Nepenthes, Cephalotus, Drosera, Heliamphora ...etc, in vitro

[pokie22]

All purpose thread for all the cultures I have in the works. Most of my cultures are Nepenthes, but I should have a couple of everything...In the last two months, I have amassed nearly 100 cultures. I have listed the media in cases where it is not clear. Here are a few of the cute sprouts:

Nepenthes Attenboroughii (Jeremiah) 2 month old seedling on 1/4MS+2.5%sucrose+1mg/L BAP, ph5.6

Nepenthes Attenboroughii (Jeremiah)2 month old seedling; compost control

Nepenthes Glabrata putative

Nepenthes Glabrata; compost control putative

Drosera Regia in vitro

Drosera Regia; compost control

Pinguicula Lusitanica on ¼ MS + 1x Vits ph 5.75

Dionaea Fused Fuzzy on ⅓ MS + 1X Vits+ 3% sucrose ph 5.6

Dionaea Sawtooth II on ⅓ MS + 1X Vits+ 3% sucrose ph 5.6

Sarracenia Alabamensis; 24hr h2O presoak + 42hr 500ppm ga3 soak onto 1/3MS+1xvits+3%sucrose ph5.8

Sarracenia Jonesii -- red/HV;24hr h2O presoak + 42hr 500ppm ga3 soak onto 1/3MS+1xvits+3%sucrose ph5.8

Sarracenia Oreophila;24hr h2O presoak + 42hr 500ppm ga3 soak onto 1/3MS+1xvits+3%sucrose ph5.8

Darlingtonia Californica, Siskiyou Co; 42hr 500ppm ga3 soak onto 2/3KC+3%sucrose+0.5mg/L NAA ph5.8

Darlingtonia Californica, Del Norte Co; 42hr 500ppm ga3 soak onto 1/3MS+3%sucrose+1mg/L BAP ph5.6

Darlingtonia Californica, Del Norte Co; 42hr 500ppm ga3 soak onto 2/3KC+3%sucrose+1mg/L NAA ph5.6

In my hands, Darlingtonia California necessitates a ga3 treatment (or stratification) to break dormancy. My controls without ga3 +/- NAA or BAP, are not showing any signs of germination. Also, no cute sprouts on any media with NAA. Instead, they look like those white Chinese steamed buns (callus perhaps..) during the first rise.

 

[dionae]

Very good looking cultures pokie.

 

[DonH]

Very nice! It looks like everything is coming along great. Thanks for sharing your formula for success.

 

[BioZest]

Nice seedlings! I hope they do well for you

 

[pokie22]

Most of the sprouts will be for experiments. Extras will be given away. As far as the Nepenthes sprouts are concerned, I will be trying some protoplast fusion with the more abundant ones, and if a suitable SOP can be devised, then some ultra highlanders. I have made many yeast protoplast fusions for senescence experiments in the past, as well as dual mammalian species hybrids - those require sendai virus..etc. In most of those cases, one phenotype is dominant and silences the other spp by epigenetic means. Any requests Don?

 

[Vbkid]

Pokie, have you been doing this long? Any advice for someone looking to get started in it?

 

[DonH]

Most of the sprouts will be for experiments. Extras will be given away. As far as the Nepenthes sprouts are concerned, I will be trying some protoplast fusion with the more abundant ones, and if a suitable SOP can be devised, then some ultra highlanders. I have made many yeast protoplast fusions for senescence experiments in the past, as well as dual mammalian species hybrids - those require sendai virus..etc. In most of those cases, one phenotype is dominant and silences the other spp by epigenetic means. Any requests Don?

As soon as I decipher what you just wrote, I'll come up with a request.

Pokie, have you been doing this long? Any advice for someone looking to get started in it?

Pokie's a lab geek. Lol!

 

[pokie22]

Pokie, have you been doing this long? Any advice for someone looking to get started in it?

You should read my previous thread: http://www.terraforums.com/forums/showthread.php?132907-Nepenthes-Tissue-Culture

My first question is: How earnest are you in getting started?

For me, this is a past time when I am waiting on an experiment. And even so, there was an initial investment needed to acquire TC consumables that I did not have.

I started my first TC of CPs on Thanksgiving of 2012, so ~ 2 months. I have a few plants in compost, but watering is not for me. I started CP TC with Jeremiah's attenboroughii seed in late November. However, I am a scientist. It would be unfair to use me as a measure of how easily one can perform TC, as my normal work entails: cloning, mammalian tissue culture for antibody production purposes or small synthetic proteins, hESC, mESC, iPS cells, differentiation of primary human cells, phage display, algae for biodisel, anaerobic thermophillic fungi, lentivirus production....etc.etc. Therefore, sterile technique comes almost as a reflex to me, and almost no tissue culture will be as difficult as the coddling necessary to grow hESC on feeder layers. However, that is only part of the equation. The other part is to perform one's due diligence and educate yourself on what media or PGRs are necessary for the CP in question. Everything I have learned has either come from this forum, **********, WOC, ICPS boards or scientific journals. If you are unsure,there are many people with more experience that are usually happy to lend a helping hand. I know I have asked BigBella more than a few. Although, expecting someone else to just tell you some exact protocol when it is already freely available, is not the best choice. In the grand scheme of things, CP TC is rudimentary, and fairly easy. First, it is an organism which inherently has some self-defenses against potential nasties that would like to eat the jello. Second, one does not need to enumerate, count, or passage (very often). I am actually very surprised that most would wait to know the sex or spp of their Neps- it has been a great while since the dawn of molecular biology.

