CN101904305A - Encapsulation-vitrification ultra-low temperature storage method of sweet potato stem tip - Google Patents
Encapsulation-vitrification ultra-low temperature storage method of sweet potato stem tip Download PDFInfo
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- CN101904305A CN101904305A CN 201010228497 CN201010228497A CN101904305A CN 101904305 A CN101904305 A CN 101904305A CN 201010228497 CN201010228497 CN 201010228497 CN 201010228497 A CN201010228497 A CN 201010228497A CN 101904305 A CN101904305 A CN 101904305A
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Abstract
The invention relates to an encapsulation-vitrification ultra-low temperature storage method of a sweet potato stem tip, which comprises the steps of: encapsulating the stem tip without being pre-cultured by taking sweet potato seedling of China sweet potato variety as a material; pre-culturing in cane sugar MS solutions with different concentrations after encapsulating, dewatering for 90-120min in a vitrification solution, freezing and storing at ultra-low temperature; and restoring the culture after freezing, directly budding without callus to form a complete planet, and carrying out a mass of vegetative propagation. The material used in the method is derived from the China sweet potato variety, is simple in operation, and quick in restoring growth after the material is frozen for a long time.
Description
Technical field
The present invention relates to the technology of sweet potato quality saving and plant regeneration, particularly the embedding vitrification ultra-low temperature store method of sweet potato stem tip belongs to sweet potato tissue culture technique field.
Background technology
For a long time, a lot of kinds of sweet potato are degenerated rapidly promote producing the back, and the good strains of seeds of forfeiture new varieties causes very big influence to the yield and quality of sweet potato.Therefore, how to collect preferably and preserve sweet potato germ plasm resource and come into one's own gradually.At present, the method for Chang Yong protective plant germ plasm resource all has limitation separately.Numerous tests show that it is comparatively desirable a kind of method that embedding vitrifying method superfreeze is preserved the plant germplasm technology.Its function and advantage are as follows: 1, keep the germplasm vigor for a long time, 2, the genetic stability behind the frozen material is good, 3, simultaneously handle lot of materials, handle the back material recover growth fast, poison little and bud ratio is high to material, 4, simple operation and other advantages.Existing embedding vitrification ultra-low temperature is preserved technology A, referring to D.Hira, A.Sakai, Simplified cryopreservation of sweet potato [Ipomoeabatatas (L.) Lam.] by optimizing conditions for osmoprotection, Plant Cell Reports, (2003) 21:961-966.
At present existing embedding vitrifying method is applied to the report on the various plants such as apple, grape, potato and cassava.But find no the application that Chinese sweet potato variety is preserved with embedding vitrification ultra-low temperature method so far.
The embedding vitrification ultra-low temperature of the sweet potato stem tip among the present invention is preserved technology, according to domestic variety is studied, proposes a kind of ultralow temperature preservation technology of suitable Chinese sweet potato variety.
Summary of the invention
At the deficiencies in the prior art, the invention provides a kind of technology of sweet potato stem tip embedding vitrification ultra-low temperature store method and freezing back plant regeneration of suitable Chinese sweet potato variety.
The term explanation:
MS (murashige skoog): be tissue culture minimal medium commonly used, belong to term known in this field.The MS solid culture medium can be used for evoked callus, also can be used for the cultivation of embryo, stem section, stem apex and flower pesticide, and its liquid nutrient medium is used for cell suspension cultures.The quantity and the ratio of the inorganic nutrients of MS medium are proper, can satisfy plant cell nutritionally with physiological needs.
PVS (plant vitrification solution) vitrification solution: i.e. plant vitrification solution is a compound hypertonic solution of handling plant sample.
Technical scheme of the present invention is as follows:
A kind of sweet potato stem tip embedding vitrification ultra-low temperature store method, step is as follows:
1. get 1～1.5mm sweet potato stem tip from the test-tube plantlet of sweet potato; Sweet potato stem tip is moved in the MS solution of no calcium ion of sodium alginate that concentration is 3 gram/100ml and 0.2M glycerine soaking at room temperature 5～20 seconds;
2. will contain 11.1g/l CaCl
2, 2M glycerine, 0.2M sucrose the precooling 15～25 minutes in ice cube of MS solution, the sweet potato stem tip of step 1 is added wherein together with soaking solution, forming diameter is 0.5-0.8cm sweet potato stem tip bead; The sweet potato stem tip bead moved in the following pre-culture medium successively cultivates in advance:
The bead immigration is filled in the triangular flask of 50ml pre-culture medium I solution, and pre-culture medium I solution formula is: MS+30g/l sucrose+1g/l casamino acid, and shaking table was cultivated 24 hours, and 25～28 ℃, 60～90rpm;
The bead immigration is filled in the triangular flask of 50ml pre-culture medium II solution, and pre-culture medium II solution formula is: MS+0.3M sucrose, and shaking table was cultivated 16 hours, and 25～28 ℃, 60～90rpm;
The bead immigration is filled in the triangular flask of 50ml pre-culture medium III solution, and pre-culture medium III solution formula is: MS+1.6M sucrose+2M glycerine, and shaking table was cultivated 3 hours, and 25～28 ℃, 60-90rpm.
