CN107278902B - Culture medium and culture method for tissue culture of syphilis trees - Google Patents
Culture medium and culture method for tissue culture of syphilis trees Download PDFInfo
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- CN107278902B CN107278902B CN201710657718.1A CN201710657718A CN107278902B CN 107278902 B CN107278902 B CN 107278902B CN 201710657718 A CN201710657718 A CN 201710657718A CN 107278902 B CN107278902 B CN 107278902B
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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
Abstract
The invention relates to the technical field of plant tissue culture, in particular to a culture medium and a culture method for tissue culture of a plum slice tree. The invention takes the plum branch as an explant, selects a specific primary culture medium, a specific secondary culture medium and a specific rooting culture medium, utilizes a plant tissue culture technology, realizes the rapid propagation of the plum tissue culture by a new way, obviously improves the bud induction rate, the propagation coefficient and the rooting rate of the plum, shortens the propagation period, and provides a more effective way for the industrialized production of the plum.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a culture medium and a culture method for tissue culture of a plum slice tree.
Background
The natural d-borneol is widely applied in the field of medicine and health care, and the plum slice tree in the lauraceae plant is an important source of the d-borneol. The traditional cutting rooting method can also achieve the purpose of propagation, but has the defects of easy aging of plants, less years of flowering and fruiting and the like. Plant tissue culture refers to the culture of isolated parts of plants, such as root tips, stem tips, leaves, embryos, seeds, fruits, various tissues and cells, protoplasts, and the like, by using aseptic technique. The theoretical basis for plant tissue culture is totipotency of plant cells, i.e., each cell of a plant contains all the genetic information of the species, and thus has the genetic ability to develop into a complete plant.
In the Lauraceae plants, tissue culture of the plants such as cinnamomum camphora, cinnamomum camphora and the like has been studied, optimal culture mediums of various stages such as bud induction, bud proliferation, rooting and the like are obtained respectively, MS is taken as a basic culture medium, for example, in tissue culture and rapid propagation of cinnamomum camphora, the bud induction is more suitable by using improved MS +6-BA 1.2mg/L + NAA0.2mg/L, the subculture is to improve MS +6-BA 0.8mg/L + KT0.3mg/L + IBA0.2mg/L, the rooting culture medium 1/3MS + IBA0.35mg/L + NAA0.2mg/L + Ac0.2g/L, and the rooting rate can reach more than 96%; in the tissue culture and rapid propagation of the cinnamomum camphora, modified MS + BA2.0 mg/L + NAA 0.1mg/L is used as a bud induction culture medium, the induction rate is 93%, the optimal propagation culture medium is modified MS +6-BA 2.0 mg/L + NAA0.05 mg/L, the propagation coefficient is 5.57, the growth cycle is 30 days, the appropriate propagation culture condition is 25 ℃, the illumination intensity is 3000lx, the illumination time is 11 hours, the growth vigor of the adventitious bud is good, the optimal rooting culture medium is 1/2 modified MS + IBA 0.5 mg/L + IAA 0.4mg/L + sucrose 20 g/L, the rooting rate can reach 97.3%, the number of the rooting strips is 3-5, and the rooting time is 12 days.
The research on tissue culture of the syphilis tree is very little, and a complete tissue rapid propagation system is lacked. At present, only a literature reports a bud induction culture medium for tissue culture of the plum slice trees, and the formula of the bud induction culture medium is as follows: MS +6-BA 2.0 mg/L + NAA0.05 mg/L, and the bud induction rate reaches 89.13%. Although the induction rate of the medium on the buds of the prune tree is high, no report is found on the medium in the bud multiplication culture and rooting culture stages of the prune tree. Therefore, a set of complete, stable and efficient tissue culture and rapid propagation system for the plum slice trees is established to promote the economic benefit of the plum slice trees, and the method has important practical significance.
