CN102461462A - Preparation method of culture medium suitable for rapid seedling growing tissue culture of blueberry - Google Patents

Abstract

The invention relates to the field of culture mediums, and discloses a preparation method of a culture medium suitable for rapid seedling growing tissue culture of a blueberry. The preparation method comprises the following steps of 1, adding water into an empty beaker, wherein the amount of the added water is 50% of a total preparation amount, weighing various raw materials according to a formula, putting the raw materials into the beaker, stirring by a glass rod, heating for dissolution, and carrying out volume metering, 2, carrying out sub-package with continuous stirring so that agar powder in each one of test tubes is uniform; 3, after the sub-package, respectively plugging tampons in the test tubes and a conical flask, wrapping the test tubes and the conical flask with brown papers, carrying out bundling to form bundles, and noting a date, and 4, carrying out disinfection to obtain the culture medium, and placing the disinfected culture medium aside for next use, wherein before being used, the disinfected culture medium must be subjected to constant-temperature culture at a temperature of 35 to 37 DEG C for 24 hours so that an aseptic growth condition is obtained, The culture medium obtained by the preparation method provided by the invention is suitable for blueberry tissue culture.

Description

A kind of medium preparation method of suitable blueberry fast seedling growing tissue culture

Technical field

The present invention relates to the medium field, is a kind of medium preparation method of suitable blueberry fast seedling growing tissue culture.

Background technology

Blueberry (Blueberry) belongs to Ericaceae (Ericaceae) Vaccinium (Vaccinium spp), and the Vaccinium plant has more than 400 kind approximately, originate in main product in U.S.'s U.S.'s blueberry that is otherwise known as, mean the meaning of blue berry.Blueberry is a kind of little berry, and it is blue that fruit is, and color and luster is beautiful, pleasing, pulp is fine and smooth, and seed is minimum.The blueberry fruit on average weighs 0.5～2.5g, maximum heavy 5g, and edible rate is 100%, sweet and sour palatability, and have fragrant refreshing pleasant fragrance, for eating good merchantable brand raw.Contain rich nutrient contents in the blueberry fruit; It not only has good nutrition health-care functions; Also have and prevent that cerebral nerve is aging, cardiac stimulant, anticancer softening blood vessel, strengthen people's functions such as immunity of organism that reputation is called the gold fruit that has development prospect in the world today most.

Interrelated data is analyzed needs 800,000 tons in whole world year, but can only produce 400,000 tons every year, estimates to be difficult to realize the co-ordination of supply and marketing in 20 years; The market demand is big, but blueberry why development is slow again, main cause is the restriction that receives nursery stock bottleneck in short supply; Because expensive; Fresh fruit all is used for market, and cuttage survival rate is low, just is difficult to the popularization state because the restriction of medium preparation technique causes the seedling propagation technology to be in for a long time again.

Medium characteristics commonly used:

1, MS medium.It is 1962 be to cultivate tobacco cell to design by Murashige and Skoog.Characteristics are that mineral salt and ion concentration are higher, and pH value is high, is more stable balance solution.The quantity and the ratio of its nutrient are more suitable, can satisfy nutrition and the physiological requirements of plant.Its nitrate content is high than other medium, and the organ, flower pesticide, cell and the protoplast that are widely used for plant are cultivated, and be respond well, and some medium is developed by it.

2, B5 medium.Being nineteen sixty-eight is designed for cultivating the soybean root cells by Gamborg etc.Its main feature is to contain lower ammonium, and this possibly have inhibitory action to the growth of many cultures.Learn that from practice some plant is preferably in B5 medium growth, like dicotyledon woody plant particularly.

3, White medium.Nineteen forty-three is to cultivate the tomato tip of a root to design by White.Did improvement again in 1963, and be called the White improved culture medium, improved the concentration of MgSO4 and increased roc plain.Be characterized in that mineral salt quantity is lower, be suitable for culture of rootage.