Make pre-cultured sweet potato stem tip bead.
3. above-mentioned pre-cultured sweet potato stem tip bead is moved into and fill in the triangular flask of 20ml PVS vitrification solution 25 ℃ of cultivation 90～120min.
4. the sweet potato stem tip bead of step 3 being handled moves in the ultralow temperature pipe of 2ml specification, and 8-10 bead of dress inserted freezing preservation in the liquid nitrogen in each pipe.
In the time the sweet potato stem tip of freezing preservation need being recovered to cultivate, proceed following steps:
5. the ultralow temperature pipe that fills bead that step 4 is obtained takes out from liquid nitrogen, moves on to rapidly in 38 ℃ the water-bath, thaws 2-4 minute, and bead is put into unloading solution, and room temperature is placed 20-25min.
The unloading solution composition is: 1.2M sucrose+MS, pH5.8.
6. the bead that step 5 obtained moves into and fills in the culture dish that 25ml survives medium, survives culture medium prescription and is: MS+0.5mg/l 6-benzyl aminopurine+1mg/l gibberellin+30g/l sucrose+7.5g/l agar, pH5.8,25 ℃ of dark culturing 3 days; Cultivated 7 days at 24-28 ℃ then, and keep 3000lux illumination cultivation and the dark culturing of 8 hours every days of 16 hours every days.Must survive bead.
7. the above-mentioned bead that survives is moved into and to fill in the culture dish of 25ml regeneration culture medium,, cultivate into seedling,, will become the seedling bead to transfer in the culture dish that fills the regeneration culture medium of newly joining every three weeks the 24-28 ℃ of 3000lux illumination of cultivating and keeping 16 hours every days.Described regeneration culture medium prescription is: MS+2.5mg/l gibberellin+30g/l sucrose+7.5g/l agar, pH 5.8.When the seedling for the treatment of into the seedling bead reaches 0.4-0.6cm, change in the MS solid culture medium and take root.
The PVS vitrification solution consists of in the above-mentioned steps 3: MS+30 gram/100ml glycerine+15 gram/100ml ethylene glycol+15 gram/100ml dimethyl sulfoxide (DMSO) (DMSO)+0.4M sucrose, pH 5.8.
Sweet potato described in the inventive method is Chinese sweet potato variety, and potato 18 or Ji Xu 23 further preferably help.
The freezing preservation of step 4 can be preserved more than the 60min for a long time in the inventive method.
Excellent results of the present invention is as follows:
1, the present invention has determined the store method of Chinese sweet potato variety, all is not suitable for the quality saving of the sweet potato variety of China through the technology of the open in the past report of evidence, and potato 18 and Ji Xu 23 sweet potato varieties particularly help.Survival rate 80-90% after the inventive method superfreeze, prior art only is 30-35%.
2, the present invention is a material with the sweet potato seedling of Chinese sweet potato variety, need not pre-the cultivation its stem apex is carried out embedding.Cultivate in advance carry out embedding in the sucrose MS of variable concentrations solution after, vitrification solution dehydration processing 90 minutes, superfreeze was preserved.
3, the inventive method is after the recovery after the superfreeze is cultivated, and directly sprouting without callus forms complete plantlet, can carry out a large amount of vegetative propagations.
4, method material therefor of the present invention derives from domestic sweet potato variety, and is simple to operate, effective, and long-term frozen back material recovers growth soon.
Description of drawings
The bead of shinny white is for surviving bead, embodiment 1 step 6 among Fig. 1.
Fig. 2 cultivates the plantlet that the back generates for recovering, and seedling reaches 0.5cm, embodiment 1 step 7.