Disclosure of Invention
In view of the above, the invention provides a culture medium for tissue culture of a prune leaf tree and a culture method thereof, so that the formula of the culture medium can remarkably improve the bud induction rate, the multiplication coefficient and the rooting rate of the prune leaf tree.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a culture medium for tissue culture of a plum slice tree plant, which comprises a primary culture medium, a secondary culture medium and a rooting culture medium;
the primary culture medium is DCR culture medium +2 ~ 3mg/L6-BA +0.05 ~ 0.1.1 mg/LNAA +10g/L agar +30g/L sucrose, and the pH value is 5.8 ~ 5.9.9;
the subculture medium is DCR medium +3 ~ 5 mg/L6-BA +0.05 ~ 0.1.1 mg/L NAA +10g/L agar +30g/L sucrose, and the pH value is 5.8 ~ 5.9.9;
the rooting medium is DCR medium, 0 ~ 1 mg/L6-BA, 1.5mg/L IBA, 10g/L agar and 10g/L sucrose, and the pH value is 5.8 ~ 5.9.9.
For plant tissue culture, a culture medium is one of the most critical factors of the growth state of the plant tissue culture, and if the selection and combination and collocation of the culture medium in the three stages of primary induction differentiation, secondary proliferation and rooting are not proper, the callus induction rate, the adventitious bud differentiation rate, the proliferation coefficient and the rooting rate in each stage are not ideal. The prior art only discloses a bud induction culture medium for tissue culture of the prune tree, and researches show that the culture medium is not suitable for two stages of subculture proliferation and rooting of the prune tree, so that the rapid propagation of the prune tree cannot be effectively realized by a tissue culture technology. According to the invention, a complete and efficient rapid propagation system of the prunella vulgaris is formed by selecting the culture medium in three specific stages, so that the bud induction rate, the propagation coefficient and the rooting rate of the prunella vulgaris are improved remarkably, the propagation period is shortened effectively, and a more effective way is provided for the industrial production of the prunella vulgaris.
In the specific embodiment provided by the invention, the primary culture medium is DCR culture medium +2 mg/L6-BA +0.05mg/L NAA +10g/L agar and 30g/L sucrose, and the pH value is 5.8;
the subculture medium is: DCR culture medium +4 mg/L6-BA +0.05mg/L NAA +10g/L agar +30g/L sucrose, pH value is 5.8;
the rooting culture medium comprises: DCR medium +1.5mg/L IBA +10g/L agar +10g/L sucrose, pH 5.8.
In the specific embodiment provided by the present invention, the primary medium is: DCR culture medium +2 mg/L6-BA +0.1mg/L NAA +10g/L agar +30g/L sucrose, pH value is 5.9;
the subculture medium comprises: DCR culture medium +3mg/L6-BA +0.05mg/L NAA +10g/L agar +30g/L sucrose, pH value is 5.9;
the rooting culture medium comprises: DCR culture medium +1.5mg/L IBA +1 mg/L6-BA +10g/L agar +10g/L sucrose, pH 5.9.
In another embodiment provided by the invention, the primary culture medium is DCR culture medium +3mg/L6-BA +0.05mg/L NAA +10g/L agar and 30g/L sucrose, and the pH value is 5.8;
the subculture medium is: DCR culture medium +5 mg/L6-BA +0.1mg/L NAA +10g/L agar +30g/L sucrose, pH value is 5.8;
the rooting culture medium comprises: DCR medium +1.5mg/L IBA +10g/L agar +10g/L sucrose, pH 5.8.
The invention also provides a tissue culture method of the prune tree, which comprises the following steps:
step 1: inoculating the plum tree explants to the primary culture medium in the culture medium for primary bud induction culture, and inducing the explants to grow axillary buds;
step 2: transferring the explant with the axillary buds to a subculture medium in the culture medium for bud multiplication culture to obtain cluster buds;
and step 3: and (4) transferring the cluster buds to a rooting culture medium in the culture medium for rooting culture to obtain the tissue culture seedling.
