CN117441616B - In-vitro rapid propagation method for beef tendons - Google Patents
In-vitro rapid propagation method for beef tendons Download PDFInfo
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- 210000002435 tendon Anatomy 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 23
- 238000000338 in vitro Methods 0.000 title claims abstract description 16
- 239000001963 growth medium Substances 0.000 claims abstract description 69
- 230000035755 proliferation Effects 0.000 claims abstract description 43
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- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims description 10
- 239000008223 sterile water Substances 0.000 claims description 9
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 7
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 7
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Classifications
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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
-
- 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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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses an in-vitro rapid propagation method of beef tendons, and belongs to the technical field of plant tissue culture. The method comprises the following steps: (1) Taking tender tissue of beef tendon as an explant, sterilizing, and inoculating the explant into an induction culture medium for induction culture; (2) Inoculating the tissue culture seedlings subjected to induction culture into a proliferation culture medium for proliferation culture; (3) Dividing adventitious buds generated by multiplication culture into single buds, and inoculating the single buds into a strong seedling culture medium for strong seedling culture; (4) Inoculating the tissue culture seedlings after strengthening the seedlings into a rooting culture medium for rooting culture to obtain Niu Jintiao rooting seedlings. The culture medium for each stage of the culture of the beef tendon tissue culture seedlings is optimally prepared, the survival rate of the tissue culture seedlings is high, the induction survival rate of the explants is more than 50%, the proliferation coefficient is more than 3, the rooting rate is 100%, no variant plants are found, and the excellent properties of the mother plants can be maintained to the greatest extent.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to an in-vitro rapid propagation method of beef tendons.
Background
With the development of agricultural cultivation technology, the pursuit of fruit tree industry workers is gradually changed from high yield to high quality, and the production mode is gradually changed into mechanized and modern modes. The dwarf close planting cultivation has the advantages of high yield per unit, early fruiting, convenient operation, suitability for intensive cultivation and the like, can effectively make up the defects of the traditional arbor thin planting mode, and accords with the development direction of the future fruit tree industry.
Apple is an important rosaceous cultivated tree species in China, and the breeding of dwarf stocks of apple has been greatly progressed. The nutrition system stock M-line bred by the layering breeding method at the east-metallocene forest test station in the United kingdom and the G-line stock bred at the university of Conneler in the United states are widely used at present.
Pear (Pyrus L.) is a plant of the genus Rosaceae (Rosaceae), is an important temperate fruit tree in the world, has a cultivation area in China inferior to that of apples and oranges, and has an important production position. The pears are divided into two large cultivation species groups, namely, the American pears and the eastern pears from cultivation. The quince is mainly used as a dwarf stock, and the grafting survival can realize dwarf crowns, early fruiting, high quality, long storage resistance and the like, so that the quince has good production benefits. Dwarf stock of Oriental pear, which is used in the current production and has foreign introduced quince and domestic bred dwarf stock, but the dwarf stock of pear which is equivalent to high-yield apple stock M9 is still lacking, and the nutrition system dwarf stock suitable for Asian pear is not obtained.
In order to solve the problem, the research of strengthening the research of the dwarfing mechanism is particularly important while strengthening the excavation and optimization breeding technology of the available breeding resources of the Oriental pears.
Niu Jintiao (Dichotomanthes tristaniaecarpa) is plant of genus Niujingzhi of family Rosaceae, and is prepared from evergreen shrub to small arbor, 2-4 m high; shoots are clustered and typically grown in the slope open field heterowood forest and the edges of the evergreen oak forest. As proved by research in 1978, the beef tendon can be used as a heterogeneous stock of Yunnan pears, has good dwarfing and early fruiting effects, has high grafting survival rate with most cultivars of the system of the sand pears, the white pears and the foreign pears, has a wider variety range than the adaptation of quince stock, and is a promising dwarfing stock. However, the related report that the beef tendon is used as the pear dwarf breeding resource is rarely seen later.
