CN108719280A - A kind of store method of lily ball tissue - Google Patents
A kind of store method of lily ball tissue Download PDFInfo
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- CN108719280A CN108719280A CN201810772880.2A CN201810772880A CN108719280A CN 108719280 A CN108719280 A CN 108719280A CN 201810772880 A CN201810772880 A CN 201810772880A CN 108719280 A CN108719280 A CN 108719280A
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- lily ball
- lily
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N3/00—Preservation of plants or parts thereof, e.g. inhibiting evaporation, improvement of the appearance of leaves or protection against physical influences such as UV radiation using chemical compositions; Grafting wax
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- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Plant Pathology (AREA)
- Toxicology (AREA)
- Agronomy & Crop Science (AREA)
- Dentistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The present invention relates to plant bulb tissue preservation techniques field, especially a kind of store method of lily ball tissue.By during preculture, lily ball tissue mica being coated, the resistance and survival rate of lily ball tissue can be improved；It is added as allogenic material using starch and antifreeze glycoprotein and is used into vitrification solution, to prevent lily ball cell membrane from damage, improve the recovery percentage of lily ball tissue.
Description
Technical field
The present invention relates to plant bulb tissue preservation techniques field, especially a kind of store method of lily ball tissue.
Background technology
Lily is the herbaceous plant of Liliaceae lilium, is world-renowned flowering bulb, has that pattern is abundant, posture is excellent
Beautiful characteristic.It is ornamental etc. that lily is widely used in gardens setting, cut-flower, fresh flower and potting.China is distributed as lily
Center, germ plasm resource is abundant, but most wild species still fall within endangered plants.Lily cannot pass through as perennial bulb draft
Low temperature seed bank is preserved, and current main preserving type preserves for field planting, and field, which preserves, easily to be gone out during preserving
Existing brown stain is rotted, therefore the medium-term and long-term preservation problems demand of lily solves.
Cryopreservation is the modern germ plasm resource Plantlet to grow up the seventies in last century, is usually existed
It is preserved in liquid nitrogen, the metabolism and vegetative activity being saved in Materials Cell almost stop, and are in metastable life
Object state achievees the purpose that long-term preservation germplasm, a kind of raising lily embryo as disclosed in patent CN201410468249.5
The method of callus preservation effect uses the vitrification solution containing carbon nanomaterial to handle lily embryo callus to carry
Its high preservation effect；For another example plant genetic resources journal 2007.8 (2)：" flower lily Shoot Tips glass disclosed in 170-173
Change method cryopreservation is studied ", what ultralow-temperature glass method preserved at present is all in vitro plant callus or cell.
Cryopreservation by vitrification is that cell or tissue is placed in by a certain proportion of permeability and impermeability protection
In the vitrification solution of agent composition, material and its vitrification solution is made to be solidified into amorphous glass under sufficiently fast rate of temperature fall
Glass state, and preserved at low temperature with this glassy state.Vitrification is at low cost because simple and quick, suitable for preserving type
Extensively, material genetic stability, the advantages that preservation effect is good are preserved, but is different plant or its vitrifying of cell or tissue is molten
The selection of liquid and allogenic material will directly affect the survival rate and preservation effect of plant.
Invention content
In order to solve the above technical problems, the present invention provides a kind of store method of lily ball tissue, pass through following skill
Art scheme is achieved.
A kind of store method of lily ball tissue, includes the following steps：
1) preculture：Lily ball is cleaned up, is sterilized, slice is placed in ice bucket and preserves after wrapping up in last layer coating
0.5~4 hour；
2) it is dehydrated：Lily ball tissue is transferred in cryovial, handles 20~35min with vitrification solution at room temperature,
10~50min is handled under the conditions of 0 DEG C with PVS2 stostes again；
3) Liquid nitrogen storage：PVS2 stostes are removed, lily ball tissue is encapsulated with wax stone, is sealed in cryovial, directly throw
Enter freezen protective in liquid nitrogen；
4) it thaws：The lily ball tissue sealed up for safekeeping in liquid nitrogen is taken out, is thawed rapidly, surface paraffin layer is removed, uses cleaning solution
Washing 2~3 times carries out renewal cultivation.
