CN115413577B - Tissue culture and rapid propagation method for gleditschia horrida - Google Patents
Tissue culture and rapid propagation method for gleditschia horrida Download PDFInfo
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- CN115413577B CN115413577B CN202211052532.0A CN202211052532A CN115413577B CN 115413577 B CN115413577 B CN 115413577B CN 202211052532 A CN202211052532 A CN 202211052532A CN 115413577 B CN115413577 B CN 115413577B
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- 238000000034 method Methods 0.000 title claims abstract description 24
- 241000931143 Gleditsia sinensis Species 0.000 claims abstract description 46
- 229920001817 Agar Polymers 0.000 claims abstract description 14
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 14
- 229930006000 Sucrose Natural products 0.000 claims abstract description 14
- 239000008272 agar Substances 0.000 claims abstract description 14
- 239000005720 sucrose Substances 0.000 claims abstract description 14
- 239000001963 growth medium Substances 0.000 claims abstract description 11
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 10
- 238000012258 culturing Methods 0.000 claims abstract description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 229960002523 mercuric chloride Drugs 0.000 claims description 5
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 4
- 235000017274 Diospyros sandwicensis Nutrition 0.000 claims 1
- 241000282838 Lama Species 0.000 claims 1
- 230000008901 benefit Effects 0.000 abstract description 5
- 230000004083 survival effect Effects 0.000 abstract description 4
- 230000035784 germination Effects 0.000 abstract description 3
- 238000004161 plant tissue culture Methods 0.000 abstract description 2
- 241000196324 Embryophyta Species 0.000 description 14
- 238000004017 vitrification Methods 0.000 description 12
- 238000009472 formulation Methods 0.000 description 11
- 239000000203 mixture Substances 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 239000000463 material Substances 0.000 description 4
- 238000002791 soaking Methods 0.000 description 3
- YLUMOTIQYNIXLO-UHFFFAOYSA-N 1-morpholin-4-ylethanesulfonic acid Chemical compound OS(=O)(=O)C(C)N1CCOCC1 YLUMOTIQYNIXLO-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000220485 Fabaceae Species 0.000 description 1
- 241000218652 Larix Species 0.000 description 1
- 235000005590 Larix decidua Nutrition 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000000749 insecticidal effect Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 208000017520 skin disease Diseases 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000002522 swelling effect Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
Classifications
-
- 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
-
- 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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/22—Improving land use; Improving water use or availability; Controlling erosion
Landscapes
- 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 a method for tissue culture and rapid propagation of gleditsia sinensis lam, and relates to the technical field of plant tissue culture. The method comprises the following steps: (1) Taking the gleditsia sinensis seeds for disinfection treatment, and then physically puncturing the shell; (2) Inoculating the seeds which physically puncture the shell into a culture medium, and culturing the seeds into group seedlings; the formula of the culture medium is as follows: MS +0.5g/LMES +30-40g/L sucrose +5-6g/L agar, pH5.8. According to the invention, the gleditsia sinensis seeds are subjected to tissue culture, so that a gleditsia sinensis tissue culture rapid propagation system is established, and the survival rate reaches 100%. The method solves the problems of rare seeds, low germination rate, high pollution rate and low propagation speed, and is suitable for industrialized seedling of the gleditsia sinensis; the method provided by the invention has the advantages that the propagation speed is obviously improved, the number of germplasm resources of the gleditsia sinensis is favorably increased, the cost can be reduced, the operation is simple, and the economic benefit is high.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a gleditsia sinensis tissue culture and rapid propagation method.
Background
The Gleditsia sinensis (Gleditsia sinensis Lam.) is a Gleditsia sinensis of Caesalpiniaceae of Leguminosae, and is a larch, the Gleditsia sinensis has abundant nutritive value from seeds, gleditsia sinensis to spina gleditsiae, the polysaccharide gum contained in the embryo can be applied to food and drug industry and other industries, the spina gleditsiae has swelling and insecticidal effects and is commonly used for treating skin diseases, and the Gleditsia sinensis has strong adaptability to extreme climatic conditions, can grow rapidly, has the characteristics of economic tree species such as nitrogen fixation performance and the like. Because the damage of natural environment and the phenomena of disordering, cutting and chopping are happened occasionally, the materials such as the gleditsia sinensis lam are endangered to be extinct. The germination rate of the gleditsia sinensis is low and the growth time is long under natural conditions, so that the market demand is difficult to meet.