Tissue culture is not that difficult. One needs to be cogniscent of where your hands are at all times and everything that touches something sterile, needs to be sterile. If you lived close to me, I would invite you over. Seeing and doing, is different from reading and hearing. As I find with most of my students, learning by immersion is the best way. Of course, some media will be contaminated and there will be initial losses, but that is expected with the learning curve. It is only the idiot who does not learn from this - I have had a few of these too. After some trial and error, you will have a methodology that works for you. Have you visited the HTCG? They do sell starter kits. I do my TC where all other TC is performed - laminar flow hood. There are many DIY plans out there for. Albeit, it is not necessary, but does inhibit any contamination arising airborne spores.

I like to try new things all the time, and for the most part I am successful. Unlike most people, I like to do it right the first time. Just as I don't try to live, I never try to do anything - I just do. If you met me, you would understand.

 

[BigBella]

Very nice cultures there, Jen. I especially like those Pinguicula "cabbages" that you had mentioned the other day . . .

 

[amphirion]

Dang.... David and Jen, both active in tc and located in the Bay Area.... Us Bay Area cp growers are spoiled silly! Add in donH from SoCal---Cali is where it's at!

 

[pokie22]

Yeah, those cabbage patches are real tiny and seemingly sprout overnight. But I am pretty sure they will not be as cute as they grow, as with most things.

Amphirion, you know all bad girls are hiding some jello, somewhere.......

 

[DonH]

amph: My name doesn't belong in the same post as pokie and BigBella when it comes to TC. pokie literally does this for a living, although not for Neps. BigBella is the resident TC guru. I'm just someone who manages a research facility that has a lot of high end scientific equipment to play with and biotechs who can lend a helping hand if I have any questions.

Jen - If you want, I can replate a lowii seedling into a test tube and send it your way so you can try protoplast fusion with an attenboroughii (if you have an extra one). It would be interesting to see how it turns out.

 

[Vbkid]

Pokie,
I am very serious about getting started in TC. Much like you, I like to know my method and be fully prepared before starting anything, which is why I have been so hesitant. Having never performed TC before, there is just so much information it is hard to sort through. I think I've heard HTC broguth up enough times now that they will be my first stop.
With that being said, I am an engineer by trade with plenty of lab, contamination, and procedural experience that I am confident in my abilities to follow any appropriate method.
So if I would go the HTC route, would there be a special media I should request if I am mainly interested in nepnthes and possibly cephalotus?

 

[pokie22]

Vbkid,
If you search BigBella's numerous threads you can see that Murashige and Skoog +vitamins media and Knudsen-C orchid media will usually suffice. There is a thread specifically on TC here: http://www.flytrapcare.com/phpBB3/tissue-culture.html. I can not vouch for Cephalotus as all my jars are being stratified until mid March.

If you are hesitant, you can start off with some sacrificial lambs. For instance, I purchased some Nepenthes plants in September and tossed them outside to see if they would survive my negligence. I believe Dionae started with tomato seeds at first and a Birds of Paradise before embarking on CPs.

Following an SOP is trivial, given that there exist one. I believe that to be the most problematic for most novices as any many methods are singular or secretive. God save us.... My advice would be to read through some of the TC threads and make note of salient details such as media, PGRs, temperature, lighting, and sterilization techniques. This will serve as a basis for your standard SOP.

I would stay away from baby jars. Although, they are easily procured - and often free - they build condensation which obscures the ability to see anything, contamination or cute sprouts.

DonH - I have lowii seeds too on jello. Although, no sprouts yet. I can certainly can try that cross- it would be interesting. Of course, I will have extra attenboroughii - all sprouts are on the chopping board, so to speak. Unless, someone has specifically requested a plant. I am going to wait until the leaves are a little bigger - they are one second true leaves currently.

 

[pokie22]

As promised, the corn kernels!

Nepenthes Glabrata
These are my favorite so far. Short, pudgy, and very cute

Pinguicula Lusitanica
These are not the cute cabbages anymore.....

Drosera Regia - a comparison of in vitro vs. in vivo. These germinated at the same time and are on the same growing rack. Those oscomote pellets are working wonders.

Nepenthes Truncata Lowland

Nepenthes Truncata Lowland compost control

 

[mass]

Looking good Jen!

 

[amphirion]

sweet progress pokie! thanks for sharing.
parafilm.....fun fun fun fun....

 

[pokie22]

Parafilm was my first friend in lab. I use to wrap it around my fingers all the time. However, it did not bode well when I exhausted the roll and had to explain that I used it for finger wrapping......

Dry ice is also another favorite. I have fond memories of witch's brew from my elementary school days so I filled all the sinks with dry ice and added water. The entire lab was in smoke - lovely. That, and a couple plugged sinks later + boiling liquid Drano (10M NaOH), and everything was back to normal.

 

[pokie22]

The defunct cabbages are about to flower. See BigBella, I have patience. Almost a full circle from seed....

Pinguicula Lusitanica

 

[DonH]

Wow, those things grow fast!