Coating material described in step (1) is the mixture of mica, food-grade gelatin and water soluble chitosan, may make hundred
Corm forming superficial film is closed, the fresh-keeping effect and survival rate of lily ball are improved.
Further, the mixture of the mica, food-grade gelatin and water soluble chitosan presses 10：(5~8)：3 mixing.
The PVS2 solution that the vitrification solution of step (2) is a concentration of 60%.
Further, the PVS2 solution of described 60% is that 0.4mol/L liquid sucroses culture medium presses 4 with PVS2 solution：6 institutes
Obtain mixed liquor.
Further, the PVS2 solution compositions be 30% glycerine, 15% ethylene glycol, 15%DMSO, 3% sucrose, 3%
Starch and 0.4mol/L antifreeze glycoproteins.
Lily described in step (4) thaws method to take out lily ball from liquid nitrogen, and first water-bath is thawed, and is then removed
It is washed with cleaning solution after vitrification solution, is finally transferred to renewal cultivation in recovery media.
Further, the condition that the water-bath is thawed is the 60~120s that thaws in 30~40 DEG C of water-bath.
Further, the cleaning solution is to contain 1.0~1.5mol/L sucrose and 5~10mmol/LKNO3MS culture solutions.
Further, the renewal cultivation is that lily ball tissue obtained by step (4) is placed in solid medium, dark to train
It supports 1 week, optical culture was placed in soil or matrix and plants after 4 weeks.
The light culture refers to being cultivated under dark no light condition；The optical culture refers under sunlight or fluorescent lamp
Culture.
It is 1~365 day that the lily ball group, which is woven in time for being sealed up for safekeeping in liquid nitrogen,.
The beneficial effects of the present invention are：
The present invention is according to the physical aspect of lily ball tissue, the comprehensive store method for having formulated lily ball tissue, packet
The links such as preculture, dewatering and degree and the renewal cultivation of lily ball tissue are included, the present invention is in preculture mistake
Lily ball tissue is wrapped up in into last layer coating in journey, compared with conventional lime coating agent, coating agent good permeability of the present invention, not shadow
Seed breathing is rung, can effectively extend lily ball tissue shelf-life and improve the resistance and survival rate of bulb tissue.
The present invention is added as allogenic material and is used into vitrification solution using starch and antifreeze glycoprotein, to prevent hundred
Bulb cell membrane is closed from damage, improves the recovery percentage of lily ball tissue, method is to lily disclosed in the present invention
The preservation effect optimization of bulb tissue is notable, and facilitation is played to preservation of plant vitrification ultra-low temperature.
Specific implementation mode
Be described further below technical scheme of the present invention, but claimed range be not limited to it is described.
The formula of experiment reagent is as follows in the embodiment of the present invention：
1, vitrification solution is that a concentration of 0.4mol/L liquid sucroses culture medium and PVS2 solution press 4：6 gained mixed liquors.
2, PVS2 solution compositions be 30% glycerine, 15% ethylene glycol, 15%DMSO, 3% sucrose.
3, cleaning solution is to contain 1.0~1.5mol/L sucrose and 5~10mmol/L KNO3MS culture solutions.
Embodiment
A kind of store method of lily ball tissue, includes the following steps：
1) preculture：Lily ball is cleaned up, is sliced, disinfection wraps one layer of mica mixture coating, is placed in ice bucket
It is middle to preserve 0.5~4 hour；
2) it is dehydrated：Lily ball tissue is transferred in cryovial, handles 20~35min with vitrification solution at room temperature,
10~50min is handled under the conditions of 0 DEG C with PVS2 stostes again；
3) Liquid nitrogen storage：PVS2 stostes are removed, lily ball tissue is encapsulated with wax stone, is sealed in cryovial, directly throw
Enter freezen protective in liquid nitrogen；
4) it thaws.
