medium is MS + sucrose 30g/L + agar 6 g/L; when the culture is started, after inoculation, dark culture is carried out for 3d under the condition of 25 ℃, and then culture is carried out under the conditions that the illumination intensity is 2000lx and the photoperiod is 16 h/d;
s5, when the bud on the starting culture medium grows to 1-2cm, cutting axillary buds, inoculating the axillary buds on the enrichment culture medium for enrichment culture, inoculating 30 bottles, and inoculating 1 axillary bud in each bottle; the proliferation culture medium is: MS + sucrose 30g/L +6g/L agar + ZT3.0mg/L, and the conditions of proliferation culture are as follows: the temperature is 25 ℃, the illumination intensity is 2000lx, and the photoperiod is 16 h/d;
s6, when the sprouts in the enrichment culture grow to 2cm, cutting the sprouts into a rooting culture medium for induction culture to obtain wild tissue culture seedlings for rooting of the veneers, and inoculating 15 bottles of sprouts, wherein each bottle is inoculated with 2 sprouts; the rooting culture medium comprises: 1/2MS + sucrose 30g/L + agar 6g/L + IBA0.1 mg/L + NAA0.05 mg/L, during induction culture, dark culture is carried out for 3d under the condition of 25 ℃, and then culture is carried out under the conditions of illumination intensity of 2000lx and photoperiod of 16 h/d;
s7, moving the wild bark tree rooting tissue culture seedlings to room temperature, domesticating the seedlings for 5 days under natural light, taking out the seedlings, ensuring that the roots are not damaged when the seedlings are taken out, cleaning a root culture medium, and transplanting the seedlings to turf: vermiculite: and (3) putting the perlite in the sterilized substrate with the ratio of 3:1:1 in a shading environment after planting, and performing normal environmental condition management after seedling rejuvenation.
Example 4
Example 4 differs from example 1 in that, in S6, induction culture was performed on a rooting medium of 1/2MS + sucrose 30g/L + agar 6g/L + IBA0.1 mg/L + NAA0.1 mg/L.
Comparative example 1
Comparative example 1 differs from example 1 in that in S5, the propagation medium was: MS + sucrose 30g/L + agar 6g/L +6-BA0.5 mg/L + NAA0.1 mg/L.
Comparative example 2
Comparative example 2 differs from example 1 in that in S5, the propagation medium was: MS + sucrose 30g/L + agar 6g/L +6-BA 5mg/L + NAA0.1 mg/L.
Comparative example 3
Comparative example 3 differs from example 1 in that in S6, the rooting medium is: 1/2MS + sucrose 30g/L + agar 6g/L + NAA0.05 mg/L.
Comparative example 4
Comparative example 3 differs from example 1 in that in S6, the rooting medium is: 1/2MS + sucrose 30g/L + agar 6g/L + IBA0.05 mg/L.
Comparative example 5
Comparative example 5 differs from example 1 in that in S3, it was sterilized in 75% alcohol by volume for 10S, and in HgCl at a mass concentration of 0.1% 2 Sterilizing in solution for 8 min.
Comparative example 6
Comparative example 6 differs from example 1 in that in S3, disinfection was carried out for 30 seconds in 75% alcohol by volume and HgCl was carried out at 0.1% by mass 2 Sterilizing in solution for 12 min.
The growth results of the veneer of the above examples and comparative examples are shown in tables 1 to 3.
As can be seen from Table 1, the germination rate of the alcohol and NaClO disinfectant combination is between 42.59-90.74%, the contamination rate is between 1.85-57.41%, the mortality rate is between 0-24.07%, and the alcohol and HgCl are 2 The germination rate of the disinfection combination is between 27.70 and 90.74 percent, the pollution rate is between 0 and 62.96 percent, and the death rate is between 9.26 and 48.15 percent, so the effect of the disinfection combination of the alcohol and the NaClO is better than that of the alcohol and the HgCl 2 The disinfection effect of the disinfection combination is good, and mercury pollution is avoided.
As can be seen from Table 2, the proliferation coefficient of the proliferation medium using MS + sucrose 30g/L +6g/L agar + ZT 0.5-3.0mg/L is between 5.31-6.03, while the proliferation coefficient of the proliferation medium using MS + sucrose 30g/L +6g/L agar +6-BA0.5-5mg/L + NAA0.1 mg/L is between 0.92-2.26, and the data show that the proliferation rate obtained by using the proliferation medium provided by the invention is obviously superior to that of the proliferation medium in the prior art.
