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lesthegringo

Inermis seeds germinating

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Oh dear, this is not good - these seeds were put into the in-vitro jar on the 21st June, so everything was OK for nearly two months, then I looked this morning..... The last time I checked it was Wednesday so this happened quickly. For info, the seeds were treated to 10 minutes in a 6% H2O2 solution, then dipped in a fungicide, then left in a PPM solution for 10 minutes. Clearly wasn't enough for these seeds, though interestingly I have jars done at the same time using the same method that are absolutely clear. For info these are N Jacquelineae seeds, luckily I have had considerable success in getting these to germinate so this isn't a disaster

 

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Still, on a more positive note, I have had some success in transplanting seeds from spoiled in-vitro jars to sphagnum. I got the gel out with the seeds, rinsed them in warm tap water to remove as much of the gel and fungus as possible and then put them in a 6% H2O2 solution for a couple of minutes. I then sowed them onto live sphagnum, where they appear now to be germinating. This gives me hope that I can rescue any seeds in a similar situation in future

 

As for further progress, here is N x Pyriformis starting to germinate, there are a number of seeds showing signs of life

 

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This is one of the N Jamban coming along nicely

 

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N Inermis

 

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N Jacquelieae

 

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N Talangensis

 

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N Dubia (sorry for the rubbish photo)

 

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And here's a couple of shots of the in-vitro jar in the previous posts

 

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Hope you enjoy!

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It's absolutely wonderful to follow your post Les! I've been taking notes and actually used some of your techniques with my seeds  :Laie_98:

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It occurred to me that I had never posted general pics of the greenhouse and all the seed trays - so here you go

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There are a total of 92 of those little blue seed trays, and each tray has 8 pots, although some do have Sarracenia and Darlingtonia seed in them.

The multicoloured plastic pots are the in vitro jars, those on the left are ones that have seeds germinating, those on the right have yet to show any signs of life.

You can see some cream coloured tubing above the seed trays, these are the supply tubes to the misting heads, and were kits by a company called Holman. They are primarily for misting to cool outdoor areas, but with a good high pressure pump are perfect for a nepenthes house. A total of around 25 misting heads are distributed around the greenhouse, primarily along the roof ridge and where the roof meets the walls. The pump is connected via an electronic timer that (at the moment) turns on the pump twice a day for 3 minutes each time. In summer that will be changed to four times a day.

If anyone wants to do the same, be aware that you still have to ensure that the plants are all correctly watered, as the distribution is not easy to control, so a couple of times a week I go in and hand water everything in the pots.

Edited by lesthegringo
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Oh wow. I didn't expect this nice greenhouse!!! There are A Lott of beautiful seedlings!!!!!!!

Wow you look like a nepenthes lover. Oh look at the huge leaf of nepenthes. That is very big ! I wanna visit there. But too far. ;(

Sent from my iPhone using Tapatalk

Edited by fksdl421
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This is the biggest plant, a N Maxima x Sibuyanensis which at the moment doesn't seem keen on producing upper pitchers - it's about four feet (120cm) tall

 

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And look at this tiny little Inermis, isn't it cute?

 

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Here's a better picture of the greenhouse, this time in focus

 

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Edited by lesthegringo
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Just one very quick update today, to say that after 90 days one of the N Maxima seeds has finally germinated. As you can see the dopey thing was growing upside down, I've since gently moved it so that it is the right way up.

 

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I have to confess that after so long I had consigned it to the shelf of shame, along with a few other trays that were refusing to play ball - not that I was about to throw them away, though. I have read about people seeing seeds germinate after a year, so any trays that don't have seeds that germinate within 3 months go to the secondary germination area, AKA the shelf of shame, AKA the top of the kids bookshelf. I seal them in zip-loc bags after giving them a good watering, and then on to the top of the bookshelf they go where it is warm. The advantage is that it clears space in the greenhouse, plus it also means that I'm not checking on them so often, so when growth has happened it is obvious and a pleasant surprise.

