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Testing Seed Viability

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I'm going to trial this one in the next few weeks- anyone have any experience with it? Rev, I'll send some your way too if you'd like

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From: N. Prakash, Methods in Plant Microtechnique ( 3rd edition ), UNE Armidale School of Botany 2000

Testing seed viability with Tetrazolium Chloride:

The seeds are cut longitudinaly through the embryo into two halves and immersed completely in a small petrie dish of 0.1%- o.5% aqueous solution of 2,3,5 triphenyl tetrazolium chloride ( Sigma T8877 ) for 1.5 to 6 hours in the dark

The embryo in viable seeds is stained pink to red due to its respiratory activity while to nonviable seeds remain unstained

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hi,

i read about it..

i like it simple!

if seeds take up water, say gaining

size fast after water(or acid treat

ment,or digetive fluids of chooks)

its a good sign!

slicing one seed on inspection is re

comanded too!

one has to just knowe the protocol of

each seed. stratification, rubbing with

sandpaper,even sometimes seeds need

good ariation between wet (monsun like)

coditations.

this is a good topic

hasta la vista

wolfgang

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Guest reville

geez darklight

looks good when youve only got a hanful or are unsure of the protocols to break dormancy

Im not that high tech

I just plant them and see if they grow (after adequte prettreatment

or i rely on feedback

most of my seed is fresh as the AFSR is new but in future i will have to implement a viability test

usually 10 -100 seeds on an inert medium and tak the numbers as a percentage estimate.

Germinants can be potted on to provide the next batch of seed

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rev, there are some important applications for this.

for example, I have kilos of Griffonia seed and have tried to germinate hundreds without success. I know the seed is fresh and there is no reason why it shouldn't germinate (tried all the treatments). before I waste more time and materials it would be nice to know if the seed is actually viable.

Ditto for some native seeds, such as the pituri seed. No good wasting time and materials on seed that is unviable. However, if it is viable (which I presume it should be), then I shoulf be frugal with the numbers of seeds used in each trial until I find the right protocol.

Then there is the issue of rare seeds in TC. I have some Uncaria seed (cat's claw), but I am unsure if the storage was appropriate and the seed may have been killed by it. Proving that the conditions before they got to me killed them would mean I could stop the supplier from killing them and obtain viable seed that way. Trying to find that out by sticking them all into TC is a little expensive.

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As well as heartbreaking and a waste of my time and resources...

You really do hope the best for your seeds & plants when you put them in ( as you'd know by now Rev )and carefully plot and monitor the amount of serious mistreatment ( bleaching, fungicide etc ) you put them through to sterilise them so it won't kill them and they continue to live in culture. I get upset when my work doesn't help the plants to live.

Two of the biggest problems I've faced when starting new species from seed which have no previous protocols are identification and viability. Assessing the protocol for sterilisation is very important too, but you'd be surprised just how many spp I've begun where no seed has germinated, or plants have proved to be from an entirely different species.

Either of these mistakes can happen anywhere along the line- the seed collector, various storage points, in shipping, wholesale or retail- and just get passed along on the assumption that the person who handled the process prior to yours was capable, knowledgable and honest. Now when there's any doubt about viability, at least we can test it here.

Knowing that the seed embryos ( as opposed to the endosperm etc ) are viable means that you can change your in vitro germination technique, for example you can assume the endosperm is no longer viable and excise the seed embryo onto a media containing liquid endosperm such as coconut milk.

Or you can assume the seed needs further treatment such as stratification or extra heat or light.

If you know that the seed embryos are viable, you're in with a better chance, I reckon. This sitting round and willing the seed coat to break so your plants can grow only has so much going for it as a method, from my experience smile.gif

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Guest reville
Originally posted by Torsten:

rev, there are some important applications for this..

Sorry if the wrong message came across. I was meaning to say exactly what you said re. rare seeds and Unknown germination procedures.

Most of the time i dont deal in the rare ones, though im looking forward to it in future.

Torsten do you know the germination procedures for Sassafras albidum? or how long they might be viable for?

