Safe alternatives to induce 4N in seeds?

[orchidfever]

Hi,

I know of Colchicine. But is there any other safer alternative to achieve the same thing? Please kindly direct me to the resources.

Thank you!

[Andrew]

Oryzalin is commonly used as a safer alternative to colchicine.

[RobS]

Indeed I used Oryzalin on Nerine and Lilies when I was in university.

Oryzaline should first be disolved in DMSO (20 mg / ml) after that it can be diluted until the desired concentration. 0.001% and 0.002% (still both Oryzaline and DMSO are toxic). Treatment duration was 3 hours and after that rinsing during 1, 5, and 10 minutes. After that dry the plants so that any left over DMSO can evaporate.

Maybe you shouldn't use in on seeds but on protocorns?

Reference:

http://www.liliumbreeding.nl/oryzaline.pdf

Van Tuyl, J. M., Meijer B. and van Dien M., 1992.
The use of oryzalin as an alternative for colchicine in in-vitro chromosome doubling of Lilium and Nerine; Acta Horticulturae. vol 325 pag 625/630

[orchidfever]

Thanks Andrew and Rob for your suggesting Oryzalin!

Rob: is there any reason for using it on protocorms instead of seeds? I thought using it on seeds would guarantee conversion of all cells since an orchid seed is essentially a single cell. I Also wonder what the plant would be like if some of the cells are 4N and some are 2N in a protocorm.

Where could I obtain Oryzalin (and DMSO) ? I am located in Canada. It would be best if it is available in pre-mixed form for this particular use.

[FairyInTheFlowers]

This is a VERY interesting question orchidfever! I am also in Canada, and I plan on starting some small scale breeding in a few years, and would love to work with tetraploids. I also wonder where you could obtain Oryzalin?

[RobS]

Well maybe you're right I have no experience with seeds. It was just comming from the fact that you'd need some kind of cell division activity to be able to get 4n.

Try to find a local laboratory supplier and ask them otherwise ask a university that's working on plants.

[Royal]

Originally Posted by RobS

you'd need some kind of cell division activity to be able to get 4n.

This is the key point. You must apply the mutagen while multiplication/division is taking place. These type of treatments are usually done on developing protocorms.

There are a bunch of chemicals that cause mutations and may be used to induce polyploidy. ALL of them would be toxic to some extent. Many are used as herbicides due to the interference with normal division. Oryzalin is one of these, 2,4-D is another.

There is never a guarantee that conversion takes place - the only sure way to know is to do a chromosome count. If you are set up to do conversions, you should also be able and willing to do counts to verify.

[RobS]

Well you could gamble and just take the largest most vigorous young plants. Chances are that they are the polyploids.

[Louis_W]

Originally Posted by RobS

Well you could gamble and just take the largest most vigorous young plants. Chances are that they are the polyploids.

According to Roy Tokunaga this is the oppsite of what you should do. Tetraploids will lag behind diploods by a year or two so they will seem runty at first. They are big strong plants but they are not faster and more vigerous.

[orchidfever]

Originally Posted by Royal

This is the key point. You must apply the mutagen while multiplication/division is taking place. These type of treatments are usually done on developing protocorms.

That makes me wonder if it has been done on new buds with any success. Since new buds are growing and tiny, I would think that soaking them in Oryzalin solution could achieve conversion without the hassle of replating and so on.

There is never a guarantee that conversion takes place - the only sure way to know is to do a chromosome count. If you are set up to do conversions, you should also be able and willing to do counts to verify.

How much does it typically cost to do a count? And where can I find place to do that?

Thanks for your info!