The lily ball group is woven in the time sealed up for safekeeping in liquid nitrogen as 24 hours.
Lily ball tissue preserration is divided into experimental group and control group according to above-mentioned steps.
The coating material of experimental group 1 is that mica, food-grade gelatin and water soluble chitosan press 10：5：3 mixture, glass
Change and contains 3% starch in solution；
The coating material of experimental group 2 is that mica, food-grade gelatin and water soluble chitosan press 10：7：3 mixture, glass
Change and contains 0.4mol/L antifreeze glycoproteins in solution；
The coating material of experimental group 3 is that mica, food-grade gelatin and water soluble chitosan press 10：8：3 mixture, glass
Change and contains 3% starch and 0.4mol/L antifreeze glycoproteins in solution；
Control group 1：Control group presses 2 the difference is that coating material is mica, food-grade gelatin and water soluble chitosan：
2：1 mixture does not contain 3% starch and 0.4mol/L antifreeze glycoproteins in vitrification solution, other are identical as experimental group；
Control group 2：Control group presses 3 the difference is that coating material is mica, food-grade gelatin and water soluble chitosan：
8：3 mixture, preculture lily ball tissue are coated without mica mixture, other are identical as experimental group.
By in liquid nitrogen the lily ball tissue of freezen protective for 24 hours taken out from liquid nitrogen, solved in 30~40 DEG C of water-bath
Freeze 60~120s, washed with cleaning solution after then removing vitrification solution, the lily ball tissue after washing is placed in solid training
It supports in base, light culture 1 week, optical culture was placed in soil or matrix and plants after 4 weeks.It calculates and in comparative experiments group and control group
The relative survival of lily ball tissue, is shown in Table 1.
The relative survival of the lily ball tissue of 1 experimental group of table and control group
| Group | Experimental group 1 | Experimental group 2 | Experimental group 3 | Control group 1 | Control group 2 |
| Survival rate | 32.1% | 36.4% | 43.8% | 7.8% | 13.4% |
Experimental result
As shown in Table 1, lily ball tissue carries out preculture using coating, be added in vitrification solution 3% starch and
0.4mol/L antifreeze glycoproteins cryopreservation can make lily seedling stem tissue renewing growth rate be increased to 32.1% by 7.8%,
36.4% and 43.8%.Wherein, the most notable to add the improvement effect of starch and antifreeze glycoprotein simultaneously.
On the basis of test group 3, carry out experimental group 4, experimental group 5, experimental group 6, experimental group 7 respectively.
Experimental group 4：Lily ball group is woven in liquid nitrogen on the basis of experimental group 3 and is preserved 2 days；
Experimental group 5：Lily ball group is woven in liquid nitrogen on the basis of experimental group 3 and is preserved 60 days；
Experimental group 6：Lily ball group is woven in liquid nitrogen on the basis of experimental group 3 and is preserved 120 days；
Experimental group 7：Lily ball group is woven in liquid nitrogen on the basis of experimental group 3 and is preserved 365 days；
After empirically lily ball group is woven in and is preserved 2 days, 60 days, 120 days, 365 days in liquid nitrogen by the method for group 4~7, then
Lily ball tissue is taken out from liquid nitrogen, thaw 60~120s in 30~40 DEG C of water-bath, then removes vitrification solution
It being washed afterwards with cleaning solution, the lily ball tissue after washing is placed in solid medium, light culture 1 week, optical culture is after 4 weeks,
It is placed in soil or matrix and plants.The relative survival of calculating and more each experimental group lily ball tissue, is shown in Table 2.
The lily ball tissue relative survival of the different holding times of table 2
| Group | Experimental group 4 | Experimental group 5 | Experimental group 6 | Control group 7 |
| Survival rate | 44.1% | 43.9% | 43.6% | 43.5% |
As known from Table 2, the time of lily seedling stem freezen protective in liquid nitrogen influences lily ball tissue relative survival
Smaller, peasant household can save one-year age, and in second year, renewal cultivation is planted again.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe
The personage for knowing this technology can all carry out modifications and changes to above-described embodiment without violating the spirit and scope of the present invention.Cause
This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as
At all equivalent modifications or change, should by the present invention claim be covered.
