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Chromosome doubling experiment - so it begins :)

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Chromosome doubling experiment - so it begins :)

Post by Admin on 26th August 2011, 19:22

OK... it has started!

This is step number one in trying our chromosome doubling procedure of Rosa laevigata. My student set this up this afternoon to leave over the weekend:

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The plant pot contains a struck cutting of Rosa laevigata that we struck a few months back. It has been prepared by allowing the primary axillary buds to grow and then rubbing them off to force the secondary axillary buds to start growing. These are the buds that grow from the side of the stem, not the tip. The primary axillary bud is the first (main) bud to grow and the secondary axillary buds are the ones that grow from the same place if the primary one is damaged. The reason we chose these is that there is some evidence to suggest that the accuracy of the DNA replication in this tissue is lower than in other areas and is prone to sporting. We decided, with this in mind, that if this is so then maybe it would also be more likely to be affected by chemicals that affect cell division too. The pot was inverted and the cutting placed into a solution of caffiene and a few drops of detergent. The caffiene interupts normal cell division and with a little luck will result in tetraploid cells being made instead of the normal diploid ones. On Monday we will remove the cutting, rinse it, and put it in the glasshouse to grow normally. As the buds grow we will remove the stems and strike these as cuttings and then count the chromosomes in the roots to determine ploidy. If they are tetraploid then there may also be an increase in the size of leaves and flower parts (ever noticed why modern hybrid tea leaves are so much bigger than most species roses).

This procedure has not been tried with roses before but has been shown to be successful on plants such as wheat. If it works it will prove to be breakthrough in rose ploidy manipulation protocols because to date the chemicals used to do this are extremely dangerous (colchicine) or expensive. Caffiene is easily obtainable and safe to use. If it works we will grow the plant until it flowers to confirm polyploidy in the pollen by measuring pollen diametre and comparing it to other tetraploid pollen diametres as there is a chance that it can be tetraploid in the root tissue and not in the pollen. If successful it means this plant might be more easily crossed with modern tetraploid roses and more fertile offspring produced. Rosa laevigata is evergreen (even here in Tasmania) and extremely disease resistant and vigorous. To introduce its genes into modern roses might result in modern roses very suited to Australian conditions with superior disease resistance and vigour.

Stay tuned!


Last edited by Simon on 26th August 2011, 21:37; edited 1 time in total

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Re: Chromosome doubling experiment - so it begins :)

Post by Dave on 26th August 2011, 19:28

Cool experiment Simon, the best of luck - this is the way to go with Laevigata. Can I be the first to trial a plant on the East coast?

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Re: Chromosome doubling experiment - so it begins :)

Post by AutumnDamask on 26th August 2011, 19:58

It is a very cool experiment. Thumbsup

I suspect if all goes reasonably to plan there would be no trouble finding trial sites all over this wide, brown land.... Very Happy

Coming up Roses
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Re: Chromosome doubling experiment - so it begins :)

Post by Ozeboy on 26th August 2011, 20:44

Simon, tell them from me,they are very lucky students.

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Re: Chromosome doubling experiment - so it begins :)

Post by Admin on 26th August 2011, 21:30

Bruce... I am a lucky teacher Thumbsup

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Re: Chromosome doubling experiment - so it begins :)

Post by Admin on 26th August 2011, 21:38

This is just a test run to see if it works. If it does work then all the cuttings Rosalie sent will be treated the same way when they strike too.

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Re: Chromosome doubling experiment - so it begins :)

Post by Balinbear on 26th August 2011, 22:22

Oh my God! Its alive!!!!!!!!! (said in my best monster movie acsent)

I hope your experiment does not escape your land and threatens to take over the world.
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Re: Chromosome doubling experiment - so it begins :)

Post by Admin on 26th August 2011, 23:03

It would look cool with bolts in its neck too Wink

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Re: Chromosome doubling experiment - so it begins :)

Post by Admin on 10th September 2011, 01:04

Quick update!

The reason we used a few drops of detergent in the caffienne solution was because the outer surface of a plant is covered in a waxy coating. This helps plants reduce water loss by putting a waterproof barrier over the surface of the leaf. Wax is a type of oily substance and detergent saponifies fats and oils so will breakdown this cuticle layer and hopefully improve the penetration of the caffiene into the tissue. After 48 hours in the solution, which was being constantly stirred by the magnetic stirrer, the cutting came out fine but it seems the solution may have been a bit harsh. Most the buds have been killed, however, there is two tiny swellings starting to appear today that look like new buds forming Fingers Crossed With any luck they will continue growing so we can propagate from it.

I think we will try it a few more times now over 24 hours instead of 48 to see if that impoves bud survival rates. Then with no detergent to see how that goes too.

