CN101982046B

CN101982046B - Method for storing pear pollen germplasm for long term

Info

Publication number: CN101982046B
Application number: CN2010102562310A
Authority: CN
Inventors: 张绍铃; 陶书田; 吴俊�; 齐永杰; 齐开杰; 吴华清
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2010-08-18
Filing date: 2010-08-18
Publication date: 2012-05-23
Anticipated expiration: 2030-08-18
Also published as: CN101982046A

Abstract

The invention provides a method for reliably storing pear pollen germplasm for long term, belonging to the field of plant developmental biology. The method is characterized by putting the pear pollen into an allochroic silicagel box after drying the pear pollen, packing and sealing the box with tinfoil, freezing the pollen in liquid nitrogen and storing the pollen at ultralow temperature, placingthe stored pollen for 24 hours at minus 73 DEG C, minus 20 DEG C and 4 DEG C respectively in sequence to gradually revive the pollen when the pollen needs to be used in the coming year and finally restoring the pollen to room temperature in dark environment for later use. The method is secure and reliable, can effectively maintain the vigor of the pear pollen, is simple and easy to operate, saves space, can be used for reference to preserve other germplasm resources and has wide application value.

Description

The method of long term storage pear pollen germplasm

One, technical field

The present invention is the method for long term storage pear pollen germplasm, belongs to the development of plants field of biology.

Two, background technology

Pears (Pyrus) belong to gamete build self-incompatibillity fruit tree, and most kind self-pollinations are shaky, on producing, must dispose pollinizers, perhaps carry out artificial pollination and could obtain expected yield and quality.But on producing,, usually need gather, call pollen, even need use the pollen of storage the previous year from the other places owing to flowering asynchronism or weather reason; In the pears breeding work, according to the difference of breeding objective, the breeder also usually will call the pears germplasm pollen of the special or excellent proterties of tool from various places to enrich genetic diversity; In addition; Because pears are typical gamete build self-incompatibillity fruit trees; Usually as important research examination material, but can only adopt once in 1 year by pear pollen in the research of self-incompatibillity mechanism for pear pollen, and this must relate to the storage problem of pollen in the research cycle in 1 year.No matter from pear tree cultivation, breeding or scientific research aspect, its working foundation is that pollen must have higher vigor, the pollen with higher vigor is the key precondition that above-mentioned work is achieved success.Therefore, how in storage, transportation, prolong the pollen life-span, it is significant to pear tree production, quality saving, scientific research to keep the pollen vigor to greatest extent.The correlative study report, the vigor of pollen receives the influence of temperature and humidity, but does not form the effective storage practice that can better keep the pear pollen vigor as yet.The general normal temperature pear pollen vigor of storage down descends rapidly, loses using value.The present invention forms the pear pollen long term storage technology that comprises three key technology steps through the influence of the different storage practices of comparative studies to pear pollen sprouting and pollen tube growth.Operation is simple for this technology, saves the space, effectively kept the vigor of pear pollen after the long term storage, has important use at aspects such as pear tree production, breeding, quality saving, scientific researches and be worth.

Three, summary of the invention

Specification requirement

The objective of the invention is to solve pear pollen pollen vigor in conventional storage and reduce rapidly, so that can not satisfy the problem of production, scientific research needs.

Technical scheme

It is following that the present invention will address the above problem the new solution that is adopted:

1, pollen collecting method: adopt the big flower bud phase petal that a bud just ready to burst, take back indoorly, rub flower, flower pesticide is separated with filigree, with fine ga(u)ge screen sieve flower pesticide down.Pick flowers behind the rain or petal speckles with when revealing, dry earlier and rub flower again.Flower pesticide is even at the indoor individual layer stand of 20～25 ℃ of temperature, relative moisture 70%～80%, no sunlight direct projection, and pollen can shed after 1～2 day.The pollen of producing divides parcel to wrap with waxed paper, and every bag 5cm * 5cm * 0.5cm size imbeds in the drying receptacle that is placed with discolour silica gel, and the wrapping of external application tinfoil is neat, to reach the purpose of dry shading;

