CN109430055B - Low-temperature preservation method for gene resources of potato virus-free test-tube plantlet - Google Patents
Low-temperature preservation method for gene resources of potato virus-free test-tube plantlet Download PDFInfo
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- CN109430055B CN109430055B CN201811346500.5A CN201811346500A CN109430055B CN 109430055 B CN109430055 B CN 109430055B CN 201811346500 A CN201811346500 A CN 201811346500A CN 109430055 B CN109430055 B CN 109430055B
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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
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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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Abstract
The invention discloses a low-temperature preservation method of potato virus-free test-tube plantlet gene resources, which comprises the following steps: after the virus-free test-tube plantlet with 1 leaf bud stem segment is transferred into a test tube filled with a culture medium, the virus-free test-tube plantlet is cultured to be 2-3cm high in the environment of 21-23 ℃, the photoperiod of 16H/d and the illumination intensity of 3000-: 4-6 ℃; the illumination intensity is as follows: 1000-; the photoperiod: storing in 10-12H/d artificial climate box. The preservation period of the invention is prolonged to 1.5-2 years; the test-tube plantlet preserved by the method has strong activity and good recovery capability.
Description
Technical Field
The invention belongs to the technical field of potato virus-free test-tube plantlet storage, and particularly relates to a low-temperature storage method of a gene resource of a potato virus-free test-tube plantlet.
Background
In recent years, the loss of plant germplasm resources is more and more serious, the preservation of plant germplasm resources becomes a topic of global attention (consolidation, prosperity. plant tissue culture [ M ]. Beijing: chemical industry Press, 2007: 178-. Unlike vegetative propagation crop, potato has special germplasm resource preservation characteristic, and potato may be preserved in tuber and test tube seedling or in seedling seed.
At present, tuber preservation and tissue culture seedling preservation are the most commonly used methods for potato germplasm resources, and a large amount of manpower, material resources and financial resources are needed in the tuber cyclic preservation process, the preservation effect is not ideal, the preservation period is short, the storage space is large, and high risks such as resource degradation, mixing or loss exist; the traditional tissue culture seedling has short preservation period, large workload and large storage space, and is easy to cause high risks such as resource degradation or loss and the like; aiming at the defects of the preservation method and the characteristics of potatoes, the slow growth and in vitro preservation of the virus-free test-tube plantlet is proved to be an effective germplasm resource preservation method, based on the tissue culture technology, the cell growth is reduced to the minimum by changing the internal and external environmental conditions of the culture growth, the successive transfer interval time is prolonged, the successive transfer times are reduced, and the purpose of medium-long term preservation of plant germplasm resources is realized (Chenghui, Chengling, Chenlongqing, and the like, lily germplasm resource restriction growth method preservation research [ J ]. gardening academic newspaper, 2006,33(4):789 and 793.), the method is successfully applied to various plants (Junhong, Beili Xia, Dijie, Dingjie, the influence of different growth retardants on the preservation of the virus-free test-tube plantlet of potatoes [ J ]. Heilongjiang agricultural science, 2010 (1-2), Wang Fang, Weimin, juneberry, et al, 'deity horse' chrysanthemum isolated preservation and genetic stability [ J ]. northwest plant journal, 2007,27(7):1341-1348 ], but at present, the technology usually prolongs the storage life by adding a high-concentration plant growth inhibitor into the culture medium, still has the defects of short preservation period (no more than 1 year), low survival rate and plant vigor, weak recovery capability, disordered resource preservation, inconvenient orderly management and the like, and may cause risks such as genetic variation and the like; the ultra-low temperature preservation method is still in the laboratory research stage and cannot be used for the preservation of large-scale germplasm resources.
Therefore, it is necessary to provide a new method for low-temperature preservation of gene resources of potato virus-free test-tube plantlets.
Disclosure of Invention
In view of the above, the invention provides a low-temperature preservation method of potato virus-free test-tube plantlet gene resources.