 

One last update concerns the rescue of some seedlings that had germinated in vitro, only for the dreaded mould to appear. A jar with mixed highland seeds had six or seven seedlings that had sprouted, but a yeasty looking (and smelling) substance grew over the surface of the gel. As with the seeds that had mould in vitro, I gingerly extracted the seedlings, washed the gel off and rinsed the seedlings in a weak bleach solution and then water. I then planted the seedlings in live sphagnum and put it with the other seeds. I briefly considered sealing it in a zip-loc bag, but decided that a closed environment probably helps the mould more than the plant. I don't know if they will survive, but this way I can see if this strategy works or not. I don't have anything to lose, they would have been doomed anyway if I'd have left them. It is very labour intensive, though, not something I would want to have to do often.

 

Since the seeds still in the jar would probably have died eventually due to the mould contamination, I washed out the seeds and used the same bleach solution and rinse, and planted them on the remainder of the tray with the seedlings. With luck I may have saved them, again it is something I hope works.

 

Cheers

Edited by lesthegringo
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Well, the attempt to save the seedlings from a spoiled in-vitro jar failed. The six or seven seedlings have all died, so my method of transferring them to sphagnum doesn't work. I don't know if it was due to the seedlings not liking the root stem being moved or the fungicidal washing treatment, but whatever, my first seedling deaths have occurred, R.I.P. plantlets

On a brighter note, the seeds I rescued look to be faring better, with some germinating, so not a complete disaster.

The fact that I have had variable success with in-vitro shows that it can be done, and when it works it is a great method, but I clearly need to pin down the sterelisation technique for it to be useful going forward. Some batches of seed were awful, with every jar spoiling (Reintwardiana for example, although they are germinating on sphagnum ok) while others seem to have been easy.

I have a dionaea which is putting up a flower stalk, I will try to do some tissue culture on that and use it to try some different sterelisation techniques.

One thing I have concluded is that it is best to plant seeds on sphagnum as well as in-vitro to guarantee a greater chance of success.

Over the weekend I will try and photograph the seedlings showing the age of the plants to help you guys see how they develop and how quickly (or more accurately slowly) they grow.

As a last comment, Drosera Capensis is taking over my greenhouse; I only have a couple of plants that were in the greenhouse for a while, but while they were there they flowered, and the seeds must have been blown round the greenhouse. Most of the nepenthes seed trays have hundreds of tiny seedlings. So, if all else fails, at least I'll have those to grow!

Les

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If anyone tried to germinate seeds using Kukata's 'seedsprouter' thing, I think they will just end up with a soggy mass of dead seeds........

 

Anyway, a while back when I started setting seeds to germinate, I had purchased seeds not realising how many each pod would contain. As a result, I had more than I could realistically sow, so I realised that unless I did something with them they would eventually die and be of no use to anyone. I gave a load of seeds away to people on this forum, but was still left with some stragglers that I didn't know what to do with..... so I simply sowed them in the pots with my adult plants and also in some trays where I am growing sphagnum so that I have some live media for planting. I then promptly forgot all about them.

 

I was having a tidy up today (boy, did the greenhouse need it) and also was moving stuff around to try and improve the light getting to my adult plants, while making sure that direct sunlight wasn't going to shine on the seedlings, now that the sun finally clears next doors fence. Of course, all the adult plants had to be moved, and I saw a load of seedlings in a number of the pots. Nepenthes seedlings, from those seeds I had rather dismissively sown months ago. Which ones? No idea, but who cares, they are nepenthes.

 

This is going to sound stupid, but I think I honestly never expected any of them to germinate. Why, I don't know, maybe I though that they needed more special care that just being dumped unceremoniously on the pots. Logically, though, it's only what happens in nature, so I shouldn't have been surprised. If the conditions are right for the adult plants, why shouldn't it work for the seeds and seedlings?

 

Still, it is so cool to see them, and it finally convinced me that growing nepenthes from seed is not super hard or full of secrets so I encourage everyone to try.

