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Heli pollen observations

My slides and cover slips arrived today so I thought I would have a peek at the H. folliculata pollen I harvested on 11/6

This pollen was dried overnight in a sealed container with ground up saltine crackers as a desiccant. The pollen was then froze for a few days, thawed and used to pollinate the next flower. Since that time it has sat at room temp in its sealed container. (Wouldn't surprise me if it wasn't still viable)

My media was approx 20% sucrose by volume... (I don’t have any scales, I was hoping I wouldnt need to invest in any). I prepared a slide; applied the pollen, and made a growth chamber by placing wet paper towel in the bottom of a bowl with the slide suspended above. This was then covered in cellophane and allowed to sit at room temp for 6 hours.

At 400x the pollen grains were easy to identify but no tubes were observed.

Many of the references indicate the media may need some boric acid and calcium nitrate.

I will try the plain sucrose media again with fresh pollen in an effort to isolate the problem. If they still don’t grow I will try fresh pollen using the onion membrane method.

Still waiting on microscope imager to arrive... so no pics yet.
 
Hmmm. Much to ponder. I need to test the onion skin method at the office
 
I should be able to harvest some fresh pollen tomorrow.... that should tell me if the plain sucrose media has any hope of working
 
If only you could harness that scientific zeal for good and not evil . . .
 
If only you could harness that scientific zeal for good and not evil . . .

Give in to the dark side my friend.... we have cookies :p

Well I thought I would look one more time this morning before cleaning the slide.... no change.

On a related note, I love the microscope... much nicer than the basic Tasco I had as a kid.
But all the optics are in need of a good cleaning
 
Why not try a control with pollen from a species that is known to work on onions skin?
 
Warren,

From what all I've read on the subject I'm not sure using another type of pollen would tell me much.

It seems that what media works for one pollen type may or may not work for another.

I got a Spanish onion and some leeks today; I will try a simultaneous run with onion, leek and 20% sucrose with uber fresh pollen.

(From flower to slide)
 
20% seems like a lot of sugar. Seems it would cause cells to suffer from an osmotic imbalance .....
 
Mach,

I dunno.... almost everything Ive read on the subject state anywhere from 2-20% with most refs saying 20%

So that is why I started there... If still not results with 20% next time ill drop back to 10%

But Im wayyyyyyyyyyy out of my field of expertise, I am open to any and all suggestions.

I tried harvesting some pollen a few mins ago, only got a tiny amount... the anthers are still a little green,

So I tried the onion epidermis and 20% sucrose, the leeks are going to go well with a steak Im about to grill LOL

---------- Post added at 01:46 PM ---------- Previous post was at 01:43 PM ----------

example ref:

Sucrose concentration: various sources list concentrations from 1 per cent to 20 per cent w/v. The SAPS protocols recommend a starting point of 1.2 M sucrose, which is about 40% w/v, diluted by half on mixing with mineral salts culture medium. The optimum sucrose concentration varies with species – so it may be necessary to use trial and error to find a reliable concentration. 10% - 20% final sucrose concentration seems to be ideal for most. This solution will keep for some months in a refrigerator.

Im betting Ill end up needing the boric acid and calcium nitrate for the sucrose based media

This is from the ref that seems the most "pollen tubes for dummies" friendly :)

To Germinate the Pollen:
GERMINATION MEDIA (Since germination occurs rapidly, there is no need to autoclave unless you are preparing them in advance.)
NOTE: Media C and E are used for extensions, only.

Medium A - 10% sucrose
Medium B - 10% sucrose, 100mg/L boric acid, 300 mg/L calcium nitrate
Medium C - 10% sucrose, 100mg/L boric acid, 300 mg/L calcium nitrate, 200 mg/L
magnesium sulfate, 100 mg/L potassium nitrate
Medium D - the same as Medium B with the addition of 1% agar. Heat to boiling and pour into
petri dishes.
Medium E - the same as Medium C with the addition of 1% agar. Heat to boiling and pour into
petri dishes.
 
Last edited:
  • #10
http://www.practicalbiology.org/are...rving-the-growth-of-pollen-tubes,127,EXP.html

Says:
1 Sucrose concentration: various sources list concentrations from 1 per cent to 20 per cent w/v. The SAPS protocols recommend a starting point of 1.2 M sucrose, which is about 40% w/v, diluted by half on mixing with mineral salts culture medium. The optimum sucrose concentration varies with species – so it may be necessary to use trial and error to find a reliable concentration. 10% - 20% final sucrose concentration seems to be ideal for most. This solution will keep for some months in a refrigerator.

1.2 M of sucrose = 410.76 grams
 
  • #11
Im really hoping the onion epidermis works LOL
 
  • #12
then someone is pickling sweet onions, chopped steak and onions, grilled onions, bacon and onions :p
 
  • #13
Kula onion kimchi.
 
  • #15
If you could see the T-bone that I am letting rest at the moment

omg yum!
T-bone, sauteed Leek, broiled asparagus with olive oil and sea salt.... and cold Guinness

you two better get over here quick :p
 
  • #16
Warren,

From what all I've read on the subject I'm not sure using another type of pollen would tell me much.

It seems that what media works for one pollen type may or may not work for another.

I got a Spanish onion and some leeks today; I will try a simultaneous run with onion, leek and 20% sucrose with uber fresh pollen.

(From flower to slide)

Always the optimist! No result with pollen tubes = french onion soup on a cold Sunday night . . .
 
  • #17
Well, instant success would have been nice :p
But I do love me some good French Onion soup on a cold winter's night

I had some tatei pollen that i had froze using standard protocol with desiccant... got it out and made a slide of it with Leek epidermis

so, while not uber fresh... it was stored in accordance with accepted techniques,

surely one of these is going to show growth... umm maybe

LOL
 
  • #18
You may as well do a graduated sugar concentration set while you are at it: 5, 10, 15 and 20% sucrose. Just label your slides :)
 
  • #19
yep.... i know it, and start looking for some cheap scales...

already found cheap source for small quantities of boric acid (I probably already have it, use to use it as a flux) and calcium nitrate

meh, if it was that simple it would have already been done i guess
 
  • #20
Well if your 20% is pretty close you can take equal parts of the 20% solution and water to make 10%. And again with the 10% to make 5%. Equal parts of the 10 and 20% will give you 15%.
 
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