In order to solve the technical problem, the invention discloses a low-temperature preservation method of potato virus-free test-tube plantlet gene resources, which comprises the following steps: after the virus-free test-tube plantlet with 1 leaf bud stem segment is transferred into a test tube filled with a culture medium, the virus-free test-tube plantlet is cultured to be 2-3cm high in the environment of 21-23 ℃, the photoperiod of 16H/d and the illumination intensity of 3000-: 4-6 ℃; the illumination intensity is as follows: 1000-; the photoperiod: storing in 10-12H/d artificial climate box, wherein the culture medium comprises MS + sucrose 2-3% + agar powder 0.6% + sorbitol 3-5% + gibberellin 0.1-0.2mg/L, and pH 5.8-6.0.
Optionally, each resource is tagged with its own barcode and number before the tube enters the climatic chamber.
The invention also discloses a low-temperature preservation culture medium for the gene resources of the potato virus-free test-tube plantlet, which specifically comprises the following steps: MS + cane sugar 2-3% + agar powder 0.6% + sorbitol 3-5% + gibberellin 0.1-0.2mg/L, pH5.8-6.0.
Compared with the prior art, the invention can obtain the following technical effects:
1) the preservation period of the invention is prolonged to 1.5-2 years;
2) the test-tube plantlet preserved by the method has strong activity and good recovery capability.
3) The resource bar code is numbered and managed orderly.
Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 shows the state of the potato No. 5 of the variety of the present invention in which the resources are conserved for 37 days;
FIG. 2 shows the state of the potato No. 5 of the variety of the present invention in which the resource preservation 487d is performed;
FIG. 3 shows the state of the variety Chuanyu 117 of the present invention for resource conservation 35 d;
FIG. 4 shows the state of the resource saving 455d of the variety Chuanyu 117 of the present invention.
Detailed Description
The following embodiments are described in detail with reference to the accompanying drawings, so that how to implement the technical features of the present invention to solve the technical problems and achieve the technical effects can be fully understood and implemented.
The invention discloses a low-temperature preservation method of potato virus-free test-tube plantlet gene resources, which comprises the following steps:
the stem segment with 1 leaf bud of the virus-free test tube seedling is preferably the stem segment with terminal bud, after being transferred into a test tube filled with a culture medium, the virus-free test tube seedling is cultured to be 2-3cm high in the environment with the temperature of 21-23 ℃, the photoperiod of 16H/d and the illumination intensity of 3000-: 4-6 ℃; the illumination intensity is as follows: 1000-; the photoperiod: the artificial climate box with 10-12H/d is stored, and the artificial climate box can be stored for 1.5-2 years by the method, so that a large amount of manpower and material resources are saved, the efficiency is improved, and the resource loss caused by improper resource storage is effectively avoided.
Optionally, the preserved resource is qualified detoxified test-tube plantlets which have been detoxified and subjected to quality inspection.
Before the test tube enters the artificial climate box, each resource is pasted with a self bar code and a serial number, so that the order of resource storage and convenient retrieval can be ensured.
Wherein, the temperature is controlled at 4-6 ℃, the temperature is too low, the plant is frozen, and the temperature is too high, so that the growth of the plant can not be effectively controlled; the photoperiod is 10-12h, the time is too short, the plant quality is poor, the time is too long, and the growth of the plant cannot be effectively controlled; the illumination intensity is 1000-2000lux, the illumination intensity is too low, the plant quality is poor, the illumination intensity is too high, and the plant growth cannot be effectively controlled.
The invention also discloses a low-temperature preservation culture medium for the gene resources of the potato virus-free test-tube plantlet, which comprises 2-3% of MS + sucrose, 0.6% of agar powder, 3-5% of sorbitol, 0.1-0.2mg/L of gibberellin and has the pH value of 5.8-6.0.
Wherein, the cane sugar is used as a carbon source to provide energy and maintain osmotic pressure; gibberellin (GA) can promote shoot differentiation; sorbitol can improve osmotic pressure of a culture medium, inhibit water utilization of plants and deteriorate the growth environment of test-tube plantlets, so that the purpose of delaying growth is achieved; the concentration range is 3-5%, the low concentration can not play a role in delaying growth, and the high concentration can cause the reduction of plant vigor.
Example 1
Transferring a virus-free test tube seedling with 1 leaf bud stem segment, wherein the variety is Zhongshu No. 5, into a test tube filled with a culture medium, culturing to the height of 2-3cm under the environment of 22 ℃, 16H/d photoperiod and 3500lux illumination intensity, namely transferring to the temperature: 4-6 ℃; the illumination intensity is as follows: 1500 lux; the photoperiod: and (4) storing in an artificial climate box of 11H/d.