 

As for the in-vitro stuff, yes, it is working, but I must now confess that in the face of the success in germinating seeds in a 'normal' manner, it is hard to justify the extra expense, hassle and complexity of the in-vitro setup. Was it fun? Yes. Interesting? Definitely. Better than letting seeds do what they do in nature? Errrr, maybe not. Don't get me wrong, I enjoyed doing it and would still have done so knowing what I do now, but I don't think I would try and convice anyone else to do so unless they really were looking for a commercial setup as I think the returns for a hobby grower are not there.

 

We live and learn!

Edited by lesthegringo
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Against expectations, the Lowii seeds I sowed have germinated, along with some Aristolochioides. Not many, to be fair and I have heard that they are both slow growers, but still quite pleasing. Encouragingly so far I have not lost any seedlings to damp off or fungus gnats from the seeds sewn in traditional media so the oldest plants are approaching 3 months old. Hopefully that will continue to be the case.

 

I also bought myself a new toy, a (cheap) USB microscope so that I could look at my little plants, look at the pics I was able to take of these little Inermis seedlings! This is a three month old plant

 

 

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Edited by lesthegringo
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Adorable babies! Good luck with them! I also have four 4-month-old nep seedlings crossed by myself. Its exciting to see how they grow and gain size :)

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The in-vitro experiment is definitely polarising my opinion. Pots that go for months, with no problems and with seedlings sprouting suddenly go bad, with a kaleidoscope of different coloured moulds suddenly claiming them, One thing I am certain of is that you cannot, under any circumstances, let the sun shine on them, as this really seems to trigger the mould. However, there are some that are doing incredibly well, with absolutely no sign of any infection and with the little plantlets growing away very happily.

 

As a result of the spoiling of the in-vitro jars, I have had to try and recover quite a number now, and can report better success on this that with some of my prior attempts. On the basis that the seedlings, or more to the point the super delicate roots, don't like harsh chemicals I decided to see if I could just move the seeds and seedlings onto sphagnum after only a rinse in rainwater. Maybe the sphagnum has some anti-bacterial properties, maybe the mould doesn't grow as well outside of the in-vitro conditions, but it certainly seems to be working. I have seedlings that I was sure would be dead still alive and frankly looking good ten days after their rescue, and seeds are germinating on their new substrate.

 

On a different note, we will have to move house at the end of the year due to the owner of the house we rent wanting it back. A pain, but one I intend to turn to my advantage. I told my wife how traumatic it would be for all my poor plants to have to move them in the hottest, driest time of year, and that to dismantle the greenhouse, transport it and re-assemble it would take days, especially with everything else going on. Wouldn't it be better, I told her, to find somewhere where I can build a new greenhouse, then move all the plants over, then I could deal with the old one at my leisure....? So it looks like I will have two greenhouses soon, one for highland, one for lowland. Of course, I haven't actually got any lowland Nepenthes yet, but let's not nit-pick.

 

Lastly, a word on sphagnum vs coir as a substrate. Pure coir seems (at least from my experience) to result in slower germination that when you sow on sphagnum, but if you sow seeds on live sphagnum that is growing quickly, the seeds sort of get lost down there somewhere and in some cases never show themselves. Those that do, you have to be nifty with tweezers or modelling nippers (I use Tamiya ones) in order to clear the sphagnum to let the seedlings get some light. I have found that the best solution is to use coir, with no perlite (see a separate thread) onto which I water a slurry of really finely chopped live sphagnum. The small size of the sphagnum pieces is great to keep the seeds moist, and although the sphagnum still grows it doesn't overwhelm the seeds at the early stage. Using dried sphagnum that has been chopped up works too, though doesn't look as nice. But whatever moss you use, just a little bit as a dressing for the coir (or orchid bark) seems to be most effective. Pure coir takes longer, but it is reasonably good, with the least effective being long live sphagnum.

