Pretreatment of roots, shoots, callus and other plant organs depends upon the crop plants used. For cereals, ice cold water pretreatment at 0 degree centigrade in the refrigerator for 18 to 24 hrs accumulates a large number of metaphase cells. But for small chromosomes plants like soybean 8-hydroxyquinoline and paradichlorobenzene arrests a large number of metaphase cells as I have observed with 8 -hydroxyquinoline and Palmer and Heer (1973) with paradichlorobenzene. Soybean has about 10x smaller chromosomes that those of barley and wheat. The length, temperature and concentration can be modified with the materials. Thus, one protocol is not perfect!! Recently pretreatment of maize and soybean roots with nitrous oxide arrests condensed metaphase chromosomes in high frequency. My book just describes routinely used protocols and may be a starting point!

Its role is to accumulate metaphase cells. At the stage where it is used, the meristematic cells are still alife and the process of karyokinesis is still ongoing. It inhibits the process in such a way that the chromatids at metaphase do not proceed with anaphase or subsequent phases. Therefore its deployment helps to accumulate metaphase cells before the roo tips are rapidly killed by fixing.

Hi Dr. Blackstock.

I have spent the last several months optimizing a root tip squash protocol for use on Caltha (Ranunculaceae). In addition to Dr. Osuji's reply above, my understanding is that pretreatment in a saturated solution of PDB inhibits spindle formation (hence halting the chromosomes in metaphase) and clarifies chromosome constrictions "due to the contraction and differential hydration of chromosome segments." (Sharma and Sharma, 1980; p 21). I have had the best luck using PDB at about 8C for 20-24 hours (though longer than 3 hrs is supposed to cause fragmentation of chromosomes!) or 3 hrs at 4C. A colleague working on Solidago found that 3 hrs at room temp worked. I now just use prechilled distilled water (kept in ice bath in fridge for ~24 hours) as this works well for me.

These pretreatments were all followed by fixation in 3:1 95% ethanol to glacial acetic acid at 4C then transferred to 70% ethanol in freezer... for staining I'm using Wittmans's iron-aceto-hematoxylin mounted in 50% Hoyer's solution with excellent results (2n = 12x = 96 in some individuals). I'm happy to share protocols/resources/notes with you. Just let me know! Good luck.

Sharma, A. K. and A. Sharma. 1980. Chromosome techniques: Theory and Practice (3rd ed.). London: Butterworths.

Many thanks to Julian and Keir.  The reagents have now arrived and my students are looking forward to seeing chromosomes.

May the sedge be with you

Nige

Can anyone suggest how to prepare saturated solution of para di-chloro benzene?

Weigh 3g of PCB & add to it 200ml distilled water, incubate overnight at 600 C then cool, some crystals may remain undissolved. Shake thoroughly before using. see this book :Plant Cytogenetics", Second Edition

By Ram J. Singh,  (https://books.google.co.in/books?id=srMJQ2PvNQAC&pg=PA8&dq=paradichlorobenzene+for+chromosome+studies&hl=en&sa=X&ved=0CC8Q6AEwAmoVChMItfTmt_mbyAIVBP4sCh39zgZH#v=onepage&q=paradichlorobenzene%20for%20chromosome%20studies&f=false)

Rahul P R, I have tried the method you have mentioned above,but the PDB get crystalised as soon as I take it from oven temp.It appears as a globule of oil in water

Swetha, 

This is a good sign that there is some excess of PDB that recrystalizes. This shows that your solution is indeed saturated. You can leave it at room temp and just pull out what you need...

Best, Keir

Pretreatment of roots, shoots, callus and other plant organs depends upon the crop plants used. For cereals, ice cold water pretreatment at 0 degree centigrade in the refrigerator for 18 to 24 hrs accumulates a large number of metaphase cells. But for small chromosomes plants like soybean 8-hydroxyquinoline and paradichlorobenzene arrests a large number of metaphase cells as I have observed with 8 -hydroxyquinoline and Palmer and Heer (1973) with paradichlorobenzene. Soybean has about 10x smaller chromosomes that those of barley and wheat. The length, temperature and concentration can be modified with the materials. Thus, one protocol is not perfect!! Recently pretreatment of maize and soybean roots with nitrous oxide arrests condensed metaphase chromosomes in high frequency. My book just describes routinely used protocols and may be a starting point!

Many thanks  - I still haven't a positive result but I will play around with the protocol until I do.

I am not sure about the plants you are studying. Please let me know. I shall go from there. 

I'm looking at Carex species (Cyperaceae).  They tend to have small chromosomes with a diffuse centromere.  I'm trying to look at root tips but most work by other authors has been carried out on pollen mother cells.

Hi Dr. Blackstock, 

For what its worth: I have no experience with monocot squashes, but found that the most important thing is to get very healthy, actively growing root tips. How are you pretreating? I like the 1 degree C ice water myself, but have also had luck with PDB (and less success with 8-hydroxyquinoline and colchicine). 

I suggest trying 8 hydoxyquinoline. The method is:

Aqueous solution is prepared by dissolving 0.5g of 8-hydroxyquinoline in 1L double distilled water (ddH2O) at room temperature (RT) on cold stir plate; requires 24h or more to dissolve. Make sure chemical is dissolved. Filter in a colored bottle and store in a refrigerator.

Pretreat roots in 8-hydroxyquinoline for 2 to 3h at 15 to 16C. Warmer temperatures (RT) often cause sticky chromosomes. Pretreatment with 8-hydroxyquinoline has been very effective for plants with small-size chromosomes. It makes primary (kinetochore) and secondary constrictions (nucleolus organizer region) and cytoplasm very clear.

This woks best for the soybean the size of mitotic chromosomes 1.42-2.84 µm.

Attached is a photomicrograph.

I shall go and have a chat to my plants to encourage fresh root growth and give it a go.  Many thanks for your advice.

This pretreatment and staining should work.

Please Help me, I am in problem of working out somatic metaphase chromosome of Bryophyllum spp.(Kalanchoe spp.) using 8-Hydroxyquinoline followed by Acetic acid treatment. Please suggest me proper treatment.