TC Australian Drosera and Cephalotus

[pygmydrosera]

Hi All,

Who have here any experience with TC Australian drosera's and Cephalotus ?? I try and try and try but until now it will not work here. All the time nothing happend with the leafs I cut off and I put on the agar. So maybe my desinfecting is not good to strng to long or something of that. Please can somebody who TC Petiolaris Complex drosera or Pygmy drosera or Cephalotus help me with what is the best way for desinfect what to use and for how long. And when I will use PPM is there anybody here who tried this already on this group of drosera and how many PPM do I need for 1 Liter media ??

PLease can somebody help me ??

Regards

Tom

[coldcoffee]

What are you doing? Ie you say you have tried, what has been your attempt so far? Plant hormones, disinfection protocol, etc.... I have not done pygmys yet but am currently working in cephs.

[coldcoffee]

Oh, and when you try things, what happens?

[pygmydrosera]

Hi

I use for Drosera 1/3 Ms medium, Kinetin and IBA

and for

Cephalotus 1/2 Ms medium, 2Ip, GA3, IBA, Adenine Hemisulfate

Both with 30 /L sugar and 7g/l agar or Gelcarin GP 812 NF

The problem is I made my medium in bottles I autoclave it for 45 min at 15 PSI I use selfindicated bioindicators to see that the sterilisatie proces whas good.

I use a Tyvek overal with gloves, mounth mask and cap over my head so fully packed. I clean my hands with 70% ethanol.

I use long tweezers who sterilized first in autoclave and between two transfers I resterilize it with 70% alcohol and a bunsen burner on the blue flame the hotest.

But all the time my plants will be around with mold Penicillium like mold so somewhere in my sterilisatie proces of my ex-plant it going wrong. Today I get a sample bottle of 30 ml PPM so I put 1 Ml / L to my media I freshly made today. And I even make a bottle with 2 times MS media and 95 ml water and 5 ml PPM. They told me to desinfect there my ex-plant in and then directly without rincing putting the ex-plant parts on the surface of my media.

Please can anybody give me some advice can I use also other desinfecting stuff like

Bleach, Isopropylalcohol, Ethanol or Hydrogen Peroxide ??

For how long and how strong must the sulution are ??

Regards

Tom

[coldcoffee]

Personally, using IBA on a Drosera would not be something I would try first. I would try using no PGRs or just using the Kinetin. That is a personal thought. Your choice of PGRs may have come from experience and you may very well have the right approach.

With some droseras, I have even had success with 1/2 MS (I have a D. Binata explant which is exploding right now that I did on a 1/2 MS + Kinetin + PPM, I have not tried 1/3 MS for comparison with that species), I am not nessasarily suggesting that this is the way to go, just simply an observation.

As for the Ceph, how did you try disinfecting it? Am I reading your message correctly that someone advised that you not rinse after a disinfection soak? I would not do that myself. Try a 10% bleach solution- dip the explant in some isopropyl for a few seconds then soak in the bleach solution for 10 minutes. I have also heard of people using Physan to disinfect explants.

What are you taking for your explant?

[pygmydrosera]

Hi Ryan,

Here the text I get from Dr Assaf Guri (this is the person who made PPM)

Dear Tom.
You can try to add 1ml/l PPM but I don't think it will work.
Therefore add 5 ml PPM to 95 ml sterile water and as I mentioned to you in my first E-mail gently stir some of the the ex-plants for 4 hours some for 6 and some for 8 hours. Find out which time period is most effective. You should add 2X MS basal salts to the solution to obtain better ex-plants' protection.
You don't need to use the antibiotics at al and without rinsing place the explants on the medium. Space the explants to avoid cross contamination.
Finely see which time period gives you less contamination and better servival rate( from the treatment with PPM ).
Let me know later about the results.
Assaf

[pygmydrosera]

Hi Ryan,

I have also a question for you. You told that you have on this moment a exploding D. binata.

Please can you tell me what part you used for TC ?? and did you put it into the agar or did you placed it flat on top of the agar.

And can you describe you ex-plant desinfection method for D. binata

did you use PPM also as desinfectand for your ex-plant ?? Or did you use bleach or something else and for how long ??

I hope you will share this info.

[coldcoffee]

Jul 29, 2011 10:22:54 GMT pygmydrosera said:

Hi Ryan,

Here the text I get from Dr Assaf Guri (this is the person who made PPM)

Dear Tom.
You can try to add 1ml/l PPM but I don't think it will work.
Therefore add 5 ml PPM to 95 ml sterile water and as I mentioned to you in my first E-mail gently stir some of the the ex-plants for 4 hours some for 6 and some for 8 hours. Find out which time period is most effective. You should add 2X MS basal salts to the solution to obtain better ex-plants' protection.
You don't need to use the antibiotics at al and without rinsing place the explants on the medium. Space the explants to avoid cross contamination.
Finely see which time period gives you less contamination and better servival rate( from the treatment with PPM ).
Let me know later about the results.
Assaf

To be honest, I have never used PPM as a disinfectant, honestly the thought never occurred to me- I have been using it in my media to suppress any contamination which might get past my disinfection procedure. I might have to try this out. When I get the time (perhaps this weekend, I was thinking of cutting some dionaea explants and trying them with and without PPM in the media mixture just to see what happens).

