FORUM IN VITRO

My experiences shows that it is very hard. I kept N.sanguinea in vitro growing for 2 months, after 2 months contamination occured.....Sterylisation was 3 steps- NaOCl, NaDCC and PPM..... nepenthes tissue is strong and hard to destroy, but also very hard to sterylise... Now i'm experimenting with meristem isolation, effects are now promising... But i need microscope to better isolation- actual i did basing on my eyesight.... It's very easy to destroy meristem, but for 4 attemps only one was contaminated. Also growth is so slow, and i don't know does the plant develop, or only stay "green"....

I want to try with not in all isolated meristem, sterylised well with NaDCC and PPM- first bath with NaOCl and top part of plant, next better isolation, cuttong away everything what is possible, short bath with NaDCC and long PPM....

Do you have any experiance with that? I know that everyone have his "secrets", but i think that hobbyst are not big hazard to companies producing plants by TC.....

regards

I have suceeded one time with sterilization of stem segment of N. x Ventrata in NaDCC 0.5g/L for 24 on shaker. But i think N.x Ventrata is exception ( i have read that other people suceeded with surface sterilization of this specie with bleach ). This method does not work for any other specie i have tried.

The steps before sterilization are important. It is necessary to clean up explant by rote and treat it in antibiotic. I use Tetracycline for the duration of 24 hours. Meristem is very sensitive and it can get dry in a laminar box . It is necessary to operate quickly.
Michal

Thanks for replies.


I have only chloramphenicol- 100mg/l + tween 80 for 24h should be OK?

What will you say on that protocol:

explant- top cutting with one "node" [~2 leaves]
pre cleaning with toothbrush under running tap water
shaking in tween 80 solution for 20-30 min
20h 100mg/l chloramphenicol
20 min 5% activated bleach
cutting off oxidized parts, all excess of leaves what can be removed
4h 1,5g NaDCC/l
2h 3% PPM

putting on 1/3 MS 30g surcose/l 2ml PPM/l

Regards

Michal, what concentration of tetracycline you are using ? I guess the 24hours treatment will require pretty low concentration < 50mg/L due fytotoxicity ?

sativ:
the antibiotic treatment must be at the end. There are articles, that using antibiotic before sterilization have no effect ( bleach or any other sterilant will wash or neutralize the antibiotic residues on/in the explant ) .

I didn't use antibiotic before, thanks for the advice.

Does anyone know what exacly symbiotic funghis/bacteries lives with nepenthes?

And i'm also interested about the concentration of tetracycline You use [whith tertracycline is it? the most common- CAS=60-54-8 ?]

Chloramphenicol is toxic but tetracykline is harmful only.
I use of concentration 100 mg/l .
Michal

If you are looking for really cheap generic Tetracycline, you can buy it for example here:

http://www.pharmacy1010.com/product_buy.asp?id=461

Tommorow i made first attempt with chloramphenicol.

1.Washing tunning tap ater
2.Dip EtOH 10s
3.Tween 80 5 min. [washing]
4. 5% activated, NaOCl, 15 min
5. 30h 0,5g NaDCC/l
6. 28h 100mg/l chloramphenicol
7.Passaged on media with 2ml PPM/l

Explant looks very healthy.

Regards

Hi Sativ,

I have try with N. trichocarpa and mirabilis but fail!!! This is my method:

-wash under running water: 5 min
-dip in alcohol 70%: 5s
-Tween 20: 5 min
-rinse in sterilized water: 5 min
-H2O2 10%: 5 min
-rinse in sterilized water: 5 min
-Bleach 10%: 10 min
-rinse in sterilized water: 5 min
-NaDCC 1g/l: 20h
-Plate

Did you have any success with your method?

Regards!