The subject group screens out a part of beef tendon strains with good affinity and dwarfing effect with 'Cuiguan' pears through earlier-stage research, hopes to further propagate the beef tendon strains obtained through screening, explores the performances of the beef tendon strains in terms of grafting affinity and dwarfing with more eastern pears, screens out and obtains beef tendon clone with excellent comprehensive properties, and provides a certain reference for breeding Asian pear dwarfing stock. At present, the beef ribs are mainly bred by seed breeding and cutting breeding, but the two modes have the problems of low breeding efficiency, long period, low survival rate and the like. The in vitro rapid propagation system suitable for the beef tendons is explored, the breeding period can be shortened, the breeding efficiency can be improved, the cost can be reduced, and a foundation is laid for popularization and application of the beef tendons.
Disclosure of Invention
The invention aims to provide an in-vitro rapid propagation method for beef tendons, which shortens the breeding period, improves the breeding efficiency, has high tissue culture survival rate and maintains the excellent characters of parent plants to the greatest extent.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides an in-vitro rapid propagation method of beef tendons, which comprises the following steps:
(1) Taking tender tissue of beef tendon as an explant, sterilizing and inoculating the explant into an induction culture medium for induction culture, wherein the induction culture medium takes MS as a basic culture medium and also comprises 25-35 g/L sucrose, 7-8 g/L agar, 0.5-1.5 mg/L6-benzylaminopurine (6-BA) and 0.2-0.5 mg/L alpha-naphthylacetic acid (NAA), and the pH value is 5.75-5.80;
(2) Inoculating the tissue culture seedling subjected to induction culture into a proliferation culture medium for proliferation culture, wherein the proliferation culture medium takes MS as a basic culture medium, and further comprises 25-35 g/L sucrose, 7-8 g/L agar, 0.6-0.8 mg/L6-BA and 0.3-0.5 mg/L NAA, or further comprises 25-35 g/L sucrose, 7-8 g/L agar, 1.0 mg/L6-BA, 0.5mg/L NAA and 0.01-1.5 mg/L gibberellin, and the pH value is 5.75-5.80;
(3) Dividing adventitious buds generated by multiplication culture into single buds, inoculating the single buds into a strong seedling culture medium for strong seedling culture, wherein the strong seedling culture medium takes MS as a basic culture medium and also comprises 25-35 g/L sucrose, 7-8 g/L agar, 0.1 mg/L6-BA, 0.1-0.3 mg/L NAA and pH of 5.75-5.80;
(4) Inoculating the tissue culture seedlings after strengthening the seedlings into a rooting culture medium for rooting culture to obtain Niu Jintiao rooting seedlings, wherein the rooting culture medium takes 1/2MS as a basic culture medium and also comprises 15-25 g/L sucrose, 7-8 g/L agar and 0.3-1.0 mg/L3-indolebutyric acid (IBA), and the pH value is 5.75-5.80.
In the step (1), the beef tendon explants are inoculated into an induction culture medium for induction culture.
Further, in the step (1), the tender tissue is tender leaf at the top of the current annual branch of the beef tendon.
Further, the current-year branches of the beef tendons are cut off during germination of the spring buds. Researches show that the material collected during spring bud germination has less germ carrying amount, is favorable for improving the survival rate of explants and reducing the pollution rate. Preferably, the samples are collected in 4-5 months, and the current annual branches with good growth vigor and no plant diseases and insect pests are cut.
Further, the sterilization comprises the steps of sterilizing the explant with alcohol for 30-45 s after washing with running water, sterilizing the explant in sodium hypochlorite solution for 15-20 min after washing with sterile water, and finally washing with sterile water. After each disinfection treatment, the water is washed 3 to 4 times by sterile water. The explant can be rapidly and effectively detoxified through the steps. The volume percentage concentration of the alcohol is 75%; the volume percentage concentration of the sodium hypochlorite solution is 2%. The sodium hypochlorite solution has small toxic and side effects, and the invention adopts the 2 percent sodium hypochlorite solution as the disinfectant, and compared with the 0.1 percent mercuric chloride solution, the disinfectant can be realized under the condition of improving the safety index.
Further, after the disinfection is completed, the surface water of the explant is sucked dry, and the outermost bracts are removed and inoculated into an induction culture medium for culture.
