1、学习植物组织培养技术Learn plant tissue culture techniques学习植物组织培养技术（Learn plant tissue culture techniques）I. teaching aims1. understand the basic principles, objectives, requirements and methods of plant tissue culture techniques.2. master the operation skills of plant tissue culture.Two, reference materialsM
2、edium preparation, a common medium in plant tissue culture, is the MS medium. The preparation of the MS medium comprises the following steps.Preparation and preservation of MS culture medium containing nearly 30 kinds of nutritional components of base liquor, in order to avoid every medium preparati
3、on should be carried out on the weighing dozens of ingredients, can be cultured in the medium of various components, according to the original amount of 20 times or 200 times respectively weighing, with concentrated liquid, this concentrated solution is called culture base liquor. In this way, each
4、use, take the total amount of 1/20 (50 mL) or 1/200 (5 mL), dilute with water, into the culture medium. The quantities of various substances required for the preparation of the mother liquid of the culture medium are listed for use in preparation.A large number of elements (mother liquor I) mg / LNH
5、4NO3 33000KNO3 38000CaCl2 2H2O 8800MgSO4 7H2O 7400KH2PO4 3400Trace elements (Mother Liquor II)KI 166H3BO3 1240MnSO4 4H2O 4460ZnSO4 7H2O 1720Na2MoO4 2H2O 50CuSO4 5H2O 5CoCl2 6H2O 5Iron salt (mother liquor III)FeSO4 7H2O 5560Na2-EDTA 2H2O 7460Organic ingredients (mother liquor IV)IV AInositol 20000IV
6、BNiacin 100Pyridoxine hydrochloride (vitamin B6) 100Thiamine hydrochloride (vitamin B1) 100Glycine 400The amount of all the nutrients mentioned above was 200 times of concentrated liquid except for mother liquor, which was 20 times concentrated liquid.Each of these mother liquids shall be individual
7、ly separated into 1 L stock solution. The preparation method of mother liquor, liquor and liquor are: II IV weighing several components each in the mother liquor after respectively with distilled water to dissolve a small amount, and then they are immiscible, the final volume to 1 L.The preparation
8、method is: Liquor III will be called FeSO4 7H2O and Na2-EDTA 2H2O respectively in 450 mL distilled water, heating and stirring to dissolve them, then the two solutions are mixed, and will be adjusted to pH 5.5, the final volume to 1 L, stored in brown glass bottle.After all the mother liquor has bee
9、n prepared, they are stored in glass bottles, labeled with mothers numbers, preparation times, dates and so on. They are kept in the refrigerator.MS medium adding 2 4-, two chlorophenoxyacetic acid (2, 4-D), NAA, 6-BA (NAA) 6- (6BA) and other plant growth regulators, and respectively with mother liq
10、uor (0.1 mg/mL). The preparation method is: the 3 substances were collected every 10 mg, 2, 4-D and NAA (1 mL) with a small amount of ethanol predissolving, 6BA (1 mL) with a small amount of substance is dissolved in NaOH solution for 0.1 mol/L, the dissolution process requires constant volume heati
11、ng in a water bath, at last to 100 mL, the concentration of 0.1 mg/mL solution.Preparation of liquid or using a pipette to remove the required amount from a variety of mother liquor respectively: mother liquor was 50 mL, II, III and IV liquor A and IV B 5 mL. Take 2 4-D, 5 mL, 1 NAA mL, and all kind
12、s of liquor together into a beaker.Should pay attention to preparation of medium: in the use of advance preparation of mother liquor, should take all kinds of liquor in the amount before, gently shake the bottle containing mother liquor, if found a bottle of precipitation, suspended or microbial con
13、tamination should be immediately eliminated, the mother liquor, re preparation; using a pipette or the amount of training base liquor before, must be taken by the volume of the cylinder or pipette liquor Guan Runxi 2 times; the amount of liquor, the liquor will be all the best according to the amoun
14、t of the order written on paper, Volume 1, crossed 1, in order to avoid mistakes.Melt agar with coarse balance were weighed, steamed sugar agar 9 g 30 g 1000 mL into the enamel cup, then add 750 mL of distilled water, with electric heating, heating and stirring with a glass rod until translucent liq
15、uid. Then add the prepared mixture to the boiling agar, and finally add the distilled water to 1000 mL, stirring evenly.It is important to note that the operator must not leave the agar when it is heated and the medium is prepared, otherwise boiling agar overflows,It needs to be re weighed and prepa
16、red. In addition, if there is no enamel cups available instead of beaker. But we should pay attention to the outer surface of the bottom of the beaker cannot touch water, otherwise when heating the solution beaker easily cracked, overflow, scald.PH with the dropper absorb amount of substance concent
