Help With Plant Embryo Rescue Via Plant Tissue Culture

I am interested in what resources you have on the topic, what input you have with regards to the topic, and if you believe a particular method/mix/approach will work better than others for not simply regular cannabis/hemp embryo culture, but also for embryo rescue with older genetics in mind.

I am developing SOP’s and would appreciate input past ScienceDirect, Springer, JSTOR, and Wiley.

I am thinking on running a MT or MS medium with agar, balancing cytokinins and auxins, with other added aminos, vitamins, and carbon sources contributing to successful embryo rescue .

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By “embryo rescue” are you referring to culturing seed?

No hormones and minimal sugar was my approach when trying to rescue a tobacco line from essentially non-viable seed.

Might have used 1/2 strength MS.

Got what I was after…

I am asking at the excision of the embryo. let’s say these extremely old seeds did not germinate post-sterilization, gibberellic and moderate brix content failed, the same methods opened, “hemp seeds,” 1-5 years old without issue, but these are let’s say stubborn, or that they weren’t kept in the correct conditions for decades

How many seed can you throw at it?

I assume you have a few seeds from a large number of strains.

Dissecting out the embryo was a non-starter with tobacco, I doubt it will help with cannabis, but at least the seed is big enough that you can attempt that.

in short, yes this is one method that works, but I am interested in other’s experience, tips, tricks, methods, and never assume a thing about seed game, could be just one or two seeds of gorilla glue

It could. If you’ve tried and failed and only had two, the conversation is over…

I was able to throw a couple of hundred at the problem.

It was more than 30 years ago.

I recall being willing to induce callus rather “germination”, but I got lucky.

I believe organogenesis is best for PTC, but this is a question about Embryo rescue. I’m trying this out with as many trials as necessary, goal is under 4000 trials.

And I’m trying to understand how many seed you can afford to lose….4000 trials suggests more that a couple of GG#4

You’re certain to get strain specific germination rates with old and abused seeds…the problem is different if you have two seeds of 2000 strains vs 2000 seeds of two strains….by all means continue to obsfucate, and I’ll wander off.

Yes, I get that you’re looking at embryo rescue.

AND those guiding me 30 years ago suggested that IF I could not get “germination” on hormone free media, then trying callus inducing media to get any cell division was the next step…


Mystical wisdom observed, trials were/are of 5 different hemp varieties, nothing to beds or ground though, enough seeds to build the tech

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Have you optimized your sterilization tech? You want to be as gentle as possible without losing sterilization.

What are you using for osmoticum? Something less edible than sucrose can help when you’ve dialed sterilization down to a knifes edge.

If you’ve got hundreds or thousands of seeds from each strain, you’re at least in a place where you can actually TRY things.

First things I would try are 1/2 strength versions of published meristem culture media. MS and ??

Then I’d play with osmoticum. Both level and constituents.

The gelling agent can make a difference. Try a couple.

Then hormones. Shoot inducing/root inducing/callus inducing/gibberellin/none.

Without knowing what you’ve tried and how successful/unsuccessful you’ve been, or how far you are from your goal, it’s difficult to go beyond that.

For tissue culture in general, making progress is often about the right person wielding the scalpel & tweezers. Either they do less damage, or they recognize subtle differences in cell types/organization. If you’re dissecting your seeds, or even just removing the seed coat, this may apply.

I get why you’re obsfucating, but why then would you expect anyone who’s actually solved it to share?

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There have been enough studies published to know my way about this and sterilization isn’t an issue here. I have honed studies from protoplast to callus, and I am simply seeking input on straight from embryo

thank you for your input.
We’ll see if there is another interested contributor

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established protocols will absolutely get them sterile. My question was more “are you sure you’re not killing them?”

Hard to be certain if the result is they don’t grow.

Awesome. That should put you in good stead. You already understand the utility of all the general suggestions.

I too am curious re:other participants…I believe laying out as much detail as you can share would help…


When embryo culture fails, getting ANY cells to play is the next trick…”rescuing” the embryo requires that most of it be viable.

Culturing any viable cells from older material that doesn’t respond well to “rescue” is probably closer to going from protoplasts (which sucks more, being alone without a cell wall, or being surrounded by your dead buddies?!?)

Although you should have the remains of two meristems with pluripotent cells…primed to respond differently…which is where playing with the hormone balance (shoot vs root vs callus first) comes in.

If you’ve got the various organogenic media at your fingertips, dissecting some embryos or even cellulase treating and gently making “blobs” may help with the recalcitrant line(s).

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@Bavarian_Buds is this a rabbit hole you have explored?[

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I made some seeds in 2005 and they are popping near 100 percent but do come up weak and stunted. im not taking proper care either but most are still alive. Store your seeds in the fridge.

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Ill donate some old viable seeds if you wanna mess with them in tissue culture experiments.

I’m going through SOP’s with newer stock, moderately old stock, and extremely old

i got 100 percent roots poping, 70 percent above ground but they are weak. most damped off and out of 20 plus i have like 6

how long does it take to tissue culture a seed into a vigorous plant? Im guessing their old age and me not coddleing them set them back a month

From which stage?

from a seed thats not yet popped