Excising embryos of Rosa beggeriana yahoo!!

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johannes p
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Excising embryos of Rosa beggeriana yahoo!!

Post: # 48806Post johannes p
Sat Nov 24, 2012 10:39 pm

Hi, Guys, For me germinating Rosa beggeriana has always been a nightmare. When I have these problems I will usually excise the embryos which was pioneered by people on this forum. I believe that it states that begg. is too small with too hard of an exocarp with a too soft cotyledons. I too found that was the case,

/So to soften the outside I treated it in 0.3% H2O2. placed for two days in the fridge- important that it be in there so that metabolic rate stops protecting it since the solution becomes depleted of 02 causing the seeds to sufficate.

The now soft skin gently clips off be very gentle they softer than you think. It works !!!! Lost only 2!

Johannes

Don
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Re: Excising embryos of Rosa beggeriana yahoo!!

Post: # 48807Post Don
Sun Nov 25, 2012 2:39 am

>> 0.3% H2O2.

Interesting solution.

The next step is to double the chromosomes and then make some crosses into modern roses to create some F1 breeders. You may still find Colchicum autumnale bulbs for sale although it's getting late.
What doesn't kill them makes them stronger.

jbergeson
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Re: Excising embryos of Rosa beggeriana yahoo!!

Post: # 48808Post jbergeson
Sun Nov 25, 2012 9:14 am

"Interesting solution." Groaning quietly to myself.

Congrats, Johannes! I've never tried such excisions.
Joe Bergeson
Zone 3, NW Minnesota

Margit Schowalter
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Re: Excising embryos of Rosa beggeriana yahoo!!

Post: # 48811Post Margit Schowalter
Sun Nov 25, 2012 12:31 pm

Johannes

That's super! Are they from your own crosses or open pollinated?

Margit

johannes p
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Re: Excising embryos of Rosa beggeriana yahoo!!

Post: # 48819Post johannes p
Sun Nov 25, 2012 5:25 pm

Hi Margit,

These are crosses that I have made purposely. One is Rosa beggeriana X White Pavement, I am trying to see if I get a Snow Dwarf like plant for Paul's theory... I am also crossing it with Connie's laxa that you gave me. I am very pleased with this rose -the colour, the rebloom is good for a species.

Johannes

jbergeson
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Re: Excising embryos of Rosa beggeriana yahoo!!

Post: # 48820Post jbergeson
Sun Nov 25, 2012 5:45 pm

This is really exciting, Johannes. I hope you keep us posted. Are you germinating them right away? I'm not sure how that works with embryos.

If you get good enough at it to have an abundance of embryos, then you could try to double the chromosomes. That would be soooo exciting.

Are you willing to tell us your other beggeriana crosses besides X White Pavement and X laxa?

My R. beggeriana grew into a handsome 4' shrub this year, but had nary a blossom.
Joe Bergeson
Zone 3, NW Minnesota

johannes p
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Re: Excising embryos of Rosa beggeriana yahoo!!

Post: # 48822Post johannes p
Sun Nov 25, 2012 8:26 pm

I have no desire to work with a tetraploid. I love my diploids too much since recovering recessive genes is so much easier. I used to work with tetraploids but found that they lack fertility just as much as a wide cross diploid.

It is only a matter of time before someone gets a dark yellow rebloomer (I have my crosses partly done Wink Wink!)

[attachment 1264 959.JPG]

Don
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Re: Excising embryos of Rosa beggeriana yahoo!!

Post: # 48825Post Don
Sun Nov 25, 2012 10:04 pm

What is this one?
What doesn't kill them makes them stronger.

johannes p
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Re: Excising embryos of Rosa beggeriana yahoo!!

Post: # 48826Post johannes p
Sun Nov 25, 2012 11:22 pm

Hi Don, The breeding is (Rosa rugosa alba X Rosa woodsii) X Leonie Lasmesh. This plant plant is the most successful of the polyantha crosses that I did 5 yr ago. It has some potential but I am working with with a diffent set of plants now. Johannes It is not cold hardy at al

Don
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Re: Excising embryos of Rosa beggeriana yahoo!!

Post: # 48828Post Don
Mon Nov 26, 2012 12:54 am

Too bad it is not cold hardy. F1 species hybrids that express carotenoids are difficult to come by, let alone two species.
What doesn't kill them makes them stronger.

johannes p
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Re: Excising embryos of Rosa beggeriana yahoo!!

Post: # 48829Post johannes p
Mon Nov 26, 2012 1:37 am

Yes, Don

It would be nice for this plant to be non-caractenoid cleaving but it is- the yellow fades but all the genes to produce it are there.



This year I went out to indentify for a noncleaving parent. I extracted tonnes of caratine from Persian Yellow.and placed dots on chromatography papers. I than collected many diploid roses. On these dot I scwiched a small amount of liquid from the petals.. Only on cultivar did not whiten the dots by cleaving. I suspect that this rose was developed from a triploid HT. I am doing crosses now with this plant.I am sorry that I can not tell you what it is but there is 10 yrs of work in here. My negative control was Rosa altaica.

Johannes

Don
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Re: Excising embryos of Rosa beggeriana yahoo!!

Post: # 48831Post Don
Mon Nov 26, 2012 2:03 am

>> I extracted tonnes of caratine from Persian Yellow.and placed dots on chromatography papers. I than collected many diploid roses. On these dot I scwiched a small amount of liquid from the petals.. Only on cultivar did not whiten the dots by cleaving.