Preferably, before the inoculation of the prune tree explant in the step 1, the method further comprises the steps of selecting annual healthy shoots with buds of the prune tree collected in month 4 ~ 5, removing leaves, preserving petioles, cutting into small segments with the length of 1 ~ 3cm and a pair of buds or a terminal bud, and performing disinfection treatment.
In an embodiment provided by the present invention, the sterilization treatment is specifically: and (3) carrying out alcohol disinfection, water cleaning, mercuric chloride solution disinfection and water cleaning on the prune tree branches with the leaves removed once.
Preferably, the alcohol accounts for 75 percent by volume, the alcohol disinfection time is 1 ~ 2min, the mercuric chloride solution accounts for 0.1 percent by mass, and the disinfection time is 8-12 min.
Preferably, the light irradiation period of tissue culture of the prune tree is 16h/d, the light irradiation intensity is 3500lx, the culture temperature is 24 ~ 26 deg.C, and the relative humidity is 30 ~ 40%.
In the embodiment provided by the invention, the illumination period of the plum tree tissue culture is 16h/d, the illumination intensity is 3500lx, the culture temperature is 25 ℃, and the relative humidity is 34%.
Preferably, the tissue culture method provided by the invention further comprises a step of hardening off the tissue culture seedlings after the tissue culture seedlings are obtained.
In the specific embodiment provided by the invention, the seedling exercising is as follows: closing the bottle, placing in weak light for 5 days, and shading; standing under strong light for 5 days, opening the cover of the tissue culture seedling, ventilating for 5-7 days, covering with a film for moisturizing, and spraying water properly; spraying sufficient root fixing water after transplanting, covering the film for moisture preservation, properly supplementing water according to the humidity, gradually reducing the water spraying amount and gradually removing the covering film, and transferring to normal maintenance after 30-40 days.
The invention takes the plum branch as an explant, selects a specific primary culture medium, a specific secondary culture medium and a specific rooting culture medium, utilizes a plant tissue culture technology, realizes the rapid propagation of the plum tissue culture by a new way, obviously improves the bud induction rate, the propagation coefficient and the rooting rate of the plum, shortens the propagation period, establishes a stable and efficient tissue culture rapid propagation system, and provides a more effective way for the industrialized production of the plum.
Drawings
FIG. 1 is a diagram showing a plum tree explant for inoculation in example 1 of the present invention;
FIG. 2 is a diagram showing axillary bud induction of a plum tree explant in example 1 of the present invention;
FIG. 3 is a diagram showing a plum tree explant used for subculture in example 1 of the present invention;
FIG. 4 is a diagram showing the proliferation of clumped buds of plum blossom in example 1 of the present invention;
FIG. 5 is a diagram showing the rooting of plum tree seedlings according to example 1 of the present invention;
FIG. 6 is a diagram showing the tissue culture seedlings of plum trees before transplantation in example 1 of the present invention.
Detailed Description
The invention discloses a culture medium and a culture method for tissue culture of a plum slice tree, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
The invention is further illustrated by the following examples:
example 1 tissue culture of Meretrix Linnaeus
(1) Collecting healthy branches of plum trees with buds growing in one year near the base in month 4 ~ 5 as explants, avoiding collecting in rainy days, removing leaves and retaining petioles, and soaking in detergent to remove surface impurities.
(2) Cutting the explant into stem segments of about 2 ~ 3cm with 1-2 axillary buds in an ultraclean workbench, soaking with 75% alcohol for 1min, washing with sterile water for 6 times (each time for 1 min), soaking with 0.1% mercuric chloride solution for sterilization for 10min, and washing with sterile water for 6 times (each time for 1 min).
