CN104957038A - In-vitro preservation method of fragaria ananassa germplasm - Google Patents
In-vitro preservation method of fragaria ananassa germplasm Download PDFInfo
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- CN104957038A CN104957038A CN201510398994.1A CN201510398994A CN104957038A CN 104957038 A CN104957038 A CN 104957038A CN 201510398994 A CN201510398994 A CN 201510398994A CN 104957038 A CN104957038 A CN 104957038A
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Abstract
The invention discloses an in-vitro preservation method of fragaria ananassa germplasm. The in-vitro preservation method of fragaria ananassa germplasm comprises the following steps in sequence: (1) taking healthy fragaria ananassa creeping stems without disease spot and insect gall, and washing the fragaria ananassa creeping stems with running water; (2) wringing 0.4 to 0.6mm of shoot apical meristems from the fragaria ananassa creeping stems, and inoculating the shoot apical meristems to MS medium for culturing; (3) transferring plantlets to a proliferation medium for culturing after the shoot apical meristems grow to 0.5cm; (4) inoculating adventitious buds into a rooting preservation medium for rooting culturing when the adventitious buds obtained in the step (3) grow to 1.5cm; and (5) putting fragaria ananassa tissue culture plantlets obtained in the step (4) and the rooting preservation medium in a refrigeration box for preserving at 0 to 4 DEG C, culturing under weak light with illumination intensity being 5 to 10 micromoles per square meter in one second. Through the adoption of the method, a series of problems of influence of diseases and insect pests and variety degeneration in the conventional preservation of seed nurseries can be solved.
Description
Technical field
The present invention relates to a kind of method of conservation in vitro of strawberry germplasm.
Background technology
Strawberry (Fragaria ananassa Duch.) belongs to the rose family (Rosaceae) Fragaria (Fragaria) perennial evergreen herbaceous plant, is the Important Economic crop of extensively cultivation in the world.But domestic strawberry main breed is single, current deterioration of variety phenomenon is serious, therefore needs seed selection improved seeds, and resolve the first step work of strawberry good variety selection, just be to set up strawberry germplasm storehouse, preserve germ plasm resource cost-effectively, for the breeding work of strawberry lays the foundation.Strawberry generally nourishes and generates by stolon, traditional field gene bank store method needs plantation throughout the year and management strawberry, not only easily by the impact of the various factors such as damage by disease and insect, liquid manure in preservation process, and long-term preservation may make the merit of original kind degenerate, and therefore utilizes biotechnology to find suitable preserving seed technology to be the task of top priority.
Obtain high-quality Plantlets of Strawberry by strawberry stem tip meristematic tissue, utilize and suppress test-tube plantlet growth and Cryopreservation to preserve strawberry germplasm for a long time.
According to Literature Consult, the in-vitro conservation method of current strawberry germplasm is generally selects Developing restraint type medium, and namely add the inhibitor such as paclobutrazol, mannitol, abscisic acid in the medium, normal temperature is preserved; Add these inhibitor in the medium and may have certain harmful effect after kind of matter restoration ecosystem.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of method utilizing group training detoxification technology to preserve strawberry germplasm in vitro cost-effectively.