Embodiment
The present invention will be further described below in conjunction with embodiment.The MS medium that uses among the embodiment is source, Shanghai leaf bio tech ltd product.Prescription is as table 1
Table 1.MS prescription
Embodiment 1: Chinese sweet potato variety is Ji potato 18
A kind of sweet potato stem tip embedding vitrification ultra-low temperature store method, step is as follows:
1. get the 1.5mm sweet potato stem tip from the test-tube plantlet of sweet potato (Ji potato 18); Sweet potato stem tip is moved into 3% (in the MS solution of the no calcium ion of sodium alginate of gram/ml) and 0.2M glycerine, soaking at room temperature 15 seconds;
2. will contain 11.1g/l CaCl
2, 2M glycerine, 0.2M sucrose the precooling 20 minutes in ice cube of MS solution, the sweet potato stem tip of step 1 is added wherein together with soaking solution, forming diameter is 0.5-0.6cm sweet potato stem tip bead; The sweet potato stem tip bead moved in the following pre-culture medium successively cultivates in advance:
The bead immigration is filled in the triangular flask of 50ml pre-culture medium I solution, and pre-culture medium I solution formula is: MS+30g/l sucrose+1g/l casamino acid, and shaking table was cultivated 24 hours, and 25 ℃, 90rpm;
The bead immigration is filled in the triangular flask of 50ml pre-culture medium II solution, and pre-culture medium II solution formula is: MS+0.3M sucrose, and shaking table was cultivated 15-16 hour, and 25 ℃, 90rpm;
The bead immigration is filled in the triangular flask of 50ml pre-culture medium III solution, and pre-culture medium III solution formula is: MS+1.6M sucrose+2M glycerine, and shaking table was cultivated 3 hours, and 25 ℃, 90rpm.
Make pre-cultured bead.
3. above-mentioned pre-cultured bead is moved into and fill in the triangular flask of 20ml PVS vitrification solution, cultivate 90min for 25 ℃, the PVS vitrification solution consists of: (((gram/ml) dimethyl sulfoxide (DMSO) (DMSO)+0.4M sucrose, pH 5.8 for ethylene glycol+15% of gram/ml) for glycerine+15% of gram/ml) for MS+30%.
4. the bead of step 3 being handled moves in the ultralow temperature pipe of 2ml specification, and 8-10 bead of dress inserted in the liquid nitrogen in each pipe, keeps 1 hour;
In the time the sweet potato stem tip of freezing preservation need being recovered to cultivate, directly carry out following steps:
5. the ultralow temperature pipe that will fill bead takes out from liquid nitrogen, moves on to rapidly in 38 ℃ the water-bath, thaws 3 minutes, and bead is put into unloading solution, and room temperature is placed 20min.
The unloading solution composition is: 1.2M sucrose+MS, pH5.8
6. bead is moved into and fill in the culture dish that 25ml survives medium, survive culture medium prescription and be: MS+0.5mg/l6-benayl aminopurine+1mg/l gibberellin+30g/l sucrose+7.5g/l agar, pH5.8,25 ℃ of dark culturing 3 days; Cultivated 7 days at 25 ℃ then, and keep 3000lux illumination cultivation and the dark culturing of 8 hours every days of 16 hours every days.Must survive bead.
7. will survive the bead immigration and fill in the culture dish of 25ml regeneration culture medium,, cultivate into seedling 25 ℃ of 3000lux illumination of cultivating and keeping 16 hours every days.Every three weeks, bead is transferred in the fresh culture dish that fills regeneration culture medium.Described regeneration culture medium prescription is: MS+2.5mg/l gibberellin+30g/l sucrose+7.5g/l agar, pH 5.8.
When treating that seedling reaches 0.5cm, change in the MS solid culture medium and take root.
Embodiment 2: Chinese sweet potato variety is Ji Xu 23
Step is as described in the embodiment 1, and different is:
1. get the 1.0mm sweet potato stem tip from the test-tube plantlet of sweet potato (Ji Xu 23); Sweet potato stem tip is moved into 3% (in the MS solution of the no calcium ion of sodium alginate of gram/ml) and 0.2M glycerine, soaking at room temperature 10 seconds;
2. will contain 11.1g/l CaCl
2, 2M glycerine, 0.2M sucrose the precooling 20 minutes in ice cube of MS solution, the sweet potato stem tip of step 1 is added wherein together with soaking solution, forming diameter is 0.6-0.8cm sweet potato stem tip bead; The sweet potato stem tip bead moved in the following pre-culture medium successively cultivates in advance:
The bead immigration is filled in the triangular flask of 50ml pre-culture medium I solution, and pre-culture medium I solution formula is: MS+30g/l sucrose+1g/l casamino acid, and shaking table was cultivated 24 hours, and 28 ℃, 60rpm;
The bead immigration is filled in the triangular flask of 50ml pre-culture medium II solution, and pre-culture medium II solution formula is: MS+0.3M sucrose, and shaking table was cultivated 15-16 hour, and 28 ℃, 60rpm;
The bead immigration is filled in the triangular flask of 50ml pre-culture medium III solution, and pre-culture medium III solution formula is: MS+1.6M sucrose+2M glycerine, and shaking table was cultivated 3 hours, and 28 ℃, 60rpm.
