CN109362718A - A kind of method of Siraitia grosvenorii pollen Cryopreservation - Google Patents
A kind of method of Siraitia grosvenorii pollen Cryopreservation Download PDFInfo
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- CN109362718A CN109362718A CN201811406256.7A CN201811406256A CN109362718A CN 109362718 A CN109362718 A CN 109362718A CN 201811406256 A CN201811406256 A CN 201811406256A CN 109362718 A CN109362718 A CN 109362718A
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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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Abstract
The invention discloses a kind of methods of Siraitia grosvenorii pollen Cryopreservation, belong to Siraitia grosvenorii storage technique field.The method of the Siraitia grosvenorii pollen Cryopreservation, includes the following steps: step 1: the Collecting and dealing of pollen；Step 2: the drying of pollen；Step 3: Cryopreservation.The method of Siraitia grosvenorii pollen Cryopreservation of the invention, both solved male and female flowering asynchronism in Pollination of Luohanguo in the prior art, because place of production full-bloom stage it is different caused by pollen supply it is uneven the problems such as, the resource of Siraitia grosvenorii pollen is taken full advantage of again, the service life for extending Siraitia grosvenorii pollen reduces the labor intensity of Pollination of Luohanguo.In addition, the Siraitia grosvenorii pollen that the present invention obtains, compares other germplasm materials, pollen does not carry virus and worm's ovum, can avoid crossbreeding and introduces pest and disease damage.
Description
Technical field
The present invention relates to a kind of methods of Siraitia grosvenorii pollen Cryopreservation, belong to Siraitia grosvenorii storage technique field.
Background technique
Siraitia grosvenorii (Siraitia grosvenorii (Swingle) C.Jeffrey) is many years that Curcurbitaceae Siraitia grosvenorii belongs to
Raw liana.Siraitia grosvenorii is traditional one of the exporting of the famous specialty in Guangxi and China, in Southeast Asia, Japan, America and Europe
Equal countries have long enjoyed a good reputation, and are known as " east mind fruit ", supply falls short of demand in the market.Due to its property do it is cool, have moistening lung to arrest cough, heat-clearing
Cool blood is relieved summer heat, relax bowel and defecation and other effects, and the fields such as medicine, beverage and flavouring are widely used in.
With the continuous development in Siraitia grosvenorii market, Siraitia grosvenorii cultivated area constantly expands, and the yield and quality of Siraitia grosvenorii is but
Do not have greatly improved, Siraitia grosvenorii studies serious lag, the degeneration of Siraitia grosvenorii variet complexity, breediness is seriously one of reason.
And Siraitia grosvenorii breed breeding, cultivation technique backwardness are the main reason for causing variety deterioration.Therefore carry out crossbreeding, excellent list
It selects good strains in the field for seed and the work such as educates and seem more urgent.Pollen includes all genes of species, has genetic diversity abundant, is germplasm
One of preservation and the important materials of exchange.The service life of Siraitia grosvenorii pollen is only capable of survival 1-2 days under field conditions (factors), uses household ice
The storage transport of case preservation by low temperature method, also can only extend to 4-5 days.It is greatly inconvenient that this comes to work belts such as crossbreeding, while
It is unfavorable for relevant genetic research, the research such as accelerates breeding process and improve germplasm resource preservation method.
In addition to Siraitia grosvenorii choosing, breeding work, pollen can also be had an important influence on Siraitia grosvenorii.Since Siraitia grosvenorii is
Female, male dioecian plant, staminiferous plant have viscosity completely not as a result, its pollen is born on the inside of the lower gulf ditch of " s " shape anther, both
It is not easy to be touched by insect, is also not easy to be blowed by wind, female flower spontaneous pollination rate is extremely low, therefore must be by artificial in Siraitia grosvenorii cultivation
Supple-mentary pollination could be solid.The florescence of Siraitia grosvenorii is longer, and it is Siraitia grosvenorii production upper one that section carries out artificial pollination daily during the blossom season
Item conventional technique work.