In order to solve the problems of the technologies described above, the invention provides a kind of strawberry in-vitro conservation method, comprising the following steps:
1), get without scab, healthy strawberry stolon tap water (such as put into beaker to wash, washing time is 1 hours) without insect gall;
2), step 1) gained strawberry stolon through sterilization (be routine disinfection, i.e. volumetric concentration 75% alcohol disinfecting 30 seconds, mass concentration 0.1% mercuric chloride solution sterilization 10min) after, strip the shoot apical meristem 0.4 ~ 0.6mm (being such as 0.5mm) of strawberry stolon, be seeded on MS medium and cultivate; Condition of culture is: 16 h light, intensity of illumination 10 ~ 30 μm of olm
-2.s
-1(be preferably 15 ~ 25 μm of ol m
-2.s
-1), temperature is 26 ~ 28 DEG C; 8 hours light culture, temperature is 20 ~ 22 DEG C; Above-mentioned illumination and light culture hocket;
3), when shoot apical meristem grows up to 0.5cm seedling, be forwarded on proliferated culture medium and cultivated; Condition of culture is: 16 h light, intensity of illumination 10 ~ 30 μm of ol m
-2.s
-1(be preferably 15 ~ 25 μm of ol m
-2.s
-1), temperature is 26 ~ 28 DEG C; 8 hours light culture, temperature is 20 ~ 22 DEG C; Above-mentioned illumination and light culture hocket;
4), until step 3) indefinite bud of gained is when growing into 1.5cm, and be inoculated on Storaged media of taking root and carry out culture of rootage, incubation time is 6 ~ 8 days (namely about one week); Condition of culture is: 16 h light, intensity of illumination 10 ~ 30 μm of ol m
-2.s
-1(be preferably 15 ~ 25 μm of ol m
-2.s
-1), temperature is 26 ~ 28 DEG C; 8 hours light culture, temperature is 20 ~ 22 DEG C; Above-mentioned illumination and light culture hocket;
5), by step 4) the Strawberry Plantlets seedling of gained is together with Storaged media of taking root (that is, whole bottle) in refrigerating box 0 ~ 4 DEG C preservation, and the low light level is cultivated, intensity of illumination 5 ~ 10 μm of ol m
-2.s
-1.
Remarks illustrate: preserves about 6 months, the growing way of seedling, length, radical and root long substantially unchanged (that is, preserve starting date, preservation expiration date foregoing is consistent substantially).
Improvement as the Plantlet in vitro method of strawberry germplasm of the present invention: preserve and change Storaged media continuation 0 ~ 4 DEG C of preservation of taking root for 1 year afterwards, the low light level is cultivated, intensity of illumination 5 ~ 10 μm of ol m
-2.s
-1.
Further improvement as the Plantlet in vitro method of strawberry germplasm of the present invention: described step 2) in MS medium be MS minimal medium+sugared 30g/l+ agar 8g/l, pH be 5.5 ~ 6.0.
The preparation method of this MS medium is: based on MS minimal medium, adds 30g sugar, 8g agar Homogeneous phase mixing in the MS minimal medium of every 1L, utilizes the HCl of KOH or 1mol/L of 1mol/L to regulate pH to be 5.5 ~ 6.0.
Further improvement as the Plantlet in vitro method of strawberry germplasm of the present invention: described step 3) in proliferated culture medium be: MS minimal medium+6-benzyl aminopurine (6-BA) 0.3 ~ 0.5mg/l+ sugar 30g/l+ agar 8g/l, pH is 5.5 ~ 6.0.
The preparation method of this proliferated culture medium is: based on MS minimal medium, 0.3 ~ 0.5mg 6-benzyl aminopurine, 30g sugar, 8g agar is added in the MS minimal medium of every 1L, Homogeneous phase mixing, utilizes the HCl of KOH or 1mol/L of 1mol/L to regulate pH to be 5.5 ~ 6.0.
Further improvement as the Plantlet in vitro method of strawberry germplasm of the present invention: it is characterized in that step 4) in Storaged media of taking root be: containing organic component 1/2MS minimal medium (the 1/2MS minimal medium of improvement)+sugared 15g/l+ agar 10g/l, pH are 5.5 ~ 6.0.
The preparation method of this Storaged media of taking root is: based on the 1/2MS minimal medium not containing organic component, every 1L does not add 15g sugar, 10g agar containing in the 1/2MS minimal medium of organic component, Homogeneous phase mixing, utilizes the HCl of KOH or 1mol/L of 1mol/L to regulate pH to be 5.5 ~ 6.0.
Not containing the 1/2MS minimal medium of organic component, in ie in solution, the content of macroelement, trace element, molysite is the half of MS minimal medium, not containing the organic principle of any content.
