Open Source Plant Breeding Forum

General Category => Seed Saving => Topic started by: Joseph Lofthouse on 2020-11-11, 08:09:55 PM

Title: Germinating super old seeds
Post by: Joseph Lofthouse on 2020-11-11, 08:09:55 PM
I recently traveled to a seed bank in Colorado, and acquired seeds for the Beautifully Promiscuous and Tasty Tomato Project. A plant breeder named GW Denna had started a project like that in about 1971 to 1974, then he died young. The seeds sat in a warehouse in Colorado until they made a new home in the seed bank a few years ago. I was able to tell the custodian of the seed bank why Denna's project was important, and about the meaning of the labels on the seed packets.

The seeds are:

F1 hybrids between Solanum peruvianum.
F2 hybrids between domestic tomatoes and Solanum peruvianum.
A domestic OP tomato that was highly attractive to bumblebees.
LA128 L hirsutum glabratum from Baos Equador.  Self fertile.
LA387 L hirsutum from Santa Apolonia Peru. Mixed fertility.
"Wild Tomato Crosses" with large-ish seeds indicating domestic ancestors.
L peruvianum. SI F2 Intercrosses with large-ish seeds indicating domestic ancestors.

The seeds are 45 years old and were stored haphazardly. I have about 500 seeds to work with.

Can you recommend germination protocols for old seeds?

Some ideas that have been suggested include.

I'm currently fascinated with the idea of making a tea from tomato seeds, and watering with dilute hydrogen peroxide, on some type of hydroponics mat. The peroxide is also an antibiotic.

Anyone want to recommend your favorite method for germinating old seeds?

Title: Re: Germinating super old seeds
Post by: William S. on 2020-11-11, 10:18:25 PM
Rough Surface sterilization protocol:

10% bleach with a drop of free and clear dish soap stir
Them 3% H202 stir to rinse of the bleach

Then sterile water rinse.

A product called PPM or Plant Protective Material can reduce contamination rates.

If used media sans sugar to soak germination paper wouldn't really need agar and without glucose less risk of mold etc.

Can also sterilize everything in a pressure cooker or microwave depending.

Carol Stiff wrote up detailed instructions for home tissue culture very helpful.

Kinda want to just load up my plant tissue culture kit and come help but probably can't.

Surface sterilization generally increases germination rates

Liquid smoke generally increases germination rates (choose one without added sugar). One of the neat things about forest and chaparral fires is the wildflower flush after. Precedes massive germination events. The smoke may help replace the solutes that leak out and or suppress damping off organisms. Speculating in part as to how it works, Google probably knows more by now than I do about it.

Title: Re: Germinating super old seeds
Post by: Garrett Schantz on 2020-11-11, 10:32:48 PM
The oxygen boost is pretty important considering what you are working with. So I would incorporate that no matter what.
Nitrogen has been found to increase germination in old seeds, unsure if this applies to tomato seed though.
Letting the seeds soak in diluted sugar could improve germination as well - be sure to sterilize the seeds in diluted hydrogen peroxide to reduce chances of mold if you go this route. Diluted Hydrogen Peroxide can help with oxygen for the seed as well, to revitalize enzymes and such.
Some sources mention diluted Gibberellic Acid as an option as well, unsure though.
I would try all of these and see what works best. 45 years combined with not so good storage conditions, the seeds probably need all the help they can get.
 William posted a lot of good information / ideas.
Title: Re: Germinating super old seeds
Post by: S.Simonsen on 2020-11-12, 12:53:15 AM
How to manage risk and make best use of time and resources germinating different batches depends a lot on the size of each batch as well, and depends on how much time/resources/attention you have to spare.

Personally I would start out with the largest batches where you have the most chances to get it right, and take out a small (5-10%) sample. A germination test on damp paper would be my first step, just to get a sense of standard levels of viability, especially since you know very little about how the seed has fared during the long storage time. If you get absolutely no sign of germination then repeat with a slightly larger percentage of seed. If you get poor germination with sprouts too weak to establish under normal treatment only then would I consider going down the route of sterilisation and boosting treatments or tissue culture. Then you can figure out a successful protocol on small portions of the larger batches before applying the method to the smaller and more valuable batches.
Title: Re: Germinating super old seeds
Post by: Ocimum on 2020-11-12, 03:17:22 AM
If they were kept dry, first keep them for a few hours/up to days at high humidity, otherwise they soak up the water too fast and may die. At least this is for some species, no idea if it's the same with tomatoes. I germinated seeds which, according to life expectancy in books, should not have sprouted that way.

