Different types of media and established protocols are available for in vitro culture of plant embryos. Typical of these media are Murashige and Skoog (MS) medium, N6 medium and White's medium.
The subsequent paragraphs state instructions for processing Y3 medium used in culturing varieties of Cocos nucifera. The compounds can be purchased from any agricultural specialty store or ordered from large laboratory chemical suppliers. Despite the simplicity of brewing this solution for plant culture, laboratory safety measures should still be followed for a smooth and non-hazardous preparation.
Y3 MACRO STOCK SOLUTION
Analytical balance : Graduated cylinder : dark-colored 1 liter bottles
Chemicals : volumetric flask : refrigerator
Magnetic spinning bar : magnetic stirrer
Beakers : doubly distilled water
Initially, an analytical balance is used to weigh the following compounds:
• 5.35 g of Ammonium Chloride (NH4Cl)
• 2.94 g of Calcium Chloride Dihydrate (CaCl2•2H2O)
• 2.47 g of Magnesium Sulfate Heptahydrate (MgSO4•7H2O)
• 20.20 g of Potassium Nitrate (KNO3)
• 14.92 g of Potassium Chloride (KCl)
• 3.12 g of Sodium Dihydrogen Phosphate Dihydrate (NaH2PO4•2H2O)
Fill a 1000 mL-capacity beaker with 500 mL of doubly distilled water. Use the magnetic stirrer as you dissolve the chemicals in the water in the particular order: CaCl2•2H2O, then MgSO4•7H2O, KCl. Put in the rest of the substances, and increase the volume of the mixture to 1000 mL by adding in more doubly distilled water. The preparation should be put in the dark-colored bottle and stored at 4˚C.
The Y3 macro solution above is complemented by its micronutrient counterpart:
• 0.31 g of Boric Acid (H3BO3)
• 0.024 g Cobalt Chloride Hexahydrate (CoCl2•6H2O)
• 0.025 g of Copper Sulfate Pentahydrate (CuSO4•5H2O)
• 1.12 g of Manganese Sulfate Tetrahydrate (MnSO4•4H2O)
• 0.0024 g of Nickel Chloride Hexahydrate (NiCl2•6H2O)
• 0.024 g of Sodium Molybdate Dihydrate (Na2MoO4•2H2O)
• 0.83 g of Potassium Iodide (KI)
• 0.72 g of Zinc Sulfate Heptahydrate (ZnSO4•7H2O)
The same process is used to prepare this stock solution, except that the beaker is replaced by a 1000 mL volumetric flask. Another requirement for a complete Y3 culture medium is given below.
Y3 Iron Source Stock Solution
• 1.39 g of Iron Sulfate Heptahydrate
• 3.73 g of Sodium EDTA Dihydrate
The chemicals should be dissolved separately in 500 mL of doubly distilled water in a 1000 mL volumetric flask. Make 1 liter of the solution by adding doubly distilled water, transfer to a dark-colored bottle and store at 6˚C.
All of the given stock solutions, good for one liter each, make for a more convenient preparation of modified Y3 culture media. What's left for the biotech-savvy plant cultivator to do is just combine them along with some other ingredients.
Modified Y3 Culture Medium
Other ingredients are required to prepare the complete set:
• 2.50 g of Activated Carbon
• 6.00 g of Agar for initial solid medium preparation
• 0.1 M Hydrochloric Acid (HCl)
• 0.10 g of myoinositol
• 0.1 M Sodium Hydroxide (NaOH)
• 55.00 g of sucrose
• 100 mL of Vitamin mix (available from agro-chemical shops)
Apparatuses to be added include:
• Rubber stopper #4 with 2mm hole in middle stuffed with cotton
• Hot plate stirrer
• pH meter
• 20 mL large syringe without needle
• Heat-resistant plastic
• Culture bottle and test tubes
In this guide a solid medium is preferred for the primary culture, but the agar component can be omitted for succeeding cultures to save money and difficulty in handling. Liquid Y3 medium lessens damage to roots of plantlets before potting, while solid Y3 medium assists in keeping the embryo upright during incubation.
To begin, mix the 100 mL of the macronutrient solution with 10 mL of micronutrients, iron-EDTA solution, and vitamins in a 1000 mL beaker. Dissolve the sucrose and myoinositol with the help of the stirring apparatuses, and add doubly distilled water to make 1 liter.
Transfer the nutrient solution to a volumetric flask and stir continuously while adjusting pH to 5.8 using base or acid and a pH meter.
Add the activated carbon and stir for five minutes; blend in the agar for a solid solution.
Divide 15 mL of the medium equally to test tubes while constantly agitating it for even dispersal of the activated carbon. For older cultures, dispense 100 mL of medium in bottles. Cover bottles and tubes with rubber stopper and seal with plastic.
Autoclave the containers and their contents for 25 minutes at 121˚C, 15 PSI. Before cooling, shake the medium thoroughly to prevent settling of activated carbon. For immediate solidification, dip the setup in an ice bath after it is left to achieve room temperature.
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