CN104604705A - Large-scale production method of AA basal solid culture medium - Google Patents

Abstract

The invention discloses a large-scale production method of an AA basal solid culture medium, and belongs to the technical field of plant biology. The method comprises the following steps: firstly calculating the required 1000-fold increased amounts of 18 kinds of elements contained in the AA basal culture medium; accurately weighing the fold-increased amounts of medicines, wherein CuSO4.5H2O and CoCl2.6H2O need to be accurately weighed by using a 0.1 milligram-scale electronic balance; mixing the 18 kinds of elements according to the required amounts from the small amount to the large amount, i.e., firstly mixing the smallest amounts of two elements, and adding the second smallest amounts of elements to be uniformly mixed, and so on; sealing every 250g of the solid culture medium in a fixed plastic container; and respectively unsealing to use according to the needs. The 1000-fold increased amount of AA basal solid culture medium can be prepared by the method. The method has the characteristics of convenience in operation, quickness, simplicity in application, and small errors of products from different batches and inside a batch. The phenomena of large error, precipitation, mildew and the like caused by the manual operation such as calculation and weighing and external factors such as weather, temperature and moisture in the production of an AA liquid culture medium are avoided. The method can be applied to the plant tissue culture and other biotechnology applications.

Description

The scale preparation method of the basic solid culture matrix of a kind of AA

Technical field

The invention discloses the scale preparation method of the basic solid culture matrix of a kind of AA, belong to plant biotechnology field.Be more particularly one include accurate calculating, accurate weighing, first few after how mix, seal, the five steps such as use of breaking seal, to obtain different batches and batch between the minimum culture matrix of error, minimizing experimental error method.

Background technology

Plant tissue culture technique or cultivation technique without soil need preparation medium mother liquor usually, and namely preparation expands certain multiple, liquid containing all kinds reagent.But, because mother liquor contains dissimilar reagent, be usually divided into the large class of macroelement, trace element, molysite and organic element four, and the multiple that four large classes expand is different, causes medicine service property (quality) inconsistent, easily occurs the error of calculation first; Second the producer of medicine, quality are inconsistent, even if to calculate, weigh consistent, also can affect result of the test; Three due to mother liquor be liquid condition, at the end of spring and the beginning of summer season, because the temperature rises and humidity increases, extremely easily cause mother liquor bacterial infection, mould, cause the phenomenon presenting different colours in mother liquor or occur precipitation, waste raw material; Comparatively big error can be there is to the difference of the cognition of container scale (especially trace element), instrument precision in four fundamental rules because of weighing person.

In view of the above-mentioned shortcoming of mother liquor; with the mother liquor medium of routine; usually there will be the failed phenomenon that maybe cannot repeat of the result of the test caused because of factors such as weighing person, medicine, seasons; have impact on the process using Plant Tissue Breeding or cultivation technique without soil to carry out test greatly, hit the confidence of experimenter.

Summary of the invention

A scale preparation method for the basic solid culture matrix of AA, is characterized in that, include in described method accurate calculating, accurate weighing, first few after how mix, seal, the five steps such as use of breaking seal.

Method according to claim 1, is characterized in that, described five steps concrete operations are as follows:

1. accurately calculate: element in 18 contained by basic for AA solid-based is expanded 1000 times, i.e. calcium chloride dihydrate 440g, potassium dihydrogen phosphate 170g, epsom salt 370g, potassium chloride 2940g, potassium iodide 0.83g, boric acid 6.2g, Sodium Molybdate Dihydrate 0.25g, white vitriol 8.6g, four water manganese sulphate 22.3g, CoCL2 6H2O and each 0.025g of cupric sulfate pentahydrate, iron edetate 36.5g, inositol 100g, thiamine hydrochloride 0.5g, glycine 75g, Glu 877g, L-Aspartic acid 266g, L-arginine 228g;

2. accurate weighing: according to the result precise of 1. middle calculating, especially with CoCL2 6H2O and the cupric sulfate pentahydrate of each 0.025g of ten thousand/balance precise;

3. how to mix after first few: mix according to few priority of carrying out of amount more, namely mix according to the order of CoCL2 6H2O, cupric sulfate pentahydrate, Sodium Molybdate Dihydrate, thiamine hydrochloride, potassium iodide, boric acid, white vitriol, four water manganese sulphates, iron edetate, glycine, inositol, potassium dihydrogen phosphate, L-arginine, L-Aspartic acid, epsom salt, calcium chloride dihydrate, Glu, potassium chloride, amount to 5541.23g;

4. seal: the requirement of mixed solid matrix according to 250g every bottle is sub-packed in plastic containers, and seals;

5. to break seal use: as required, add the above-mentioned matrix 5.54g of precise in every 1L distilled water, heating, after it dissolves completely, adds reagent needed for other.

Beneficial effect of the present invention: without the error of calculation between the matrix of different batches; Because all reagent all expands 1000 times, the weighing error between the matrix of different batches is minimum; In theory, owing to being the usage amount (calculating with each 1L) of 1000 times at every turn, namely can prepare the above-mentioned formula of 1000L, batch inside there will not be the human error caused because using the reagent of different manufacturers, different times; Matrix after expansion is that every 250g is sealed in plastic containers, can avoid because the impact of the extraneous factor such as weather, temperature, moisture causes occurring the common precipitation of liquid mother liquor, the phenomenon such as mouldy; Above-mentioned plurality of advantages is all the follow-up test carried out for matrix with this medium, obtains result of the test accurately and lays the foundation.