 

I'd be interested in hearing others experience of the last bit

 

Cheers

Edited by lesthegringo

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On a different note, we will have to move house at the end of the year due to the owner of the house we rent wanting it back. A pain, but one I intend to turn to my advantage. I told my wife how traumatic it would be for all my poor plants to have to move them in the hottest, driest time of year, and that to dismantle the greenhouse, transport it and re-assemble it would take days, especially with everything else going on. Wouldn't it be better, I told her, to find somewhere where I can build a new greenhouse, then move all the plants over, then I could deal with the old one at my leisure....? So it looks like I will have two greenhouses soon, one for highland, one for lowland. Of course, I haven't actually got any lowland Nepenthes yet, but let's not nit-pick.

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I've called it a day with my first in-vitro experiment, those pots that didn't have any fungus were starting to show seedlings that were dying off. Rather than wait for them to all go, I bit the bullet and transferred all the remaining seeds and seedlings into my stock coir/LFS/live sphagnum dressing soil. I don't bother washing them in anything specific, other than rainwater, and even then only in cases where there is evidence of mould contamination. The live sphagnum seems to do a sterling job of keeping them alive, if any have died due to the transfer the number is small enough that it isn't noticeable.

 

I don't regret doing it at all, I learned a lot, and will be attempting it again. Next time I will try and be a bit more scientific about it, and try and aim for a more controlled environment in which to keep the pots, as I am convinced that sun and high light levels trigger the mould and fungus that would otherwise have stayed dormant. However I also need to pin down a better method for pre-sterelising the seeds, as this is the one factor that can help avoid spoilage in the first place. I need to do some research, and would also welcome comments from anyone that has successfully grown seed in vitro.

 

On a different note, the search for space in the greenhouse is now an ongoing issue, as the increasing number of seedlings and seedling pots has taken up a lot of the shelf space. Thankfully the sun is now shining into the greenhouse again, so I was able to rearrange the plants so that the seedling trays were all on the lower shelves, knowing that they would still get sufficient light. A collateral benefit is that the adult plants provide shade from direct sun. As you can see it is all quite neat

 

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There are over 100 of those seeds trays, and they are arranged by the date they were sowed. A red tip to the label indicates that some of the seeds have germinated, and I am pleasantly surprised by the proportion that have. Curiously I have trays sown on the same day, with the same soil mix with the same seeds, and one will have loads of seeds germinate, and the one next to it has none. Can't explain it, and won't try!

 

I always have a thrill when I see a new species germinate, although I know that you don't really know what they are until the adult pitchers form, and that is still a long way off; the oldest seedlings I have are just under five months old (from being sown), and are about three and a half months from germination. Still babies, but I have not (yet) had the infant mortality rate other people have been unfortunate to suffer. Time will tell if that continues.

 

In a previous post I showed a picture of my rather gangly N Sibuyanensis X Maxima, which had put out loads of leaves and reached about 4 feet long, but since producing 1 upper pitcher stopped pitchering. Well, a first in my experience, some of the pitcher buds that have been doing nothing for at least six months have woken up and are now developing into upper pitchers. Normally if they don't form pitchers almost immediately, my experience is that they never develop. I was going to cut the stem for cuttings, but now I will leave it to let the four of five uppers develop.

 

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I think the fact that it has warmed up outside (even though my greenhouse is heated) plus the vastly better quality of light afforded by the sun has triggered the development. Hopefully that will translate across to all the others in the house too. I'm going to repot the adult plants into plastic hanging pots, as I am not convinced that the wire basket with a fibre liner is the best, plus they are so large diameter that they block the light. A fellow grower here in Aus has suggested that an orchid mix produced by Osmocote is good to add as it is an open mix that also contains a little fertilizer. I'll give it a go, it may spur the plants on a little.