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YEAH, I do the 2,3,5-triphenyltetrazolium chloride (TTC) test on some of my seeds. Works great. In my experience you DO NOT want to slice up the embryo. If the seeds are small and white (like capsicum seed) you can use the whole seed, but if the seed coat is a dark color (like watermelon seed) you should cautiously remove the seed coat and do it that way (removing the coat before the test makes the test go faster. But scaring the coat before the test, and removing the coat after the test (after the seeds swell, don't use non-swollen seeds) is far more accurate because it minimizes pretest stress and false positives due to physical damage and bacteria. If you REALLY want to you can use just the embryo, but the embryo must not be exposed to air for more than a minute before placing in the TTC solution.

I use 0.1 to 0.25% TTC for my simple seed tests. Some dudes use a 0.05 M phosphate buffer at pH 5.80 with upto 1% TTC for cell culture testing. And if your testing pollen viability under a microscope I have been told it is required to use a filter sterilized sucrose/TTC solution with some incubation to activate pollen.

Also, some alternatives to TTC have been developed. One more common one is MTT [3-(4,5-dimethylthiazolyl-2-)-2,5-diphenyl tetrazolium bromide] This is often used for the above mentioned pollen test. I have never used MTT, so don't ask me how well it works.

Any questions?

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Originally posted by Auxin:

If you REALLY want to you can use just the embryo, but the embryo must not be exposed to air for more than a minute before placing in the TTC solution.

Thanks for that, that information was omitted from my protocol.

I'm working on large dark seeds with a dark endosperm and a poorly defined embryo- but I have a few of them so I can try all the options

Some dudes use a 0.05 M phosphate buffer at pH 5.80 with upto 1% TTC for cell culture testing.

The purpose of which is? And what do they adjust the pH of TTC with?

Any questions?

I didn't think I did, but I do smile.gif Just asked them. Thanks Auxin I really appreciated that!

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"And what do they adjust the pH of TTC with?"

Glad you asked, the buffer adjusts the TTC to a final sol'n pH of about 4.9

Check this out: http://www.agro.agri.umn.edu/plant-tc/list...1/msg00048.html

I presume that the buffer is to optimize the TTC test by making sure that the test reagent is not so acidic as to kill off more sensitive cells. Remember, this specific test was engeneered for microscopic (post cryo) cell culture testing, maby the buffer is not used in seed tests because seeds have so many cells that even if the TTC kills some cells in a viable seed, it wont kill 'em all, so you still get a positive result.

Or maby the dudes were just aiming for the pH of a specific ringer sol'n or something and that info later got filtered out somehow.

The plant-tc archive has a fair amount of info on TTC tests if you have the time to go through it.

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Originally posted by reville:

Sorry if the wrong message came across. I was meaning to say exactly what you said re. rare seeds and Unknown germination procedures.

OK. I thought it was a bit odd for you to think differently wink.gif

Torsten do you know the germination procedures for Sassafras albidum? or how long they might be viable for?

Considering this is a weed I am almost embarrassed about the lack of success with this species. I know that the berries are viable longer than the clean seed, as the seed dries out easily. I think clean seed is only viable for a few weeks, while the berries are supposedly viable for a year or more. I doubt it though. I've had a lot of berries from different sources, stratified them properly, and still no success. I won't even try the seeds.

I have a small plant now, which I bought as a plant. i also know the location of a 3m tree and think that it might be possible to grow from dormant cuttings. It's still a couple of years away, but we will be able to offer this species eventually.

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re,

lets not forget about polarity,some

seeds hate it beeing planted wrong

side up! seeds are tested with the

wet towel methode and watching the

different stages of development.

catha edulis have to be planted with

the wing facing up.hemp with anus

up.seeds placed wrong still can do it

if lots of energy is still stored.

taking the coat off can be enough to

germinate old seeds.starting an in vitro culture would be certainly the way to go

for those difficult ones. but costs

forbidde that and tissue culture certainly

cant make some hard to culture plants

suddenly easy to propagate.

but by the way your pausinystalia

are halve way hardent out meaning the domes

are of since a week.

plant babys are like human ones...

wash your hands before inspection,

clean enviro.

how long before a yohieba sets seeds?

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Has it ever occured to anyone that we are lucky to have some very smart people here on this forum!

I learn so much. thanks all!

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Originally posted by Darklight:

The embryo in viable seeds is stained pink to red due to its respiratory activity while to nonviable seeds remain unstained

Do the seeds respire if they're dormant?

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seeds, hardly breath, if cold !

some longtime storage can be achived

by using dehydration sachets and

freezing. re moistering is the trick

in this case!

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