Embodiment:

Below in conjunction with specific embodiment, the present invention is conducted further description.

The scale preparation method of the basic solid culture matrix of a kind of AA in the present embodiment, include accurate calculating, accurate weighing, first few after how mix, seal, the five steps such as use of breaking seal.

Above-mentioned five steps concrete operations are as follows:

1. accurately calculate: element in 18 contained by basic for AA solid-based is expanded 1000 times, i.e. calcium chloride dihydrate 440g, potassium dihydrogen phosphate 170g, epsom salt 370g, potassium chloride 2940g, potassium iodide 0.83g, boric acid 6.2g, Sodium Molybdate Dihydrate 0.25g, white vitriol 8.6g, four water manganese sulphate 22.3g, CoCL2 6H2O and each 0.025g of cupric sulfate pentahydrate, iron edetate 36.5g, inositol 100g, thiamine hydrochloride 0.5g, glycine 75g, Glu 877g, L-Aspartic acid 266g, L-arginine 228g;

2. accurate weighing: according to the result precise of 1. middle calculating, especially with CoCL2 6H2O and the cupric sulfate pentahydrate of each 0.025g of ten thousand/balance precise;

3. how to mix after first few: mix according to few priority of carrying out of amount more, namely mix according to the order of CoCL2 6H2O, cupric sulfate pentahydrate, Sodium Molybdate Dihydrate, thiamine hydrochloride, potassium iodide, boric acid, white vitriol, four water manganese sulphates, iron edetate, glycine, inositol, potassium dihydrogen phosphate, L-arginine, L-Aspartic acid, epsom salt, calcium chloride dihydrate, Glu, potassium chloride, amount to 5541.23g;

4. seal: the requirement of mixed solid matrix according to 250g every bottle is sub-packed in plastic containers, and seals;

5. to break seal use: as required, add the above-mentioned matrix 5.54g of precise in every 1L distilled water, heating is after it dissolves completely.

1L solution after above-mentioned substrate preparation is added sucrose and the 5g agar powder of 30g, and then add the KOH of 1N of 1ml, dissolve completely and be sub-packed in the vial of 30 200ml, after 121 DEG C of sterilizing 20min.On superclean bench, inoculate photinia glabra callus, after cultivating 30d under 25 DEG C of dark conditions, callus disperses and well-grown, in milk yellow.

Claims (3)

1. a scale preparation method for the basic solid culture matrix of AA, is characterized in that, include in described method accurate calculating, accurate weighing, first few after how mix, seal, the five steps such as use of breaking seal.

2. method according to claim 1, is characterized in that, described five steps concrete operations are as follows:

1. accurately calculate: element in 18 contained by basic for AA solid-based is expanded 1000 times, i.e. calcium chloride dihydrate 440g, potassium dihydrogen phosphate 170g, epsom salt 370g, potassium chloride 2940g, potassium iodide 0.83g, boric acid 6.2g, Sodium Molybdate Dihydrate 0.25g, white vitriol 8.6g, four water manganese sulphate 22.3g, CoCL2 6H2O and each 0.025g of cupric sulfate pentahydrate, iron edetate 36.5g, inositol 100g, thiamine hydrochloride 0.5g, glycine 75g, Glu 877g, L-Aspartic acid 266g, L-arginine 228g;

2. accurate weighing: according to the result precise of 1. middle calculating, especially with CoCL2 6H2O and the cupric sulfate pentahydrate of each 0.025g of ten thousand/balance precise;

3. how to mix after first few: mix according to few priority of carrying out of amount more, namely mix according to the order of CoCL2 6H2O, cupric sulfate pentahydrate, Sodium Molybdate Dihydrate, thiamine hydrochloride, potassium iodide, boric acid, white vitriol, four water manganese sulphates, iron edetate, glycine, inositol, potassium dihydrogen phosphate, L-arginine, L-Aspartic acid, epsom salt, calcium chloride dihydrate, Glu, potassium chloride, amount to 5541.23g;

4. seal: the requirement of mixed solid matrix according to 250g every bottle is sub-packed in plastic containers, and seals;

5. to break seal use: as required, add the above-mentioned matrix 5.54g of precise in every 1L distilled water, heating, after it dissolves completely, adds reagent needed for other.

3. method according to claim 1, is characterized in that, the scale preparation method of this culture matrix has following characteristics, i.e. without the error of calculation between the matrix of different batches; Because all reagent all expands 1000 times, the weighing error between the matrix of different batches is minimum; In theory, owing to being the usage amount (calculating with each 1L) of 1000 times at every turn, namely can prepare the above-mentioned formula of 1000L, batch inside there will not be the human error caused because using the reagent of different manufacturers, different times; Matrix after expansion is that every 250g is sealed in plastic containers, can avoid because the impact of the extraneous factor such as weather, temperature, moisture causes occurring the common precipitation of liquid mother liquor, the phenomenon such as mouldy; Above-mentioned plurality of advantages is all the follow-up test carried out for matrix with this medium, obtains result of the test accurately and lays the foundation.