Wherein the transfer material is a virus-free test-tube plantlet with 1 leaf bud stem segment or a virus-free test-tube plantlet with a stem segment of a terminal bud.
The culture medium comprises MS + sucrose 2.5% + agar powder 0.6% + sorbitol 4% + gibberellin 0.15mg/L, and pH 5.9.
As shown in FIGS. 1 and 2, the effect of the culture medium and the culture conditions on the retardation of the growth of the test-tube plantlet is obvious; and under the culture condition, the growth state of the virus-free seedlings is good after about 500 days of culture.
Example 2
Transferring a virus-free test-tube plantlet with 1 leaf bud stem segment, wherein the variety is Szechwan yam 117, into a test tube filled with a culture medium, culturing to 2cm high in an environment with the photoperiod of 16H/d and the illumination intensity of 4000lux at the temperature of 21 ℃, and then transferring to a temperature: 4 ℃; the illumination intensity is as follows: 2000lux; the photoperiod: and (5) storing in a 10H/d artificial climate box.
Wherein the transfer material is a virus-free test-tube plantlet with 1 leaf bud stem segment or a virus-free test-tube plantlet with a stem segment of a terminal bud.
The culture medium comprises MS, sucrose 2%, agar powder 0.6%, sorbitol 5%, gibberellin 0.1mg/L, and has pH of 5.8.
As shown in FIG. 3, the effect of the medium and the culture conditions on the retardation of the growth of the test-tube plantlet is demonstrated.
Example 3
Transferring a virus-free test-tube plantlet with 1 leaf bud stem segment, wherein the variety is Szechwan yam 117, into a test tube filled with a culture medium, culturing to 3cm high in an environment with 23 ℃, 16H/d photoperiod and 3000lux illumination intensity, and then transferring to a temperature: 6 ℃; the illumination intensity is as follows: 1000 lux; the photoperiod: and storing in a 12H/d artificial climate box.
Wherein the transfer material is a virus-free test-tube plantlet with 1 leaf bud stem segment or a virus-free test-tube plantlet with a stem segment of a terminal bud.
The culture medium comprises MS, sucrose 3%, agar powder 0.6%, sorbitol 3%, gibberellin 0.2mg/L, and has pH of 6.0.
As shown in FIG. 4, it was demonstrated that the growth of the virus-free seedlings cultured for 450 days or more was good under the above culture conditions.
Table 1 comparison of the effects of the present invention and the prior art
While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (3)
1. A low-temperature preservation method for potato virus-free test-tube plantlet gene resources is characterized by comprising the following steps: after the virus-free test-tube plantlet with 1 leaf bud stem segment is transferred into a test tube filled with a culture medium, the virus-free test-tube plantlet is cultured to be 2-3cm high in the environment of 21-23 ℃, the photoperiod of 16H/d and the illumination intensity of 3000-: 4-6 ℃, illumination intensity: 1000-: storing in 10-12H/d artificial climate box, wherein the culture medium comprises MS + sucrose 2-3% + agar powder 0.6% + sorbitol 3-5% + gibberellin 0.1-0.2mg/L, and pH 5.8-6.0.
2. The cryopreservation method of claim 1 wherein each resource is labeled with its own barcode and number before the tube enters the climatic chamber.
3. A low-temperature preservation culture medium for potato virus-free test-tube plantlet gene resources is characterized by comprising the following specific steps: MS + cane sugar 2-3%, + agar powder 0.6%, + sorbitol 3-5%, + gibberellin 0.1-0.2mg/L and pH 5.8-6.0.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113785774A (en) * | 2021-09-17 | 2021-12-14 | 内蒙古中加农业生物科技有限公司 | Preservation method of potato germplasm resources |
| CN113841612B (en) * | 2021-09-17 | 2022-06-17 | 中国农业科学院作物科学研究所 | Potato test-tube plantlet growth-limiting storage method |
| CN115211369A (en) * | 2022-07-18 | 2022-10-21 | 古丽米拉·热合木土拉 | Method for slowly growing potato germplasm resources by using biological regulator |
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