Oh, and some use Lactobacillus to improve germination in seeds

Title: Re: Germinating super old seeds
Post by: Joseph Lofthouse on 2020-11-12, 09:30:14 AM
I looked more carefully at the seeds. There are about ~300 seeds from the wild ancestor. ~300 seeds from the F2 of a cross between domestic tomatoes and Solanum peruvianum. And ~40 elite seeds that are exactly what I'm looking for in my breeding project. (These might be G4). So it's looking like I'll be able to successively experiment to maybe find suitable germination conditions. 
Title: Re: Germinating super old seeds
Post by: Adrian on 2020-11-12, 12:43:11 PM
I choose a potting soil and i add sand in.
The best is a potting soil with mychoryze muschroom
What would the effect of lactobacillus mixed with the coffee ground ?  I will try a mix with potting soil, ash,sand and coffee ground for create a very good seedling potiing soil! For me the most important is the coating of the seed by the potting soil.
The problem of the potting soil alone is the difficulty to humidifated this and have a bad water penetration.
Title: Re: Germinating super old seeds
Post by: Steve1 on 2020-11-13, 04:17:38 AM
If it was worth alot to me, I'd be going the tissue culture route. Probably with some gibberellic acid. Seems to me with weak seeds that damping off/fungus get hold before germination occurs. Surface sterilisation at least if done properly halts that process. If you have a clean hepa filtered room, and good sterile technique then you may get months before the plate gets infected which means best chance. The 32,000 year old seeds from the Russian permafrost recently germinated were done in tissue culture. Guessing there would be a paper that might help with details.
Title: Re: Germinating super old seeds
Post by: Adrian on 2020-11-13, 07:16:11 AM
For a weak plant i think that the most dangerous is the fly of the seedling and the pythium muschroom.Its very difficult to save a plant after their attacks. The alone solution is the cutting.
I don't know if its a good idea but i hace try to put gibbeleric acid at the germ position before the germination.
Title: Re: Germinating super old seeds
Post by: Adrian on 2020-11-28, 08:24:46 AM
This year i have try the mychorize poting soil N=6 P=7 K=8 with coffe ground above the synergy with the coffe and the mychorize is for me miraculous.I have see of lentil in and i have never see a lentil plant so beautiful!
The result is best with a mulch above the coffe ground for fight against the drought of the germ if she is long to germinate.
Title: Re: Germinating super old seeds
Post by: Richard Watson on 2020-11-28, 10:01:34 PM
I had a sack of Mangle beet seed that would have to have been 10years old and was not stored in a ideal location really. Thinking bugger all would germinate I sowed thickly, surprised to find i must have had 90%+ success rate, got a big job now thinning out.
Title: Re: Germinating super old seeds
Post by: Adrian on 2020-12-13, 05:30:47 AM
Has the coffe ground contain an inhibitor of germination,
 i have been an idea for have a best germination with the coffe ground!
I will did a solution of C6H12O6, glucosis with H2O water in my coffe ground.I may stimulated the cellular growing and feed more easily my muschrooms mychorizes.
 have mixed the cofee ground with of granule manure.
Title: Re: Germinating super old seeds
Post by: Joseph Lofthouse on 2020-12-13, 06:45:22 PM
I started some preliminary testing with the old tomato seeds...

Attempting germination in petri dishes with quilt batting and coffee filter paper. Three different sets of conditions. In all cases with 0.15% hydrogen peroxide.

Water only
Tomato seed extract
M/S Gamborg fertilizer and vitamins, commonly used in plant tissue culture.

Using my standard germination conditions: 90F and lighted for 16 hours per day. 60F and dark the rest of the time.

The tomato seed extract is encouraging the growth of microbes.

I'm expecting that surface sterilizing of the seed will become part of the procedure for next time. I autoclaved the petri dishes and solutions. 

Title: Re: Germinating super old seeds
Post by: Steve1 on 2020-12-15, 07:57:51 PM
Joseph, the tomato seed extract could be interesting / helpful. You could mix that with your medium / GA and then sterilize the plates then sow seed after surface sterilization. We use V8 mixed fruit/vegetable juice in medium when tissue culturing specific fungal plant pathogens. Good luck. 
Title: Re: Germinating super old seeds
Post by: Steph S on 2020-12-30, 11:07:26 AM
Wow, Joseph, what a find!   Hope you have had some success by now?

I've heard of using dilute N ferts in the seed soak for old tomato seeds.  I like the point of humidifying them before soaking.  I have only used an overnight soak in water, for some 15yr old tomato seeds and I did get seedlings.   But 45 years...  I guess much depends on unknown conditions during that time.
One thing I've noticed with old pepper seeds is that they take much longer to germinate and then can suddenly surprise you, weeks after you gave up.  I always soak pepper seeds overnight, even if they're fresh.  I had some hot pepper seed given me, marked "old/low germination".  I put their soaking cup on the heat mat for the overnight soak.  Ended up with seedlings too many to count in just a couple of days.
Tomato seeds that stubbornly don't germinate also have a tendency to pop up much later, unexpectedly.
So whatever treatment/soak you used, I would treat, plant, and then wait at least a month.  Maybe 45 days for 45 years?

