Tooling around this evening, I ran across this and thought it might be of interest here. [flickr_photo src=http://farm9.staticflickr.com/8049/8095870063_1e2d8df9de.jpg nsid=67995840@N04 id=8095870063]1948 ars embryo culture (1)[/flickr_photo][flickr_photo src=http://farm9.staticflickr.com/8188/8095875344_68b0767a1a.jpg nsid=67995840@N04 id=8095875344]1948 ars embryo culture (2)[/flickr_photo]
At first, 4-5-6 or so years back, EC gave me some spectacular results, however on reflection the numbers I was initially dealing with were tiny.
The “true success rate” for me came out when I got really good at extractions and had a hugely bigger number of embryos to deal with, several years later. That rate was horribly low, totally unacceptable. My methods for culture were experimental and not derived from publications.
You need to follow tried and tested methods, and you need a huge amount of time to do it, time I certainly do not have for it.
Does anyone out there with a chemistry background know what type of mixture the formula printed in this article should approximate? Would there be some type of “off the counter” item that could be used instead?
Andy
Hi Andy: KCl is potassium chloride - the bag I use to desalt my walk-way in the winter.
CaSO4 is calcium sulfate, or gypsum
MgSO4 is magnesium sulfat, or epsom salt
FePO4 is ferric phosphate, or Sluggo to kill slugs
KNO2 is potassium nitrate, it’s 75% in gunpowder, a bit in toothpaste to desensitize pain (Sensodyne)
I think a fertilizer with gypsum, epsom salt, iron, sulphate of potash is good-enough as substitute.
I would start by checking with Don’s handbook which I believe is posted at the RHA site. Key thing is sterility if you are going to supply sugar (glucose aka dextrose) which will grow mold. Also each seed gets its own little container, so a bad one doesn’t spoil the lot.
I think you’d find this similar to some of the things that Murashige and Skoog developed a few years later, sold as MS medium. But you’d have to leave out the growth hormones from their recipe. I’ve not taken time to do the calculations, but its likely that a good soluble fertilizer (Miracle -Gro, Peters) properly diluted and thickened with agar could work, in principle for the micronutrients which are sorely lacking in this formulation but may be found in technical grade fertilizer chemicals. Fertilizer formulation and marketing has advanced tremendously since the 1940s. But unfortunately the ammonium nitrate (Timothy McVey etc) scare has screwed up most formulations and they may well be toxic with urea and ammonium replacing nitrate.
With enough light there’s no need for the sugar, and probably fine vermiculite would work well as a support medium if kept moist, not flooded. After all, seeds germinate in dirt with few added nutrients. These folks just tried to accelerate growth a bit by adding sugar. Plain sucrose might work fine. It does for soybean seeds I know.
As I recall, one cultivar was introduced in the late 1940s derived from this kind of tissue culture (mentioned in my review on RHA site). But it’s too much work for ordinary hybridization programs to afford.
Regarding the formula. When I was doing tissue culture http://home.roadrunner.com/~kuska/mytissueculture.htm , I checked the formulation of powedered baby milk substitute products against the murashige and skoog (ms) tissue culture media formulation. Some were rather close. (It is too long ago to remember what sucess I had. I am sure I tried some.)
My full tissue culture (probably out of date) page is at: http://home.roadrunner.com/~kuska/tissue_cultureindex.htm
I missed your post last week, Kim, interesting find and one I will include when I update the manual.
Hi Don, great! I’m glad you enjoyed it.
It would be good to put together a bank of variations that people have had success with.
So far I have had some success by using ziplock bags as in Don’s manual but I tend to take them out before Don recommends. The two most successful extractions occurred when I removed the embryos from the bags just as they turned green and the radicle had started to elongate. Then I sterilised normal potting mix (next time I would sieve it) with boiling water in the microwave (forget for how long) which was drained and put into sterilised medicine cups and allowed to cool. The embryo was placed radicle down in the potting mix, sprayed down with a contact fungicide like Mancozeb and covered in cling wrap. I gave the embryos a light spray with Mancozeb every day. I found the embryos seemed to take off faster when in contact with the soil. I assumed this was due to access to nutrients (macro and micro) in the potting mix. I also figured the last part is essentially a race against the fungus so I needed to get it going as quick as possible and out into a more open environment. The cover was taken off the medicine cup after about 5-7 days to begin hardening off the seedling.
We use MS at work with potato tissue culture and in my most recent attempt to culture my (hopefully) doubled laevigata (3 weeks into it and touch wood they look pretty good).
[center][attachment 1193 sinowilsoniixviolette1.jpg]
Approx 5 days after transfer to the cup[/center]
It is amazing how a tiny percentage of embryos will grow just simply being ploncked into soil from the outset (not recommended of course). I will never forget an embryo I had chucked (immediately post extraction) into soil from Knock Out X OP… I forgot about it, thought it was a goner after seeing no germination at about the 10 day mark, then after roughly two weeks it sprouted …and sooooo vigorously !
One thing I have learnt over the years of experimentation with EC, is that there are embryos and then there are SUPER embryos !
LOL