The research of the invention shows that compared with IBA, the combination of NAA and 6-BA adopted in the plant hormone in the culture medium is more favorable for keeping the good growth state of the explant and is convenient for subsequent proliferation, seedling strengthening and rooting.
Preferably, the induction culture medium takes MS as a basic culture medium, and further comprises 30g/L sucrose, 7.5g/L agar, 1.0 mg/L6-BA and 0.3mg/L NAA, wherein the pH value is 5.75-5.80.
The condition of the induction culture is that the temperature is 23-28 ℃, the humidity is 65-75%, the illumination time is 12-16 h/d, the illumination intensity is 1500-2000 lx, and the induction culture is 30-35 days.
In the step (2), the explants which are successfully started to culture are divided into single buds, and inoculated into a proliferation medium for proliferation culture. Researches show that the proliferation coefficient of adventitious buds of beef tendons is high by adding 0.6-0.8 mg/L of 6-BA and 0.3-0.5 mg/L of NAA into a proliferation culture medium or adding 1.0mg/L of 6-BA, 0.5mg/L of NAA and 0.01-1.5 mg/L of GA 3, and the phenomena that plants are vitrified and the like and are unfavorable for continuous culture are not seen.
Preferably, the proliferation culture medium MS is a basic culture medium, further comprises 30g/L sucrose, 7.5g/L agar, 0.8 mg/L6-BA and 0.3mg/L NAA, or further comprises 30g/L sucrose, 7.5g/L agar, 1.0 mg/L6-BA, 0.5mg/L NAA and 0.01mg/L gibberellin (GA 3), and has a pH of 5.75-5.80.
The conditions of proliferation culture are that the temperature is 23-28 ℃, the humidity is 65-75%, the illumination time is 12-16 h/d, the illumination intensity is 1500-2000 lx, and the proliferation culture is carried out for 45-50 days.
In the step (3), the adventitious buds generated by multiplication culture are divided into single buds and transferred to a strong seedling culture medium for strong seedling culture. Researches show that the average plant height of the plant seedlings can reach 2-2.5cm by adding 0.1mg/L of 6-BA and 0.1-0.3 mg/L of NAA into the strong seedling culture medium, the leaf color is dark green, the leaf is large, the growth condition is good, and the follow-up culture and growth are facilitated.
Researches show that the tissue culture seedlings obtained after the proliferation culture of the beef tendon explants are weak in growth vigor and unsuitable for rooting, or the survival rate of transplanting after rooting is low, and the rooting rate of the follow-up rooting culture is remarkably improved through the strong seedling culture.
Preferably, the seedling strengthening culture medium takes MS as a basic culture medium, and further comprises 30g/L sucrose, 7.5g/L agar, 0.1 mg/L6-BA and 0.1mg/L NAA, wherein the pH value is 5.75-5.80.
The conditions of strong seedling cultivation are that the temperature is 23-28 ℃, the humidity is 65-75%, the illumination time is 12-16 h/d, the illumination intensity is 1500-2000 lx, and the strong seedling cultivation is carried out for 45-50 days.
In the step (4), the plants after seedling strengthening are inoculated into a rooting culture medium for rooting culture. Researches show that the rooting rate of Niu Jintiao can reach 100% by taking 1/2MS as a basic culture medium and adding 0.3-1.0 mg/L IBA, and the plant has large leaves and dark green leaves.
Further, strong seedlings with the plant height of more than or equal to 2cm are selected and inoculated into a rooting culture medium.
Preferably, the rooting culture medium takes 1/2MS as a basic culture medium, further comprises 20g/L sucrose, 7.5g/L agar and 0.5mg/L IBA, and the pH is adjusted to be 5.75-5.80.
The rooting culture condition is that the temperature is 23-28 ℃, the humidity is 65-75%, the illumination time is 12-16 h/d, the illumination intensity is 1500-2000 lx, and the rooting culture is carried out for 30-35 days.
The invention has the beneficial effects that:
The invention provides an in-vitro rapid propagation method of beef ribs, which is characterized in that the explant is subjected to induction, proliferation, seedling strengthening and rooting culture to successfully obtain the beef rib tissue culture seedling, the tissue culture seedling has high survival rate, and the excellent character of a mother plant can be maintained to the greatest extent. In the embodiment of the invention, the induction survival rate of the explant is more than 50%, the proliferation coefficient is more than 3, the rooting rate is 100%, and no variant plants are found.
