# Chlorosis



## Trithor (Mar 30, 2014)

I have been trying out a number of flasking mediums and a few 'hybrid' versions as well. I have one medium which is showing fantastic potential, but has a nasty side effect, .... chlorosis of a high percentage of the germinating seedlings.
I have tried out around 15 mainstream media, and have resorted to hybridising existing and now adding a few spins to these. 
Of the 'main-stream' media, my best results are currently with old fashioned M&S at half strength with adjusted micros and vitamins, plus coconut water and a 1/20 addition of Svante Malmgren medium. With this I am getting good germination of paphs and other species.
My new favourite is a mix using the above base with Tryptone substituted for the Peptone, and a liquidised endosperm of an African nut which is occurs on my farm. With this I am getting fantastic germination (glaucophylum, villosum and Maudiae are my current test subjects). The seedlings germinate around 5 weeks ahead of the same seed on 3 different mainstream media and show accelerated growth. The only downside is that 50% (of the bigger seedlings) are chlorotic. I replated the flasks to a general maintenance medium and a full strength version of the germinating medium, with adjusted NH4NO3.
My question is, .... does anyone have any experience with flask seedlings and them greening up? Is there some other adjustment that I can make? The seedlings are doing fantastically, only ice white! Predictably it is the biggest plants that are affected.
PLEASE any suggestions will be welcome!


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## Ryan Young (Mar 30, 2014)

Perhaps the addition of Triacontanol to the medium would aid in this problem, you could also spray on seedlings in intervals. It acts as a plant growth booster by aiding absorption of nutrients, increasing growth and the production of chlorophyll. There are a few papers written on it. It is from the waxy coating of alfalfa.


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## Ryan Young (Mar 30, 2014)

Also a little goes a long way 2.5-10 ppm range is sufficient according to the papers that I've read.


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## TyroneGenade (Mar 30, 2014)

Can you provide some pictures? Chlorosis can have several causes and the pattern of chlorosis can suggest the underlying problem. See http://tomatoheadquarters.com/wp-content/uploads/2013/03/disease-graphic.png for a quick summary.

This is a little bit more indepth: http://www.420magazine.com/forums/problems-pests-disease-control/153954-some-plant-deficiencies.html (Amazing how much research has gone into growing Cannabis.)


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## PaphMadMan (Mar 30, 2014)

Not much experience with orchid seed flasking, but some thoughts from plant tissue culture work a lifetime ago.

That it occurs on the largest fastest growing seedling suggests there may be a nutrient uptake problem. The slow seedlings can keep up with their nutrient needs, the larger ones can't. What size are the seedlings when you first see this? Is root growth inhibited? Are the seedlings otherwise healthy in appearance, not glassy or waterlogged?

Alternatively, there could be a borderline deficiency or some nutrient is getting sequestered in an unavailable form. Something incompatible in the hybrid formulations you're trying? Something unexpected in the endosperm that is unique to your formulations?


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## gonewild (Mar 30, 2014)

Are all three varieties showing the problem?

What is in the nut that made you think it would be good to use?
In nature do a lot of plants grow around and under the nut tree? Or is the ground void of competing plants?


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## Trithor (Mar 31, 2014)

As I am still testing and comparing various media, both for germination and maintenance, I make two mother flasks on three different media of each seed set (6 in total). Then one month later I assess the flasks and if contamination was a problem, then I re flask the affected cross in the same way (not much contamination problems, so the second flasking does not happen very often)
The chlorosis appears to affect the glaucophylum the worst, but out of the three paph test subjects, the glaucophylum are the fastest growing. I have 2 Catt hybrids and a stanhopea species (seed from JohnM) on the same set of media, and these are growing like grass, and are green like grass too.
The seedlings affected are the paphs only, with the glaucophylum being the worst affected. The flasks have both green and white plants in the same flask, with the biggest plants being chorotic. The root growth is good on both the green and chorotic plants. The seedlings are not very far advanced yet. 
The glaucophylum was flasked on 4 jan 14, and spent 2 weeks in the dark prior to being moved to the racks. By mid feb seed swelling had occurred in all 6 of the flasks. By end feb, the seed on P668 half strength, and M&S third strength with adjusted micro and vitamins were still only swollen, but showed no further development. The seed on the hybrid medium had germinated and was starting to show the beginnings of root and leaf. As of yesterday the hybrid medium had fully differentiated seedlings with good root and leaf starts. The only problem is the larger ones are chlorotic. The development in the other 4 flasks are fully 2months behind. I replated the two flasks to a general maintenance medium (duchefa orchidmax with charcoal and banana)

I started using the nut as an additive, because (rather empirically) they look like mini coconuts and the antelope seem to love to eat them, as do the monkeys. There is no less or more plant growth under these trees in comparison to others in the same area. Only the large trees have a reduced plant growth below them, but that is because they are used as shade trees by the game, and so everything including grass is trampled to dust.

Tyrone, there is no discernible pattern, the plants are just chlorotic (completely), I will see if there is a change in the replated plants, also if the seedlings on the mainstream media show the same affliction (if and when they eventually start growing)


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## TyroneGenade (Mar 31, 2014)

Hi Gary,

If the complete leaf is green-less then the issue is nitrogen. Reflask the big seedlings to medium with more nitrogen and they should green-up nicely.

You may want to figure out a way to get some more nitrogen into the mix or aim to replate sooner or more frequently. 

You seem to have hit on the secrete to good germination but for growth maybe reflasking on to P668 is a good idea.


