Tissue Culture

[Vincent]

Does anyone know where i can find info on carnivorous plant tissue?(list of ingredients, preparations,etc.)This is my final frontier, and once i learn, i know i will make unspeakable amounts of CPs!!!(and i have to,)Speaking of which, i think its time to harvest D. capensis seeds. I have ridiculous amounts of seeds,and more flowers to come! anyone willing to trade or buy please ask.note i am buisy, so it might take me at most a day to get back with yas.

[arvin555]

Very short time to reply now, sorry, but try googling KitchenTissueCulture.

I bought a kit from them, but haven't tried it yet

I have several links to websites about TC for CP, but it's in my home PC I think.

TTFN
Arvin

[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

[Vincent]

Yeah i have looked at the kitchen culture kits, and i want one!tell me how it looks when u recieve it =)

Wow! thank u for that info! gonna keep a note book and write everything down.

[arvin555]

KitchenTC kits looks quite nice, I got the kit of course which includes a manual on how to do TC. The only things you need that are not in the Kit are usually easily found household chemicals and stuff. The hardest part for me is to find the Baby food jars, as it is not popular here. They sell them in Groceries, but hey my two kids never even tasted one. I was thinking to buy some and donate the contents, but quite difficult I think. So I am not able to use the nice jar caps that the kit contains with.

From the manual I think that PPM is one big factor for the kitchen Culture thing, it inhibits contaminant.

Jason, thanks for posting your system, really great help and info, because the Kitchen Culture manual has only a bit of a section about CP, there aren't any info about Nepenthes, only VFT and Drosera.

TTFN
Arvin

[jason840220]

As far as I am concern, leafs micropropagating nepenthes or sarracenia are fairly difficult. These plants develop such symbiosis relationship with the microbs contains inside their traps. In such, it's hard to get 'em sterilized. Perhaps TC using seeds is the best option. One of our forum member had tried but to no avail. Nevertheless, I never tried it on pitcher plants though.

I don't know 'bout the chemical contain in your kit. But it should come with phytohormones and PPM (or at least equivalent). The ratio of auxin to cytokinin in the media determines initiation of root versus shoot buds. Hence TC'ng using ready made kit yield higher success rate. Often, home made TC set struggle to have plantets to root.

[TimeEve]

I am interested in this. Maybe can learn from orchid tissues culture.

[Vincent]

I have an idea for making hybrids of nepenthes.Either seed or tissue would work, and you would need a big clear plastic tube about 4 ft tall. Basically grow the plants from seed or tissue to adult in these Tubes with 2 t-5 lighting units hanging down opposite from each other. hard to tell when they would flower,especially the two you wanna make seed from, but then if u had room u could have more pods. U would need probably 4 different size of chambers, 2 each,and different mixtures for eah set up pod.once you got to the 2nd pod, you would maybe induce normal leaf developement by laying off some of the hormones. at pod 3 you could add hormones to induce flowering(i think potassium?) whalla! harvest seeds, tissue culture em up! save a few for the collection, and use the rest for hybridizing more plants. Now my question is, will this little fantasy i played out in my mind work? Can you grow a plant to a fully flowering state, invitro? thank u's =P