The saga continues Smile


Last edited by Simon on 3rd October 2011, 22:24; edited 1 time in total

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Re: Chromosome doubling experiment - so it begins :)

Post by Admin on 3rd October 2011, 22:23

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These are secondary axillary buds that grew out from caffienne treated buds. They died right back to nothing and then these little green buds started to push out. I am hopeful of success with these. Next step sis to grow them on, take cuttings material from them, remove some root tips and count the chromosomes Smile

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This is a batch of Rosalie's laevigata cuttings that we have treated. Their buds shot and started growing so I decided to treat them anyway and see what happened. Not as much leaf damage as I was expecting. Hoping the growing points have been affected.

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Re: Chromosome doubling experiment - so it begins :)

Post by OzRose on 4th October 2011, 22:34

WOW
I'm impressed. OMG!!!
Are you sure they are not going to turn into Triffids ??? those cuttings already have a taste for human blood !!!
I think you will find when you get further into your genetic testing , you will find them quite closely related to Carcharodon carcharias
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Re: Chromosome doubling experiment - so it begins :)

Post by Admin on 4th October 2011, 22:38

lol - I shall call it 'The Great White Rose' Wink

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Re: Chromosome doubling experiment - so it begins :)

Post by OzRose on 5th October 2011, 01:54

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Re: Chromosome doubling experiment - so it begins :)

Post by Schuler on 18th October 2011, 22:27

This experiment is really amazing!
Were the cuttings completely in the caffein solution?
I'm thinking about trying this experiment with passiflora cuttings this (european) winter and maybe with 'Agnes' next year.

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Re: Chromosome doubling experiment - so it begins :)

Post by Admin on 18th October 2011, 22:38

they were emersed about half-way. The shoots have now really taken off. The experiment is looking good so far.

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Re: Chromosome doubling experiment - so it begins :)

Post by Ozeboy on 18th October 2011, 22:43

Can't wait for the next episode, well done Simon, will cross my fingers for success.

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Re: Chromosome doubling experiment - so it begins :)

Post by Schuler on 19th October 2011, 02:08

This is a quotation from Hall Love, he wrote this on Facebook in the Group "Passiflora - Passion Flowers Online"

"Caffeine inhibits cytokinesis during cell division. The result is binucleated cells. After several rounds of division, the two nuclei often fuse to produce a single tetraploid nucleus. From all I have read, it does not work well with vegata...tive cells and is not effective at inducing tetraploidy. However, it does work well in some cases, when injected into the developing flower buds of sterile hybrids. In this case, it induces the formation of unreduced gametes, making it possible to cross the previously sterile diploid with other tetraploids. I don't think it has ever been tested in Passiflora. The timing is very critical. The concentration is typically 0.2-0.3%."

I really hope your experiment works well and the fingers are strongly crossed too Very Happy

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Re: Chromosome doubling experiment - so it begins :)

Post by Admin on 8th December 2011, 17:52

Update time:

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I have been in contact with Dr David Zlesak about what we are seeing and he has said this agrees with what he was seeing when he doubled species using trifluralin. He was saying he often observed reduced vigour and a reduction, not an increase, in the size of body parts. The treated on on the right is the same age and from the same plant as the control on the left. I am hopeful, but not certain yet, that this means it has been doubled, at least in the part of the plant (note... doubling in sections such as the leaves and roots does not guarantee doubling in the tissue that makes pollen and ovules). The reduced vigour, so far, does not seem to have affected it's disease resistance. No mildew has been noted in either the treated cuttings or the controls in the greenhouse.

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Re: Chromosome doubling experiment - so it begins :)

Post by AutumnDamask on 8th December 2011, 18:03

Interesting the colour difference too.
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Re: Chromosome doubling experiment - so it begins :)

Post by Ozeboy on 8th December 2011, 20:13

Simon, thanks for the update. Hardly a week goes by that I don't think of this experiment.

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Re: Chromosome doubling experiment - so it begins :)

Post by The Lazy Rosarian on 7th February 2012, 06:46

Simon, was just wondering how the experiment is going. I relise you are not back at school yet scratch
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Re: Chromosome doubling experiment - so it begins :)

Post by Admin on 7th February 2012, 11:02

At this point it is just growing and putting on size. The next step, once some size has been put on, is to remove some stems and strike them and once roots have formed cut the tips off the roots, squash them, stain them, and see if we can count the chromosomes. I have bought a microscope attachment for my dSLR so if we can confirm tetraploidy we can take good high resolution photos to show people.

Right now, the success or failure of the experiment is largely guess-work. I can't actually see any difference at all between the growth on this one compared to the growth of the control plants except that maybe the leaves are a darker ricker green and that growth is not as rampant. Laevigata tends to throw suckers and lots of sub-soil basal shoots. I have been rubbing these off because they were not treated with the caffiene and my concern is that if I allow these to keep growing, eventually they will consume the treated portions due to differences in vigour. Only the top buds were. These are the only ones I have allowed to grow.

My wonderful student has gone off and graduated to college now but is coming back to perform the ploidy tests throughout the coming term. I go back to [paid] work this Friday. I repotted the plant into a larger pot with better soil over the holidays and have been giving it plenty of tlc.

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Re: Chromosome doubling experiment - so it begins :)

Post by OzRose on 9th February 2012, 14:22

Keep the updates coming . This is fascinating.
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