Remarks illustrate: MS minimal medium is made up of macroelement, trace element, molysite, organic component, and this is routine techniques (that is, composition, content all belong to known technology).
Inventive point of the present invention is mainly: the formula being provided with suitable Storaged media of taking root, and is provided with suitable preservation intensity of illumination and storage temperature.Thus achieve the long-term Plantlet in vitro of strawberry germplasm, that is, extend the holding time.
Conservation in vitro of strawberry germplasm method of the present invention, belongs to the in-vitro conservation method in kind of matter strange land store method.According to the principle of cellular omnipotency in Plant Tissue Breeding, the high quality seedling (seed) that a large amount of genetic background is identical, growing way is consistent can be produced at short notice, then test-tube plantlet growth (Storaged media of taking root suppresses Strawberry Seedlings growth) is suppressed, utilize strawberry hibernation temperature simultaneously, pass through Cord blood, medium Developing restraint, namely adopts the measure of compound Developing restraint, obtains the conservation in vitro of strawberry germplasm technology of complete set.Adopt method of the present invention, each germ plasm resource of strawberry can be made (namely, each different strawberry cultivars) be inoculated in the suitable culture medium (Storaged media of taking root) of Developing restraint and be stored in laboratory freezer more than 1 year, several years can be preserved by replaced medium, therefore this biotechnology conservation is not only economical, convenient, can also solve conventional seed garden preserve in run into affect by damage by disease and insect, the series of problems such as deterioration of variety.
Embodiment
Embodiment 1: a kind of method of conservation in vitro of strawberry germplasm, carry out following steps successively:
1), get without scab, without insect gall health " red cheek " strawberry stolon, put into beaker, tap water 1 hour;
2), step 1) stolon of gained is through routine disinfection, i.e. 75% (volume %) alcohol disinfecting 30 seconds, 0.1% (quality %) mercuric chloride sterilization 10min, strips strawberry stem tip meristematic tissue about 0.5mm, is seeded on MS medium and cultivates; Condition of culture is: 16 h light, intensity of illumination 15 ~ 25 μm of ol m
-2.s
-1, temperature is 26 ~ 28 DEG C; 8 hours light culture, temperature is 20 ~ 22 DEG C; Above-mentioned illumination and light culture hocket;
MS medium is: MS minimal medium+sugared 30g/l+ agar 8g/l, pH is 5.5 ~ 6.0.
3), when above-mentioned shoot apical meristem grows up to about 0.5cm seedling, transferred in being inoculated on proliferated culture medium and cultivated; Condition of culture is: 16 h light, intensity of illumination 15 ~ 25 μm of ol m
-2.s
-1, temperature is 26 ~ 28 DEG C; 8 hours light culture, temperature is 20 ~ 22 DEG C; Above-mentioned illumination and light culture hocket;
Proliferated culture medium is: MS minimal medium+6-BA0.5mg/l+ sugar 30g/l+ agar 8g/l, pH is 5.5 ~ 6.0.
4), treat that indefinite bud grows into about 1.5cm, be inoculated on Storaged media of taking root and carry out culture of rootage one week; Condition of culture is: 16 h light, intensity of illumination 15 ~ 25 μm of ol m
-2.s
-1, temperature is 26 ~ 28 DEG C; 8 hours light culture, temperature is 20 ~ 22 DEG C; Above-mentioned illumination and light culture hocket;
Storaged media of taking root is: containing organic component 1/2MS minimal medium (the 1/2MS minimal medium of improvement)+sugared 15g/l+ agar 10g/l, pH are 5.5 ~ 6.0.
5), by the whole bottle of Strawberry Plantlets seedling (that is, together with Storaged media of taking root) of above step gained be positioned in refrigerating box and preserve, the low light level is cultivated, and preservation condition is: temperature 0 ~ 4 DEG C, intensity of illumination 5 ~ 10 μm of ol m
-2.s
-1.