4, N6 medium.Be that the extremely clear grade of Zhu in 1974 designs for the cultivation of cereal crops such as paddy rice.Be characterized in that composition is simpler, KNO3 is with (NH4) 2SO4 content is high.The anther culture and other tissue culture that have been widely used in wheat, paddy rice and other plants at home.

5, KM-80 medium.It is to cultivate for protoplast in 1974 to design.Be characterized in that organic principle is complicated, it has comprised all monose and vitamin, is widely used in the cultivation that protoplasm merges.

Medium has numerous species, need select different culture medium for use according to different plants and cultivation position and different culture purpose.But the tissue culture of above-mentioned all kinds of medium incompatibility blueberries.

Summary of the invention

The objective of the invention is to provide a kind of preparation method of blueberry culture medium for tissue culture.

Technical scheme of the present invention is:

(1) prescription of minimal medium and weighing:

(2) culture medium preparation method and step:

A, earlier in empty flask, add to prepare about 50% water of total amount;

B, weighing;

Correctly taking by weighing various raw materials according to prescription is put in the beaker.If synthetic medium, each composition will dissolve one by one;

When claiming insoluble solid, should claim separately, if few can the claiming of amount with mineral salt, but must mixing packing again;

C, dissolve:

Glass rod stirs, heating for dissolving, last constant volume;

Constant volume: can add the low amounts of water dissolving earlier, pour graduated cylinder into, moisturizing is to scale again;

D, adjust pH: transfer pH with 1N NaOH or 1N HCl, contrast with the pH test paper;

E, add agar powder: divide in the process of assembling and will constantly stir, so that the agar powder of every pipe can both be even;

F, packing: 1/4 of test tube length overall, 1/2 (loading amount refers to solid culture medium) of triangular flask;

G, wrapping are sealed: test tube and conical flask be tampon and wrap brown paper beyond the Great Wall respectively, wrap up bundled, dated which kind of medium, time;

H, the sterilization subsequent use;

Must confirm asepsis growth at 35-37 ℃ of following constant temperature culture 24h behind the medium sterilization, can use.

Owing to adopted technique scheme, the medium of being made adapts to the tissue culture of blueberry.

Embodiment

Below in conjunction with embodiment the present invention is further described.

(1) prescription of minimal medium and weighing:

(2) culture medium preparation method and step:

A, earlier in empty flask, add to prepare about 50% water of total amount;

B, weighing;

Correctly taking by weighing various raw materials according to prescription is put in the beaker.If synthetic medium, each composition will dissolve one by one;

When claiming insoluble solid, should claim separately, if few can the claiming of amount with mineral salt, but must mixing packing again;

C, dissolve:

Glass rod stirs, heating for dissolving, last constant volume;

Constant volume: can add the low amounts of water dissolving earlier, pour graduated cylinder into, moisturizing is to scale again;

D, adjust pH: transfer pH with 1N NaOH or 1N HCl, contrast with the pH test paper;

E, add agar powder: divide in the process of assembling and will constantly stir, so that the agar powder of every pipe can both be even;

F, packing: 1/4 of test tube length overall, 1/2 (loading amount refers to solid culture medium) of triangular flask;

G, wrapping are sealed: test tube and conical flask be tampon and wrap brown paper beyond the Great Wall respectively, wrap up bundled, dated which kind of medium, time;

H, the sterilization subsequent use;

Must confirm asepsis growth at 35-37 ℃ of following constant temperature culture 24h behind the medium sterilization, can use.

The high pressure steam sterilization step:

At first internal layer sterilization bucket is taken out, in outer pot, add an amount of water again.Put back to the sterilization bucket, and pack into and treat sterilizing article.Add a cover, with two the relative bolts of screwing simultaneously of symmetrical manner in twos.The water boiling is to get rid of the cold air in the pot.After treating that cold air drains fully, shut air bleeding valve.When pot inner pressure was raised to required pressure, the control thermal source was kept pressure to required time.When manometric pressure reduces to 0, open air bleeding valve, unscrew bolt, uncap is put into 37 ℃ of incubators with the sterilising medium that takes out and was cultivated 24 hours, on inspection as if no varied bacteria growing, can be for use.

