阅读说明：本技术 一种马铃薯种质资源的保存方法 (Preservation method of potato germplasm resources ) 是由 刘广晶 吕文霞 赵玉平 候健花 包美丽 孙翠翠 李燕 田艳花 于冬梅 秦昊 于 2021-09-17 设计创作，主要内容包括：本发明属于马铃薯技术领域,且公开了一种马铃薯种质资源的保存方法,S1、先进行种薯的挑选,选择新鲜、光泽好且腐烂率低的,接着用刀马铃薯切块,并观察有无环腐病,选择无环腐病的健康植株并截取3～4cm有腋芽的茎段若干个。本发明通过设置有种薯挑选环节,可保证挑选的种薯新鲜且健康,并保证截取的茎段无环腐病基因,利用蒸馏水和灭菌水对截取的茎段进行清洗,通过调控温度和光照强度可增强幼苗对不同生长环境的适应性并加强抵抗力,可挑选根茎较为粗壮且活性较好的试管苗并通过不断的转接,可保证培育出来的试管苗质量较好,且存活率更高,可保证品种较为优秀的马铃薯种质资源能一直长期保存。(The invention belongs to the technical field of potatoes and discloses a preservation method of potato germplasm resources, S1, firstly, selecting potato seeds, selecting fresh potatoes with good luster and low rotting rate, then cutting the potatoes into blocks by using a knife, observing whether ring rot exists, selecting healthy plants without ring rot and cutting a plurality of stem sections with 3-4 cm axillary buds. According to the invention, through arranging the seed potato selection link, the selected seed potatoes can be ensured to be fresh and healthy, the intercepted stem segments are ensured to have no ring rot genes, the intercepted stem segments are cleaned by using distilled water and sterilized water, the adaptability of the seedlings to different growth environments and the resistance can be enhanced by regulating and controlling the temperature and the illumination intensity, the test-tube seedlings with thicker rootstocks and better activity can be selected and continuously transferred, the quality of the cultivated test-tube seedlings can be ensured to be better, the survival rate is higher, and the potato seed quality resources with more excellent varieties can be ensured to be stored for a long time all the time.)

1. A preservation method of potato germplasm resources is characterized by comprising the following steps:

s1, selecting seed potatoes, selecting fresh potatoes with good luster and low rotting rate, cutting the potatoes into blocks by using a knife, observing whether ring rot exists, selecting healthy plants without ring rot and cutting a plurality of 3-4 cm stem sections with axillary buds;

s2, washing the cut stem segments with clear water, then clamping out the stem segments with sterile forceps, and placing the stem segments in sterile water for cleaning for a plurality of times to prepare for the stem segments to grow into sterile seedlings in the future;

s3, placing the sterilized stem segments into an MS culture medium for culture, sealing the bottle mouth for storing the culture medium by using a protective film, then transferring the bottle mouth to a tissue culture seedling growing room, adjusting the illumination intensity of the tissue culture seedling growing room to 2600 and 3000lx, and controlling the temperature to be kept at 20-24 ℃;

s4, regular detection and sterilization during growth of the aseptic seedlings, adjusting the illumination intensity of the tissue culture growth room to 3200-3600 lx when the aseptic seedlings are cultured for 1-2 months, and controlling the temperature to be kept at 23-25 ℃ for continuous culture;

s5, when aseptic seedlings are formed, cutting 4-5 stem sections of the aseptic seedlings under an aseptic condition, placing the cut stem sections in an MS culture medium for continuous culture and preservation, sealing the bottle openings by using a protective film, moving the cut stem sections to a germplasm resource preservation room for preservation, controlling the temperature to be 15-20 ℃, controlling the illumination intensity to be 2800-3200 lx, and regularly sterilizing the germplasm resource preservation room, so that new test-tube seedlings can be continuously formed and can be preserved for a long time.

2. The method of claim 1, wherein the method comprises: the protective films in S3 and S5 are preservative films.

3. The method of claim 1, wherein the method comprises: the MS medium appeared in S5 and contained 3% mannitol, pyridoxine hydrochloride and thiamine hydrochloride.

4. The method of claim 1, wherein the method comprises: the clean water in the S2 is distilled water and needs to be indirectly cleaned for 0.5-1.5 h.

5. The method of claim 1, wherein the method comprises: and the stem sections need to be cleaned for 3-4 times by the sterilized water in the S2.