Preserve the growing way of 180 days seedlings, length, radical and root long substantially unchanged.Specifically as described in Table 1.Three repetitions, often repeat ten seedlings, average.
Table 1
| Seedling growing state | Original | Preserve 180 days | Preserve 13 months |
| Growing way | Well | Well | Well |
| Length (cm) | 2.33 | 2.45 | 2.69 |
| Radical | 3.67 | 3.78 | 4.01 |
| Root long (cm) | 1.58 | 1.61 | 1.79 |
6), preserve 1 year replacing root media and continue preservation, the conditional synchronization rapid 5 that preservation, the low light level are cultivated).
When preserving 13rd month, detect the growing way of seedling, length, radical and root progress row, acquired results is as shown in table 1.
Therefore, according to said method, " red cheek " strawberry germplasm successfully can be preserved, more more convenient, more economical than conventional field gene bank preserving seed.
Embodiment 2: " red cheek " the strawberry stolon in embodiment 1 is renamed as " a chapter Ji " strawberry stolon; All the other are with embodiment 1.
Acquired results is as shown in table 2 below.
Table 2
| Seedling growing state | Original | Preserve 180 days | Preserve 13 months |
| Growing way | Well | Well | Well |
| Length (cm) | 2.02 | 2.18 | 2.34 |
| Radical | 3.98 | 4.11 | 4.19 |
| Root long (cm) | 1.42 | 1.61 | 1.73 |
Comparative example 1-1, the 1/2MS minimal medium (the 1/2MS minimal medium of improvement) of organic component " not containing " in " Storaged media of taking root " of embodiment 1 to be made into " conventional 1/2MS minimal medium "; All the other are with embodiment 1.
Remarks illustrate: conventional 1/2MS minimal medium, and in ie in solution, the content of macroelement, trace element, molysite, organic principle is the half (this is known technology) of MS minimal medium.
Comparative example 1-2, " the agar 10g/l " of taking root in Storaged media in embodiment 1 to be made into " agar 4g/l "; All the other are with embodiment 1.
Comparative example 2-1, by embodiment 1 step 5) in intensity of illumination by " intensity of illumination 5 ~ 10 μm of ol m
-2.s
-1" make " intensity of illumination 20 ~ 25 μm of ol m into
-2.s
-1".All the other are with embodiment 1.
Comparative example 2-2, by embodiment 1 step 5) in intensity of illumination by " intensity of illumination 5 ~ 10 μm of ol m
-2.s
-1" make " intensity of illumination 1 ~ 2 μm of ol m into
-2.s
-1".All the other are with embodiment 1.
Comparative example 3-1, by embodiment 1 step 5) in temperature make " 5 ~ 8 DEG C into by " 0 ~ 4 DEG C ".All the other are with embodiment 1.
Comparative example 3-2, by embodiment 1 step 5) in temperature make "-4 ~-1 DEG C into by " 0 ~ 4 DEG C ".All the other are with embodiment 1.
The result of above-mentioned comparative example is as shown in table 3 below.
Table 3
Finally, it is also to be noted that what enumerate above is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be had.All distortion that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, all should think protection scope of the present invention.