It is usually that 100 plants of female plants configure 3-5 plants of male plants in Siraitia grosvenorii production.In recent years by Global climate change
Etc. the factors such as natural conditions influence, can also meet that female, male plant cannot bloom simultaneously or male plants are held in production sometimes
Flower amount is not able to satisfy the problem of female flower pollination demand.Therefore, Siraitia grosvenorii bloom pollinate season usually occur male flower shortage, plantation
Family looks about the phenomenon that male flower, and some new planting areas are also often because flowering asynchronism needs strange land tune powder.Even some times,
Every male flower can sell for 0.5 yuan -0.8 yuan of price.Pollen supply in time be Siraitia grosvenorii production on a big problem, in some years
Factor of the timely supply of part pollen also at left and right yield.
In consideration of it, it is necessary to provide a kind of methods of Siraitia grosvenorii pollen Cryopreservation, so as to solve the deficiencies in the prior art.
Summary of the invention
The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of method of Siraitia grosvenorii pollen Cryopreservation.
The method of Siraitia grosvenorii pollen Cryopreservation of the invention, solve male and female flowering asynchronism in Pollination of Luohanguo in the prior art,
Because place of production full-bloom stage it is different caused by pollen supply it is uneven the problems such as, make full use of the resource of Siraitia grosvenorii pollen, extend sieve
In the service life of Chinese fruit pollen, reduce the labor intensity of Pollination of Luohanguo.
The technical scheme to solve the above technical problems is that a kind of method of Siraitia grosvenorii pollen Cryopreservation,
Include the following steps:
Step 1: the Collecting and dealing of pollen
In the morning of Siraitia grosvenorii male flower season of flowers, male flower is picked, stamen pollen is taken, is laid on clean paper,
It is placed at room temperature shady and cool ventilation.
Step 2: the drying of pollen
Step 1 is spread polliniferous paper to be put into the hermetically drying device equipped with desiccant, under room temperature it is dry for 24 hours-
72h, the dry water content to pollen is 10%-40%, then pollen is quickly fitted into the cryopreservation tube of 5ml-10ml and is sealed, pollen
Volume be no more than cryopreservation tube capacity 2/3；
Step 3: Cryopreservation
It step 2 is filled into polliniferous cryopreservation tube is put into -86 DEG C of ultra low temperature freezers or is put into -196 DEG C of liquid nitrogen and store, obtain
To the pollen of Cryopreservation.
The principle of the present invention:
First point: large batch of pollen is in drying process process because pistil water content is high, moisture loss in the prior art
Slowly, it is easy mouldy, influences pollen quality.And the present invention is that pollen is laid in on paper and is put into the sealing container equipped with desiccant
Pollen is divided in multiple cryopreservation tubes by interior drying again after dry, and staff can according to need, dispense according to quantity, store and
It takes, not only flexibly and easily, but also does not influence pollen quality.
Second point: the present invention uses Cryopreservation Siraitia grosvenorii pollen, when can greatly prolong the preservation of Siraitia grosvenorii pollen
Between, make it possible the storing and exchange of pollen, while breeding work can also be served very well, improves breeding efficiency.
Based on the above technical solution, the present invention can also be improved as follows.
Further, in step 1, the male flower is derived from the staminiferous plant that robust plant, flower are big, flower quantity is more, pollen is full.
Be using above-mentioned further beneficial effect: the quality for the male flower that above-mentioned staminiferous plant obtains is higher.
Further, in step 2, the desiccant is silica gel or anhydrous calcium chloride.
It is using above-mentioned further beneficial effect: uses above-mentioned desiccant, dry effect is preferable.
Further, it in step 3, is stored in the liquid nitrogen, every liquid nitrogen of two months supplements in storage period.
Using above-mentioned further beneficial effect is: can be with the storage environment of stable pollen.
Further, the measuring method of the viability of the pollen for the Cryopreservation that the step 3 obtains, including walk as follows
It is rapid:
(1) dyeing liquor is prepared: being weighed 0.1g thiazolyl blue, is dissolved in the 50g/L sucrose solution of 5ml, shakes up, dyed
Liquid is placed in 4 DEG C of refrigerators and is kept in dark place；
(2) it dyes: instilling the dyeing liquor that drop step (1) obtains on glass slide, dipped with tweezers and take a small amount of pollen, put
Enter in dyeing liquor, stir evenly, place 1min-5min at room temperature, obtains pollen staining slide；
(3) microscopy is observed: the pollen staining slide obtained with 100 times of biomicroscope observation of steps (2) has life
The pollen of power in black, darkviolet, aubergine, pink round shaped grain shape, do not have viable pollen to be transparent or translucent
Shrivelled shape, pollen viability is stronger, and color is deeper；Pollen viability is weaker, and color is more shallow, chooses 3-4 100, visual field flower
Powder counts pollen tinctorial yield.