(3) Inoculating the processed explant into a subpackaged primary culture medium, wherein the primary culture medium is as follows: DCR culture medium +2 mg/L6-BA +0.05mg/L NAA +10g/L agar and 30g/L sucrose, and the pH value is 5.8. Placing in a culture room with illumination for 16h/d, maintaining the temperature at 25 + -1 deg.C, illumination intensity at 3000lx, and relative humidity at 34%. After 30 days of culture, the explants grow axillary buds (as shown in figure 2), the bud emergence condition is recorded, and the bud induction rate is counted.
(4) Selecting an explant with high-quality axillary buds in the induction of the primary buds and the axillary buds growing to 1.5-2cm (as shown in figure 3), gently taking the axillary buds down from the original explant in a clean bench, and transferring the axillary buds into a subculture medium. The subculture medium is: DCR culture medium +4 mg/L6-BA +0.05mg/L NAA +10g/L agar +30g/L sucrose, pH value is 5.8. Culturing for 30 days in the same culture environment as that in step (3), recording the proliferation condition of the buds (as shown in FIG. 4), and calculating the proliferation coefficient of the cluster buds.
(5) Selecting green healthy and strong plantlets with lateral buds growing to more than 1.5cm from the cluster buds, gently taking out the buds from an original culture medium by using forceps in a super clean workbench, cutting off lower-end callus, and inoculating the buds into a rooting culture medium, wherein the rooting culture medium is as follows: DCR medium +1.5mg/L IBA +10g/L agar +10g/L sucrose, pH 5.8. After 20 days of culture, the plantlets begin to root, and are cultured for three weeks, the rooting condition is observed (as shown in figure 5), and the rooting rate is calculated.
(6) Selecting strong tissue culture seedlings with developed root systems (as shown in figure 6), closing the bottles, placing the seedlings outside the bottles for 5 days in low light, and appropriately shading the seedlings; standing under strong light for 5 days, opening the cover of the tissue culture seedling, ventilating for 5-7 days, covering with a film for moisturizing, and spraying water properly; spraying sufficient root fixing water after transplanting, covering the film for moisture preservation, properly supplementing water according to the humidity, gradually reducing the water spraying amount and gradually removing the covering film, and transferring to normal maintenance after 30-40 days.
Example 2 tissue culture of Meretrix Linnaeus
(1) Collecting healthy branches of plum trees with buds growing in one year near the base in month 4 ~ 5 as explants, avoiding collecting in rainy days, removing leaves and retaining petioles, and soaking in detergent to remove surface impurities.
(2) Cutting the explant into stem segments (about 2 ~ 3 cm) with 1-2 axillary buds in an ultraclean workbench (as shown in figure 1), soaking in 75% ethanol for 2min, washing with sterile water for 6 times (each time for 1 min), soaking in 0.1% mercuric chloride solution for sterilization for 12min, and washing with sterile water for 6 times (each time for 1 min).
(3) Inoculating the processed explant into a subpackaged primary culture medium, wherein the primary culture medium is as follows: DCR culture medium +2 mg/L6-BA +0.1mg/L NAA +10g/L agar +30g/L sucrose, pH value is 5.9. Placing in a culture room with illumination for 16h/d, maintaining the temperature at 25 + -1 deg.C, illumination intensity at 3000lx, and relative humidity at 34%. After culturing for 30 days, the explants grow axillary buds, the bud emergence condition is recorded, and the bud induction rate is counted.
(4) Selecting an explant with high-quality axillary buds in the induction of the primary buds and the axillary buds growing to 1.5-2cm, gently taking the axillary buds down from the original explant in a super-clean workbench, and transferring the axillary buds to a subculture medium. The subculture medium is: DCR culture medium +3mg/L6-BA +0.05mg/L NAA +10g/L agar +30g/L sucrose, pH 5.9. And (4) culturing for 30 days in the same culture environment in the step (3), recording the bud proliferation condition, and calculating the proliferation coefficient of the cluster buds.
