What Is a Plant Tissue Culture?

Preparation and storage of the mother liquor of the medium. The MS medium contains nearly 30 kinds of nutrients. Weigh 20 times or 200 times respectively to prepare a concentrated solution. This concentrated solution is called the culture mother liquor. In this way, each time, take 1/20 (50 mL) or 1/200 (5 mL) of the total amount and dilute with water to make a culture solution. The quantities of various substances required to prepare the mother liquor of the culture medium are now listed for use in the preparation.

Plant tissue culture method

Right!

Preparation and storage of the mother liquor of the medium. The MS medium contains nearly 30 kinds of nutrients. In order to avoid weighing these dozens of ingredients each time the medium is prepared, the various ingredients in the medium can be weighed according to the original amount. Weigh 20 times or 200 times respectively to prepare a concentrated solution. This concentrated solution is called the culture mother liquor. In this way, each time, take 1/20 (50 mL) or 1/200 (5 mL) of the total amount and dilute with water to make a culture solution. The quantities of various substances required to prepare the mother liquor of the culture medium are now listed for use in the preparation.

Chinese name

Plant tissue culture method

Major hormone

Auxin

Principle

Plant totipotency

Select a part

Young plant part

1 MS media preparation involves the following steps.

Preparation and storage of the mother liquor of the medium. The MS medium contains nearly 30 kinds of nutrients. In order to avoid weighing these dozens of ingredients each time the medium is prepared, the various ingredients in the medium can be weighed according to the original amount. Weigh 20 times or 200 times respectively to prepare a concentrated solution. This concentrated solution is called the culture mother liquor. In this way, each time, take 1/20 (50 mL) or 1/200 (5 mL) of the total amount and dilute with water to make a culture solution. The quantities of various substances required to prepare the mother liquor of the culture medium are now listed for use in the preparation.

Large amount of elements (mother liquor I) mg / L

NH4NO3 33 000

KNO3 38 000

CaCl2 · 2H2O 8 800

MgSO4 · 7H2O 7 400

KH2PO4 3 400

Trace elements (mother liquor )

KI 166

H3BO3 1 240

MnSO4 · 4H2O 4 460

ZnSO4 · 7H2O 1 720

Na2MoO4 · 2H2O 50

CuSO4 · 5H2O 5

CoCl2 · 6H2O 5

Iron salt (mother liquor )

FeSO4 · 7H2O 5 560

Na2-EDTA2H2O 7 460

Organic ingredients (mother liquor IV)

IVA

Inositol 20 000

IVB

Niacin 100

Pyridoxine hydrochloride (vitamin B6) 100

Thiamine hydrochloride (vitamin B1) 100

Glycine 400

The dosages of the above various nutritional ingredients are 200-fold concentrated solution except mother liquor I is 20-fold concentrated solution.

The above-mentioned mother liquors should be separately prepared into a 1 L stock solution. Among them, the mother liquor I, mother liquor II and mother liquor IV are prepared by weighing several components in each mother liquor, thoroughly dissolving them with a small amount of distilled water, and then dissolving them to a final volume of 1 L.

The mother liquor is: put the weighed FeSO4 · 7H2O and Na2-EDTA · 2H2O into 450 mL of distilled water, and keep stirring while heating to dissolve them, then mix the two solutions and adjust the pH to 5.5 Make up to 1 L and store in a brown glass bottle.

After the various mother liquors are prepared, they are stored in glass bottles, labeled with the mother liquor number, preparation multiple, date, etc., and stored in the refrigerator freezer.

Plant growth regulators such as 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA), and 6-benzylpurine (6BA) need to be added to the MS medium, and formulated into mother liquor ( 0.1 mg / mL). The preparation method is: Weigh out 10 mg of each of the three substances, pre-dissolve 2,4-D and NAA with a small amount (1 mL) of absolute ethanol, and 6BA with a small amount (1 mL) of the substance. The 0.1 mol / L NaOH solution was dissolved, and the dissolution process required heating in a water bath. Finally, the volume was adjusted to 100 mL to obtain a mother liquor with a mass concentration of 0.1 mg / mL.

Prepare the required amount of the culture liquid from the various mother liquors using a measuring cylinder or pipette: mother liquor I is 50 mL, and mother liquor II, III, IVA, and IVB are each 5 mL. Then take 2,4-D 5 mL and NAA 1 mL and put them into the beaker together with various mother liquors.

When preparing the culture solution, please pay attention to the following: When using the mother solution prepared in advance, before measuring all kinds of mother solution, shake the bottle containing the mother solution gently. If there is sediment, suspended matter or contamination with microorganisms in the bottle, immediately Eliminate this kind of mother liquor, and remake it; Before measuring the mother liquor of the medium with a measuring cylinder or pipette, you must rinse the measuring cylinder or pipette twice with the measured mother liquor; When measuring the mother liquor, it is best to Write the various mother liquors on the paper in the order you want to measure them. Measure 1 and cross out 1 to avoid mistakes.

Weigh 9 g of agar and 30 g of steamed sugar with a crude balance, put them into a 1000 mL enamel measuring cup, add 750 mL of distilled water, heat in an electric furnace, and stir with a glass rod while heating until the liquid is translucent shape. Then add the prepared mixed culture solution to the boiled agar, add distilled water to the final volume to 1 000 mL, and stir well.

Adjust the pH. Use a dropper to suck the NaOH solution with a concentration of 1 mol / L, dropwise into the dissolved medium, stir while dropping, and measure the medium at any time with a precision pH test paper (5.4 7.0). pH until the pH of the medium is 5.8 (the pH of the medium must be strictly controlled at 5.8).

2 BA is cytokinin NAA is naphthaleneacetic acid, a kind of auxin

3 hormones suitable for lily root have not been found

The following are the hormones of leaves and embryos: Aseptic culture of LiliumlongiflorumThunb leaves can be obtained in vitro. The test results show that the medium MS + BA 0 1 mg / L + NAA 10 mg / L is suitable for primary culture and helps the differentiation of roots and shoots; MS + BA 0 5 mg / L + NAA1 5 mg / L is suitable for proliferation culture, Conducive to the generation of callus and promote the differentiation of shoots; rooting medium is 1/2 MS + NAA 10 mg / L + PP333 100 mg / L; when the test tube seedling root length is 1 cm, it is easy to survive transplantation

Cultured on MS medium containing 1 mg / L IAA, 1 mg / L GA3, 600 mg / L CH, and sucrose content of 6% to 8%, lily embryo embryo growth was the best, cultured for 30 days Normal embryos can be obtained later. Pale green callus can be formed on MS medium containing 0.1 mg / L NAA, 1 mg / L BA, and these callus can be transferred to 1/2 MS hormone-free culture A large number of adventitious buds can be formed by culturing on the basis.After adventitious buds have been transplanted, their survival rate can reach more than 90%.