Drawings
FIG. 1 shows the induction of tissue culture seedlings in induction medium combination 1 (A) and induction medium combination 2 (B), wherein the tissue culture seedlings die after 2-3 passages of transfer in A.
FIG. 2 shows the growth of tissue culture seedlings in proliferation medium A4 (A) and strong seedling medium B1 (B).
FIG. 3 shows rooting condition of tissue culture seedlings under the condition of 1/2MS culture medium, wherein A is a view angle of the bottom of a tissue culture bottle, and B is a view angle of the side face of the tissue culture bottle.
Detailed Description
The invention will be further illustrated with reference to specific examples. The following examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
MS media used in the following examples were purchased from beijing hua vietnam M519; the 1/2MS culture medium is used by halving the MS culture medium; the 1/4MS culture medium is MS culture medium, and the adding amount is reduced to 1/4.
NN69: coolaber (custom) formulation as shown in table 1:
TABLE 1 NN69 media formulation
The 6-BA (CAS: 1214-39-7) used in the examples below was purchased from Alatine; IBA (CAS: 133-32-4) was purchased from microphone; NAA (CAS: 86-87-3) was purchased from a source foliar organism; GA 3 (CAS: 77-06-5) was purchased from Soy pal.
Example 1
1. Materials and methods
1. Test materials
The test materials of the beef ribs are taken from scientific research bases of the national academy of sciences in Wu Xing area of Huzhou, zhejiang, and are sampled respectively in 11 months 2022, 4 months 2023 and 5 months 2023. In the test, the current annual branches with good growth vigor and no plant diseases and insect pests are cut off and brought back to a laboratory for standby.
2. Test method
2.1 Sterilization of explants and initiation of culture
And shearing tender leaves at the tops of the branches to serve as explants, washing the explants with tap water added with a proper amount of detergent, washing the explants with running water for 1h, removing dust and impurities on the surfaces of the explants, and placing the explants into an ultra-clean workbench for sterilization.
Sterilizing with 75% alcohol for 30s, washing with sterile water for 3 times, sterilizing with 2% sodium hypochlorite solution for 15 and 20min respectively, and washing with sterile water for 3-4 times. The bottle body is slightly shaken during the process, so that the disinfection is sufficient. The surface water of the explant is sucked by sterile filter paper, and the outermost bract is removed and inoculated into an induction culture medium for culture. After 30d of culture, the pollution rate, death rate and survival rate are counted. The branches sheared at different stages are disinfected.
Culture conditions: the temperature is 25 ℃, the humidity is about 70 percent, and the illumination time is 16h/d. The culture conditions are not particularly described below.
Induction medium: MS is taken as a basic culture medium, 30g/L sucrose and 7.5g/L agar are added, a certain concentration of plant hormone is added, and the pH is regulated to 5.75-5.80.
TABLE 2 Induction Medium additional hormone composition and concentration
Under the conditions of two culture media, the starting rate of the beef tendons can reach more than 80%, and the difference from expectations is that most pear varieties are suitable for growing and breeding in the culture media containing the IBA, but after the beef tendons are transferred for 2-3 generations on the culture media containing the IBA, more than 80% of tissue culture seedlings can fade, yellow and the like, as shown in a figure 1 (A), the beef tendons are not suitable for further culture, so that the IBA is not suitable for being used as a hormone variety for a subsequent test.
2.2 Proliferation culture
Explants that successfully initiated culture were divided into individual shoots and transferred to different proliferation media.
2.2.1 Effect of different hormone combinations on proliferation of bovine tendons
The test was set up with a total of 6 proliferation media: MS is taken as a basic culture medium, 30g/L sucrose and 7.5g/L agar are added, plant hormones with different concentrations are added, the pH is regulated to 5.75-5.80, and the mixture is respectively marked as combination A1-A6.