(5) Selecting green healthy and strong plantlets with lateral buds growing to more than 1.5cm from the cluster buds, gently taking out the buds from an original culture medium by using forceps in a super clean workbench, cutting off lower-end callus, and inoculating the buds into a rooting culture medium, wherein the rooting culture medium is as follows: DCR culture medium +1.5mg/L IBA +1 mg/L6-BA +10g/L agar +10g/L sucrose, pH 5.9. Culturing for about 20 days, starting the seedling to root, culturing for three weeks, observing the rooting condition, and calculating the rooting rate.
(6) Selecting strong tissue culture seedlings with developed root systems, closing the bottles, placing the seedlings outside the bottles for 5 days in low light, and appropriately shading; standing under strong light for 5 days, opening the cover of the tissue culture seedling, ventilating for 5-7 days, covering with a film for moisturizing, and spraying water properly; spraying sufficient root fixing water after transplanting, covering the film for moisture preservation, properly supplementing water according to the humidity, gradually reducing the water spraying amount and gradually removing the covering film, and transferring to normal maintenance after 30-40 days.
Example 3 tissue culture of Meretrix Linnaeus
The primary culture medium comprises DCR culture medium, 3mg/L6-BA, 0.05mg/L NAA, 10g/L agar and 30g/L sucrose, and the pH value is 5.8;
the subculture medium is: DCR culture medium +5 mg/L6-BA +0.1mg/L NAA +10g/L agar +30g/L sucrose, pH value is 5.8;
the rooting culture medium comprises: DCR medium +1.5mg/L IBA +10g/L agar +10g/L sucrose, pH 5.8.
The other operations were the same as in example 1.
Test example 1
Tissue culture of the prune tree was performed by the test method of example 1-2, and the bud induction rate, the multiple bud proliferation coefficient and the rooting rate were counted 4 control groups 1 ~ 3 were set up in the test, specifically divided into:
control group 1: the primary culture medium is MS culture medium +2 mg/L6-BA +0.05mg/L NAA +10g/L agar +30g/L sucrose, and the secondary culture medium is: MS culture medium, 4 mg/L6-BA, 0.05mg/L NAA, 10g/L agar and 30g/L cane sugar, wherein the rooting culture medium comprises: MS culture medium +1.5mg/L IBA +10g/L agar +10g/L sucrose.
Control group 2: taking an improved MS culture medium (the content of ammonium nitrate is reduced by half, namely the concentration of ammonium nitrate is 825 mg/L) as a basic culture medium, wherein the primary culture medium is as follows: improved MS culture medium +2 mg/L6-BA +0.0.05mg/L NAA +10g/L agar +30g/L sucrose, and the subculture medium is: modified MS culture medium +4 mg/L6-BA +0.05mg/L NAA +10g/L agar +30g/L sucrose, rooting culture medium is: modified MS culture medium +1.5mg/L IBA +10g/L agar +10g/L sucrose.
Control group 3: an improved DCR culture medium (the content of ammonium nitrate and potassium nitrate is reduced by half, namely the concentration of ammonium nitrate is 200mg/L and the concentration of potassium nitrate is 170 mg/L) is taken as a basic culture medium, wherein the primary culture medium comprises: the modified DCR culture medium +2 mg/L6-BA +0.05mg/L NAA +10g/L agar +30g/L sucrose, the subculture medium is: the improved DCR culture medium, 4 mg/L6-BA, 0.05mg/L NAA, 10g/L agar and 30g/L sucrose, and the rooting culture medium comprises: modified DCR medium +1.5mg/L IBA +10g/L agar +10g/L sucrose.
