CN106689121B - A kind of spindle tree pollen cryopreservation and the production method of regeneration - Google Patents
A kind of spindle tree pollen cryopreservation and the production method of regeneration Download PDFInfo
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- CN106689121B CN106689121B CN201611032560.0A CN201611032560A CN106689121B CN 106689121 B CN106689121 B CN 106689121B CN 201611032560 A CN201611032560 A CN 201611032560A CN 106689121 B CN106689121 B CN 106689121B
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- pollen
- spindle tree
- cryopreservation
- sucrose
- boric acid
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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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- Engineering & Computer Science (AREA)
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- Toxicology (AREA)
- Agronomy & Crop Science (AREA)
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- Agricultural Chemicals And Associated Chemicals (AREA)
- Pretreatment Of Seeds And Plants (AREA)
Abstract
The invention provides a kind of spindle tree pollen cryopreservation and the production method of regeneration：The well-developed pollen that spindle tree full-bloom stage is gathered is fitted into 1.8ml cryopreservation tube；Then cryovial is put into the drying basin for filling silica-gel desiccant；Put into 196 DEG C of liquid nitrogen containers of ﹣ and preserve after drying；Then thawed；Pollen after thawing is sprouted in sucrose and boric acid nutrient solution.The present invention has simple to operate, safety and stability, greatly prolongs the holding time of pollen；Also make it possible the long-term storing and exchange of pollen, provided conveniently for interzone, international kind mass transter；Ultra-low temperature surroundings also avoid the advantages that introducing of pest and disease damage.The method spindle tree produce and scientific research in terms of on there is important dissemination.
Description
Technical field
The present invention relates to a kind of spindle tree pollen cryopreservation and the production method of regeneration, belongs to a kind of gardens life
Production, plasm resource protection field.
Background technology
Spindle tree, also known as white Du, winterberry euonymus herb, it is a kind of dungarunga of Celastraceae Euonymus, being that China is common views and admires
One of class flower garden seeds, its is leaf beautiful, and plant is in great numbers, is coated with the fruit of red aril and can harbor for a long time in end of the branch, arrives
In winter, white haw of avenging is set each other off, very beautiful, and many colors are added for the northern gardens in winter.Spindle tree is cold-resistant, anti-
Drought, wind resistance etc. have good adaptability；Spindle tree fruit total saposins are to Bel7402-SMMC, human cervical carcinoma
Cell line-HeLa and breast cancer cell line-MCF7 has obvious suppression growth；The seed of spindle tree is containing abundant fat
Class material, oil content are up to more than 40%, can be used as iundustrial oil；Timber exquisiteness is tough and tensile, available for carve, boom processed or coaster
Deng；Seed and root are medicinal, available for diseases such as the treatment pain of the knee joint, dermatitis rhus.As can be seen here, spindle tree be a kind of collection industry,
It is medicinal, view and admire applied to one plant, there is higher promotional value.Preserved according to traditional plantation, easily make its by
To germ or the influence of insect pest, the loss of great manpower financial capacity is in turn resulted in, ripe pollen contains all heredity of plant
Quality information, pollen cryopreservation is simple to operate, is a new way for carrying out Germ-plasma resources protection, is currently the only feasible
The long-term and method that stably preserves germ plasm resource.
Plant germplasm resource cryopreservation refer to by plant cell, tissue or organ liquid nitrogen (- 196 DEG C) ultralow temperature
Under the conditions of preserved.At such lower temperatures, participated in vegetable material metabolism various biology enzymes activity by
Greatly suppress, organism metabolism is basic to be stopped, and in " seemingly-dead " state, plant germplasm resource can be realized using this method
Persistence.The material type for successfully carrying out cryopreservation at present has seed, resting bud, shoot apical meristem, pollen, conjunction
Sub- embryo (plumular axis), somatic embryo, suspension cell, callus, protoplast etc..Wherein pollen cryopreservation is simple to operate,
Specific genotype can be kept.
Cryopreservation technology is preserved applied to pollen, its advantage：Operation is simple, occupies little space, be safe steady
Surely cost low holding time that can greatly prolong pollen is preserved, makes it possible the storing and exchange of pollen, while can also be very
Breeding work is served well, and its genetic integrity and stability low temperature can be kept by the pollen of Cord blood by improving breeding efficiency
Under the conditions of can also avoid the introducing of pest and disease damage, be interzone, international kind mass transter provider just.
A kind of spindle tree pollen cryopreservation and the production method of regeneration, yet there are no report.Therefore, invention peach
Leaf winged euonymus pollen cryopreservation and the production method of regeneration are very important.