Make pre-cultured bead.
3. above-mentioned pre-cultured bead is moved into and fill in the triangular flask of 20ml PVS vitrification solution, cultivate 120min for 25 ℃, the PVS vitrification solution consists of: (((gram/ml) dimethyl sulfoxide (DMSO) (DMSO)+0.4M sucrose, pH 5.8 for ethylene glycol+15% of gram/ml) for glycerine+15% of gram/ml) for MS+30%.
4. with step 4 among the embodiment 1.
5. with step 5 among the embodiment 1.
6. bead is moved into and fill in the culture dish that 25ml survives medium, survive culture medium prescription and be: MS+0.5mg/l6-benayl aminopurine+1mg/l gibberellin+30g/l sucrose+7.5g/l agar, pH5.8,28 ℃ of dark culturing 3 days; Cultivated 7 days at 28 ℃ then, and keep 3000lux illumination cultivation and the dark culturing of 8 hours every days of 16 hours every days.Must survive bead.
7. will survive the bead immigration and fill in the culture dish of 25ml regeneration culture medium,, cultivate into seedling 28 ℃ of 3000lux illumination of cultivating and keeping 16 hours every days.Every three weeks, bead is transferred in the fresh culture dish that fills regeneration culture medium.Described regeneration culture medium prescription is: MS+2.5mg/l gibberellin+30g/l sucrose+7.5g/l agar, pH 5.8.
When treating that seedling reaches 0.5cm, change in the MS solid culture medium and take root.
The experimental result of embodiment 1-2 is as follows:
Annotate: prior art A is D.Hira, A.Sakai, Simplified cryopreservation of sweet potato[Ipomoea batatas (L.) Lam.] by optimizing conditions for osmoprotection, Plant Cell Reports, (2003) 21:961-966.
Claims (4)
1. sweet potato stem tip embedding vitrification ultra-low temperature store method, step is as follows:
(1) gets 1～1.5mm sweet potato stem tip from the test-tube plantlet of sweet potato; Sweet potato stem tip is moved in the MS solution of no calcium ion of sodium alginate that concentration is 3 gram/100ml and 0.2M glycerine soaking at room temperature 5～20 seconds;
(2) will contain 11.1g/l CaCl
2, 2M glycerine, 0.2M sucrose the precooling 15～25 minutes in ice cube of MS solution, the sweet potato stem tip of step 1 is added wherein together with soaking solution, forming diameter is 0.5-0.8cm sweet potato stem tip bead; The sweet potato stem tip bead moved in the following pre-culture medium successively cultivates in advance:
The bead immigration is filled in the triangular flask of 50ml pre-culture medium I solution, and pre-culture medium I solution formula is: MS+30g/l sucrose+1g/l casamino acid, and shaking table was cultivated 24 hours, and 25～28 ℃, 60～90rpm;
The bead immigration is filled in the triangular flask of 50ml pre-culture medium II solution, and pre-culture medium II solution formula is: MS+0.3M sucrose, and shaking table was cultivated 16 hours, and 25～28 ℃, 60～90rpm;
The bead immigration is filled in the triangular flask of 50ml pre-culture medium III solution, and pre-culture medium III solution formula is: MS+1.6M sucrose+2M glycerine, and shaking table was cultivated 3 hours, and 25～28 ℃, 60-90rpm;
Make pre-cultured sweet potato stem tip bead;
(3) above-mentioned pre-cultured sweet potato stem tip bead immigration is filled in the triangular flask of 20ml PVS vitrification solution, cultivate 90～120min for 25 ℃;
(4) the sweet potato stem tip bead of step (3) being handled moves in the ultralow temperature pipe of 2ml specification, and 8-10 bead of dress inserted freezing preservation in the liquid nitrogen in each pipe.