2, liquid nitrogen cryopreservation pollen: the sizeable liquid nitrogen container of how much selecting according to the Tibetan pollen amount, the pollen that bandages is placed in the liquid nitrogen container, be submerged in liquid nitrogen.For preventing the liquid nitrogen volatilization totally, whenever replenished a liquid nitrogen in the duration of storage at a distance from two months;

3, pollen recovery: when waiting to need to use pollen, open jar mouthful, topple over the liquid nitrogen in jar, the pollen of must guard against this moment immediately wrapping directly is exposed in the normal temperature environment.Can cause the pollen brown stain too soon because heat up, make and reduce the pollen vigor greatly by pollen protein enzyme property inactivation.Should the pollen of storage be placed respectively in-73 ℃ ,-20 ℃, 4 ℃ successively and progressively revive in 24 hours, in dark surrounds, return to room temperature at last, subsequent use.

Beneficial effect

Storage practice of the present invention has been kept the pear pollen vigor through long term storage effectively, has important beneficial effect for aspects such as the production of pear tree, breeding, quality saving, scientific researches, and concrete manifestation is following:

The safety of gathering when (1) having ensured artificial pollination, allocating and transporting pollen in the pollen process makes and preserves pollen in the production in order to using the coming year, ensures that the pollen sufficient supplies becomes possibility;

(2) the high vigor preservation for source, different region pollen required in the breeding work provides technical support, for carrying out the pears quality saving through pollen a new approach is provided simultaneously;

(3) can be the pear pollen that the scientific research of pears self-incompatibillity mechanism provides the high vigor of tool in the anniversary, overcome the limitation in season of correlative study;

(4) investment of this storage practice is little, simple to operate, can in very little space, preserve a large amount of pollen, and can use for reference other germ plasm resources such as being used to preserve seed, and wide practical value is arranged.

Four, description of drawings:

Fig. 1 ' abundance of water ' variation of pear pollen pollen vigor under-20 ℃ ,-73 ℃ ,-196 ℃ three kinds of storage environments;

The schematic flow sheet of Fig. 2 long term storage pear pollen;

Five, embodiment:

(1) experimental technique

1, pollen collection: this patent serves as to study the examination material with ' abundance of water ' pears, gathers the big flower bud phase petal that a bud just ready to burst, collects flower pesticide, and dry quick-fried powder under the room temperature is treated to wrap with waxed paper after pollen sheds, and presses diverse ways and preserves pollen, and regularly detect;

2, pollen store method:

(1) gradient of pollen storage temperature is handled: same bag pollen is divided into five parts; Every part of about 5cm * 5cm * 0.5cm size; Imbed and be placed with discolour silica gel (a kind of granulated dried agent; Blueness bleaches after the suction) drying receptacle in, under room temperature, 4 ℃ ,-20 ℃ ,-73 ℃ ,-196 ℃ conditions, preserve respectively.Wherein every under the room temperature at a distance from 5 days, by above-mentioned pollen germination rate of pollen cultivation measurement and pollen tube length.4 ℃ every measured once at a distance from 10 days, and the pollen of preserving under-20 ℃ ,-73 ℃ ,-196 ℃ conditions will be from preserve be measured in the 30th day, 60 days, 90 days, 180 days, 270 days, 360 days;

(2) gradient of pollen storage humidity is handled: wrap the pollen of gathering in the airtight box that is placed on air humidity 15%, 32%, 52%, 66%, 81% with waxed paper; And box placed under dark, the room temperature condition; After storing 1,6,12,18 day respectively, cultivate and measuring method mensuration pollen germination rate and pollen tube length by above-mentioned pollen;

3, pollen vitality test: this research adopts solid culture medium to cultivate pollen.The component of medium is the agar of 10% sucrose, 0.01% boric acid and 1%.With pollen equably program request on medium, under dark, 25 ℃ of conditions, cultivate 24h.On medium, drip several methyl blue dyeing liquors, examine under a microscope the sprouting and the growing state of pollen.In statistics during pollen germination rate, with the pollen tube length of eruption surpass the pollen grain diameter for germinateing, otherwise be the pollen that do not germinate, add up more than 100; Pollen germination rate multiply by 100% for the pollen number of having sprouted divided by total pollen number of observing; Its mean value is calculated in 20 of pollen tube growth linear measure longimetrys.Each handles repetition 3 times.