Claims (10)
1. a kind of store method of lily ball tissue, it is characterised in that：Include the following steps：
1) preculture：Lily ball is cleaned up, is sterilized, after wrapping up in last layer coating, is placed in ice bucket and preserves 0.5~4 hour；
2) it is dehydrated：Lily ball tissue is transferred in cryovial, handles 20~35min with vitrification solution at room temperature, then use
PVS2 stostes handle 10~50min under the conditions of 0 DEG C；
3) Liquid nitrogen storage：PVS2 stostes are removed, lily ball tissue is encapsulated with wax stone, is sealed in cryovial, direct plunges into liquid
Freezen protective in nitrogen；
4) it thaws：The lily ball tissue sealed up for safekeeping in liquid nitrogen is taken out, is thawed rapidly, surface paraffin layer is removed, 2 is washed with cleaning solution
~3 times, carry out renewal cultivation.
2. the store method of lily ball tissue as described in claim 1, it is characterised in that：Coating material described in step (1)
Material is that mica, food-grade gelatin and water soluble chitosan press 10：(5~8)：3 mixture.
3. the store method of lily ball tissue as described in claim 1, it is characterised in that：The vitrification solution of step (2)
It is a concentration of 60% PVS2 solution.
4. the store method of the lily ball tissue as described in claim 1 or 3, it is characterised in that：The vitrification solution
4 are pressed for 0.4mol/L liquid sucroses culture medium and PVS2 solution：6 gained mixed liquors.
5. the store method of lily ball tissue as claimed in claim 4, it is characterised in that：The PVS2 solution compositions are
30% glycerine, 15% ethylene glycol, 15%DMSO, 3% sucrose, 3% starch and 0.4mol/L antifreeze glycoproteins.
6. the store method of lily ball tissue as described in claim 1, it is characterised in that：Described thawing is by lily kind
Ball tissue takes out from liquid nitrogen, and first water-bath is thawed, and is washed with cleaning solution after then removing vitrification solution, is finally transferred to recovery training
Support renewal cultivation in base.
7. the store method of lily ball tissue as claimed in claim 6, it is characterised in that：The water-bath defrosting is 30
Thaw 60~120s in~40 DEG C of water-bath.
8. the store method of lily ball tissue as claimed in claim 6, it is characterised in that：The cleaning solution be containing
1.0~1.5mol/L sucrose and 5~10mmol/L KNO3MS culture solutions.
9. the store method of lily ball tissue as described in claim 1 or 6, it is characterised in that：The renewal cultivation is
Lily ball tissue obtained by step (4) is placed in solid medium, light culture 1 week, optical culture is placed in soil or base after 4 weeks
It is planted in matter.
10. such as the store method of claim 1~9 any one of them lily ball tissue, it is characterised in that：The lily
Bulb tissue, the time sealed up for safekeeping in liquid nitrogen are 1~365 day.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810772880.2A CN108719280A (en) | 2018-07-14 | 2018-07-14 | A kind of store method of lily ball tissue |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810772880.2A CN108719280A (en) | 2018-07-14 | 2018-07-14 | A kind of store method of lily ball tissue |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115606586A (en) * | 2022-10-28 | 2023-01-17 | 华中农业大学 | Method for prolonging low-temperature storage time and regulating flowering phase of tulip/lily bulbs |
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| CN106258070A (en) * | 2016-08-10 | 2017-01-04 | 国俊伍 | A kind of seed freezing storage device and method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115606586A (en) * | 2022-10-28 | 2023-01-17 | 华中农业大学 | Method for prolonging low-temperature storage time and regulating flowering phase of tulip/lily bulbs |
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Application publication date: 20181102 |