My thought about soaking with ferts or kelp is that I didn't really want something that is decomposing or promoting decomposition in the vicinity of the pre-germination seed.
Old seeds definitely do need ferts as soon as they have germinated though.   Brassica seed a case in point which I find deteriorates after the third year or so - they still germinate but the cots will come out yellow and they don't thrive unless you feed them immediately.  Same was true of the old tomato seeds - some cotyledons less than prime condition, so a bit of a handicap in ability to nourish itself.  This is because the nutrients saved in the seed for the purpose of getting started are no more - used up in seed respiration iirc or simply deteriorated over time.

I know lactobacillus is supposed to discourage pathogens on the ungerminated seed surface.  I used whey to soak some tomato seeds before planting.  They didn't mind it, were healthy.

Here's a thought:  Some time ago I read about the microbiome around seed germination - apparently the exudates of the seed promote a very specific and transitory microbial community which assists in getting to the seedling stage.   Maybe an extract could be made by ? washing germinating seeds to collect the exudates and associated microbes, to soak the seeds in before planting and/or to water them with, once they're in the soil.  If you had a big batch of seeds you don't plan to grow out, you could sprout them for the purpose...

That is, if you don't have luck with the agar.  Hope you did. :)
Title: Re: Germinating super old seeds
Post by: WayneA on 2020-12-31, 06:04:48 AM
Hoping to piggyback on this topic; my wife just found some dried bean seeds in a (very old) handbag whilst clearing an old cupboard. She thinks they are likely to be between 20 and 15 year's old, they're very dried and wrinkled and they may be her family's personal strain of beans from the Czech republic (the only explanation she can cook up is she must have put some in there whilst visiting in the early 2000s and forgot em.

Obviously big seeds like these are rather different to Joseph's toms; any hints or tips for giving them a go please (or is it beyond hope)?
Title: Re: Germinating super old seeds
Post by: Adrian on 2020-12-31, 02:26:43 PM
You can try to sow one seed in pot at a temperature above 15C and toward a window. A grow-light can  help you.
The bean is a plant of long days, he prefer the days above 12 hours.
This seed will be sensitive at pythium, a drain soil is important for fight against this.
The last year i have try of old seeds of mungo beans. The seed has germinate but the cotyledons was stayed yellow, i have did the error to sow this seed in a room too cold (between 13 and 18C)and a potting soil too poor, it was of sand.
Title: Re: Germinating super old seeds
Post by: Adrian on 2021-01-29, 08:14:55 AM
Test of my seedling with cofee ground without mychorize.
Tested with phacelia and lentil.
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Title: Re: Germinating super old seeds
Post by: Ferdzy on 2021-01-29, 10:19:52 AM
WayneA, I often soak beans and peas in a hydrogen peroxide solution before planting them. It might help and it's not likely to hurt. It is known to kill surface pathogens and speed up germination.
Title: Re: Germinating super old seeds
Post by: Plantman on 2021-01-29, 03:33:21 PM
I think you should use the gibberellin, and be prepared for a seedling with very elongated internode.
When you dilute peroxide into water and it contacts carbon ( soil ) it will give up an oxygen molecule.
Not sure it does it in solution though.
If you are going to scarify your seed  by soaking, then an aquarium air pump with an airstone inside a jar will stop the soak becoming aneorobic.
The plant can produce its own immune response also, I'd consider soaking in a weak nutrient solution (400-600ec) that has kelp and fulvic acid.
The embryo will require carbs that have degraded over time.
Some people germinate in bean soak juice solution.
Almond and bean soak juice, is similar to besan flour and macca extract, they provides saponins that work as a surfacant, and will allow the seed raising mix to wet fully.

If the seed is as precious to you as you say, maybe go and browse some canna breeders' growing forum to see how they treat older seeds.
That industry has a bit of experience with germinating older cherished small seed batches.
Good luck.
Title: Re: Germinating super old seeds
Post by: Ocimum on 2021-03-19, 03:24:17 AM
I have got some amazing but super old Chickpea seeds, which don't germinate normally.

I am trying with hydrogen peroxide 1.5%, no idea if it will work. I have some old seed with a weak germination, I can try a few different treatments there.