(2) result and analysis

1, different reserve temperatures are to the influence of pollen germination and pollen tube growth

Find that through data analysis the pollen germination rate and the pollen tube growth difference in length of storage are remarkable under different reserve temperatures.Under the room temperature condition, it is very fast that the pollen vigor descends, preserve germination rate after 20 days from 0 day 98.7% drop to 4.3%, preserve after 30 days, pollen loses vigor fully.But along with the reduction of reserve temperature, the time that pollen maintains vigour is more and more longer, under liquid nitrogen cryopreservation (196 ℃) condition, the germination rate of pollen storage pollen after 360 days still can reach 68.9% and pollen tube length still longer, energetic (Fig. 1) is described.

2, storage humidity is to the influence of pollen germination and pollen tube growth

There are very big-difference (table 1) in pollen germination and pollen tube growth under difference storage humidity; Pollen loses vigor rapidly under high humidity, and along with the reduction of humidity, it is also long more that pollen is kept time of vigor; During like relative moisture 15%; After the room temperature storage 18 days, divide germination rate and still reach 58.2%, pollen pipe range under the also higher appropriate condition of pollen tube length.

Reserve temperature and humidity and pollen vigor retention time are negative correlativing relation, explain that low temperature, low humidity help the long preservation of pear pollen, are beneficial to keeping of pear pollen vigor.

According to above conclusion content of the present invention (Fig. 2) is proposed: pear pollen is loaded in the discolour silica gel box after drying; External application tinfoil wrapping sealing; Carry out the nitrogen ultra low temperature storage; Need to wait the time spent that the pollen of storage is placed respectively in-73 ℃ ,-20 ℃, 4 ℃ successively and progressively revived in 24 hours, in dark surrounds, return to room temperature at last, subsequent use.This storage practice insurance is reliable, and operation is simple and save the space, has solved pear pollen pollen vigor in conventional storage and has reduced rapidly, so that can not satisfy the problem of production, scientific research needs.

Germination rate and the pollen tube length of table 1 ' abundance of water ' pollen after the different humidity storage

Claims

1. the method for long term storage pear pollen comprises:

1) pollen collection: adopt the big flower bud phase petal that a bud just ready to burst, take back indoorly, rub flower; Flower pesticide is separated with filigree, and flower pesticide under sieving with aperture 1.43mm fine ga(u)ge screen is picked flowers behind the rain or petal speckles with when revealing; Dry earlier and rub flower again, flower pesticide is even at the indoor individual layer stand of 20～25 ℃ of temperature, relative moisture volume ratio 70%～80%, no sunlight direct projection, and pollen shed after 1～2 day; The pollen of producing divides parcel to wrap with waxed paper, and every bag 5cm * 5cm * 0.5cm size imbeds in the drying receptacle that is placed with discolour silica gel; Said drying receptacle external application tinfoil wrapping is neat, to reach the purpose of dry shading;

2) liquid nitrogen cryopreservation pollen: the pollen that bandages is placed in the liquid nitrogen container, be submerged in liquid nitrogen,, whenever replenished a liquid nitrogen in the duration of storage at a distance from two months for preventing the liquid nitrogen volatilization totally;

3) pollen is recovered: when waiting to need to use pollen, open a jar mouth, topple over a liquid nitrogen that in the jar, the pollen of storage is placed respectively in-73 ℃ ,-20 ℃, 4 ℃ successively progressively revived in 24 hours, in dark surrounds, return to room temperature at last, and subsequent use.