TABLE 3 hormone combinations for proliferation culture of beef tendons
15 Explants were inoculated to each medium, and after 45d of culture, proliferation was counted.
2.2.2 Effect of different concentrations of GA 3 added on proliferation of bovine tendons
The experiment was set up with 8 proliferation media: MS is taken as a basic culture medium, 30g/L sucrose, 7.5g/L agar, 1.0 mg/L6-BA and 0.5mg/L NAA are added, and the pH is regulated to 5.75-5.80. After high temperature sterilization, GA 3 at 0.01, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0mg/L was added, respectively, and the resulting mixture was designated as combination A7-A14.
15 Explants were inoculated to each medium, and after 45d of culture, proliferation was counted.
2.3 Cultivation of strong seedlings
Dividing adventitious buds generated by multiplication culture into single buds and transferring the single buds to a strong seedling culture medium. In this experiment, 9 strong seedling culture mediums were set up: MS is taken as a basic culture medium, 30g/L sucrose and 7.5g/L agar are added, 6-BA (0.1, 0.3 and 0.5 mg/L), NAA (0.1, 0.2 and 0.3 mg/L) and GA 3 (0, 0.5 and 1.0 mg/L) with different concentrations are added, orthogonal tests are set, and the pH of each culture medium is adjusted to 5.75-5.80 and respectively recorded as a combination B1-B9.
15 Explants are inoculated to each culture medium, and after 45d of culture, the growth condition of the tissue culture seedlings is counted.
TABLE 4 hormone combinations for the cultivation of strong seedlings of beef tendons
2.4 Rooting culture
Transferring the strong seedlings with the length of more than 2cm into rooting culture medium: the test uses 4 different basic culture mediums, adding 20g/L sucrose, 7.5g/L agar and 0.5mg/L IBA, adjusting pH to 5.75-5.80, and respectively marking as combination C1-C4. Each medium was inoculated with 12 explants and rooting was counted after 30d of culture.
TABLE 5 basic culture Medium for rooting culture of beef tendon
2. Data statistics and processing
Contamination rate = number of contaminated explants/total number of explants x 100%;
mortality = number of dead explants/total number of explants x 100%;
Survival = number of uncontaminated and sprouted explants/total explants x 100%;
proliferation coefficient = number of shoots after proliferation/number of inoculated explants;
Rooting rate = number of rooting explants/number of inoculated explants x 100%;
Average root number = total root number/number of rooted explants.
3. Results and analysis
1. Explant disinfection and sterilization
1.1 Effect of different sampling times on explant growth
As a result of using the same sterilization method for explants obtained at different times, it is clear from Table 6 that the survival rate of explants obtained by cutting in the year 4 of 2023 is highest, the survival rate of material obtained in the year 11 is lowest, and the number of times of 5 months is highest. The method shows that the quantity of the carried germs is small when the buds sprout in spring, and the shearing of the test materials plays a positive role in reducing the pollution rate.
TABLE 6 statistics of explant growth taken at different times
1.2 Effect of different disinfection times on explant growth
2 Different sterilization times were used. As shown in Table 7, the survival rates were not greatly different in the two sterilization treatments, and the contamination rate of the explants was high when the sterilization was carried out for 15min, but the death rate was increased when the sterilization time was increased to 20 min.
TABLE 7 statistics of explant growth at different sterilization times
2. Proliferation medium selection
2.1 Effect of different hormone combinations on proliferation culture of beef tendons
The experiment used 6 different hormone combinations. As shown in Table 8, the proliferation factor of the beef tendon was 3.18 at the highest in the case of the combination of the hormones A4, and the plants were not vitrified or the like in the case of the combination of the hormones, which was not conducive to further cultivation, as shown in FIG. 2A. Thus, the hormone combination most suitable for proliferation of beef tendons under the test conditions is 0.8 mg/L6-BA+0.3 mg/L NAA.
TABLE 8 influence of different hormone combinations on proliferation culture of beef tendons
2.2 Effect of different concentrations of GA 3 on proliferation culture of beef tendons
The experiment used 8 different hormone combinations. As shown in Table 9, after GA 3 was added in the different concentrations based on the combination A1, the vitrification ratio was effectively reduced. The proliferation coefficient of beef tendon is highest after 0.01mg/L GA 3 is added, and is 3.22, and when the concentration of GA 3 exceeds 1.0mg/L, the proliferation coefficient can be reduced or vitrification phenomenon of a certain proportion occurs along with the increase of the concentration of GA 3.