Control 1 ~ 3 the procedure of example 1 was followed except that the medium was different from that of example 1, and the test results are shown in Table 1 ~ 3:
TABLE 1 bud Induction
| Method of producing a composite material | Number of explants inoculated | Number of buds | Germination percentage (%) | Growth state |
| Example 1 | 118 | 106 | 89.83 | ++++ |
| Example 2 | 107 | 88 | 82.24 | ++++ |
| Control group 1 | 140 | 103 | 73.57 | +++ |
| Control group 2 | 160 | 129 | 80.62 | +++ |
| Control group 3 | 140 | 109 | 77.86 | ++ |
Note that + indicates that the bud length is between 0.5-1cm, the growth state is normal, + indicates that the bud length is above 1cm ~ 1.5.5 cm, the growth state is good, and + indicates that the bud length is above 1.5cm, the leaf is green and spread, and the growth state is good
TABLE 2 bud proliferation
| Method of producing a composite material | Number of inoculated buds | Number of buds | Coefficient of proliferation | Growth state |
| Example 1 | 124 | 283 | 2.28 | ++++ |
| Example 2 | 140 | 287 | 2.05 | ++++ |
| Control group 1 | 120 | 195 | 1.63 | ++++ |
| Control group 2 | 130 | 242 | 1.86 | +++ |
| Control group 3 | 120 | 183 | 1.53 | ++ |
TABLE 3 rooting conditions
| Method of producing a composite material | Number of inoculated plants | Root number of 30 days | Rooting percentage (%) | Rooting time (d) (from the beginning of the experiment to rooting) | Root number after 60 days |
| Example 1 | 51 | 36 | 70.59 | 21 | ≥3 |
| Example 2 | 42 | 34 | 80.95 | 23 | ≥3 |
| Control group 1 | 39 | 12 | 30.77 | 36 | 1~2 |
| Control group 2 | 48 | 17 | 35.41 | 31 | 1~2 |
| Control group 3 | 38 | 17 | 44.74 | 31 | 1~2 |
As can be seen from the data in table 1 ~ 3, compared with the culture medium of control group 1 ~ 3, the present invention selects a specific primary culture medium, secondary culture medium and rooting culture medium, realizes rapid propagation of tissue culture of the prune leaf trees, and significantly improves the bud induction rate, propagation coefficient and rooting rate of the prune leaf trees.
When the medium of the invention in the embodiment 3 is used for carrying out tissue culture on the prune leaf trees, the results of the bud induction rate, the propagation coefficient and the rooting rate are the same as or similar to those of the embodiment 1 ~ 2, and no significant difference exists (P is more than 0.05).
Test example 2
The invention takes a DCR culture medium as a basic culture medium, researches the influence of other culture mediums except the hormone combination of the application on induction, bud proliferation and rooting conditions of the plum tree buds, and the result is shown in a table 4 ~ 6:
TABLE 4 Effect of different hormone combinations on shoot Induction in plum blossom
TABLE 5 Effect of different hormone combinations on shoot proliferation in plum blossom
TABLE 6 Effect of different hormone combinations on rooting of plum blossom Tree
The data in Table 4 ~ 6 show that when the explant of the mei tree is cultured by using other culture media except the hormone combination, the bud induction rate, the propagation coefficient and the rooting rate are obviously lower than those of the explant of the mei tree in the application.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A culture medium for tissue culture of plum slice trees is characterized by comprising a primary culture medium, a secondary culture medium and a rooting culture medium;
the primary culture medium is DCR culture medium +2 ~ 3mg/L6-BA +0.05 ~ 0.1.1 mg/L NAA +10g/L agar +30g/L sucrose, and the pH value is 5.8 ~ 5.9.9;
the subculture medium is DCR medium +3 ~ 5 mg/L6-BA +0.05 ~ 0.1.1 mg/L NAA +10g/L agar +30g/L sucrose, and the pH value is 5.8 ~ 5.9.9;
the rooting culture medium is DCR culture medium, 0 ~ 1 mg/L6-BA, 1.5mg/L IBA, 10g/L agar and 10g/L cane sugar, and the pH value is 5.8 ~ 5.9.9;
the explant inoculated on the primary culture medium is obtained by selecting healthy shoots with buds and annual growth of plum tree collected in month 4 ~ 5, removing leaves and preserving petioles;
inoculating axillary buds obtained by induction of the primary culture medium on the secondary culture medium;
and the cluster buds obtained by the culture of the subculture medium are inoculated on the rooting medium.