Be using above-mentioned further beneficial effect: in the prior art, Siraitia grosvenorii pollen viability measuring method is to sprout in vitro
Hair method has the disadvantage that operation is more complicated, and pollen smears uneven, waiting pollen germination and the quantity system for sprouting pollen
Time-consuming for meter.Acetic red dyeing stained pollen grain and the pollen grain that is unstained distinguish little, TTC (2,3,5-chlorinated triphenyl bases
Tetrazole) the pollen grain dyeing time is long, it is unobvious to dye.And the present invention is dyed using thiazolyl blue (MTT), it can be quick
Identify the substantially viability of storage pollen, the pollen grain for catching black is more, illustrates that the viability of pollen is higher.
Further, the defreezing method of the pollen for the Cryopreservation that the step 3 obtains are as follows: polliniferous cryopreservation tube will be filled
It is taken out from -86 DEG C of ultra low temperature freezers or in -196 DEG C of liquid nitrogen, 12h-36h is placed in extremely -20 DEG C prior to -18 DEG C, in 4 DEG C
12h-36h is placed, room temperature is then placed into and opens the pipe lid of cryopreservation tube, the pollen after being thawed.
Using above-mentioned further beneficial effect is: the temperature of defrosting is -18 DEG C to -20 DEG C or 4 DEG C, in household ice
It can be realized in case.Both the slow defrosting to the pollen of Cryopreservation may be implemented, reduced injury of the quick-thawing to pollen；
It is also flexible and convenient in the extreme, strong operability.
Further, the application method of the pollen after the defrosting are as follows: after being clamped with pincet or bamboo stick or dipped defrosting
Pollen, touch female chapiter and pollinate, or by the pollen after defrosting be configured to pollination nutrient solution, be sprayed to female chapiter into
Row pollination.
Further, the preparation method of the pollination nutrient solution are as follows: (1) it prepares nutrient solution: taking boric acid 0.1g-0.3g, 2,
4-D (2,4- dichlorphenoxyacetic acid) the 0.02g-0.06g and water-soluble class foliar fertilizer 0.2g of amino acid first dissolves boric acid with water, uses
95% ethyl alcohol dissolves 2,4-D (2,4- dichlorphenoxyacetic acid), then by dissolved boric acid, 2,4-D (2,4- Dichlorophenoxy second
Acid) and the water-soluble class foliar fertilizer mixing of amino acid, it adds water quantitatively to 1L, is sufficiently shaken up after preparing solution, obtain nutrient solution；(2) will
Pollen after defrosting pours into nutrient solution, covers tightly bottle cap, sufficiently rocks, and pollen is made to fall off in nutrient solution, after 2min, nutrient solution
It becomes cloudy, is visible to the naked eye and is suspended in interior pollen little particle, fall style and anther with filter screen filtration, obtain pollination nutrient solution.
Beneficial effects of the present invention:
(1) method of Siraitia grosvenorii pollen Cryopreservation of the invention, when can greatly prolong the preservation of Siraitia grosvenorii pollen
Between, make it possible the storing and exchange of pollen, while breeding work can also be served very well, improves breeding efficiency.
(2) by the Siraitia grosvenorii pollen of Cryopreservation of the present invention, the genetic integrity of Siraitia grosvenorii pollen can be kept very well
And stability, it can also avoid introducing germ and worm's ovum because of crossbreeding, for ground section, international kind mass transter provider
Just.
(3) in the prior art large batch of pollen in drying process process because pistil water content is high, moisture loss is slow, hold
It is easily mouldy, influence pollen quality.And the present invention is to be laid on paper and be put into the sealing container equipped with desiccant to do pollen
It is dry, it is divided in multiple cryopreservation tubes again after dry, staff can according to need, and dispenses, stores according to quantity, quantitative when needed
It takes, flexibly and easily.