At present, stem sections are commonly used as explants in gleditsia sinensis tissue culture at home and abroad, but the defects are high pollution rate, high browning rate and long growth period. Therefore, the provided gleditsia sinensis tissue culture method for reducing the pollution rate and improving the rapid propagation efficiency has important significance for new variety breeding and industrial large-scale low-cost production of gleditsia sinensis.
Disclosure of Invention
The invention aims to provide a gleditsia sinensis tissue culture and rapid propagation method, which aims to solve the problems in the prior art, and can efficiently and rapidly convert gleditsia sinensis seeds into plants, wherein the rooting rate can reach 100% at most.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a gleditsia sinensis tissue culture rapid propagation method, which comprises the following steps:
(1) Taking the gleditsia sinensis seeds for disinfection treatment, and then physically puncturing the shell;
(2) Inoculating the seeds after the shells are physically punctured into a culture medium, and culturing the seeds into group culture seedlings; the formula of the culture medium is as follows: MS +0.5g/LMES +30-40g/L sucrose +5-6g/L agar, pH5.8.
Further, the disinfection treatment comprises alcohol solution disinfection and mercury mercuric chloride solution disinfection.
Further, the volume fraction of the alcohol solution is 70%, and the mass fraction of the mercuric chloride solution is 0.1%.
Further, in the step (1), the physical pricking specifically includes: the inoculation clamp is placed on the outer flame of an alcohol lamp to burn and then puncture the shell of the gleditsia sinensis seeds, and the hilum and the seed hole positions are not damaged during operation.
Preferably, in step (2), the formulation of the culture medium is: MS +0.5g/L MES +30g/L sucrose +5g/L agar, pH5.8.
Further, in the step (2), the culture conditions are: temperature 25 ℃ and light and dark ratio 15/9.
The invention discloses the following technical effects:
according to the method, the gleditsia sinensis seeds are subjected to tissue culture, so that a gleditsia sinensis tissue culture rapid propagation system is established, and the rooting rate can reach 100% as high as possible. The culture medium used by the invention can successfully realize the tissue culture of the gleditsia sinensis seeds, has proper osmotic potential, and can effectively reduce the vitrification rate in the culture process.
The establishment of the method for tissue culture and rapid propagation of the gleditsia sinensis lam solves the problems of rare seeds, low germination rate, high pollution rate and low propagation speed, and is suitable for industrialized seedling of the gleditsia sinensis lam; the method provided by the invention has the advantages that the propagation speed is obviously improved, the germ plasm resource quantity of the gleditsia sinensis is favorably enlarged, the cost can be reduced, the operation is simple, and the economic benefit is high.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every intervening value, to the extent any stated value or intervening value in a stated range, and any other stated or intervening value in a stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the documents are cited. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including but not limited to.
Example 1
1) Collecting a plurality of mature gleditsia sinensis seeds on the original gleditsia sinensis stock plant in the current year as explants, taking the gleditsia sinensis seeds back to a laboratory for cleaning, putting the gleditsia sinensis seeds in a shade place for air drying, then putting the seeds on a super clean bench, soaking the seeds in alcohol with the volume fraction of 70% for 30s, washing the seeds with clear water for 3 times after soaking, then soaking the seeds in mercuric chloride with the mass fraction of 0.1% for 30min for disinfection, and washing the seeds with clear water for 5 times;
2) And putting the disinfected seeds on filter paper of a sterile culture dish to absorb moisture on the surfaces of the seeds, and carrying out high-temperature physical puncture, specifically, placing an inoculation clamp on outer flame of an alcohol lamp to burn until red is discharged, puncturing a hard shell of the gleditsia sinensis seeds, and avoiding the inoculation clamp from damaging the hilum and the positions of the seed holes during operation. Obliquely inserting and inoculating the seeds subjected to high-temperature physical puncture into a culture medium, and inoculating 3 seeds into each bottle; the formula of the culture medium is as follows: MS +0.5g/L (N-morpholinyl) ethanesulfonic acid (MES) +30g/L sucrose +5g/L agar, pH adjusted to 5.8.