TABLE 9 influence of different concentrations of GA 3 on proliferation culture of beef tendons
3. Influence of different strong seedling culture mediums on growth and culture of beef tendons
The experiments used orthogonal designs, with a total of 9 hormone combinations. As shown in Table 10, the strain of the beef tendon can be cultured for 45 days in the B1 culture medium, the strain height can reach 2-2.5 cm, the leaf color is dark green, the growth condition is good, the strain height can reach about 2cm after being cultured in the B2 and B3 culture mediums, but the strain is vitrified or other conditions which are unfavorable for continuous culture and growth exist in part, and under the condition of the rest culture mediums, the strain height of the beef tendon is lower than 2cm, and the strain is not suitable for being used as a strong seedling culture medium.
TABLE 10 influence of different seedling-strengthening culture media on growth and culture of beef tendons
4. Rooting culture
And (5) transferring the strong seedlings to a rooting culture medium for culturing for 30 days, and counting the rooting rate. As shown in Table 11, the rooting rate of Niu Jintiao can reach 100% by adding 0.5mg/L IBA by using 1/2MS as the basic culture medium, the root is white, the average root number is 7.17, the plant leaves are large, and the leaf color is dark green.
TABLE 11 influence of different minimal Medium on the rooting of the beef tendons
4. Conclusion(s)
Taking new branches sprouting in spring for later use, cutting top buds in test, washing with tap water added with detergent, washing with running water for 1h, soaking in 75% alcohol in an ultra-clean bench for 30s, washing with sterile water for 3 times, sterilizing with 2% sodium hypochlorite solution for 15/20min, washing with sterile water for 3-4 times, sucking up water, and inoculating to an induction culture medium (MS+1.0 mg/L6-BA+0.3 mg/L NAA) for culture; successful initiation of the cultured shoots, the proliferation factor of the adventitious shoots was highest in the A4 (MS+0.8 mg/L6-BA+0.3 mg/L NAA) or A7 (MS+1.0 mg/L6-BA+0.5 mg/L NAA+0.01mg/L GA 3) medium; b1 Under the condition of (MS+0.1 mg/L6-BA+0.1 mg/L NAA) culture medium, the average plant height of the beef tendons can reach 2-2.5cm, the leaf color is dark green, and the leaf is large, thus being suitable for being used as a strong seedling culture medium; the rooting rate in rooting culture medium (1/2MS+0.5 mg/L IBA) can reach 100%, and the average root number reaches 7.17.
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 (9)
1. The ex-vivo rapid propagation method of the beef ribs is characterized by comprising the following steps of:
(1) Taking tender leaves at the top of a current annual branch of beef ribs as explants, sterilizing, and inoculating to an induction culture medium for induction culture, wherein the induction culture medium takes an MS culture medium as a basic culture medium and further comprises 25-35 g/L sucrose, 7-8 g/L agar, 0.5-1.5 mg/L6-benzylaminopurine, 0.2-0.5 mg/L alpha-naphthylacetic acid and pH of 5.75-5.80;
(2) Inoculating the tissue culture seedling subjected to induction culture into a proliferation culture medium for proliferation culture, wherein the proliferation culture medium takes an MS culture medium as a basic culture medium, and further comprises 25-35 g/L of sucrose, 7-8 g/L of agar, 0.6-0.8 mg/L of 6-benzylaminopurine, 0.3-0.5 mg/L of alpha-naphthylacetic acid, or further comprises 25-35 g/L of sucrose, 7-8 g/L of agar, 1.0 mg/L of 6-benzylaminopurine, 0.5-mg/L of alpha-naphthylacetic acid, 0.01-1.5 mg/L of gibberellin, and the pH value is 5.75-5.80;
(3) Dividing adventitious buds generated by multiplication culture into single buds, inoculating the single buds into a strong seedling culture medium for strong seedling culture, wherein the strong seedling culture medium takes an MS culture medium as a basic culture medium and further comprises 25-35 g/L sucrose, 7-8 g/L agar, 0.1-mg/L6-benzylaminopurine, 0.1-0.3 mg/L alpha-naphthylacetic acid and pH of 5.75-5.80;
(4) Inoculating the tissue culture seedlings after strengthening seedlings into a rooting culture medium for rooting culture to obtain Niu Jintiao rooting seedlings, wherein the rooting culture medium takes 1/2 MS culture medium as a basic culture medium and further comprises 15-25 g/L sucrose, 7-8 g/L agar, 0.3-1.0 mg/L3-indolebutyric acid and the pH value is 5.75-5.80.
