CN104719139A

CN104719139A - Massive preparation method of a high-precision 1/2 MS (Murashige & Skoog) culture substrate

Info

Publication number: CN104719139A
Application number: CN201510082511.7A
Authority: CN
Inventors: 钱夕刚
Original assignee: Individual
Current assignee: Individual
Priority date: 2015-02-16
Filing date: 2015-02-16
Publication date: 2015-06-24

Abstract

The invention discloses a massive preparation method of a high-precision 1/2 MS (Murashige & Skoog) culture substrate, and belongs to the technical field of plant biology. The method comprises the following steps: calculating 1000-times expanded quantity demand of 16 elements contained in the substrate and 10000-times expanded quantity demand of CuSO4.5H2O and CoCl2.6H2O; accurately weighting drug quantity; sequentially mixing the 1000-times expanded 16 elements according to the quantity demand from small to large; sealing; dissolving CuSO4.5H2O and CoCl2.6H2O with 1L of distilled water for later use; respectively opening to use as required. According to the method, the prepared substrate is high in precision; the method has the characteristics of being convenient and fast to operate, simple to apply, and small in error of different batches and the same batch, and is applicable to plant tissue culture and other biological applications.

Description

A kind of scale preparation method of high accuracy 1/2MS culture matrix

Technical field

The invention discloses a kind of scale preparation method of high accuracy 1/2MS culture matrix, belong to plant biotechnology field.Be more particularly one include accurate calculating, accurate weighing, first few after how mix, seal, mother liquor preparation separately, six steps such as use of breaking seal, to acquisition 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 high accuracy 1/2MS culture matrix, is characterized in that, include in described method accurate calculating, accurate weighing, first few after how mix, seal, prepared by independent mother liquor, six steps such as use of breaking seal.

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

1. accurately calculate: 16 kinds of elements contained by MS minimal medium are expanded 1000 times, i.e. thiamine hydrochloride 0.1g, Sodium Molybdate Dihydrate 0.25g, nicotinic acid 0.5g, puridoxine hydrochloride 0.5g, potassium iodide 0.83g, glycine 2g, boric acid 6.2g, white vitriol 8.6g, four water manganese sulphate 22.3g, iron edetate 36.5g, inositol 100g, potassium dihydrogen phosphate 85g, epsom salt 185g, calcium chloride dihydrate 220g, ammonium nitrate 825g, potassium nitrate 950g; 2 kinds of element CoCL2 6H2Os and cupric sulfate pentahydrate expand 10000 times, i.e. each 0.25g;

2. accurate weighing: according to the result precise of 1. middle calculating, especially with CoCL2 6H2O and the cupric sulfate pentahydrate of each 0.25g of ten thousand/balance precise;

3. how to mix after first few: mix according to few priority of carrying out of amount more, namely according to thiamine hydrochloride, Sodium Molybdate Dihydrate, nicotinic acid, puridoxine hydrochloride, potassium iodide, glycine, boric acid, white vitriol, four water manganese sulphates, iron edetate, inositol, potassium dihydrogen phosphate, epsom salt, calcium chloride dihydrate, ammonium nitrate, the order of potassium nitrate mixes, and amounts to 2242.78g;

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

5. mother liquor preparation separately: CoCL2 6H2O and cupric sulfate pentahydrate respectively weigh 0.25g, are dissolved in 1L distilled water, are mixed with 10000 times of mother liquors;

6. to break seal use: as required, add the above-mentioned solid matrix 2.24g of precise in every 1L distilled water, heating adds the independent mother liquor of 100 μ L CoCL2 6H2Os and cupric sulfate pentahydrate after it dissolves completely, and then adds reagent needed for other.

Beneficial effect of the present invention: the scale preparation method of this high precision culture matrix has following characteristics, i.e. without the error of calculation between the matrix of different batches; Because 16 kinds of reagent all expand 1000 times, 2 kinds of reagent expand 10000 times, and the weighing error between the matrix of different batches is minimum, and precision is high; In theory, due to the usage amount (calculating with each 1L) that solid matrix is 1000 times at every turn, namely the above-mentioned formula of 1000L can be prepared, liquid mother liquor only needs 100 μ L at every turn, and batch inside there will not be the human error caused because using the reagent of different manufacturers, different times; Solid 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, and fluid matrix is only the mixing of two kinds of elements.After expanding 10000 times, precision is high; 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 a kind of high accuracy 1/2MS (carbonaceous sources and mounting medium) solid culture matrix in the present embodiment, include accurate calculating, accurate weighing, first few after how mix, seal, prepared by independent mother liquor, six steps such as use of breaking seal.

Above-mentioned six step concrete operations are as follows:

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 stevia stem section, after cultivating 20d under 25 DEG C of illumination 12h conditions, take root good.

Claims

1. a scale preparation method for high accuracy 1/2MS culture matrix, is characterized in that, include in described method accurate calculating, accurate weighing, first few after how mix, seal, prepared by independent mother liquor, six steps such as use of breaking seal.

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

3. method according to claim 1, is characterized in that, the scale preparation method of this high precision culture matrix has following characteristics, i.e. without the error of calculation between the matrix of different batches; Because 16 kinds of reagent all expand 1000 times, 2 kinds of reagent expand 10000 times, and the weighing error between the matrix of different batches is minimum, and precision is high; In theory, due to the usage amount (calculating with each 1L) that solid matrix is 1000 times at every turn, namely the above-mentioned formula of 1000L can be prepared, liquid mother liquor only needs 100 μ L at every turn, and batch inside there will not be the human error caused because using the reagent of different manufacturers, different times; Solid 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, and fluid matrix is only the mixing of two kinds of elements; After expanding 10000 times, precision is high; 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.