How to Establish a Culture System Under Home Conditions

EstablishingOrchidThe working conditions for a rapid propagation system are not unattainably high; amateur enthusiasts can make do with simple facilities, complete all or most of the operations, and cultivate their ownorchidtest-tube plants. 1. Minimum Essential Supplies and Space Without the most basic conditions and supplies, tissue culture cannot be done. Some of these essentials must be purchased, while others can be made at home. 1. Essentials (1) One household pressure cooker, for sterilizing culture media, sterile water, etc. under high pressure; having a medical portable autoclave would be even better. (2) One inoculation box (sterile inoculation box); how to make it (method and requirements are given later); (3) One ordinary balance with a capacity of 100 grams is sufficient; (4) One stainless steel or aluminum pot (capacity 1-2 liters) for boiling and preparing culture media; (5) Several bottles and test tubes of various types for culturing; (6) One alcohol lamp, for flaming sterilization during inoculation; (7) 1-2 forceps 20-25 cm long for inoculation; (8) 1-2 scalpels and several blades for inoculation; (9) 5-10 small dishes for inoculation; (10) Several bottles for holding solutions; (11) One each of 1, 2, and 5 ml pipettes; (12) One each of 10, 50, 100, and 500 ml graduated cylinders; (13) One fruit juice extractor, for squeezing natural fruit juice to use in culture media; (14) Some absorbent cotton, rubber bands, kraft paper, etc.; With the above items, work can basically be carried out. A household pressure cooker has no pressure gauge. When sterilizing, first do not put on the valve; heat until boiling, and after 10 minutes put on the valve. When steam vents from the valve, start timing; after 15-20 minutes turn off the heat, slowly release the pressure, and sterilization is complete. 2. Basic Chemicals and Water Water does not need to be excessively pure, because in a sense the culture medium only plays a role similar to soil. As long as it contains few impurities, clean river water, well water, stream water, or tap water will all do. For basic chemicals, you only need to buy the most essential, most commonly used, and larger-quantity items. (1) The macronutrients of MS medium (5 chemicals in total); (2) Ordinary alcohol (for the lamp and for surface sterilization); (3) KOH, NaOH, and HCl for adjusting pH value, and precision pH test paper (pH 5.4-7.0); (4) One bottle of bleaching powder or bleaching powder concentrate; (5) Agar; (6) White sugar; (7) One bottle each of formalin and potassium permanganate. Other chemicals are used in very small amounts and require an analytical balance for weighing, so there is no need to prepare them yourself; you can buy pre-measured portions from relevant research or production departments and store them in a household refrigerator. Inorchidcultivation, if the Kyoto formula is commonly used, the chemicals are even simpler; you only need to buy 1-2 packs of Hyponex No. 1, plus peptone or tryptone, and you can avoid the trouble of preparing MS stock solutions. 3. How to Culture When carrying out aseptic orchid sowing or culturing under amateur conditions, natural conditions should be fully utilized. After inoculation, culture bottles should be placed in a place without direct sunlight, but they need relatively strong diffused light. During dark culture, cover them with a cardboard box. You can also make a small light culture box yourself from wooden strips and glass; when winter temperatures are low, place one or two light bulbs inside to increase the temperature. 4. Homemade Inoculation Box With few materials, a proper inoculation box can be made. The materials can be wood, plywood, glass, or acrylic, etc. The size depends on actual needs; generally 600-700 mm in length, 400-450 mm in width, and 500 mm in height. There are two holes on the front for inserting hands to operate, with sleeves fitted inside. There is a removable cover on the outside. It is best to install one 8-20 w ultraviolet lamp on the inside top for sterilization. Also install an alcohol lamp, so leave one ventilation hole with a diameter of 10 cm at the lower right of the back of the box and one at the upper part of the left side. Cover the lower hole with 5-6 layers of gauze (air inlet) and the upper hole with only 2-3 layers of gauze (air outlet), to facilitate air circulation. Otherwise, because the space inside the box is small and oxygen is insufficient, the alcohol lamp will soon go out. During fumigation sterilization, all ventilation holes must be closed and sealed tightly. The operation method for fumigation sterilization is: 2-10 hours before each use, place 2-4 grams of potassium permanganate in a small wide-mouth bottle, and pour 10-20 ml of formalin into the bottle. A reaction occurs immediately, and formalin gas fills the space inside the box, playing a sterilizing role. Remove the small bottle before operating. If it can be combined with ultraviolet lamp sterilization, the effect is better and it is safe to use.