(3), the adjustment of the acid-base value of medium:

In the culture medium preparation process, often need adjust the acid-base value of medium with potassium hydroxide or sodium hydroxide and hydrochloric acid.

The preparation of a, mol KOH liquid: take by weighing 57.1 gram KOH, adding distil water to 1000 milliliter.

The preparation of b, mol KOH liquid: get 90 milliliters of 10 milliliter of 1 mol KOH liquid adding distil waters.

The preparation of c, mol NaOH liquid: take by weighing 40 gram NaOH, adding distil water to 1000 milliliter.

The preparation of d, mol NaOH liquid: get 90 milliliters of 10 milliliter of 1 mol NaOH liquid adding distil waters.

The preparation of e, mol HCL liquid: get 82.5 milliliters of adding distil waters to 1000 of concentrated hydrochloric acid (proportion 1.19) milliliter.Earlier concentrated hydrochloric acid is slowly added in about 800 milliliters distilled water during preparation, complement to 1000 milliliters with distilled water then.

The preparation of f, mol HCL liquid: get 90 milliliters of 10 milliliter of 1 mol HCL liquid adding distil waters.

G, alcohol are thimerosals commonly used in the group training work, are widely used in the sterilization of explant material, inoculating appliance and inoculation personnel both hands, also are used for the alcohol spraying disinfection of culturing room etc.

(4), plant hormone formulation types hormone prescription, regeneration and purposes in the common medium.

A, no plant hormone: root induction, asexual embryo, callus form.

B, singly add growth hormone: root induction, callus, asexual embryo, indefinite plumule.

C, singly add the cell mitogen: evoking adventive bud, lateral bud propagation, callus form.

D, the low basic element of cell division of high growth hormone: induced bud, protocorm propagation, callus form.

E, the low high basic element of cell division of growth hormone: induced bundle bud, callus form.

F, the low basic element of cell division of low growth hormone: evoking adventive bud, lateral bud propagation.

G, equivalent growth hormone and the basic element of cell division: induce lateral bud propagation.

H, with growth inhibitor (paclobutrazol, chlormequat etc.): strong sprout, retarding of growing, be beneficial to test-tube plantlet and preserve.

I, add GA ₃: break seed, bud dormancy, promote elongation growth.

Claims (1)

1. the medium preparation method of a suitable blueberry fast seedling growing tissue culture is characterized in that:

(1) prescription of minimal medium and weighing:

(2) culture medium preparation method and step:

A, earlier in empty flask, add to prepare about 50% water of total amount;

B, weighing;

Correctly taking by weighing various raw materials according to prescription is put in the beaker.If synthetic medium, each composition will dissolve one by one;

When claiming insoluble solid, should claim separately, if few can the claiming of amount with mineral salt, but must mixing packing again;

C, dissolve:

Glass rod stirs, heating for dissolving, last constant volume;

Constant volume: can add the low amounts of water dissolving earlier, pour graduated cylinder into, moisturizing is to scale again;

D, adjust pH: transfer pH with 1N NaOH or 1N HCl, contrast with the pH test paper;

E, add agar powder: divide in the process of assembling and will constantly stir, so that the agar powder of every pipe can both be even;

F, packing: 1/4 of test tube length overall, 1/2 (loading amount refers to solid culture medium) of triangular flask;

G, wrapping are sealed: test tube and conical flask be tampon and wrap brown paper beyond the Great Wall respectively, wrap up bundled, dated which kind of medium, time;

H, the sterilization subsequent use;

Must confirm asepsis growth at 35-37 ℃ of following constant temperature culture 24h behind the medium sterilization, can use.