As can be seen from Table 3, the rooting rate using the rooting medium comprising IBA and NAA was significantly better than that using the rooting medium comprising IBA or NAA alone.
As shown in FIG. 1, the tissue culture seedling obtained by the culture medium and the culture method in the example 1 of the present application has 5-8 roots with root hairs, the root hairs are 2-3cm long, and the roots are many and robust, as shown in FIG. 3, the tissue culture seedling obtained by the culture medium and the culture method in the comparative example 4 has 2-4 roots and is slender; as shown in fig. 2, the tissue culture seedlings cultured in example 1 had better growth conditions after transplantation, and statistics revealed that the survival rate of transplantation was more than 90%, as shown in fig. 4, the tissue culture seedlings of comparative example 4 had a root system that fell off very easily during transplantation and had a low survival rate.
TABLE 1 germination, contamination, and mortality of test materials under different sterilization treatments
Note: the lower case in the table indicates significant differences (P < 0.05). Capital letters indicate very significant variability (P < 0.01)
TABLE 2 proliferation rates and shoot growth conditions using different proliferation media
Note: the lower case in the table indicates significant differences (P < 0.05). Capital letters indicate very significant variability (P < 0.01)
TABLE 3 rooting percentage and root growth status using different rooting media
It should be noted that when the following claims refer to numerical ranges, it should be understood that both ends of each numerical range and any numerical value between the two ends can be selected, and the preferred embodiments of the present invention are described for the purpose of avoiding redundancy.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. The tissue culture and rapid propagation culture medium for the veneer comprises a starting culture medium, a propagation culture medium and a rooting culture medium, and is characterized in that the propagation culture medium takes the starting culture medium as a basic culture medium, and 0.5-3.0mgZT is added into each liter of the basic culture medium;
the rooting culture medium takes 1/2MS as a basic culture medium, and 20-30g of sucrose, 5-7g of agar, 0.1-0.5mg of IBA and 0.05-0.1mg of NAA are added into 1/2MS per liter.
2. The tissue culture and rapid propagation medium of the veneer wood according to claim 1, characterized in that the start medium takes MS as a basic medium, and 20-30g of sucrose and 5-7g of agar are added in each liter of MS.
3. The tissue culture and rapid propagation method of the veneer by using the tissue culture and rapid propagation culture medium of the veneer as claimed in claim 1, which is characterized by comprising the following steps:
s1, taking the stem section with axillary buds of the veneer as a test material, cleaning and disinfecting;
s2, after disinfection, the test material is inoculated into a starting culture medium for induction culture, and then is sequentially inoculated into the proliferation culture medium and the rooting culture medium;
and S3, domesticating and hardening the rooted test material, and transplanting the test material into a matrix for management.
4. The tissue culture and rapid propagation method of veneer trees according to claim 3, wherein in S1, the method for obtaining axillary bud stem segments comprises the following steps: removing the leaves of the veneer, and cutting the tender branches into small sections with 2-4 axillary buds.
5. The tissue culture and rapid propagation method of veneerwood according to claim 3, wherein in S1, the disinfection method is as follows: sterilizing with 75% alcohol for 10-30s, washing with sterile water, and sterilizing with 5% NaClO for 8-12 min.
6. The tissue culture and rapid propagation method of Pimpinella chinensis Benth as claimed in claim 3, wherein in S2, after the start culture is performed in the start culture medium, the non-contaminated and well-grown test material is inoculated into the proliferation culture medium for proliferation culture, and when the bud grows to 2-3cm, the bud is inoculated into the rooting culture medium for rooting induction.
7. The tissue culture and rapid propagation method of the veneer wood according to claim 3, wherein in S2, the initiation culture is performed by first dark culture for 3d at 25 +/-2 ℃ and then culture under the conditions of illumination intensity of 2000lx and photoperiod of 16 h/d.
8. The tissue culture and rapid propagation method of veneer trees according to claim 3, wherein in S2, the propagation culture conditions are as follows: temperature: 25 +/-2 ℃, the illumination intensity is 2000lx, and the light period is 16 h/d.
9. The tissue culture and rapid propagation method of veneer wood according to claim 3, wherein in S2, the rooting induction is: culturing at 25 + -2 deg.C for 3 days in the dark, and culturing under the conditions of illumination intensity of 2000lx and photoperiod of 16 h/d.
10. The tissue culture and rapid propagation method of the veneer wood according to claim 3, wherein in S3, the matrix is composed of the following components: vermiculite: perlite is 3:1: 1.
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