 

Lastly an obligatory pitcher shot, this is my Veitchii X Burbidgeae - not huge, but a lovely colour and shape

 

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Edited by lesthegringo
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Try sterilizing your seeds by soaking them in a 1:10 mix of PPM and water (1ml ppm and 10ml DI water). I let the nepenthes seeds soak in this PPM solution for 24 hours, then drain the PPM off and place the seeds on culture media containing 1ml/litre of PPM. I've had zero contamination problems since moving to this method. The PPM sterilization solution can be reused i you keep it refrigerated. 

 

Germinating Nepenthes seeds in tissue culture really is worth the extra effort, once you sort out any contamination problems. 

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Thanks Persivore, I did use PPM as advised by Stoker128, whom I bought the in-vitro stuff from. Unfortunately it wasn't as effective as I'd hoped and makes me think that a bit more intensive sterelisation may be necessary next time.

One avenue of enquiry is to use either bleach or hydrogen peroxide solutions to complement the PPM. Trouble is that I am not sure which to use first, the PPM or the bleach/peroxide solution.

The first attempt wasn't a disaster by any means, and comparing the seedlings from in-vitro with similar aged seedlings I'm convinced that the in-vitro grown plants are bigger. It's just that I seemed to have a roaring success in growing some great moulds too - maybe I should take up mushroom farming. Seeds planted in-vitro did seem to generally take longer to germinate though, I'm not sure if this is a product of my method or a normal phenomenon using this technique.

One important lesson I did learn was once you see signs of mould or spoilage, you have to act fast, especially if there are any sprouted seeds, and get them onto live sphagnum. I reduced my losses that way.

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I used to use bleach (calcium hypochlorite) for seed sterilization, but have moved away from it since PPM seems to be much more effective. Bleach would be successful about 90% of the time, whereas PPM has been 100% successful (so far). Strangely, i found N.inermis seed to be the most difficult to sterilize - I went through a few failed batches of these, sterilizing with bleach, before moving to PPM. If you catch the contamination early enough you can usually save most of the batch by picking out the affected seeds, re-sterilizing and re-plating. I tend to move any seedlings which germinate to a separate container once they are free of the seed casing, in case contamination hits.

 

These are my sterilization methods if you get a chance to give tissue culture another go - 

 

Calcium hypochlorite 
Seeds are soaked in 250 parts per million gibberellic acid/distilled water for 24 hours. I typically use about 20ml of this 250ppm GA3 solution. The GA3 itself doesn't sterilize anything, but the soaking probably softens up the seeds a bit and helps to bring any fungus spores out of dormancy to make them easier to kill.
After 24 hours, I add 100mg to 150mg of calcium hypochlorite powder to this 20ml to give 0.5-0.75% bleach solution. This is shaken until most of the calcium hypochlorite is dissolved. After 10-15 minutes, the seeds are rinsed in sterile distilled water 3 times and moved to culture jars. Household bleach can be used as an alternative to calcium hypochlorite powder, but the quantities will need adjusting based on the bleach strength.
 
PPM 
Seeds are again soaked in 250ppm GA3/water solution for 24 hours. After 24 hours the GA3 solution is drained and a 1:10 PPM:water solution (1ml PPM + 10ml DI water) is added to the seeds and left for a further 24 hours. The PPM solution is then drained and the seeds moved to culture jars without rinsing. The PPM solution can be re-used if kept refrigerated. 
 
Keep up the good work - you'll soon have more Nepenthes than you can cope with!
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One of my Ventricosas is putting out a flower spike, which looks like it will be a male flower.

 

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Hopefully I will be able to give the pollen to someone nearby to get some seeds

Edited by lesthegringo

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It's been a while since I last posted any updates, so here are a couple of pictures of some seedlings. These are just mixed hybrids of totally unknown parentage, that were in vitro for four months prior to being transferred to traditional media in October.

 

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Of all the seedlings these are the biggest, about 12mm across.

 

I am going to try and find a suitable fertilizer here in Australia to try and bring the seedlings along. They frankly are growing very slowly, which I expected, but I am starting to wonder if they are growing slower than what would be considered normal. Any suggestions would be welcome

 

Les

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