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## Trithor (Mar 31, 2014)

Tyrone, thanks for the input. A nitrogen shortage is my suspicion as well and I am glad to have that confirmed by someone else. I have been doing quite a bit of reading today, and have noticed that perhaps the addition of ammonium nitrate to the medium at around 720mg per liter is called for. My initial gut feel was that this was too much nitrogen, but it would appear that the big labs go for an even higher level up to 848mg for some orchid media. I think I will start with a 1/4 strength and add around 200mg. I plan to re flask the glaucophylum seed onto my new hybrid medium with 1/4, half and full strength added and see what happens. In the meantime I will adjust my hybrid medium with quarter strength as a start for all seed going forward, till I have the results of the glaucophylum test.


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## naoki (Mar 31, 2014)

Gary, this could be completely off, but different stages of endosperm development seems to have different composition of hormones. I came across  this paper, and you might want to take a look at Table 2. It seems that mature coconut endosperms contains more auxin than cytokinins. As you know, the ratio of cytokinin:auxin is important for proper developments in tissue culture. Also cytokinin is a positive regulator of chloroplast development and brassinosteroids are a negative regulator (Check this). So if your nuts were collected at a wrong time, or if it had wrong sets of hormones, it may interfere. The sensitivity to hormones could differ among species (and glaucophyllum might be really sensitive).


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## Trithor (Apr 1, 2014)

That is really interesting, thanks for the info. It will take me a bit of time to digest the details. The trees are in fruit at the moment, and the nuts are still immature. Of note is that the game do not eat them at this stage, any that fall from the tree are left on the ground untouched. I will have to watch for when the squirrels and animals first start eating them, and harvest them then, and give it a try at that stage?


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## naoki (Apr 1, 2014)

I don't know what would be the best stage of your nuts, Gary... With Coconut, it seems that immature fruits are more suitable with more cytokinin. I came across the paper when I was looking for the concentration of plant hormones in coconut water (I'm experimenting with spraying coconut water for stubborn plants). Do you happen to know the name of the species?


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## Ray (Apr 1, 2014)

naoki said:


> I don't know what would be the best stage of your nuts, Gary...



Whoa!!! Jumping to the most recent post, and reading that gave me an entirely incorrect grasp on this thread!!!


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## eteson (Apr 1, 2014)

Hi Gary. I had the same problem with phrags and some paphs months ago. My problem was related with high temperature in the growing room and, too low amount of agar in the media. I installed a air coditining system and started to use a slightly harder media and the problem gone with the wind.


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## Trithor (Apr 2, 2014)

Hi Eliseo, thanks for the input. My media gel is firm, and easily supports the growing plants. I did have a problem with a different brand of agar, but after a batch of flasks were soft, I switched back to the original brand from Duchefa in the Netherlands. My temperatures in the growing room are regulated between 25 and 28 degrees, and the laminar flow/flasking room at 20 degrees (for my comfort, as when the laminar flow hood is turned on and running, the temp rises by about 4 degrees)
I suspect that the problem lies with cutting the strength of the base medium. M&S is quite a saline medium, so I have cut the original, adjusted the vitamins and micros, and added an amino acid base plus organics. On further reading, I have come to realize that a number of the big labs reduce the maros, but add ammonium nitrate. So I have adjusted my formula to include ammonium nitrate at 200mg per liter of mother flask/germination medium and 540mg per liter of maintenance medium


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## Trithor (Apr 2, 2014)

naoki said:


> I don't know what would be the best stage of your nuts, Gary... With Coconut, it seems that immature fruits are more suitable with more cytokinin. I came across the paper when I was looking for the concentration of plant hormones in coconut water (I'm experimenting with spraying coconut water for stubborn plants). Do you happen to know the name of the species?



Sorry no, I have tried to identify the tree, but not too diligently. They only occure in one patch on the farm (in a ravine formed at the edge of a prehistoric lava flow, which has resulted in this particular area to have a elevated rainfall/runoff relative to the surrounding areas) this is where the leopard has its cave and raises its cubs every few seasons. It was on one of these visits that I found the nuts while I was collecting porcupine quills from the leopard kill. The hind quarters had been eaten, but the gut and contents were relatively intact and the nuts featured prominently in the stomach contents. To be honest, when I am in the area I don't spend too much time looking around, it is a very densely overgrown area, dark and has too many potential hiding places for leopard. It is passing toms that I am most wary of.


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## eteson (Apr 2, 2014)

Trithor said:


> My temperatures in the growing room are regulated between 25 and 28 degrees



28°C seems to me too high... my growing room is set a 24°C and I feel it too hot for the optimal development of some phrags...
Eliseo


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## Trithor (Apr 4, 2014)

Perhaps for phrags, but according to a paper I read on paphs, optimum enzyme activity is achieved at 26 to 28 degrees, with an implication that seedlings may need a degree or two higher. I have set the temperature control with the findings of that study in mind.


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## eteson (Apr 4, 2014)

true... I have much more experience with phrags. Please keep us posted about your findings.

Enviado desde mi SGH-I337M mediante Tapatalk


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## Trithor (Apr 5, 2014)

eteson said:


> true... I have much more experience with phrags. Please keep us posted about your findings.
> 
> Enviado desde mi SGH-I337M mediante Tapatalk



I cant really remember where I read that, so my info might well be distorted and inaccurate (? one of J Arditti's reviews?)

I have replated the affected flasks into the new range and will keep you updated.


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