What I read here are:

Surface disinfection (H2O2 or javel)
Soaking in H2O2 solution. Different sources state anything from 1 - 6 % for different crops, and different mechanisms of helping with germination.
Sugar (which concentration?)
Nitrogen (which concentration?)
Other nutrients (Plantman suggests 400-600 ec, difficult to measure without equipment)
Title: Re: Germinating super old seeds
Post by: Klaus Brugger on 2021-03-20, 04:30:42 AM
I'd be careful when using H2O2 on really old seeds. After all, in seed aging, oxidative damage plays a big role, so you're already dealing with e. g. rather fragile membranes. This is why when treating old seeds with H2O2 to increase oxygen bioavailabilty, Liu et al. (2012)* used a Calcium source to protect membranes. And they weren't even working with really old seeds (max. 6 years).

*Liu, G., Porterfield, D., Li, Y., & Klassen, W. (2012). Increased Oxygen Bioavailability Improved Vigor and Germination of Aged Vegetable Seeds, HortScience horts, 47(12), 1714-1721. Retrieved Mar 20, 2021, from
Title: Re: Germinating super old seeds
Post by: Adrian on 2021-03-20, 10:12:21 AM
I have put of seeds of lentils and barley  in a water pot.
I have did one seed of tetsukabuto, one seed of tetsukabuto x violino rugosa and one seed of violino rugosa.
I think that the auxin rejected during the germination of the lentils and the barley will help  the germination of the seed tetsukabuto x  violino rugosa
Title: Re: Germinating super old seeds
Post by: Ocimum on 2021-03-25, 02:40:20 PM
Here the result of a germination test with old Vigna unguiculata seeds in hydrogen peroxyde.

Control: soaked for 24 h in H2O
Treatment: soaked for 24 h in 1.5% H2O2

Then put into petri dishes.

Results a few days later:
Control: 10/30 germinated. Seeds covered in molds
Treatment: 18/30 germinated. No molds, seeds lighter due to oxidation.

Other concentrations and/or soaking times may work even better.
Title: Re: Germinating super old seeds
Post by: Diane Whitehead on 2021-03-25, 08:07:34 PM
From a 2012 article that hints, but doesn't give direct instructions:

A procedure to re-invigorate seeds is currently under development for worldwide application in situations where large seed stores are maintained in poorly controlled storage environments.

The technique involves bathing the seeds in a chemical solution with an ionic balance crucial for absorption of the molecules required to re-activate or re-introduce gibberellins.

Title: Re: Germinating super old seeds
Post by: Adrian on 2021-03-26, 09:45:09 AM
Coverd the seedling pot with a wintering veil increase the germination rate and avoid that the seed not stick at the cotyledons.Thr humidity and thr temperatur under the veil is more stabile.The light is less strong and she help to lengthen the germ.
Title: Re: Germinating super old seeds
Post by: Joseph Lofthouse on 2021-04-12, 11:05:23 PM
Update on the 47-49 year old tomato seeds.

I tried a number of germination strategies with the seeds. Sterile conditions. Gradual rehydration. Tea from fresh seeds. M/S-Gamborg. Natural soil. Tried about 110 seeds total, with zero germination.

Today was tomato planting day. Therefore, I planted nearly all of the (700?) remaining seeds, under the same conditions as I've started tomatoes for years:  coconut coir, with a hint of peat, pearlite, and some slow release fertilizer. In a germination chamber at 90F while LED light is on 16 hours per day, cooling to room temperature at night. The plants like the LED much better than the old fluorescent tubes.

Title: Re: Germinating super old seeds
Post by: Woody Gardener on 2021-04-13, 11:27:32 AM
I've got some old seeds I'd like to germinate, also some hard to germinate seeds that failed this spring. I just got a Clone-King Aeroponic machine and will try to germinate the seeds on a screen mesh just smaller than the seeds.
Title: Re: Germinating super old seeds
Post by: Adrian on 2021-04-13, 04:05:27 PM
Joseph with 16h of light per day do you arrive to controlated the humidity?
What is your humidity rate?
Title: Re: Germinating super old seeds
Post by: Joseph Lofthouse on 2021-04-14, 06:53:39 PM

Humidity is very high, because the pots are in a sealed wooden box.
Title: Re: Germinating super old seeds
Post by: Adrian on 2021-04-15, 02:48:48 PM
I use a cold frame coverd of one wintering veil fold in 3 and one wintering veil above the pots.
In the background i have put of clay balls.
The night the temperature is up 4C > at out (minimum 2C) and the humidity  is at 99%.
The day the temperature is up to 31C above the pots and the minimal humidity is 30%.
I note that the humidity is more important than the temperature for the germination
It was a great sucess for the germination of my tetsukabuto seeds.Some seeds who look liked totaly flat are germinate.
But the more important before this stape is to put the seeds in water up to they sink or get fat.
Title: Re: Germinating super old seeds
Post by: Andrew Barney on 2021-04-23, 09:46:55 PM
I have nothing to contribute on the seed germination front. I just wanted to say way to go Joseph! And way to go for someone saving those seeds despite his early death!

Keep us up to date on the success of this!