(4) pass through 1 year Siraitia grosvenorii pollen of Cryopreservation of the present invention, the female plant of blooming of artificial pollination current year, setting percentage
It is identical as the setting percentage for female plant of blooming of fresh Siraitia grosvenorii pollen artificial pollination current year is used for 80%-95%.
(5) present invention is dyed using thiazolyl blue (MTT), can be contaminated with the substantially viability of Rapid identification storage pollen
The pollen grain of upper black is more, illustrates that the viability of pollen is higher.
(6) operating method of the invention is simple and easy, at low cost, safety and stability, wide market, and suitable scale pushes away
Wide application.
Specific embodiment
Principles and features of the present invention are described below in conjunction with specific embodiment, example is served only for explaining this hair
It is bright, it is not intended to limit the scope of the present invention.
Embodiment 1
The method of the Siraitia grosvenorii pollen Cryopreservation of the present embodiment, includes the following steps:
Step 1: the Collecting and dealing of pollen
In the morning of Siraitia grosvenorii male flower season of flowers, picking robust plant, flower is big, flower quantity is more, pollen is full
Pollen in male flower pistil is laid on clean paper, is placed at room temperature shady and cool ventilation.
Step 2: the drying of pollen
Step 1 is spread polliniferous paper to be put into the hermetically drying device equipped with desiccant, drying for 24 hours, is done under room temperature
The dry water content to pollen is 10%, then pollen is quickly fitted into the cryopreservation tube of 5ml and is sealed, and the volume of pollen, which is no more than, to be frozen
The 2/3 of tube capacity amount；
Step 3: Cryopreservation
It step 2 is filled into polliniferous cryopreservation tube is put into -86 DEG C of ultra low temperature freezers and store, obtain the flower of Cryopreservation
Powder.
The measuring method of the viability of the pollen of the Cryopreservation, includes the following steps:
(1) dyeing liquor is prepared: being weighed 0.1g thiazolyl blue, is dissolved in the 50g/L sucrose solution of 5ml, shakes up, dyed
Liquid is placed in 4 DEG C of refrigerators and is kept in dark place；
(2) it dyes: instilling the dyeing liquor that drop step (1) obtains on glass slide, dipped with tweezers and take a small amount of pollen, put
Enter in dyeing liquor, stir evenly, place 1min at room temperature, obtains pollen staining slide；
(3) microscopy is observed: the pollen staining slide obtained with 100 times of biomicroscope observation of steps (2) has life
The pollen of power in black, darkviolet, aubergine, pink round shaped grain shape, do not have viable pollen to be transparent or translucent
Shrivelled shape, pollen viability is stronger, and color is deeper；Pollen viability is weaker, and color is more shallow, chooses 3-4 100, visual field flower
Powder counts pollen tinctorial yield.
The defreezing method of the pollen of the Cryopreservation are as follows: polliniferous cryopreservation tube will be filled from -86 DEG C of ultra low temperature freezers
Middle taking-up, prior to -18 DEG C in place 36h, place 12h in 4 DEG C, be then placed into room temperature and open the pipe lid of cryopreservation tube,
Pollen after being thawed.
The application method of pollen after the defrosting are as follows: the pollen after defrosting is configured to pollination nutrient solution, is sprayed to female
Chapiter is pollinated.The preparation method of the pollination nutrient solution are as follows: (1) prepare nutrient solution: taking boric acid 0.1g, 2,4-D (2,4-
Dichlorphenoxyacetic acid) 0.02g and the water-soluble class foliar fertilizer 0.2g of amino acid, first dissolve boric acid with water, with 95% ethyl alcohol dissolution 2,4-D
(2,4- dichlorphenoxyacetic acid), then by dissolved boric acid, 2,4-D (2,4- dichlorphenoxyacetic acid) and the water-soluble class leaf of amino acid
Leaven mixes, and adds water quantitatively to 1L, sufficiently shakes up after preparing solution, obtain nutrient solution；(2) pollen after defrosting is poured into nutrition
In liquid, bottle cap is covered tightly, is sufficiently rocked, so that pollen is fallen off in nutrient solution, after 2min, nutrient solution is become cloudy, suspension of being visible to the naked eye
Pollen little particle inside, falls style and anther with filter screen filtration, obtains pollination nutrient solution.