2. The culture medium of claim 1, wherein the primary culture medium is DCR culture medium +2 mg/L6-BA +0.05mg/L NAA +10g/L agar and 30g/L sucrose, and has a pH of 5.8;
the subculture medium comprises: DCR culture medium +4 mg/L6-BA +0.05mg/L NAA +10g/L agar +30g/L sucrose, pH value is 5.8;
the rooting culture medium comprises: DCR medium +1.5mg/L IBA +10g/L agar +10g/L sucrose, pH 5.8.
3. The culture medium of claim 1, wherein the primary culture medium is: DCR culture medium +2 mg/L6-BA +0.1mg/L NAA +10g/L agar +30g/L sucrose, pH value is 5.9;
the subculture medium comprises: DCR culture medium +3mg/L6-BA +0.05mg/L NAA +10g/L agar +30g/L sucrose, pH value is 5.9;
the rooting culture medium comprises: DCR culture medium +1.5mg/L IBA +1 mg/L6-BA +10g/L agar +10g/L sucrose, pH 5.9.
4. A tissue culture method of a prune tree is characterized by comprising the following steps:
step 1, inoculating the plum tree explant into a primary culture medium in a culture medium of any one of claims 1 ~ 3 for primary bud induction culture, and inducing the explant to grow axillary buds;
step 2, transferring the axillary buds to a subculture medium of any one of the culture media of claim 1 ~ 3 to perform bud multiplication culture to obtain cluster buds;
step 3, transferring the cluster buds to a rooting culture medium in the culture medium of any one of claim 1 ~ 3 for rooting culture to obtain tissue culture seedlings.
5. The tissue culture method according to claim 4, wherein the step 1 of inoculating the plum tree explant further comprises collecting the bud-bearing annual healthy shoot of the plum tree in month 4 ~ 5, removing the leaf and retaining the petiole, cutting into 1 ~ 3cm long segments having a pair of buds or a terminal bud, and sterilizing.
6. The tissue culture method according to claim 5, wherein the sterilization treatment is in particular: and (3) sequentially carrying out alcohol disinfection, water cleaning, mercuric chloride solution disinfection and water cleaning on the prune tree branches with the leaves removed.
7. The tissue culture method of claim 6, wherein the alcohol is 75% by volume, the alcohol disinfection time is 1 ~ 2min, the mercuric chloride solution is 0.1% by mass, and the disinfection time is 8-12 min.
8. The tissue culture method according to any one of claims 4 to 7, wherein the light irradiation period of the tissue culture of the prune tree is 16h/d, the light irradiation intensity is 3500lx, the culture temperature is 24 ~ 26 ℃, and the relative humidity is 30 ~ 40%.
9. The tissue culture method according to claim 4, further comprising a step of hardening off the tissue culture seedlings after the tissue culture seedlings are obtained.
10. The tissue culture method of claim 9, wherein the hardening off is: closing the bottle, placing in weak light for 5 days, and shading; standing under strong light for 5 days, opening the cover of the tissue culture seedling, ventilating for 5-7 days, covering with a film for moisturizing, and spraying water properly; spraying sufficient root fixing water after transplanting, covering the film for moisture preservation, properly supplementing water according to the humidity, gradually reducing the water spraying amount and gradually removing the covering film, and transferring to normal maintenance after 30-40 days.
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- 2017-08-03 CN CN201710657718.1A patent/CN107278902B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| Clonal propagation of Cinnamomum zeylanicum Breyn. by tissue culture;V. RAVI SHANKAR RAI & K.S. JAGADISH CHANDRA;《Plant Cell, Tissue and Organ Culture》;19871231;第9卷;第81-88页 * |
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