6. The method of claim 1, wherein the method comprises: in the S1, when the seed potatoes are cut into blocks by the knife, if the seed potatoes are found to have ring rot, the other seed potatoes can be cut into blocks after the knife is disinfected and cleaned, wherein the ring rot refers to the ring rot of the potatoes.

7. The method of claim 1, wherein the method comprises: and a plurality of stem sections appearing in S1 refer to 6-10 stem sections.

Technical Field

The invention belongs to the technical field of potatoes, and particularly relates to a preservation method of a potato germplasm resource.

Background

The potatoes belong to solanaceous plants, tubers are edible, the growth cycle of the potatoes can be roughly divided into four stages, namely, the mature tubers can be used as seed potatoes for sowing after a period of dormancy, and the potatoes are only inferior to grain crops, namely wheat, rice and corn, which are called as fourth major and middle parts of the world, namely potatoes, yam, taros, ground eggs, potatoes and the like; secondly, after sowing, the seedlings germinate and grow out by relying on self nutrition; thirdly, after seedling emergence, organic matters are produced through photosynthesis, and a complete plant growth system is formed by utilizing the root system to absorb water and inorganic elements; and fourthly, after blooming, the overground part stops growing, tubers rapidly expand, and nutrients are accumulated until the tubers are mature, so that the method is extremely important for preserving the seed potatoes with better germplasm resources, and the production capacity in the next year can be increased.

The traditional preservation method of the potato germplasm resources is basically divided into two types, one type is field planting preservation, and the other type is test-tube plantlet preservation, because the field planting preservation is easily influenced by temperature to damage potato seeds, the survival rate of the potato seeds preserved by the test-tube plantlets is higher, and the photosynthesis of chloroplasts is poorer due to insufficient sunlight of the existing test-tube plantlets; the number of air holes of the blade is small, so that the activity is poor, and the blade cannot be popularized and used generally; in addition, long-term storage at low temperature affects the growth of test-tube plantlets, which are relatively delicate and cause death due to underdeveloped cuticle of stems and leaves.

Disclosure of Invention

The invention aims to solve the problems, and provides a preservation method of potato germplasm resources, which has the advantages of high survival rate, adaptability to stress and high resistance.

In order to achieve the purpose, the invention provides the following technical scheme: a preservation method of potato germplasm resources comprises the following steps:

s1, selecting seed potatoes, selecting fresh potatoes with good luster and low rotting rate, cutting the potatoes into blocks by using a knife, observing whether ring rot exists, selecting healthy plants without ring rot and cutting a plurality of 3-4 cm stem sections with axillary buds;

s2, washing the cut stem segments with clear water, then clamping out the stem segments with sterile forceps, and placing the stem segments in sterile water for cleaning for a plurality of times to prepare for the stem segments to grow into sterile seedlings in the future;

s3, placing the sterilized stem segments into an MS culture medium for culture, sealing the bottle mouth for storing the culture medium by using a protective film, then transferring the bottle mouth to a tissue culture seedling growing room, adjusting the illumination intensity of the tissue culture seedling growing room to 2600 and 3000lx, and controlling the temperature to be kept at 20-24 ℃;

s4, regular detection and sterilization during growth of the aseptic seedlings, adjusting the illumination intensity of the tissue culture growth room to 3200-3600 lx when the aseptic seedlings are cultured for 1-2 months, and controlling the temperature to be kept at 23-25 ℃ for continuous culture;

s5, when aseptic seedlings are formed, cutting 4-5 stem sections of the aseptic seedlings under an aseptic condition, placing the cut stem sections in an MS culture medium for continuous culture and preservation, sealing the bottle openings by using a protective film, moving the cut stem sections to a germplasm resource preservation room for preservation, controlling the temperature to be 15-20 ℃, controlling the illumination intensity to be 2800-3200 lx, and regularly sterilizing the germplasm resource preservation room, so that new test-tube seedlings can be continuously formed and can be preserved for a long time.