Embodiment 2
The method of the Siraitia grosvenorii pollen Cryopreservation of the present embodiment, includes the following steps:
Step 1: the Collecting and dealing of pollen
In the morning of Siraitia grosvenorii male flower season of flowers, picking robust plant, flower is big, flower quantity is more, pollen is full
Pollen in male flower pistil is laid on clean paper, is placed at room temperature shady and cool ventilation.
Step 2: the drying of pollen
Step 1 is spread polliniferous paper to be put into the hermetically drying device equipped with anhydrous calcium chloride desiccant, under room temperature
Dry 48h, the dry water content to pollen is 25%, then pollen is quickly fitted into the cryopreservation tube of 8ml and is sealed, the volume of pollen
No more than the 2/3 of cryopreservation tube capacity.
Step 3: Cryopreservation
It step 2 is filled into polliniferous cryopreservation tube is put into -196 DEG C of liquid nitrogen and store, mended in storage period every two months
A liquid nitrogen is filled, the pollen of Cryopreservation is obtained.
The measuring method of the viability of the pollen of the Cryopreservation, includes the following steps:
(1) dyeing liquor is prepared: being weighed 0.1g thiazolyl blue, is dissolved in the 50g/L sucrose solution of 5ml, shakes up, dyed
Liquid is placed in 4 DEG C of refrigerators and is kept in dark place；
(2) it dyes: instilling the dyeing liquor that drop step (1) obtains on glass slide, dipped with tweezers and take a small amount of pollen, put
Enter in dyeing liquor, stir evenly, place 3min at room temperature, obtains pollen staining slide；
(3) microscopy is observed: the pollen staining slide obtained with 100 times of biomicroscope observation of steps (2) has life
The pollen of power in black, darkviolet, aubergine, pink round shaped grain shape, do not have viable pollen to be transparent or translucent
Shrivelled shape, pollen viability is stronger, and color is deeper；Pollen viability is weaker, and color is more shallow, chooses 3-4 100, visual field flower
Powder counts pollen tinctorial yield.
The defreezing method of the pollen of the Cryopreservation are as follows: polliniferous cryopreservation tube will be filled and taken from -196 DEG C of liquid nitrogen
Out, prior to -19 DEG C in place the pipe lid for placing in 4 DEG C for 24 hours and being then placed into room temperature for 24 hours and opening cryopreservation tube, obtain
Pollen after defrosting.
The application method of pollen after the defrosting are as follows: the pollen after defrosting is configured to pollination nutrient solution, is sprayed to female
Chapiter is pollinated.The preparation method of the pollination nutrient solution are as follows: (1) prepare nutrient solution: taking boric acid 0.2g, 2,4-D (2,4-
Dichlorphenoxyacetic acid) 0.04g and the water-soluble class foliar fertilizer 0.2g of amino acid, first dissolve boric acid with water, with 95% ethyl alcohol dissolution 2,4-D
(2,4- dichlorphenoxyacetic acid), then by dissolved boric acid, 2,4-D (2,4- dichlorphenoxyacetic acid) and the water-soluble class leaf of amino acid
Leaven mixes, and adds water quantitatively to 1L, sufficiently shakes up after preparing solution, obtain nutrient solution；(2) pollen after defrosting is poured into nutrition
In liquid, bottle cap is covered tightly, is sufficiently rocked, so that pollen is fallen off in nutrient solution, after 2min, nutrient solution is become cloudy, suspension of being visible to the naked eye
Pollen little particle inside, falls style and anther with filter screen filtration, obtains pollination nutrient solution.
Embodiment 3
The method of the Siraitia grosvenorii pollen Cryopreservation of the present embodiment, includes the following steps:
Step 1: the Collecting and dealing of pollen
In the morning of Siraitia grosvenorii male flower season of flowers, picking robust plant, flower is big, flower quantity is more, pollen is full
Pollen in male flower pistil is laid on clean paper, then is placed at room temperature shady and cool ventilation.
Step 2: the drying of pollen
Step 1 is spread polliniferous paper to be put into the hermetically drying device equipped with anhydrous calcium chloride desiccant, under room temperature
Dry 72h, the dry water content to pollen is 40%, then pollen is quickly fitted into the cryopreservation tube of 10ml and is sealed, the body of pollen
Product is no more than the 2/3 of cryopreservation tube capacity；
Step 3: Cryopreservation
It step 2 is filled into polliniferous cryopreservation tube is put into -86 DEG C of ultra low temperature freezers and store, obtain the flower of Cryopreservation
Powder.
