# Unusual rothschildianum photograph



## eteson (Apr 20, 2013)

I would like to share with you an unusual picture:
It is a rothschildianum leaf imprint showing the stomata.
We use guard cell size to know if a plant is 2N or 4N after oryzalin treatment.
Hope you enjoy!


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## Ozpaph (Apr 20, 2013)

very interesting.
Can you tell us more about what you do???


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## gnathaniel (Apr 20, 2013)

Great photos! I would also love to hear more.


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## Trithor (Apr 21, 2013)

Unusual aint the half of it! That is incredible! Please tell more (Dont you just love this forum?)


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## tcw (Apr 21, 2013)

Wow! Very interesting.


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## TDT (Apr 21, 2013)

Fascinating! So what stomata traits differentiates 2N from 4N?


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## NYEric (Apr 21, 2013)

Interesting.


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## John Boy (Apr 21, 2013)

Impressive. What's a guard-cell please? I'd love to hear all there is to it!


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## eteson (Apr 22, 2013)

The guard cells are a pair of highly specialized cells which are intended open-close the stomatal pores to allow gas exchange.

The 4N test is quite simple:

Cell size is proportional to ploidy, so the guard-cell size from a 4N plant is higher of the same kind of cell from a 2N plant.

We keep some plants untreated (as a control) of each cross to know the cell size of a "normal" plant, then, we compare the guard-cell size of plants treated and untreated of each cross.

This method is not a "direct" test to certify a plant as 4N since we are not counting the number of cromosomes but it works quite fine.

Eliseo


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## goldenrose (Apr 22, 2013)

so what is this one? 2N?4N? None of the above?


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## SlipperKing (Apr 23, 2013)

I thought someone way back in time claimed orchid stomata where fixed "open" Clearly in your photos that is not true, at least for roth.


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## eteson (Apr 23, 2013)

SlipperKing said:


> I thought someone way back in time claimed orchid Clearly in your photos that is not true, at least for roth.



As far as I know, the stomata are fixed "open" only during a few days after deflasking... but soon the plant is able to open/close the stomas normally.

I have not made direct observations but that would be really easy to test.


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## Drorchid (Apr 23, 2013)

Funny, I was just showing a customer of ours last week how to do this. He has a bunch of Cattleya seedlings, and this is an easy and cheap way to determine which of the seedlings have converted to being tetraploid.

Robert


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## Trithor (Apr 24, 2013)

SlipperKing said:


> I thought someone way back in time claimed orchid stomata where fixed "open" Clearly in your photos that is not true, at least for roth.



Birk certainly wrote that in his revised growers manual


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## likespaphs (Apr 24, 2013)

cool!


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## eaborne (Nov 28, 2013)

Can you tell the difference between a 3n and 4n plant using this method?


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## eteson (Nov 28, 2013)

eaborne said:


> Can you tell the difference between a 3n and 4n plant using this method?



Not with total confidence... the cell size is significatively different in 2N and 4N plants and it is more or less easy to see the difference... but some plants are "dubious"... In any case...cromosome count is much more confident... and expensive


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## Rick (Nov 28, 2013)

goldenrose said:


> so what is this one? 2N?4N? None of the above?



I think we need a side by side pic of a normal 2N plant next to it to compare?


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## Rick (Nov 28, 2013)

SlipperKing said:


> I thought someone way back in time claimed orchid stomata where fixed "open" Clearly in your photos that is not true, at least for roth.



I also heard that, but since then I've heard the refinement that most orchid stomata are open during light exposure regardless of humidity conditions.

Since then I've come across a decent article by Zotz that indicated that maybe up to 40 ~+/-% of orchids are CAM or C3/CAM switch hitter plants which open stomata at night (to conserve water). But at this time I haven't come across any documentation that indicates that any slipper species are anything but simple C3 plants, and will open stomata during the day regardless of hydration conditions.

I think the bulk of Cattleya are CAM plants.


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## abax (Nov 28, 2013)

I agree with Rick. A side-by-side comparison would be very helpful for those
of us who are basically clueless.


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## TyroneGenade (Nov 29, 2013)

It is pretty easy making these imprints if you want to try it at home. You will need some clear nail polish. Just apply the polish to the leaf. Repeat a once or twice if need (i.e. the first item fails). When the polish has set, you just carefully peal off the polish from the leaf. You can then examine the stomata (pores in the leaf for gaseous exchange) under a light microscope.


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## Ozpaph (Nov 29, 2013)

TyroneGenade said:


> It is pretty easy making these imprints if you want to try it at home. You will need some clear nail polish. Just apply the polish to the leaf. Repeat a once or twice if need (i.e. the first item fails). When the polish has set, you just carefully peal off the polish from the leaf. You can then examine the stomata (pores in the leaf for gaseous exchange) under a light microscope.



That sounds very interesting. Please show us an example.


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## Leo_5313 (Nov 29, 2013)

This is cool. Great post and pic.


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## eaborne (Jan 9, 2014)

Rick said:


> I think we need a side by side pic of a normal 2N plant next to it to compare?



Eliseo,
Is it possible to post a side by side picture of a 2N next to the 4N? Thanks.


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## eteson (Jan 9, 2014)

Sure. I do not have pictures of slippers converted because I started few months ago with Phrags and some Paphs, but I do have some nice pictures of Cattleyas. Let me search.


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## eteson (Jan 10, 2014)

Here we go:
This is the best example I could find in my files.
Both pictures are from the same cross. It is a selfing of Catt. mendeli var. concolor.
To the left is a plant not treated and to the right a plant that I think was converted to 4N. Scale Bar is about 100 micrometers







The first Phrags. and Paphs. are in the pipeline right now. I expect to see the first results soon.
As example here a picture of andreettae untreated (left) and treated (right). During the treatment I lost about 60% of the PLBs


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## Migrant13 (Jan 10, 2014)

Thanks for the botany lesson and photo's.


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## TDT (Jan 11, 2014)

Fascinating!


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## eaborne (Jan 13, 2014)

Thank you! How exactly would I go about doing the treatment when flaking?


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## eteson (Jan 14, 2014)

eaborne said:


> Thank you! How exactly would I go about doing the treatment when flaking?



The lab procedure is quite easy, but there are some factors affecting the final results, for example:

-Oryzaline concentration
-Time of exposition
-Age of the PLBs.

I am still experimenting with those factors in slippers so I cannot give you exact numbers.

First of all, you have to prepare liquid media: I use the same media I use for seed sowing but without agar nor activated charcoal. Over the sterilized liquid media I put the oryzalin at the desired concentration. I use a filter sterilized oryzalin solution because I do not know about the thermal stability of the compound.

Then put the PLB´s in this media and start the treatment in a rotary shaker for the desired time.

After treatment I wash the PLB´s with distilled water three or four times and sow in the replate media. In about 40 days you will see that some of the PLBs are dying while others are starting to recover. Then you can select the good ones to be transfered to fresh media and continue replating as you do with untreated flasks

You must leave at least one flask without treatment to use the untreated plants as reference when measuring guard cells.


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