Claims (5)
1. the Plantlet in vitro method of strawberry germplasm, is characterized in that comprising the following steps successively:
1), get without scab, healthy strawberry stolon tap water without insect gall;
2), step 1) gained strawberry stolon through sterilization after, strip the shoot apical meristem 0.4 ~ 0.6mm of strawberry stolon, be seeded on MS medium and cultivate; Condition of culture is: 16 h light, intensity of illumination 10 ~ 30 μm of ol m
-2.s
-1, temperature is 26 ~ 28 DEG C; 8 hours light culture, temperature is 20 ~ 22 DEG C; Above-mentioned illumination and light culture hocket;
3), when shoot apical meristem grows up to 0.5cm seedling, be forwarded on proliferated culture medium and cultivated; Condition of culture is: 16 h light, intensity of illumination 10 ~ 30 μm of ol m
-2.s
-1, temperature is 26 ~ 28 DEG C; 8 hours light culture, temperature is 20 ~ 22 DEG C; Above-mentioned illumination and light culture hocket;
4), until step 3) indefinite bud of gained is when growing into 1.5cm, and be inoculated on Storaged media of taking root and carry out culture of rootage, incubation time is 6 ~ 8 days; Condition of culture is: 16 h light, intensity of illumination 10 ~ 30 μm of ol m
-2.s
-1, temperature is 26 ~ 28 DEG C; 8 hours light culture, temperature is 20 ~ 22 DEG C; Above-mentioned illumination and light culture hocket;
5), by step 4) the Strawberry Plantlets seedling of gained arises from refrigerating box 0 ~ 4 DEG C preservation together with Storaged media one of taking root, and the low light level is cultivated, intensity of illumination 5 ~ 10 μm of ol m
-2.s
-1.
2. the Plantlet in vitro method of strawberry germplasm according to claim 1, is characterized in that: preserve and within 1 year, change Storaged media continuation 0 ~ 4 DEG C of preservation of taking root afterwards, the low light level is cultivated, intensity of illumination 5 ~ 10 μm of ol m
-2.s
-1.
3. the Plantlet in vitro method of strawberry germplasm according to claim 1 and 2, is characterized in that: step 4) in Storaged media of taking root be not containing organic component 1/2MS minimal medium+sugared 15g/l+ agar 10g/l, pH are 5.5 ~ 6.0.
4. the Plantlet in vitro method of strawberry germplasm according to claim 3, is characterized in that: described step 2) in MS medium be MS minimal medium+sugared 30g/l+ agar 8g/l, pH be 5.5 ~ 6.0.
5. the Plantlet in vitro method of strawberry germplasm according to claim 4, is characterized in that: described step 3) in proliferated culture medium be: MS minimal medium+6-benzyl aminopurine 0.3 ~ 0.5mg/l+ sugar 30g/l+ agar 8g/l, pH is 5.5 ~ 6.0.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105210868A (en) * | 2015-10-08 | 2016-01-06 | 新疆生产建设兵团第六师农业科学研究所 | The acquisition methods of strawberry detoxification breeder's stock seedling and store method |
| CN112005823A (en) * | 2020-07-27 | 2020-12-01 | 重庆市农业科学院 | Ecological planting method for strawberry variety resource preservation |
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| JP2009118835A (en) * | 2007-11-13 | 2009-06-04 | Kankyo Saien:Kk | Liquid culture method in plant seedling production which raises seedling from runner |
| CN102550418A (en) * | 2012-01-16 | 2012-07-11 | 北京农学院 | Preparation method for culture medium dedicated to normal-temperature preservation of strawberry tissue-cultured seedling germplasms |
| CN104604685A (en) * | 2015-01-31 | 2015-05-13 | 四川农业大学 | Ultralow temperature detoxification method for strawberry stem tip |
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2015
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Patent Citations (3)
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| JP2009118835A (en) * | 2007-11-13 | 2009-06-04 | Kankyo Saien:Kk | Liquid culture method in plant seedling production which raises seedling from runner |
| CN102550418A (en) * | 2012-01-16 | 2012-07-11 | 北京农学院 | Preparation method for culture medium dedicated to normal-temperature preservation of strawberry tissue-cultured seedling germplasms |
| CN104604685A (en) * | 2015-01-31 | 2015-05-13 | 四川农业大学 | Ultralow temperature detoxification method for strawberry stem tip |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105210868A (en) * | 2015-10-08 | 2016-01-06 | 新疆生产建设兵团第六师农业科学研究所 | The acquisition methods of strawberry detoxification breeder's stock seedling and store method |
| CN112005823A (en) * | 2020-07-27 | 2020-12-01 | 重庆市农业科学院 | Ecological planting method for strawberry variety resource preservation |
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