The measuring method of the viability of the pollen of the Cryopreservation, includes the following steps:
(1) dyeing liquor is prepared: being weighed 0.1g thiazolyl blue, is dissolved in the 50g/L sucrose solution of 5ml, shakes up, dyed
Liquid is placed in 4 DEG C of refrigerators and is kept in dark place；
(2) it dyes: instilling the dyeing liquor that drop step (1) obtains on glass slide, dipped with tweezers and take a small amount of pollen, put
Enter in dyeing liquor, stir evenly, place 5min at room temperature, obtains pollen staining slide；
(3) microscopy is observed: the pollen staining slide obtained with 100 times of biomicroscope observation of steps (2) has life
The pollen of power in black, darkviolet, aubergine, pink round shaped grain shape, do not have viable pollen to be transparent or translucent
Shrivelled shape, pollen viability is stronger, and color is deeper；Pollen viability is weaker, and color is more shallow, chooses 3-4 100, visual field flower
Powder counts pollen tinctorial yield.
The defreezing method of the pollen of the Cryopreservation are as follows: polliniferous cryopreservation tube will be filled from -86 DEG C of ultra low temperature freezers
Middle taking-up, prior to -20 DEG C in place 12h, place 36h in 4 DEG C, be then placed into room temperature and open the pipe lid of cryopreservation tube,
Pollen after being thawed.
The application method of pollen after the defrosting are as follows: clamp or dip the pollen after thawing with pincet or bamboo stick, touch
Female chapiter is touched to pollinate.
Comparative example 1
With unlike embodiment 2, in comparative example 1, in step 3 not by step 2 fill polliniferous cryopreservation tube be put into-
It is stored in 196 DEG C of liquid nitrogen, but uses domestic refrigerator frozen coating (- 18 DEG C to -20 DEG C), remaining step is all identical.
Using the method for comparative example 1, the Siraitia grosvenorii pollen of storage 1 year, the female plant of blooming of artificial pollination current year, setting percentage
For 50%-60%.
Using the method for embodiment 2, the Siraitia grosvenorii pollen of storage 1 year, the female plant of blooming of artificial pollination current year, setting percentage
For 80%-95%.
It can be seen that the method for Siraitia grosvenorii pollen Cryopreservation of the invention, can greatly prolong Siraitia grosvenorii pollen
Holding time, make it possible the storing and exchange of pollen, while breeding work can also be served very well, improves breeding efficiency.
Comparative example 2
Unlike embodiment 2, in comparative example 2, the measurement side of the viability of the pollen of Cryopreservation in step 3
Method, using germination in vitro method, remaining step is all identical.
Using the method for comparative example 2, is observed after 1h, still there is pollen to germinate successively, pollen tube persistently extends.Most need fastly
It just can determine that pollen viability after 60min, germination pollen grain is more, illustrates that the viability of pollen is higher.
Using the method for embodiment 2,5-10min after dyeing, i.e. observable, the pollen grain for catching black is more, illustrates to spend
The viability of powder is higher.
It can be seen that the present invention is dyed using thiazolyl blue (MTT), it can be with the substantially life of Rapid identification storage pollen
Power, the pollen grain for catching black is more, illustrates that the viability of pollen is higher.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (8)
1. a kind of method of Siraitia grosvenorii pollen Cryopreservation, which comprises the steps of:
Step 1: the Collecting and dealing of pollen
In the morning of Siraitia grosvenorii male flower season of flowers, male flower is picked, stamen pollen is taken, is laid on clean paper, is placed in
At room temperature shady and cool ventilation.
Step 2: the drying of pollen
Step 1 is spread polliniferous paper to be put into the hermetically drying device equipped with desiccant, dry -72h for 24 hours, does under room temperature
The dry water content to pollen is 10%-40%, then pollen is quickly fitted into the cryopreservation tube of 5ml-10ml and is sealed, the volume of pollen
No more than the 2/3 of cryopreservation tube capacity；
Step 3: Cryopreservation
It step 2 is filled into polliniferous cryopreservation tube is put into -86 DEG C of ultra low temperature freezers or is put into -196 DEG C of liquid nitrogen and store, surpassed
The pollen of cryopreservation.