This is a brilliant idea.

Can you give us details for extracting carotenoids? Do you concentrate them by evaporation?

Do you assay cultivars that do not already express carotenoids in the expectation these would make good breeders? I think it would be the most imporant use for the method. Do you wait for a particular stage of bloom to assay? Do you treat with UV also?

Do you have any idea if you are measuring dioxygenase activity with this assay?

So many questions. What a good idea you have.
What doesn't kill them makes them stronger.

johannes p
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Re: Excising embryos of Rosa beggeriana yahoo!!

Post: # 48835Post johannes p
Mon Nov 26, 2012 7:00 am

Hi Don thank you for correcting my spelling:)

This is dangerous.... I filled up 1/2 a blender with nice frozen yellow petals from Persian Yellow and water. On top of this I put 5ml of WD-40 and blend at the highest speed until the petals are completely destroyed and become white.

If you survived without igniting the WD-40 you then place the solution onto a orbital sander. The oil should rise to the top if the mass was sufficiently broken up to avoid impeding the separations of the liquids. The oil is picked up with a pasteur pipet and placed on the chromatography paper. I found that it was worth it to be generous and make a large 3cm dia circle.....

WD-40 evaporates quickly and you are left with a non-polar residue that should be yellow. I have no idea which carotanoid was extracted of if they are fluorescent. I thought that it was more flavanoids that do this

.

Part ll can tell how to do the actual cleavage (i'm tired and want to go to bed)

Johannes

aeckstein
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Re: Excising embryos of Rosa beggeriana yahoo!!

Post: # 48840Post aeckstein
Tue Nov 27, 2012 12:20 am

And my wife would say "Seriously do you want to burn the house down?"

Don
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Re: Excising embryos of Rosa beggeriana yahoo!!

Post: # 48841Post Don
Tue Nov 27, 2012 5:24 am

Yes, caveat emptor - or use at your own risk.

I checked the WD40 msds which says it is mostly light fractional petroleum distillate so ligroin or petroleum ether should work, or heptane or Coleman lantern fuel. My concern over WD40 would be that there is a small amount of non-volatile oil left behind.

>> place the solution onto a orbital sander.

Well, would swirling in a separatory funnel or similar container work, with a little patience?

BTW. WD-40 is an acronym for... water displacment formula #40. It was originally used to dry condensate in the nose cones of Atlas missiles.
What doesn't kill them makes them stronger.

johannes p
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Re: Excising embryos of Rosa beggeriana yahoo!!

Post: # 48843Post johannes p
Tue Nov 27, 2012 8:30 am

Hi Don

Why I used WD40 I don't know but it was on hand and while there is a residue I still get some anyways from the plants petals. I tried to isolate the carotanoids with paper chromatography but I found it redundant. I kept using the paper because it absorbs both polar and non solutions you see I have never been able to tell where the cleaving enzyme is in the test plant petals cells.

Funnel I would expect to be very slow and the solution of interest would be at the top and the petal I still leave in the solution, A flask lets the wd40 raise up into the neck for the easiest isolation when vibrated.

I try my best in my little lab in the basement. Corner sometimes have to be cut. If you can come up with any other recommendation I would appreciate it. I will try different solution that you recommend.

One interesting thing is you can actually smell the reaction when the enzyme cleaves but I am sorry I have no Idea what the reaction is.

Thanks fir the input Johannes

Don
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Re: Excising embryos of Rosa beggeriana yahoo!!

Post: # 48849Post Don
Tue Nov 27, 2012 10:00 pm

I took out my orbital sander and gave it a whirl. The sparks from the motor are more than hot enough to ignite petroleum vapors. My advice is to find another way to stir your flask, Johannes. If your flask were to break or the stopper to pop off even five ml of any given form of light petroleum would burn like gasoline - fast, furious and injurious.

Fortunately there is plenty of time to explore some options for extraction and separation methods.
What doesn't kill them makes them stronger.

Don
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Re: Excising embryos of Rosa beggeriana yahoo!!

Post: # 48852Post Don
Tue Nov 27, 2012 11:39 pm

Use this paper in conjunction with the diagram below to start thinking about how to develop an emulsion-based screening assay for testing carotenoid degradation in rose petals.

Carotenoids in biological emulsions: solubility, surface-to-core distribution, and release from lipid droplets.

The late stage carotenoids are the strongest, most stable ones (labeled #8 and higher on the chart). There are some (diogygenase) enzymes that cleave these in half which causes much of the the fade in yellow roses. A comprehensive discussion on carotenoids in roses can be found in Eugster but these will get you started.

[attachment 1268 RosecarotenoidsfromEugsterbiosynthesismap.jpg]
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johannes p
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Re: Excising embryos of Rosa beggeriana yahoo!!

Post: # 57580Post johannes p
Mon Sep 08, 2014 8:25 pm

You guys have scared me. This year I used a teflon homogenator (sp?). Slow but effective, many hrs on the bus while people think I am making crack grind away.
Unfortunately, I identified the white/yellow plants that do not cleave. I wrote up a protocol. If I need it ever again.

Don
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Re: Excising embryos of Rosa beggeriana yahoo!!

Post: # 57584Post Don
Tue Sep 09, 2014 8:26 am

You could post the protocol here. I would like to see it and I think other people would too.
What doesn't kill them makes them stronger.

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