3) The inoculated seeds are placed in a tissue culture room with the temperature of 25 ℃ and the illumination of 15/9 (namely 5 hours of illumination and 9 hours of darkness). Culturing for 10-15 days under the condition to root, and continuously culturing to obtain the tissue culture seedling.
The tissue culture seedlings cultured in the embodiment have developed root systems and tall and straight plants, the height of each tissue culture seedling is 9-11 cm, the rooting length is 6-11 cm, the leaves are greenish, and part of the leaves are vitrified yellow.
In this example, the rooting percentage was 100% and the vitrification ratio was 27.7%.
Example 2
The difference from example 1 is that, in step (2), the medium formulation is: MS +0.5g/L MES +35g/L sucrose +5g/L agar, and the pH was adjusted to 5.8.
The tissue culture seedlings cultured in the embodiment have developed root systems and tall and straight plants, the height of each tissue culture seedling is 9-11 cm, the rooting length is 5-11 cm, the leaves are greenish, and part of the seedlings are vitrified pale yellow.
In this example, the rooting percentage was 98.9% and the vitrification percentage was 26.4%.
Example 3
The difference from example 1 is that, in step (2), the medium formulation is: MS +0.5g/L MES +40g/L sucrose +5g/L agar, and the pH was adjusted to 5.8.
The tissue culture seedlings cultured in the embodiment have developed root systems and tall and straight plants, the height of each tissue culture seedling is 7-11 cm, the rooting length is 5-11 cm, the leaves are greenish, and a small part of the seedlings are light green and are vitrified pale yellow.
In this example, the rooting percentage was 96.4% and the vitrification percentage was 23.1%.
Example 4
The difference from example 1 is that, in step (2), the medium formulation is: MS +0.5g/L MES +30g/L sucrose +5.5g/L agar, pH adjusted to 5.8.
The tissue culture seedlings cultured in the embodiment have developed root systems and tall and straight plants, the height of each tissue culture seedling is 7-11 cm, the rooting length is 5-11 cm, the leaves are greenish, and a small part of the seedlings are light green and are vitrified pale yellow.
In this example, the rooting percentage was 96.4% and the vitrification percentage was 23.1%.
Example 5
The difference from example 1 is that, in step (2), the medium formulation is: MS +0.5g/L MES +35g/L sucrose +5.5g/L agar, and the pH value was adjusted to 5.8.
The tissue culture seedlings cultured in the embodiment have developed root systems and tall and straight plants, the height of each tissue culture seedling is 7-11 cm, the rooting length is 5-11 cm, the leaves are greenish, and a small part of the seedlings are vitrified in light yellow.
In this example, the rooting percentage was 98.3% and the vitrification percentage was 25.7%.
Example 6
The difference from example 1 is that, in step (2), the medium formulation is: MS +0.5g/L MES +40g/L sucrose +5.5g/L agar, and the pH value was adjusted to 5.8.
The tissue culture seedlings cultured by the embodiment have developed root systems, tall and straight plants, the height of each tissue culture seedling is 7-11 cm, the rooting length is 5-11 cm, the leaves are greenish, and a small part of the leaves are vitrified in light green.
In this example, the rooting percentage was 95.8% and the vitrification percentage was 22.6%.
Example 7
The difference from example 1 is that, in step (2), the medium formulation is: MS +0.5g/L MES +30g/L sucrose +6g/L agar, pH adjusted to 5.8.
The tissue culture seedlings cultured in the embodiment have developed root systems and tall and straight plants, the height of each tissue culture seedling is 7-11 cm, the rooting length is 5-11 cm, the leaves are greenish, and part of the seedlings are vitrified in light green.
In this example, the rooting percentage was 97.4% and the vitrification percentage was 26.8%.
Example 8
The difference from example 1 is that, in step (2), the medium formulation is: MS +0.5g/L MES +35g/L sucrose +6g/L agar, pH adjusted to 5.8.
The tissue culture seedlings cultured in the embodiment have developed root systems and tall and straight plants, the height of each tissue culture seedling is 7-11 cm, the rooting length is 5-11 cm, the leaves are greenish, and a small part of the seedlings are vitrified in light green.
In this example, the rooting percentage was 97.1% and the vitrification percentage was 25.7%.