[edit: Ah! I see now what you meant when you said no rinsing. You were disinfecting using PPM- I had it in my head you were using bleach for some reason, that actually makes a little more sense then. I have never done that, but I will certainly be trying that out. Sometimes at the end of a disinfection, I like to dip in hydrogen peroxide and then not rinse it off- I have tried this particularly with seeds and have had some success- not sure if it is because of that step though...]

[coldcoffee]

Ok, I have my notebook here. Here is the procedure I got from a paper that I was given by Carol at the HTCG (Home tissue culture group):
1cm long shoots are taken as the explant and first shook in soapy water (Her source publication actually calls for shaking them in some Physan for 5 minutes, I believe they mean this or something similar:
www.amazon.com/Orchids-R-Us-Inc-Physan/dp/B000I2UTAQ/ref=sr_1_1?ie=UTF8&qid=1311946588&sr=8-1)
2) Disinfect in a 10% bleach solution for 10 minutes
3) Soak in sterile water for 5 minutes (I think when did this I rinsed 2 or 3 times in sterile water)
4) Dip in 70% Isopropyl then blot on a sterile paper towel

5) Them cut the explants down as you like and innoculate the media.

[coldcoffee]

Jul 29, 2011 10:26:59 GMT pygmydrosera said:

Hi Ryan,

I have also a question for you. You told that you have on this moment a exploding D. binata.

Please can you tell me what part you used for TC ?? and did you put it into the agar or did you placed it flat on top of the agar.

And can you describe you ex-plant desinfection method for D. binata

did you use PPM also as desinfectand for your ex-plant ?? Or did you use bleach or something else and for how long ??

I hope you will share this info.

I will comment on this more when I get to work (and advantage of being a software developer is that I always have a computer with me ), but yes the D. Binata has been my second most dramatic success thus far. I am waiting on a D. multifidea 'extreme' explant which has not formed a callus yet, and a few other drosera.

For the D. Binata, I either used a very young leaf tip or a young flower bud. I thought I used a flower bud but I second guessed myself on that last night. I will try to find that in my notes. I always place my initiation explants on top of the agar, not in.

I will comment on the disinfection in the near future.

[coldcoffee]

I can't download stuff onto my computer at work, so when I get home tonight I will forward to you the papers that Carol sent me on Cephalotus. I imagine you will find them very helpful

Now, regarding the D. Binata. Embarrasingly, my record sheet with the details of that tissue culture session are not in my binder where they are supposed to be :S I will have to find it. But I am pretty sure I did something of this sort:

For the disinfection, I may or may not have first dipped the explant in isopropyl alcohol that time, usually I do but there have been exceptions. I generally count to 5 then pull it out. I used a 10% bleach solution (with a drop or 2 of dish-washing detergent mixed in as a wetting agent- commonly in the literature you see references to "Tween" or "tween 20", dish-washing detergent serves this purpose for us amateurs) and probably let it soak for 10 minutes. Then I definitely did 3 rinses with autoclaved water- the first rinse was definitely 5 minutes, the last two were either both 5 minutes, or less, I am not sure off the top of my head. Sometimes with the last rinses I do them quick.

After that, take the explant out and prepare it. I have always cut the severed end off about a quarter to half of the way up the explant in case there is any residual disinfectant still in the explant. Then it is time for innoculation.

For media, I am pretty sure I used 1/2MS + standard dose Kinetin + standard dose PPM + 1TBS sugar/liter. I adjusted the pH to about 5.5 or 5.6 (when I find my notes I can post exactly what the reading was, I think it was around 5.62 or something like that... but don't quote me on that)

The explant was placed on top of the agar as I mentioned before- not inside of it.

Three things of interest:
1) I found a paper in my notebook that Carol sent to me in which the authors used low doeses of BA and NAA instead of standard dose of Kinetin (pH was 5.7 in that paper).
2) During this particular incubation, my explant turned almost completely black. This is usually bad because poisons are leached into the media, but there was just enough good material to send out little plantlets. (I will post pix later)
3) Most of this is standard procedure- nothing original on my part, (although some protocols I have used are original and are currently being tested by me, mostly new ways of doing old stuff). In fact, I will fully admit that I am not a TC expert and have a lot to learn. Please take any of my suggestions for what they are- based on my personal experience which is quite limited at this point. (The people at HTCG are very good at what they do- you are in good company there)
I hope this helps. I will send those docs off to you later if Carol has not already.

Ok, now, since I typed all this up- I want to see pics of your plants (if you have any, if not no worries)!

[pygmydrosera]

Hi coldcoffee,

I don't know what pictures you mean on my website where I working on at the moment you can see some pictures www.australian-drosera.com

But I have a question for you when you put invitro plants on peat do you first sterilize the peat by autoclaving it for 2 hours or do you directly transfer the sterile new growen plants from the bottle to non sterile peat

I ask you this because sometimes I have trouble with some insects