2. The method for in vitro rapid propagation of beef tendon as claimed in claim 1, wherein in the step (1), the current annual branch of beef tendon is cut off during spring bud germination.
3. The method for in vitro rapid propagation of beef ribs according to claim 1, wherein in the step (1), the sterilization comprises sterilizing the explant with alcohol for 30-45 s after washing with running water, sterilizing in sodium hypochlorite solution for 15-20 min after washing with sterile water, and finally washing with sterile water.
4. The method for in vitro rapid propagation of beef ribs according to claim 1, wherein in the steps (1) to (3), the culture conditions are that the temperature is 23-28 ℃, the humidity is 65-75%, the illumination time is 12-16 h/d, and the illumination intensity is 1500-2000 lx.
5. The method for in vitro rapid propagation of beef tendon as claimed in claim 1, wherein the induction medium is a basic medium of MS medium, and further comprises 30g/L sucrose, 7.5 g/L agar, 1.0 mg/L6-benzylaminopurine, 0.3 mg/L alpha-naphthylacetic acid, and the pH is 5.75-5.80; and (5) performing induction culture for 30-35 days.
6. The method for in vitro rapid propagation of beef tendon as claimed in claim 1, wherein the propagation medium MS is a basic medium, further comprising 30 g/L sucrose, 7.5 g/L agar, 0.8 mg/L6-benzylaminopurine, 0.3 mg/L alpha-naphthylacetic acid, or further comprising 30 g/L sucrose, 7.5 g/L agar, 1.0 mg/L6-BA, 0.5 mg/L alpha-naphthylacetic acid, 0.01 mg/L gibberellin, and pH 5.75-5.80; and carrying out proliferation culture for 45-50 days.
7. The method for in vitro rapid propagation of beef tendon as claimed in claim 1, wherein the seedling strengthening culture medium uses MS culture medium as basic culture medium, and further comprises 30g/L sucrose, 7.5 g/L agar, 0.1 mg/L6-benzylaminopurine, 0.1 mg/L alpha-naphthylacetic acid, and the pH is 5.75-5.80; and culturing the strong seedlings for 45-50 days.
8. The method for in vitro rapid propagation of beef tendon according to claim 1, wherein the rooting medium is 1/2MS as a basic medium, and further comprises 20 g/L sucrose, 7.5 g/L agar, 0.5mg/L IBA, and the pH is adjusted to 5.75-5.80; rooting culture is carried out for 30-35 days.
9. The method for in vitro rapid propagation of beef ribs according to claim 1, wherein in the step (4), strong seedlings with the plant height of more than or equal to 2cm are inoculated into rooting culture medium.
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| USPP14456P2 (en) * | 2002-09-30 | 2004-01-13 | Florfis Ag | Geranium plant named ‘Fiscaseye’ |
| CN106857265A (en) * | 2017-04-14 | 2017-06-20 | 云南省农业科学院园艺作物研究所 | A kind of tissue culture enrichment procedure of white Cowhells wood |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| USPP14456P2 (en) * | 2002-09-30 | 2004-01-13 | Florfis Ag | Geranium plant named ‘Fiscaseye’ |
| CN106857265A (en) * | 2017-04-14 | 2017-06-20 | 云南省农业科学院园艺作物研究所 | A kind of tissue culture enrichment procedure of white Cowhells wood |
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| Title |
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| 柠条锦鸡儿组织培养技术;邵玲玲;李毅;赵庆龙;李禄军;;农业系统科学与综合研究;20111115(第04期);第469-474页 * |
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