Example 9
The difference from example 1 is that, in step (2), the medium formulation is: MS +0.5g/L MES +40g/L sucrose +6g/L agar, and the pH value was adjusted to 5.8.
The tissue culture seedlings cultured in the embodiment have developed root systems and tall and straight plants, the height of each tissue culture seedling is 7-11 cm, the rooting length is 5-11 cm, the leaves are greenish, and a small part of the seedlings are vitrified in light green.
In this example, the rooting percentage was 96.2% and the vitrification percentage was 22.4%.
Comparative example 1
The difference from example 1 is that, in step (2), the medium formulation is: 1/2MS + IAA0.4mg/L + NAA0.1mg/L.
The tissue culture seedlings cultured in the comparative example have developed root systems and tall and straight plants, the height of each tissue culture seedling is 7-9 cm, the rooting length is 3-7 cm, the leaves are green, and most parts of the yellow brown are vitrified.
The rooting percentage of this comparative example was 68.3%, and the vitrification percentage was 31.7%.
Comparative example 2
The difference from example 1 is that, in step (2), the medium formulation is: MS + NAA0.5mg/L + IAA0.25mg/L.
The tissue culture seedlings cultured in the comparative example have developed root systems, tall and straight plants, the height of each tissue culture seedling is 6-9 cm, the rooting length is 3-7 cm, the leaves are green, and most parts of the yellow brown are vitrified.
The comparative example had a rooting percentage of 66.7% and a vitrification percentage of 29.8%.
The tissue culture seedlings obtained in examples 1 to 9 and comparative examples 1 to 2 were transplanted, and the survival rate was counted after 30 days, and the results are shown in table 1.
TABLE 1 survival rates of tissue culture seedlings
| Examples/comparative examples | Survival rate of tissue culture seedling transplantation (%) |
| Example 1 | 99.2 |
| Example 2 | 98.3 |
| Example 3 | 97.7 |
| Example 4 | 96.9 |
| Example 5 | 96.4 |
| Example 6 | 95.8 |
| Example 7 | 94.9 |
| Example 8 | 92.6 |
| Example 9 | 93.7 |
| Comparative example 1 | 92.1 |
| Comparative example 2 | 91.8 |
In conclusion, the method for tissue culture and rapid propagation of the gleditsia sinensis lam has the advantages that the efficiency of converting the gleditsia sinensis lam seeds into plants is high, the rooting rate can reach 100% at most, and large-scale and batch production of the gleditsia sinensis lam tissue culture seedlings can be achieved. Compared with the existing reported gleditsia sinensis tissue culture technology, the method has the technical characteristics of high efficiency, high speed, high seedling rate and high technical practicability.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (6)
1. A method for tissue culture and rapid propagation of gleditsia sinensis, which is characterized by comprising the following steps:
(1) Taking the gleditsia sinensis seeds for disinfection treatment, and then physically puncturing the shell;
(2) Inoculating the seeds which physically puncture the shell into a culture medium, and culturing the seeds into group seedlings; the formula of the culture medium is as follows: MS +0.5g/LMES +30-40g/L sucrose +5-6g/L agar, pH5.8.
2. The tissue culture and rapid propagation method of gleditsia sinensis lamas, claim 1, wherein said disinfection treatment includes alcohol solution disinfection and mercuric chloride solution disinfection.
3. The tissue culture and rapid propagation method of gleditsia sinensis lam as claimed in claim 2, wherein the volume fraction of the alcohol solution is 70%, and the mass fraction of the mercuric chloride solution is 0.1%.
4. The tissue culture and rapid propagation method of Gleditsia sinensis as claimed in claim 1, wherein in step (1), the physical puncturing comprises: the inoculation clamp is placed on the outer flame of an alcohol lamp to burn and then puncture the shell of the gleditsia sinensis seeds, and the hilum and the seed hole positions are not damaged during operation.
5. The tissue culture and rapid propagation method of gleditsia sinensis lam as claimed in claim 1, wherein in step (2), the formula of said culture medium is: MS +0.5g/LMES +30g/L sucrose +5g/L agar, pH5.8.
6. The tissue culture and rapid propagation method of gleditsia sinensis lam as claimed in claim 1, wherein in step (2), the culture conditions are: the temperature was 25 ℃ and the ratio of light to dark was 15/9.
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