Selecting seed potato stem sections with good selectable genes and without ring rot, cleaning by combining distilled water and sterilized water, thereby removing bacteria and germs, avoiding harmful substances from being contaminated by the bacteria and germs by using the clamping of sterile tweezers, providing required nutrient substances for the growing stem segment by the MS culture medium, matching with comfortable temperature and required illumination intensity in the growing room of the tissue culture seedling, thereby leading the seedling to grow stably, leading the test-tube seedling to adapt to different growing environments and strengthening the self resistance by regulating and controlling the temperature change, so that the seedlings are healthy and thrive, test tube seedlings with thicker rootstocks and better activity are selected and continuously transferred, can ensure that the quality of the cultivated test-tube plantlets is better, the survival rate is higher, and the excellent potato germplasm resources of the variety can be always preserved for a long time.

As a preferable technical scheme of the invention, the protective films in the S3 and S5 are preservative films which are cheap and good to use and can isolate the invasion of bacteria and germs, the preservative films are replaced periodically to ensure that the oxygen in the test tubes is sufficient, and the preservative films can be abandoned optionally along with the continuous growth of the test-tube seedlings, so that the resistance of the seedlings can be exercised and enhanced.

As a preferable technical scheme of the invention, the MS culture medium appeared in S5 contains 3% of mannitol, pyridoxine hydrochloride and thiamine hydrochloride, and the mannitol is equivalent to a regulator of osmotic pressure, can play a role in buffering osmotic pressure, can prevent cells from bursting, protects gene cells from being damaged, and ensures that test-tube plantlets with good genes can be preserved all the time.

As a preferable technical scheme of the invention, the clean water appearing in S2 is distilled water and needs indirect cleaning for 0.5-1.5 h, the distilled water does not contain impurities and can not pollute the just-cut rhizome, and the sterile water is prepared by high-temperature sterilization and is suitable for long-time sealed sterile storage by cleaning the rhizome, so that the carried germ rate is reduced, and the rhizome can grow into healthy seedlings.

As a preferable technical scheme of the invention, the stem segments need to be cleaned for 3-4 times by the sterilizing water in the S2, and the distilled water does not contain impurities and can not pollute the rhizome which is just cut into segments, so that the rhizome keeps the original cell structure and is very suitable for wound treatment.

In a preferred embodiment of the present invention, in S1, when the seed potatoes are diced by the knife, if a ring rot is found, the other seed potatoes can be diced after the knife is sterilized and cleaned, wherein the ring rot refers to a ring rot of the potato, when the ring rot potato is cut, the potato is thrown away, and the knife is sterilized by alcohol, otherwise other good quality potato seeds are infected, which results in waste, and the dead potato seeds appear when the potato seeds with germs grow into seedlings, and pollute the tissue culture seedling growth room.

As a preferable technical scheme of the invention, the stem segments in the S1 refer to 6-10 stem segments, and the survival rate of the potato seeds can be ensured to be increased by cutting the stem segments, because the phenomenon that some seedlings may die during the cultivation period is considered, so that the long-term storage of the potato seed quality resources is influenced.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention can ensure the freshness and health of the seed potatoes selected by arranging the seed potato selection link, and ensures that the intercepted stem segment has no ring rot gene, the intercepted stem segment is cleaned by distilled water and sterilized water, can remove bacteria and germs carried by the stem segments, lays a foundation for cultivating aseptic seedlings, the MS culture medium can provide sufficient nutrient elements for the stem segments needing to be cultivated, the preservative film can isolate external germs and bacteria from entering, the tissue culture growth room can provide sufficient sunlight and comfortable temperature for the stem segments to grow into seedlings, the adaptability of the seedling to different growth environments can be enhanced and the resistance can be enhanced by regulating and controlling the temperature and the illumination intensity, can select the test-tube plantlet with thicker rootstock and better activity and ensure better quality of the cultivated test-tube plantlet through continuous switching, the survival rate is higher, and the excellent potato germplasm resources can be ensured to be stored for a long time all the time.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides a preservation method of potato germplasm resources, which comprises the following steps:

s1, selecting seed potatoes, selecting fresh potatoes with good luster and low rotting rate, cutting the potatoes into blocks by using a knife, observing whether ring rot exists, selecting healthy plants without ring rot and cutting a plurality of 3-4 cm stem sections with axillary buds;

s2, washing the cut stem segments with clear water, then clamping out the stem segments with sterile forceps, and placing the stem segments in sterile water for cleaning for a plurality of times to prepare for the stem segments to grow into sterile seedlings in the future;