17、ration of NaOH solution was 1 mol/L, each medium drops into the melt, while drops while stirring, and at any time with the pH test precision (5.4 7) measuring the pH of the medium, until the pH of the medium was 5.8 so far (the pH of the medium must be strictly control in 5.8).Medium should be packe
18、d separately when heated. When loading, the medium is poured into the beaker and the medium in the beaker is poured into a conical flask (50 mL or 100 mL). Be careful not to leave the culture medium to the mouth of the bottle and the wall of the bottle. The amount of the medium in a conical flask is
19、 about 1/5 to 1/4 of the capacity of a conical flask. Each 1000 mL medium can be divided into 2530 bottles.After the medium is packed, the bottle mouth should be sealed in time. 2 pieces of parchment paper (each about the size of 9 cm * 9 cm) sandwiched between 1 thin layers of kraft paper cover the
20、 bottle, and binding. Finally, label the outer cone of the conical flask.Autoclaving of high-pressure sterilization medium involves the following steps.First, put the flask. A conical flask containing a culture medium is placed in a small metal basket and placed in a autoclave. If there is no small
21、metal basket, it can be separated by a glass plate between the two conical bottles.Second place other items that need to be sterilized. Other items also need sterilization in high pressure steam sterilization pot, such as conical flask, filled with distilled water with a screw top glass bottle, beak
22、er, jar (the above items are to be wrapped in kraft paper, kraft paper coated with sealing) Petri dishes, scissors, tweezers, scalpel, filter paper and pencil etc.Third, sterilization. To be sterilized items stacked after cover. At 98 kPa and 121.3 DEG C, 20 min was sterilized.After sterilization, r
23、emove the conical flask and allow the medium to cool and solidify naturally. Better place 1 d before using.Fumigation sterilization inoculation box will be Potassium Permanganate in a glass or ceramic container, and then pour the formaldehyde. Two the amount is usually 6 8 per cubic meter of space w
24、ith the mass fraction of mL is 16% of Formaldehyde Solution (known as Faure Marin 40%), 4 5 g Potassium Permanganate. The fumigation method is irritating to the respiratory tract and eyes, so the operator should leave immediately after the mixture has been mixed.When the explants are required to use
25、 carrot roots as explants, the carrots should be washed and peeled before being sterilized, and be left in the middle and cut into small pieces. When the chrysanthemum is used as explants, its young leaves and tender stems are recommended. The explants must pass through the surface disinfectant befo
26、re use, in addition to the use of surface disinfectant sodium hypochlorite, can also be used with mass fraction of 9% 10% calcium hypochlorite solution 5 30 min, or with the volume fraction of 10% 12% hydrogen peroxide treatment 5 15 min sterilization.Vaccination requirements and precautionsThe expl
27、ants were prepared under sterile conditions and the sterilized experimental materials were cut into small pieces to prepare explants. For example, preparation method of carrot root is: with a scalpel short of carrot root around the left layer to form part of the intermediate zone, cut into 1 cm * 1
28、cm slices. As another example, preparation method is: Cut Chrysanthemum petiole, leaf veins from the cut, then cut into 3 4 mm2 needs to cut small pieces; tender stem internodes between two sections, about 1 cm. The excised explants should be put into sterile culture dishes.Attention should be paid
29、to the inoculation. First, each inoculum should be disinfected 1 times in an alcoholic solution of 75% alcohol, and then burned on the flame of an alcohol lamp, then cooled and then inoculated. Note that the alcohol stained tweezers wait until the alcohol evaporates and then burn on the alcohol ligh
30、t flame.Second, explants should be leveled when placed in culture medium. The number of explants placed in each bottle should be determined according to the size of the cone. Generally placed 34 carrots, 68 stems or leaves of chrysanthemum. Note that explants are not placed too small to take full ad
31、vantage of the nutrients in the culture medium.Third, the alcohol used for vaccination, the flame should not be transferred too high, vaccination should be close to the alcohol lamp flame operation, vaccination speed.Fourth, vaccination to prevent fire in the box.Culture of calli and PlantletsCallus
32、 culture usually takes 2 weeks from inoculation of explants to the emergence of callus. You can see the explant grow gradually nodular callus of white or yellowish white for 2 weeks. When callus is first cultivated, the door of the incubator should be closed without light, because the callus grows faster without light. 2 weeks later,Since the nutrient content in the culture medium is nearly exhausted, it is necessary to replace the medium for subculture.The subculture medium for callus subculture was the same as the medium used for callus culture. U