The content of the invention
The purpose of the present invention is to be achieved through the following technical solutions：
The spindle tree fresh pollen that different time gathers is put into 1.8ml cryopreservation tube, the volume of pollen is no more than
The 2/3 of cryopreservation tube capacity, then cryovial is put into after drying the different time in the drying basin for fill silica-gel desiccant and obtained
The pollen of different moisture content, drying basin specification are V=410mm × 410mm × 310mm, Φ=300mm；Polliniferous jelly will be filled
Deposit and preserved in pipe input 196 DEG C of liquid nitrogen containers of ﹣；Take out cryopreservation tube and carry out difference and thaw processing；By pollen in various concentrations after thawing
Sucrose and boric acid nutrient solution in determine germination rate.
The different acquisition time, the pollen germination rate that different time is gathered in 1d is different, and we acquire 6 respectively:
00、8:00、10:00、12:00、14:00、16:00、18:00 spindle tree pollen.
The drying different time is respectively to dry 2h, 4h, 6h, 8h, 10h, 12h.
The different moisture content, the time dried for spindle tree pollen is different, obtained water content.
The difference is thawed mode, respectively three kinds：(1) room temperature is thawed 30min；(2) 40 DEG C of water-baths are thawed 2min；(3)
Running water rinses 8min.
The different sucrose and boric acid concentration, using spindle tree pollen as material, deionized water adds different as solvent
The sucrose and boric acid of concentration, using the method for two factor completely randoms, 16 kinds of different culture medias are devised altogether.Respectively：Sucrose
50g/L+ boric acid 0mg/L；Sucrose 50g/L+ boric acid 100mg/L；Sucrose 50g/L+ boric acid 150mg/L；Sucrose 50g/L+ boric acid
200mg/L；Sucrose 100g/L+ boric acid 0mg/L；Sucrose 100g/L+ boric acid 100mg/L；Sucrose 100g/L+ boric acid 150mg/L；Sugarcane
Sugared 100g/L+ boric acid 200mg/L；Sucrose 150g/L+ boric acid 0mg/L；Sucrose 150g/L+ boric acid 100mg/L；Sucrose 150g/L+
Boric acid 150mg/L；Sucrose 150g/L+ boric acid 200mg/L；Sucrose 200g/L+ boric acid 0mg/L；Sucrose 200g/L+ boric acid 100mg/
L；Sucrose 200g/L+ boric acid 150mg/L；Sucrose 200g/L+ boric acid 200mg/L.
A kind of optimal spindle tree pollen cryopreservation and regeneration are found out by carrying out experiment to above technical scheme
Production method, for claim.
Embodiment：
The present invention is described in further detail with reference to specific example below, and introduces the correlative study knot of the present invention
Fruit.
1. by taking the spindle tree pollen of Northeast Forestry University in the school as an example, the present invention is described in further detail.
1) influence of the different acquisition time to spindle tree pollen germination rate
The selected different period from one day that spindle tree is bloomed, i.e., 6:00、8:00、10:00、12:00、14:00、
16:00、18:00 gathered pollen every two hours, observed its germination rate.
Influence of the different acquisition time of table 1 to spindle tree pollen germination rate
2) germination rate before and after different moisture content pollen cryopreservation is obtained through different drying times
After measuring the maximum water holding capacity of spindle tree pollen using oven drying method first, pollen is carried out not using silica dehydrator method
With the germination rate of the preparation of water content gradient, then measure pollen；Put into liquid nitrogen container after preserving a period of time and taken out afterwards,
Germination rate of the spindle tree pollen after cryopreservation is determined after thawing.
Table 2 obtains germination rate before different moisture content pollen cryopreservation through different drying times
The germination rate after different drying times obtain different moisture content pollen cryopreservation of table 3
3) different influences of the mode to pollen germination rate of thawing
3 kinds of mode of thawing (1) room temperatures are employed to thaw 30min；(2) 40 DEG C of water-baths are thawed 2min；(3) running water rinses
8min compares 3 kinds of modes of thawing to pollen germination power to carrying out experiment of thawing after spindle tree pollen cryopreservation 24h
Influence.
The difference of table 4 is thawed influence of the mode to pollen germination rate
4) influence of different sucrose and boric acid concentration to spindle tree pollen germination rate
Using spindle tree pollen as material, deionized water adds the sucrose and boric acid of various concentrations, using two as solvent
The method of factor completely random, 16 kinds of different culture medias are devised altogether.Spindle tree pollen is equably broadcasted sowing on nutrient solution, so
After place it in 25 DEG C of growth cabinets illumination cultivation and observe pollen germination situation afterwards for a period of time, according to the sprouting of pollen
Rate is so that it is determined that the most suitable nutrient solution of spindle tree pollen germination.