s3, placing the sterilized stem segments into an MS culture medium for culture, sealing the bottle mouth for storing the culture medium by using a protective film, then transferring the bottle mouth to a tissue culture seedling growing room, adjusting the illumination intensity of the tissue culture seedling growing room to 2600 and 3000lx, and controlling the temperature to be kept at 20-24 ℃;

s4, regular detection and sterilization during growth of the aseptic seedlings, adjusting the illumination intensity of the tissue culture growth room to 3200-3600 lx when the aseptic seedlings are cultured for 1-2 months, and controlling the temperature to be kept at 23-25 ℃ for continuous culture;

s5, when aseptic seedlings are formed, cutting 4-5 stem sections of the aseptic seedlings under an aseptic condition, placing the cut stem sections in an MS culture medium for continuous culture and preservation, sealing the bottle openings by using a protective film, moving the cut stem sections to a germplasm resource preservation room for preservation, controlling the temperature to be 15-20 ℃, controlling the illumination intensity to be 2800-3200 lx, and regularly sterilizing the germplasm resource preservation room, so that new test-tube seedlings can be continuously formed and can be preserved for a long time.

Selecting seed potato stem sections with good selectable genes and without ring rot, cleaning by combining distilled water and sterilized water, thereby removing bacteria and germs, avoiding harmful substances from being contaminated by the bacteria and germs by using the clamping of sterile tweezers, providing required nutrient substances for the growing stem segment by the MS culture medium, matching with comfortable temperature and required illumination intensity in the growing room of the tissue culture seedling, thereby leading the seedling to grow stably, leading the test-tube seedling to adapt to different growing environments and strengthening the self resistance by regulating and controlling the temperature change, so that the seedlings are healthy and thrive, test tube seedlings with thicker rootstocks and better activity are selected and continuously transferred, can ensure that the quality of the cultivated test-tube plantlets is better, the survival rate is higher, and the excellent potato germplasm resources of the variety can be always preserved for a long time.

The protective films in the S3 and S5 are the preservative films, the preservative films are cheap and good to use, invasion of bacteria and germs can be isolated, the preservative films are replaced regularly to ensure that oxygen in the test tubes is sufficient, the preservative films can be abandoned optionally along with continuous growth of test-tube seedlings, and the resistance of the seedlings can be exercised and enhanced.

Wherein, the MS culture medium appeared in S5 contains 3% of mannitol, pyridoxine hydrochloride and thiamine hydrochloride, and the mannitol is equivalent to regulator of osmotic pressure, and has effects of buffering osmotic pressure, preventing cell from swelling, protecting gene cell from damage, and ensuring that test-tube plantlet with good gene can be preserved all the time.

The clear water in the S2 is distilled water which needs to be indirectly cleaned for 0.5-1.5 h, and the distilled water contains no impurities, so that the cut rhizome is not polluted, the original cell structure of the cut rhizome is kept, and the wound dressing is very suitable for wound treatment.

The stem sections are required to be cleaned for 3-4 times by the sterile water in the S2, the sterile water is prepared by high-temperature sterilization, and the stems are cleaned to be suitable for long-time sealed aseptic storage, so that the rate of germs carried by the stems and the stems is reduced, and the stems grow into healthy seedlings.

Wherein, when the seed potato is observed to the cutting with the sword in S1, if it has ring rot to discover, just can carry out the cutting to other seed potatoes after the washing to the cutter disinfection, wherein ring rot means that the potato is cyclic annular rotten, when cutting the potato that has ring rot, this kind of potato kind will throw away, and the cutter will disinfect with alcohol, otherwise can infect other better quality potato kind, thereby cause the waste, the phenomenon that can appear the dead seedling when this kind of potato kind that carries the germ grows into the seedling to can pollute group banks up seedling growth room.

Wherein, a plurality of stem segments appearing in S1 refer to 6-10 stem segments, and the cut stem segments can ensure that the survival rate of the potato seeds is increased, because the phenomenon that some seedlings may die during the cultivation period is considered, the long-term preservation of the potato seed quality resources is influenced.

