[jason840220]

Not the expert, but here's how I do it. I devided micropropagation into 2 main processes:




(A) Media - Tools & Ingredients

• You need an empty jar as a TC vessel. Often you'd find others using baby food jar. I tried using peanut butter's but ended up melting the plastic lid. Galvanized metal cover is a good option.
• You need a media that can provide the neccessary nutrients on plant growth. Unless you have access to all those reagents, getting MS (Murashige & Skoog) is much prefered. There are various types of MS. As long as it's listed as "plant tissue culture tested", you're safe to go. 2 common MS used in TC is; (1) purely inorganic & (2) with organic. I got mine from Sigma-Aldrich.
• You need sucrose as food source since the cell is not able to photosynthesis in micropropagation. I tried both table sugar (white) and brown sugar while the latter yield contamination.
• You need agar as an agent that can provide enough surface tension to stand on the media. I bought 'em from local food store. If your pocket is deep enough, getting analytical grade agar isn't bad at all. Bottom line, it should be a neutral substrate. Don't buy those with flavor and color.
• You need either autoclave or pressure cooker to sterilize all items mentioned above. In my case, I use home pressure cooker.

(A) Media - Preparing TC Media

• Clean the empty jar/s and rinse thoroughly with DI water. Subsequently, steam-cleanse the open jar/s with the cover/s in normal cooking pot for approx 30mins. Let dry naturally.
• Since CP require less micro and macronutrients, to use MS as media, it generally needs to be diluted in strength. I use 1/2 strength. If you're buying MS from Sigma-Aldrich, each packet contain enough ingredient to prepare 1L of media. To get 1/2 MS, you can use half of the packet to make up 1L of salt. In my case, I divided MS into 4 equal portions in seperate jars.
• As for surcose, 20~30g/L generally works fine. If you're buying MS with organic (with sugar and agar), then the calculation must take into account how much sugar and agar is already in the packet. You only add back in the balance needed to get to the desired final concentration. Don't forget the amount of surcose mentioned above is in g/L. If you do as I did, each jar contains 12g of table sugar (in the case of 24g/L).
• You don't want too much agar in the media. As long as it provides enough surface tension, that'll be it. 6g/L is sufficient.
• Now you have 4 jars; each with 1/2MS strength, 12g of table sugar and 3g of agar.
• Lastly, I poured 500mL of boiling water into each jar while stirring vigorously. Becareful not to get any media on the rim of sides of jar as it might provide path to contamination. To play safe, you can mix 'em up before transfering the media into TC vessel. Close the lid/s and proceed for sterilization.
• You only need roughly 2~3cm media depth each jar. Each is approx 50~70mL of media. Thus, from each portion, you can make 'em into 10 seperate vessels.
• I put all of 'em into home pressure cooker. Parameter is set to 15psi at 120 degree Celsius for 20mins. When it's done, do not open the jars unless you want to have MS foam all over your place. I usually leave 'em overnight to cool down. This also has the advantage that the agar will be fully solidified.

(B) TC Sample - Tools & Reagents

• Isopropyl phenol or commonly known as rubbing alcohol. I use 90%.
• Bleach solution. I use Chlorox.
• Wetting agent. I use dish detergent.
• DI water.
• Other desired tools such as twisser, scissor, glove, TC chamber etc.
• TC sample - seeds / leafs / flower stalk

(B) TC Sample - Getting TC Sample/s

• Seeds - seeds are the easiest to propagate as they're the easiest to be sterilized.
• Leafs - if you intend to TC using leafs like I do, get the healthy ones. In case of Drosera, try getting those with dew. No insect please.
• Flower stalk - I am yet to try it out. Sorry can't help.

(B) TC Sample - Sterilization

• Dead or alive all depends on your technique. If your sample/s contain the slightest amount of microspores that could lead to contamination, I would say 10% success rate then. For this vital part, I performed it in closed room without air movement. Meaning, no fan nor air cond. You don't want much ventilation that could carry any contaminant onto your sterilized items. With years of working in laboratory, my skin has developed rather high tolerance on alcohol, otherwise wear gloves. I sterilize my lower amrs by spraying alcohol until the tip of my fingers then let dry (ooh~ tingling...). Whatever tools you might be using, do sterilize 'em. You should have all required items ready within hand-reach, including TC vessels. I also spray some alcohol up in the air before commencement (just a ritual practice).
• Seeds - Transfer some seeds on a filter paper and soak it with alcohol for 2~4mins. A flat tip forcep eases the process and filter paper serves as holder. After given time, transfer the paper and soak it into 10% bleach (dilution prior performed). 2 drops of dish detergent are added into the solution for better penetration. Soak for 2~4mins. Both process need to be accompanied by gentle shakes. DI water as final rinse.
• Leafs - I rinse off any dew (in case of Drosera) with DI water. Put 'em in a small bottle with DI water and gently shake it. Transfer the leafs and soak with alcohol (without filter paper) for 5mins. 10% bleach with wetting agent for 4mins. Final rinse with DI water.
• Once your samples are ready, you can transfer 'em carefully into your TC vessels. Let samples sit naturally on top of media. Close lid/s and seal with parafilm.
• I place 1 of the jars in my cultivation setup and never get started. The lights must have boosted up surrounding temperature so much that leads to my TC failure. Relatively, 1 of the jars that I put in my kitchen closet seemed to form callus in 2 weeks time. Temperature should be maintained around 25~28 degree Celsius. I learn this the hard way.
• After your plantlets have reached the size of a pea, you have a choice of further multiplying them in-vitro, or transferring them out to grow in regular soil/media. If you choose to multiply your plants further, you initiate the process by simply cutting up your sterile material and moving it into new media. At each stage, you may be able to increase the number of flasks by over 10 fold. Of course, all dissection work must be done under sterile conditions. A laminar-flow hood is really handy here, as the plant material will be exposed to possible contaminants for an extended period of time.
• When your plants start producing roots, you can think of taking 'em out from vessels (or you can continue growing 'em in there). Wash off any agar that might stick onto your plants and prepare propagation chambers. Freshly transfered plantets are rather fragile and need to be hardened. Provide 90~100% humidity for first few weeks then subsequently decrease until they can be treated as other "normal" plants.

Due to budget constrain, I have never tried these:

• Gibberillic acid
• Phyto-hormones (auxins and cythokinins)
• PPM
• TC chamber