2. a kind of method of Siraitia grosvenorii pollen Cryopreservation according to claim 1, which is characterized in that in step 1, institute
It states male flower and is derived from the staminiferous plant that robust plant, flower are big, flower quantity is more, pollen is full.
3. a kind of method of Siraitia grosvenorii pollen Cryopreservation according to claim 1, which is characterized in that in step 2, institute
Stating desiccant is silica gel or anhydrous calcium chloride.
4. a kind of method of Siraitia grosvenorii pollen Cryopreservation according to claim 1, which is characterized in that in step 3, institute
It states in liquid nitrogen and stores, every liquid nitrogen of two months supplements in storage period.
5. a kind of method of Siraitia grosvenorii pollen Cryopreservation according to claim 1, which is characterized in that the step 3
The measuring method of the viability of the pollen of obtained Cryopreservation, includes the following steps:
(1) dyeing liquor is prepared: being weighed 0.1g thiazolyl blue, is dissolved in the 50g/L sucrose solution of 5ml, shakes up, obtains dyeing liquor simultaneously
It is placed in 4 DEG C of refrigerators and is kept in dark place；
(2) it dyes: instilling the dyeing liquor that drop step (1) obtains on glass slide, dipped with tweezers and take a small amount of pollen, be put into dye
It in color liquid, stirs evenly, places 1min-5min at room temperature, obtain pollen staining slide；
(3) microscopy is observed: the pollen staining slide obtained with 100 times of biomicroscope observation of steps (2) has viable
Pollen in black, darkviolet, aubergine, pink round shaped grain shape, do not have viable pollen to be transparent or translucent dry
Flat shape, pollen viability is stronger, and color is deeper；Pollen viability is weaker, and color is more shallow, chooses 100,3-4 visual field pollen system
Count pollen tinctorial yield.
6. a kind of method of Siraitia grosvenorii pollen Cryopreservation according to claim 1, which is characterized in that the step 3
The defreezing method of the pollen of obtained Cryopreservation are as follows: will fill polliniferous cryopreservation tube from -86 DEG C of ultra low temperature freezers or -
It is taken out in 196 DEG C of liquid nitrogen, places 12h-36h in extremely -20 DEG C prior to -18 DEG C, place 12h-36h in 4 DEG C, be then placed into
Room temperature and the pipe lid for opening cryopreservation tube, the pollen after being thawed.
7. a kind of method of Siraitia grosvenorii pollen Cryopreservation according to claim 6, which is characterized in that after the defrosting
Pollen application method are as follows: clamp or dip the pollen after thawing with pincet or bamboo stick, touch female chapiter and pollinate,
Or the pollen after defrosting is configured to pollination nutrient solution, it is sprayed to female chapiter and pollinates.
8. a kind of method of Siraitia grosvenorii pollen Cryopreservation according to claim 7, which is characterized in that the pollination battalion
The preparation method of nutrient solution are as follows: (1) prepare nutrient solution: boric acid 0.1g-0.3g, 2,4-D (2,4 dichlorophenoxyacetic acid) 0.02g- is taken
The 0.06g and water-soluble class foliar fertilizer 0.2g of amino acid first dissolves boric acid with water, dissolves 2,4-D (2,4- Dichlorophenoxies with 95% ethyl alcohol
Acetic acid), then dissolved boric acid, 2,4-D (2,4- dichlorphenoxyacetic acid) and the water-soluble class foliar fertilizer of amino acid are mixed, then plus
Water is quantitative to 1L, sufficiently shakes up after preparing solution, obtains nutrient solution；(2) pollen after defrosting is poured into nutrient solution, covers tightly bottle
Lid, sufficiently rocks, and pollen is made to fall off in nutrient solution, and after 2min, nutrient solution is become cloudy, and the pollen being visible to the naked eye in being suspended in is small
Particle falls style and anther with filter screen filtration, obtains pollination nutrient solution.
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| CN111543308A (en) * | 2020-05-06 | 2020-08-18 | 中国农业科学院蜜蜂研究所 | Preparation method of pollen for pollination |
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