I hope this doesn't interrupt the flow of the thread, but for those of us reading who have never practiced embryo rescue, could anyone explain roughly what the purpose/goal of doing so is, and an example of when one might attempt it? I'm trying to follow along with this post, but lacking context I'm having difficult understanding the process.
In some crosses between distantly-related species, pollen will germinate, grow down the style, and fuse with the ovule to complete fertilization. However, for a variety of reasons, the development of the new embryo may arrest or fail because of incompatibility with the maternal plant. If this developing embryo is transferred into an artificial, supportive environment (i.e. tissue culture), it may be able to survive and grow. Having past that initial hurdle, the resulting hybrid plant may have some degree of self-fertility, or be able to be back-crossed to one of the parents. This process of embryo rescue can be used to transfer useful traits from a wild species into a normal crop variety.
Anyone experienced with this technique will agree this makes it sound easier than it is (there are plenty of complications and things that can go wrong) but that's it in a nutshell, so to speak!
Sort of like a plant preemie!
(Got distracted while writing and oxbowfarm gave you a nice explanation too before I actually posted! )
oxbowfarmphilagardener -- Thank you both very much! That clears it up immensely. I was unaware that there was a stage in seed development where things were still 'playing nicely.' I'd always just assumed that while fertilization could be successful between a wide cross, that the problem with embryo growth began immediately, which is to say there was never any hope of recovering a viable embryo from a cross that 'didn't want to play.' Knowing that now, it'll be fun to go back and read this thread again and watch things fall into place xD
Embryo rescue strikes me as super easy. Open the fruits about 31 to 35 days after pollination, and plant the seeds into sterile growing media.
Could not this method be easily used for squash or cucurbit hybrids as the seeds are so large? I guess you would have to sacrifice immature squash fruit, but maybe it would be worth it. And maybe as Joseph suggests no removing seed coats are really necessary. Or at least not the whole thing.
The larger the seed the easier it's going to be to excise the embryo if necessary. Not excising the embryo is probably dependent on that embryo being able to get some sugar to feed it's germination. That might be more difficult if there is thick wall of tissue between it and the embryo in a larger seed than in say a tomato seed.
Cool interesting stuff. One question i had is about using different types of sugars. Standard protocols probably only use table grade sugar, but would using other sugars increase the ability of success? What about glucose syrup? Pure fructose? Maltose? Xylose? Etc?
I remember reading a discussion of how sucrose works in plant tissue culture in one of my books. Breaks down into its components somehow which are glucose and fructose.
I wondered the same thing a bit and if I remember correctly the answer is that for some reason sucrose works pretty well for most plant species in tissue culture. Not to say that there might not be variation in that but in general I would stick to sucrose unless I had an protocol or paper that suggested a different sugar for a specific group of species or a certain developmental stage or some such.
That said, there are an enormous number of parameters that can be manipulated to optimize a plant tissue culture environment for a specific species or even a specific genotype and this might be one of them.
Actually that right there is one of the big problems with genetic engineering. Having to grow the transgenic ex plants in Tissue culture limits the genotypes on which transformations can be performed. Net result is that sometimes transgenes aren't inserted into the best possible conventional varieties because those varieties may not be genotypes that perform well in tissue culture. These difficulties may also extend to some conventional varieties from breeding programs that used advanced non-transformational techniques that push the seedlings through a tissue culture phase like doubled haploids. The barley breeder my wife and I took plant genetics from had a team which had just massively improved their success with recovering doubled haploids. So it isn't necessarily limiting- but it can be. There are also some limitations on using tissue culture for plant conservation for these same reasons- messes with the genetic diversity of populations Because it favors individuals that do well in an incredibly artificial environment.
Oh i had another thought / question. I've read about boiling willow branches to make your own rooting hormone liquid. 1. Does this work? 2.does it work with other than just willow? 3.could something similar work to extract the nutrients needed by a specific plant or embryo to grow? Like if you we growing a rare tomato cross, could you boil tomato clippings and use that water to make the culture media?
Could you harvest only roots and boil those for concentrated root chemicals? Could you collect sprouted squash seeds and boil those for concentrated seed sprouting hormones?
Yes willow water works, and it does work for other species. When I used to grow native plant cuttings as a seasonal tech for the park service we had some species that rooted with low levels of rooting hormone and some higher. Willow water will root other species if they can be rooted with low levels of hormone.
Some hormones are more or less sensitive to boiling- a few have to be filtered and not boiled.
Many of the hormones used are artificial chemicals- you can actually harvest hormones from herbicides like 2-4D and Picloram.
While it might be fun to play with attempting to harvest hormones as a side project or as an investigation in its own right I suspect buying hormone is worthwhile in terms of time saved if the goal is to do an embryo rescue. If money savings is the goal then harvesting hormones from herbicide might be a more profitable consideration. I don't remember the chemicals being horrifically expensive though and a little bit lasts a long time.
My experience has been that busting out the plant tissue culture kit is such an investment in time that any time savings from buying quality chemicals is very worthwhile to me. For instance I have some old chemicals- I would probably buy fresh to do a tomato embryo rescue right or at least test the old ones first on some plant tissue that didn't matter. That latter is itself an big investment of time.
On the other hand, plants do produce their own hormones. It's worth checking protocols to see if hormones are required for a particular embryo rescue.
Usually embryo rescue is required due to endosperm failure. The embryo cannot grow without the nutrients that would normally be provided by the endosperm. So, I don't see how you could pull it off in soil. Normally, the embryo is cultured on a nutrient rich medium that substitutes for the endosperm.
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Post by philagardener on Dec 26, 2017 18:28:08 GMT -5
A complication may be that all your embryos are literally in one basket - staging when to harvest the developing fruit may be a bit tricky at first, but days after pollination should be a way to standardize. Good luck and keep us posted!
Days after pollination may not be a constant, due to temperature etc. But it is often true that the embryo is good for several days. Generally, one wants to let the embryo grow naturally as long as possible, then rescue it. But the embryo might have been recuable over a few days. Depends on the cross.
billw. What you said above is generally true. One exxception I know of is Zea x Tripsicum hybrids. Sometimes the embro grows well enough, but the endosperm starts breaking down. By the time the embryo is mature enough, the endosperm is gone. By sdetting the embryo upright in sterile soil and keeping it from drying out, the light on the upper embryo can feed what is pretty much a tiny plant, though it is indeed an embryo. I learned that studying the history of Zea x Tripsicum. This was the first way to do it, other than just make the cross thousands of time and hope. Today, standard embryo rescue is done for the cross since it gives a higher % success.
Do I understand this right? Your talking about growing the new plant before an actual seed even forms? So first you have to know that a pollination has occurred but the seed either won't mature or will be deficient in some way if it does?
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Post by philagardener on Dec 27, 2017 17:56:58 GMT -5
Yes, essentially growing out the young hybrid embryo to get around genetic incompatibilities that would interfere with seed development on the mother plant.
You wouldn't use the technique for crosses that can mature seed (easier to let the plant do it!); there are often clues that fertilization has taken place (like when flower doesn't immediately drop like a failed pollination, or ovary tissue that starts to swell) that suggest when the effort is worth trying.
Embryo rescue is typically only done after the natural seed formation fails. Then one watches and tries to find out when it fails. Then one decides whether one really wants that hybrid that bad, 'cause it really is extra trouble. But often it is worth the trouble. With all the history of embryo rescue, one doesn't usually have to do testcrosses to know if embryo rescue is necessary, and if so, how. That is what google is for.