2. as the described sweet potato stem tip embedding of right claim 1 vitrification ultra-low temperature store method, it is characterized in that the PVS vitrification solution consists of in the step (3): MS+30% glycerine+15% ethylene glycol+15% dimethyl sulfoxide (DMSO)+0.4M sucrose, pH 5.8; Percentage is mass volume ratio, unit: gram/ml.
3. as the described sweet potato stem tip embedding of right claim 1 vitrification ultra-low temperature store method, when it is characterized in that sweet potato stem tip with freezing preservation recovers to cultivate, continue following steps:
(5) the ultralow temperature pipe that fills bead that step (4) is obtained takes out from liquid nitrogen, moves on to rapidly in 38 ℃ the water-bath, thaws 2-4 minute, and bead is put into unloading solution, and room temperature is placed 20-25min;
The unloading solution composition is: 1.2M sucrose+MS, pH5.8;
(6) bead that step (5) obtained moves into and fills in the culture dish that 25ml survives medium, survives culture medium prescription and is: MS+0.5mg/l 6-benzyl aminopurine+1mg/l gibberellin+30g/l sucrose+7.5g/l agar, pH5.8,25 ℃ of dark culturing 3 days; Cultivated 7 days at 24-28 ℃ then, and keep 3000lux illumination cultivation and the dark culturing of 8 hours every days of 16 hours every days, must survive bead;
(7) the above-mentioned bead that survives is moved into and to fill in the culture dish of 25ml regeneration culture medium,, cultivate into seedling,, will become the seedling bead to transfer in the culture dish that fills the regeneration culture medium of newly joining every three weeks the 24-28 ℃ of 3000lux illumination of cultivating and keeping 16 hours every days.Described regeneration culture medium prescription is: MS+2.5mg/l gibberellin+30g/l sucrose+7.5g/l agar, pH 5.8.When the seedling for the treatment of into the seedling bead reaches 0.4-0.6cm, change in the MS solid culture medium and take root.
4. as the described sweet potato stem tip embedding of right claim 1 vitrification ultra-low temperature store method, it is characterized in that described sweet potato variety is Ji potato 18 or Ji Xu 23.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102726298A (en) * | 2012-07-03 | 2012-10-17 | 中国农业科学院作物科学研究所 | Cryopreservation method for in vitro shoot tips of petunia hybrida |
| CN103202225A (en) * | 2013-03-11 | 2013-07-17 | 上海市农业生物基因中心 | Ultralow-temperature storing, thawing and recultivating method for micro-stem tips of plants |
| CN109105256A (en) * | 2018-07-30 | 2019-01-01 | 浙江大学 | Sweet potato stem tip poison-removing method |
| CN115735770A (en) * | 2022-11-29 | 2023-03-07 | 上饶师范学院 | Method for ultralow-temperature preservation of burclover by embedding vitrification method |
-
2010
- 2010-07-16 CN CN201010228497A patent/CN101904305B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| 《山东农业科学》 20100530 田霄等 甘薯茎尖超低温保存技术研究 第55页右栏第9行至第56页左栏第15行 1-4 , 第5期 2 * |
| 《植物学通报》 20051231 吴雪梅，汤浩茹 包埋玻璃化法超低温保存植物种质的研究进展 第240页第8-36行 1-4 第22卷, 第2期 2 * |
| 《植物遗传资源学报》 20091231 刘丽芳等 甘薯种质超低温保存研究进展 第325页左栏第34-37行、右栏第4-6行、10-11行、17-18行和28-31行，第326页左栏第34-37行、右栏第3-5行 1-4 第10卷, 第2期 2 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102726298A (en) * | 2012-07-03 | 2012-10-17 | 中国农业科学院作物科学研究所 | Cryopreservation method for in vitro shoot tips of petunia hybrida |
| CN103202225A (en) * | 2013-03-11 | 2013-07-17 | 上海市农业生物基因中心 | Ultralow-temperature storing, thawing and recultivating method for micro-stem tips of plants |
| CN109105256A (en) * | 2018-07-30 | 2019-01-01 | 浙江大学 | Sweet potato stem tip poison-removing method |
| CN115735770A (en) * | 2022-11-29 | 2023-03-07 | 上饶师范学院 | Method for ultralow-temperature preservation of burclover by embedding vitrification method |
| CN115735770B (en) * | 2022-11-29 | 2023-08-18 | 上饶师范学院 | Ultralow-temperature preservation method for broccoli by embedding vitrification method |
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