The first embodiment is as follows:

s1, selecting seed potatoes, selecting fresh potatoes with good luster and low rotting rate, cutting the potatoes into blocks by using a knife, observing whether ring rot exists, selecting healthy plants without ring rot and cutting a plurality of 3cm stem sections with axillary buds;

s2, washing the cut stem segments with clear water, then clamping out the stem segments with sterile forceps, and placing the stem segments in sterile water for cleaning for a plurality of times to prepare for the stem segments to grow into sterile seedlings in the future;

s3, placing the sterilized stem segments into an MS culture medium for culture, sealing the bottle mouth for storing the culture medium by using a protective film, then transferring the bottle mouth to a tissue culture seedling growing room, adjusting the illumination intensity of the tissue culture seedling growing room to 2600lx, and controlling the temperature to be kept at 21 ℃;

s4, regular detection and sterilization during growth of the aseptic seedlings, adjusting the illumination intensity of the tissue culture growth room to 3200x when the aseptic seedlings are cultured for 1-2 months, and controlling the temperature to be 23 ℃ for continuous culture;

s5, when aseptic seedlings are formed, cutting 4 stem sections of the aseptic seedlings under the aseptic condition, placing the cut stem sections in an MS culture medium for continuous culture and storage, sealing the bottle openings by using a protective film, moving the cut stem sections to a germplasm resource storage room for storage, controlling the temperature to be kept at 15 ℃, controlling the illumination intensity to be 2800lx, and regularly sterilizing the germplasm resource storage room, so that the formation and long-term storage of new test-tube seedlings can be ensured continuously.

The second embodiment is as follows:

s1, selecting seed potatoes, selecting fresh potatoes with good luster and low rotting rate, cutting the potatoes into blocks by using a knife, observing whether ring rot exists, selecting healthy plants without ring rot and cutting a plurality of 3.5cm stem sections with axillary buds;

s2, washing the cut stem segments with clear water, then clamping out the stem segments with sterile forceps, and placing the stem segments in sterile water for cleaning for a plurality of times to prepare for the stem segments to grow into sterile seedlings in the future;

s3, placing the sterilized stem segments into an MS culture medium for culture, sealing the bottle mouth for storing the culture medium by using a protective film, then moving to a tissue culture seedling growing room, adjusting the illumination intensity of the tissue culture seedling growing room to 2700lx, and controlling the temperature to be 22 ℃;

s4, regular detection and sterilization during growth of the aseptic seedlings, adjusting the illumination intensity of the tissue culture growth room to 3300lx when the aseptic seedlings are cultured for 1-2 months, and controlling the temperature to be kept at 24 ℃ for continuous culture;

s5, when aseptic seedlings are formed, cutting 4.5 stem segments under aseptic condition, placing the stem segments in MS culture medium for continuous culture and preservation, sealing the bottle mouth with a protective film, transferring to a germplasm resource preservation room for preservation, controlling the temperature to be 16 ℃, keeping the illumination intensity to be 2900lx, and regularly sterilizing the germplasm resource preservation room, thus ensuring that new test-tube seedlings are continuously formed and preserved for a long time

The third implementation: s1, selecting seed potatoes, selecting fresh potatoes with good luster and low rotting rate, cutting the potatoes into blocks by using a knife, observing whether ring rot exists, selecting healthy plants without ring rot and cutting a plurality of 4cm stem sections with axillary buds;

s2, washing the cut stem segments with clear water, then clamping out the stem segments with sterile forceps, and placing the stem segments in sterile water for cleaning for a plurality of times to prepare for the stem segments to grow into sterile seedlings in the future;

s3, placing the sterilized stem segments into an MS culture medium for culture, sealing the bottle mouth for storing the culture medium by using a protective film, then transferring the bottle mouth to a tissue culture seedling growing room, adjusting the illumination intensity of the tissue culture seedling growing room to 2800lx, and controlling the temperature to be 23 ℃;

s4, regular detection and sterilization during growth of the aseptic seedlings, adjusting the illumination intensity of the tissue culture growth room to 3400lx when the aseptic seedlings are cultured for 1-1.5 months, and controlling the temperature to be kept at 25 ℃ for continuous culture;

s5, when aseptic seedlings are formed, cutting 5 stem sections of the aseptic seedlings under the aseptic condition, placing the cut stem sections in an MS culture medium for continuous culture and storage, sealing the bottle mouth by using a protective film, moving the cut stem sections to a germplasm resource storage room for storage, controlling the temperature to be kept at 19 ℃, controlling the illumination intensity to be 3100lx, and regularly sterilizing the germplasm resource storage room, so that new test-tube seedlings can be continuously formed and stored for a long time.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.