The influence of the various concentrations sucrose of table 5 and boric acid to spindle tree pollen germination rate
2. experimental result
1) as shown in Table 1, due to collection time difference, pollen germination rate is different, and its approximate trend is：6:
00-12：00 period was downward trend, 12:00 pollen germination rate minimizes；And 12:00-16：00 period pollen germination
Rate is in rising trend, and 16:Highest is reached when 00；Subsequent 16:00-18:00 period, pollen germination rate progressively decline.
It can thus be concluded that go out, 16:00 spindle tree pollen germination rate highest.
2) by table 2, table 3 can be seen that germination rate of the spindle tree pollen of different moisture content before preservation in Liquid nitrogen storage
Preceding germination rate does not have significant difference, but when pollen is after Liquid nitrogen storage, significant difference occurs.When spindle tree is spent
When powder water content is original state (14.56%), pollen after Liquid nitrogen storage is all dead, further with pollen water content
Reduce, in 12.32%-4.14% water content section, the pollen germination rate after cryopreservation shows what is be stepped up
Trend.And when pollen water content is further reduced to 1.67% from 4.14%, the pollen germination rate after cryopreservation is opened again
Begin to reduce, and final all dead, therefore polliniferous cryopreservation tube will be filled be put into the drying basin for fill silica-gel desiccant and dry
10h, pollen water content is set to be maintained between 4%-4.5%；The germination rate highest of pollen.
3) mode of as shown in Table 4, thawing has pole to significantly affect the spindle tree pollen germination rate after preservation, using room temperature
Spindle tree pollen germination rate thaw after 30min up to 14.34%, its effect is better than 40 DEG C of water-baths and thawed 2min and room temperature
Freeze both modes of thawing of 30min.It is possible thereby to infer room temperature thaw 30min effect of thawing it is preferable.
4) as shown in Table 5, spindle tree pollen sprouting situation in the nutrient solution of different formulations is variant.Pollen is in sucrose
Germination rate reaches highest when concentration is 100g/L, and as the further increase of sucrose concentration, the germination rate of pollen reduce again.Knot
The germination rate of spindle tree pollen is closed, finally it is most suitable to filter out sucrose concentration and boric acid concentration in leaf winged euonymus pollen cultures liquid for we
Match and be：Sucrose 100g/L+ boric acid 150mg/L.
Claims (1)
1. a kind of spindle tree pollen cryopreservation and the production method of regeneration, it is characterised in that：Described production method bag
Include following steps：The first step, the well-developed pollen that spindle tree full-bloom stage is gathered are put into 1.8ml cryopreservation tube, flower
The volume of powder is no more than the 2/3 of cryopreservation tube capacity；Second step, cryovial is put into the drying basin for fill silica-gel desiccant and done
It is dry, pollen water content is maintained at 4%-4.5%；3rd step, polliniferous cryovial input 196 DEG C of liquid nitrogen of ﹣ will be filled after drying
Preserved in tank；4th step, take out cryopreservation tube and thawed pollen through room temperature 30min；5th step, the pollen after thawing is in sucrose 100g/L
Cultivated in+boric acid 150mg/L nutrient solution.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611032560.0A CN106689121B (en) | 2016-11-23 | 2016-11-23 | A kind of spindle tree pollen cryopreservation and the production method of regeneration |
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|---|---|---|---|
| CN201611032560.0A CN106689121B (en) | 2016-11-23 | 2016-11-23 | A kind of spindle tree pollen cryopreservation and the production method of regeneration |
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| CN106689121A CN106689121A (en) | 2017-05-24 |
| CN106689121B true CN106689121B (en) | 2018-03-30 |
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Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01139510A (en) * | 1987-11-27 | 1989-06-01 | Gakken Co Ltd | Storage of pollen of japanese horseradish |
| CN1298219C (en) * | 2004-11-03 | 2007-02-07 | 廖旭辉 | Technology for storing Momordica grosvenori pollen |
| CN101926323B (en) * | 2010-07-29 | 2013-06-12 | 中国热带农业科学院橡胶研究所 | Ultra-low temperature preservation method for oil palm pollen |
| CN105766893A (en) * | 2016-03-10 | 2016-07-20 | 中国林业科学研究院热带林业研究所 | Storage method of eucalyptus pollen and vitality measuring method of pollen stored by storage method |
| CN105961377B (en) * | 2016-06-06 | 2017-07-07 | 广东省林业科学研究院 | A kind of red cone pollen collecting store method that can improve germination rate and extension Pollen Activity |
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