Simple methods for embryo rescue?

[gilbert]

Are there simple methods for doing embryo rescue on wide crosses with immature embryos? I've found simple methods for mature embryos, but so far nothing for immature ones. 

[billw]

Can you be more specific about what you mean by simple? Embryo rescue is pretty straightforward, but it relies on having a few solid, well-practiced lab skills. One of the trickiest parts, at least for crops that are not well known, is just knowing when the embryo is well developed enough to survive, but not yet aborted. Figuring that out on your own can take a crazy number of attempts once you figure in the number of embryos that you kill in the process. Otherwise, it is just a matter of excising the embryo, transferring it onto the proper medium, and continuing tissue culture as normal. That sounds easy enough, but developing the necessary skills is the hard part. Excising the embryo usually needs to be done under magnification and there is no room for error so it takes a lot of practice. It is totally doable for amateurs insofar as it is not difficult to understand or too expensive to equip for, but there will be a lot of failure before success.

[steev]

Ah, lab skills; so often unknown, but so valuable.

One of my buddies was always impressed at how perfectly I could split a large bottle of ale: years of being a bench-chemist builds precision in the pour; some skills have broad application.

[gilbert]

Can you be more specific about what you mean by simple?

Well, I have no lab skills to speak of. . . 

I do have a microscope.

The main drawback is that in some protocols, there seems to be a requirement for sterile conditions; how would I keep the embryo and the culture media sterile under low tech conditions? 

Would it work to just plant immature seeds? Or place immature seeds in some sort of media? Could inhibitors be leached or soaked out of the seed some how? For small seeds, even with a microscope, I imagine excising the embryo would be maddening. 

For instance; to culture mature embryos, there is a method in which the embryo is removed (from relatively large seeds such as apples) and soaked in a hydrogen peroxide solution. Then they are placed on a hydrogen peroxide soaked paper towel, which is put in a plastic bag under a grow light. No expensive gels, no sterile conditions, etc. But that is for mature embryos. Is there anything comparable for immature ones? 

[billw]

I work in a glove box. Its virtue is that it is very cheap. Otherwise, it sucks, but you can still get the job done. I do microscopy in the box by camera. I just set a laptop on top of the box and view through that. That is also a pain in the ass, because it lags and you end up destroying stuff, but again, it works if you are determined and patient.

[oxbowfarm]

I'd say the problem with embryo rescue is its a fiddly process that requires great skill. I imagine most professional breeders are only doing it on their chosen genus/species, and any shortcuts used are probably only applicable to that species.

[keen101 (Biolumo / Andrew B.)]

i too liked reading about Joseph's early embryo rescue technique with some of the tomato hybrids by putting them in soil before finishing developing. Can't remember which thread/post talked about that but it was both enlightening and surprising. If it does indeed work then wow that sounds like a good method. I would not mind at all rereading that or having Joseph talk about it again.

I have a microscope, but sadly a compound microscope, so it is the wrong kind. I want to someday get a dissecting microscope as they have a wide platform and are better for observing plants, insects, and other large things. It could also come in handy for SMD soldering of electronics, so win-win.

I am pleased to read that sterilization may not be needed in some cases. I would like to learn how to do sterile culture techniques but it sounds like from a DIY perspective it can be done similar to DIY sterile mushroom culturing techniques or diy sterile microbiology with a cheap "clean box" and a spray bottle with H2O2 or water to mist the air and remove many airborne contaminants.

But curious to know about other potential methods. Like the soil. Can regular soil be used? Does it need to be sterile? Can dirt with sugars added to it be used instead of media? Can that gel that is used in diapers or those gel water soaking beads be used? Is there a good DIY media recipe?

What do you think about these supplies?:
www.carolina.com/living-organisms/plant-tissue-culture-and-plant-physiology/plant-tissue-culture-media/10608.ct?Nr=product.siteId%3A100001
www.carolina.com/living-organisms/plant-tissue-culture-and-plant-physiology/plant-tissue-culture-equipment-and-supplies/10607.ct?Nr=product.siteId%3A100001

[keen101 (Biolumo / Andrew B.)]

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?

[keen101 (Biolumo / Andrew B.)]

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?

[keen101 (Biolumo / Andrew B.)]

Jan 15, 2017 0:05:26 GMT -5 Joseph Lofthouse said:

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.

[Day]

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.

[nathanp]

BillW posted this in the Kenosha Potato Project group last year. Link to the thread is here:
Bill's Lab Equipment
There are more comments directly in the post and questions answered, but here is the text of his initial post.

BillW - a subject for a future blog post on Cultivariable?

"this is a brief rundown of what I have in my lab and what I use it for. I don't know how useful this will be, but you can at least Google things and learn more about what they are used for. This isn't everything, but probably covers most of the important stuff. It is worth noting that I didn't start out with all of this. I accumulated it over time. If you are interested in getting into lab work with potatoes, you should probably pick one project, get the necessary gear, get some good practice, and then move on to the next thing. This shouldn't be looked at as a shopping list. Most people won't need all of this stuff. Some of the chemicals can be dangerous if not handled properly and many are also perishable. I have all of this stuff because I grow and preserve a large number of uncommon roots and tubers that are difficult or impossible to replace, so I am highly motivated to keep them free of disease.

