Nepenthes Seed Germination

DennisZ

Carnivore
@Will20013 and I tag-teamed some Nepenthes seeds here at the lab.

I'll be posting updates if we get some germination.

Sterilization Techniques

Group 1: 10% bleach solution + tween20 for 5 minutes followed by 3 rinses in sterile water
Group 2: 7% bleach solution + tween20 for 5 minutes followed by 3 rinses in sterile water

Medium:

1/4 M/S , 30g/L sucrose, 1mL/L PPM , 2.2g/L phytagel

7360
7361
7363

7362
 

John Yates

Carnivorous Plant Addict
yep me too once is more than enough, more you rinse more chance you get contams , and may be cut down on your sugar to 20 g/Lt ,it may help :)
looks a pro set there in a real lab some one is lucky , some nep seed may need longer in the solution and look for colour changes rather
than time with 30 to 40 second agitation , as different seed have seed husk density differences and harbor deeper contaminates , some seed like rajah , atten , palawanensis , each have very short dense sheaths where lowii , villosa , jamban , dudia for example have much softer sheath and the solution penetrates quicker and cook quicker too , your 7% works but you will need from my experiments up to 12 to 15 min soak , if real fresh seed from a pod , its a lot easier , but who gets that , good luck ,please let us know your results really like to here how you go in a real lab , may make all the difference ?
 

DennisZ

Carnivore
So far, non of the seeds have any contamination and none of germinated yet. There is also a very thin layer of condensed water covering the seeds so despite the higher osmolarity-effect of our 50g/L gel, it should be fine (hopefully). I'll provide another update once we see some germination.
 

Will20013

Carnivore
Thats really interesting John! Do you wait for the seed to turn a lighter colour first, before removing them from the bleach? Around what concentrations/time in bleach do you typically begin to kill the seed?
 

Pooti Ne

Sprout
In my experience seeds will still germinate even when the seed coat (husk?) is completely transparent and embryo is light yellow to white. In some cases even that degree of sterilization can't completely eliminate contaminations.

I just got some germination from seeds plated 2-3 months ago, good luck!
 

John Yates

Carnivorous Plant Addict
Thats really interesting John! Do you wait for the seed to turn a lighter colour first, before removing them from the bleach? Around what concentrations/time in bleach do you typically begin to kill the seed?
Hi willy
as you would be ware by now nep seed can vary so much in there overall strength in the bleaching process , some seed like many high land species cook very quickly in 7 or 10 % bleach ,where others can take a lot longer soaking, and the freshness will play a big part in that process too.
looking at the seed in the bleach solution constantly with 30sec stirring , you will see color changes to the seed scape, catching it before total beaching happens is just a matter or observation and timing and some experience too, all though most seed if fresh will still germinate when totally whited out but not all ways :) using 10% bleach solution will of cause cause quicker over all bleaching round the 7 to 8 min with soft seed, and 12 to 15 min with the more harder seed, using a weaker solution you can soak longer of cause , but best to stay with one strength or you will become easily confused and get the processes wrong causing more fail's . the 7 % i have soaked up to 15 min easily with bleaching out the seed, but find the 10 % to be more effective for me over all. as we discussed looking at the chlorine now too
 
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Pooti Ne

Sprout
Hey Dennis, any updates? Did those seeds germinate for you?

John's definitely right sometimes even seeds that are harvested fresh they are unviable to begin with, especially those with a rather small embryo. A friend in US made some crosses with a beautiful singlana belirang but didn't get any germination (that clone should be fertile). Sadly, I seem to have better success rate with wild collected seeds than crosses made domestically ... however Thai and Vietnam growers constantly have seeds for sale that germinate readily, maybe you can find some during the spring!

Don't be discouraged! Germinating nepenthes seeds are rolling the dice to begin with, not to mention doing it in vitro. Since you have a flowhood PPM might be unnecessary. NaDCC + wetting agent worked really well for me since it bleaches much slower and is easier to work with. I've heard 1mg/L BAP helps germination but never tried myself, but that's a question for John ; ).
 

DennisZ

Carnivore
Thanks for the advice @Pooti Ne ! Unfortunately the seeds haven't changed since the last update and I think we are going to toss them soon. :(

@John Yates This was N.madgascariensis from a site that was soon-to-be-mined and a few other uncommon species (N.aristolochoides, N.inermis, etc).

Definitely will be looking for more seeds in the future, haven't given up yet!
 

John Yates

Carnivorous Plant Addict
In general In my experience ( IME ) :) , seed from neps have a very defined germination life in most cases , , low land seed have a general expected viable life of 3 to 4 months after that there germination rate decreases massively to where success is extremely low, in both T/c and natural , is why you will find they germinate quicker than high land seed generally most hybrid seed germinate quicker than specie, the high land seed have a slightly long life of 4 to 6 months max, depending if hybrid or specie seed , , after that there germination rate is extremely poor as well , ,so its why i tell people to get there harvest date from suppliers so you know approx how long you have viable seed for, using T/c can prolong there germination time a bit being in sterile condition's but the increase of contamination increases with Evey week there not sown , same with natural germination too , so fresher the seed the better rates of germination , this is all so helped with proper storage and packaging too ,that has been discussed by me in previous posts ,its very important to your seeds health and germination rates combined with the temps media and humidity and lighting ,all work to help you germination of nep seed quicker , other CP seed will act differently VFT, tuber, drosera ect, all have there own germination rates and seed life viability a whole other story
 

John Yates

Carnivorous Plant Addict
Have to add on reflection here in this post ,that there is no given , given time or method that is 100% right and true , what will work for one many not work as well for some one else , we all do things slightly differently ,and results will vary a lot ,so many out side factors here too ,my advice is read the info, try it and make adjustments as you see fit, ask questions there are now a good number of members here , that are very experience in CP T/c-ing , with some great and innovated ideas , the old school T/c is great but it needed a good freshening up, thinks this is going forawd now with great results ,nice work T/c-ers
 
hi, my metod for nephentes seeds is :
h2o2 10min
h2o steril 5min x3 times
1h ga3+ sterile h2o
only have 1tc with nepehentes and only have 1week, but all my test in other seeds(cepha, sarra, dionaea) is all contaminated, I not have laminar flow, I not used ppm. Now for sarra, dionaeas and cepha I used :
h2o2 5 min
7 min bleach 15%
5 min sterile H2O x3 time
24h in ga3 + sterile h2o
 

Lloyd Gordon

Cactus micrografter newbie.
Staff member
If you use GA3 add it to the medium. Sitting in a 24 hour soak invites contamination, especially after the sterilization step.
Also H2O2 is probably not harsh enough, I use it before alcohol/ bleach.
 
If you use GA3 add it to the medium. Sitting in a 24 hour soak invites contamination, especially after the sterilization step.
Also H2O2 is probably not harsh enough, I use it before alcohol/ bleach.
ok, in few days I say if is contaminated or not, I forgot say that before to introduce in tc, I soak the seed 1seg in distilled water and alcohol 99.9 (50% water/alcohol), I make a lot of test and all is contaminated
 

Lloyd Gordon

Cactus micrografter newbie.
Staff member
I'm not sure if a long presoak is good before sterilization. May weaken the seed coat and allow embryo damage. @WillyCKH , comments?
 
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