The tasks that I do in the lab fall into just a few groups: pathology (diagnosing and treating plant diseases), breeding (increasing or decreasing ploidy, primarily), morphology (examining microscopic structures, determining ploidy), genotyping (fingerprinting), and preservation (storing varieties in tissue culture). Most of what I do is pathology and preservation. I started doing ploidy manipulations and genetic fingerprinting for fun and I'm now experimenting with protoplast fusion as a possible way to eliminate male sterility. There are tons of interesting things that you can do with a modest investment in lab gear.

Essentials: A desk/table, a sink, a dishwasher, a cabinet for chemicals, small refrigerator for perishable supplies, protective gear (eyes, skin, clothing, airway) - you don't need all of them all of the time, but you should make sure that you know when you do, fire extinguisher, lab type DC power supply

Labware: Plastic pipettes, dissecting needles, scalpels, slides, coverslips, beakers in sizes from 10ml to 2L, graduated cylinders, test tubes, plastic culture vessels, Petri dishes, well plates, various forceps/tweezers.

Chemicals and supplies (What is in the cabinet and refrigerator at the moment): Agar, Murashige and Skoog medium, Logol's solution (dye), toluidine blue (dye), safranine (dye), immersion oil, gram stain set, bleach, ethanol, oryzalin, potassium hydroxide, glacial acetic acid, giberellic acid, potassium nitrate, various ELISA reagents, Percoll, 2,4-D, ribavirin, 6-BAP, NAA, IAA, IBA, IPA, kinetin, salicylic acid (aspirin), zeatin, DMSO, trisodium phosphate, thiourea, 2-chloroethanol, colchicine.

Microscopes: A 1600X, trifocal Olympus microscope with a cheap USB camera. This is primarily used for counting chromosomes, guard cell chloroplasts, evaluating pollen fertility, and gathering morphological details of bacteria.

A cheap Amscope 1500X microscope modified for darkfield. This is primarily useful for examining pollen.

A cheap dissecting microscope. This is primarily used to look at fungal structures in order to identify diseases. Also useful for looking at insects and damage to plants.

A laptop computer to use as a terminal for the microscope cameras.

Chemistry gear: Heat pad/magnetic stirrer, centrifuge, fume hood (crucial for working with chemicals that break tuber dormancy, among other things), propane burner, milligram scale, centigram scale, 1000 watt microwave

Biology gear: Electrophoresis set, DNA thermal cycler (both for genetic fingerprinting), incubator, glove box (a large plastic box with ports cut into the side for doing sterile work)

Others: Hot towel cabinets with external thermostats for performing tuber thermotherapy. Small chest freezer with external thermostat set to 34F for tuber cryotherapy. Growth chambers (essentially insulated boxes with lights, small heaters and humidifiers installed for starting temperamental seeds or promoting a particular growth phase in cultured plants).

Where to get this stuff? Ebay is a great place for used lab gear. Phytotech Laboratories is a good place for media and chemicals.

With this setup, I can diagnose many viral diseases and most fungal and bacterial diseases. I can rid tubers of all the diseases that I can test for, using a combination of cryotherapy (cold storage), thermotherapy (hot storage), meristem culture, and exposure to ribavirin. I can keep cleaned varieties growing in tissue culture indefinitely so that I can restart from clean plants in the event that disease reappears. I can produce haploids by anther culture. I can increase ploidy by application of antimitotic agents such as oryzalin and colchicine. I can break dormancy of most tubers by application of GA3, thiourea, or 2-choloroethanol. I can determine the ploidy of varieties directly by counting chromosomes or indirectly by counting guard cell chloroplasts. I can use two different methods of genetic fingerprinting to compare varieties and determine of they are duplicates. The only problem is finding the time to do everything when weeds are growing.

What this list lacks is a list of all the sources of information necessary to use this stuff. That is much more difficult. Perhaps a post for another day."

[oxbowfarm]

Dec 24, 2017 13:09:21 GMT -5 Day said:

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.

Day Embryo rescue is a technique for salvaging an embryo that will most likely not survive to form a mature seed. Often this occurs when a wide cross between species is done, or between different ploidy levels of a species. Sometimes you can get an embryo to form from such a cross, but the endosperm won't form, or the endosperm is incompatible with the embryo. This would result in the embryo aborting naturally. So embryo rescue is a lab technique where the embryo gets removed from the ovary prior to the stage where it would abort, and is cultured in vitro. Pretty specialized technique.

[philagardener]

Dec 24, 2017 13:09:21 GMT -5 Day said:

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!  )

[Day]

oxbowfarm philagardener -- 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