DO READ SOME OF RELIVENT PAPERS

http://www.jstor.org/stable/2437761

http://www.sciencedirect.com/science/article/pii/0012160667900413

  Colchicine is a toxic chemical that is often used to induce polyploidy in plants.  Basically, the colchicine prevents the microtubule formation during cell division, thus the chromosomes do not pull apart like they normally do.  The end result is a cell that now has double the number of chromosomes that it would normally have.  If this cell divides again in the future, then the doubled number of chromosomes are passed to the offspring cells.  Plants that have more than the normal two sets of chromosomes are termed “polyploidy” in general, although specific names are given to the certain chromosome numbers (e.g. tetraploid or 4N plants have four sets of chromosomes).

            Polyploid plants are generated in an effort to create new plants that have new characteristics.  Sometimes the polyploidy plants are sickly and not viable, but sometimes the polyploid plants have larger leaves and flowers.  Orchid growers will often sell polyploidy plants that are larger or have larger flowers.  Often the polyploid nature of the plant is included in the cultivar name (G. species ‘big flower 4N’).  Colchicine is also used to try to make fertile hybrids between species with different numbers of chromosomes.  The use of colchicine to make hybrids is well documented in ICPS cultural section of this web site.

            Unfortunately, colchicine is very toxic and hazardous to handle.  It is acutely toxic and has been responsible for many accidental poisonings by people or pets consuming the “autumn crocus” (Colchicum sp.) that are sometimes used in gardens.  In addition to its acute toxicity, it also causes chromosomal defects.  Overall, this chemical in it pure form is best handled in a chemistry lab with a fume hood with gloves and other personal protective equipment.  Once it is dissolved and diluted into water, it can be handled outside the fume hoods but gloves are still a necessity since the chemical may be adsorbed through the skin.  A more comprehensive review of colchicine toxicity can be found in the cultural of this web site .

The objective of this project was to assess what concentrations of colchicine are necessary to have biological effects on different carnivorous plant species.  Seeds of different CP species were obtained from the seed bank and divided into different treatments.  For all species, there was a “control group” and a 500 ppm colchicine treatment group.  Some species with abundant seed availability had additional treatment concentrations.  The number of germinations were then counted after it was clear the control group no longer had any new germinations, which was two months for most species.  The goal is to create tetraploid plants with different characteristics, but the confirmation of polyploidy requires chromosome counting, which is beyond the scope of this project.

Methods:

            Seeds from 5 different genera were obtained from the ICPS seed bank.  The seed from each species was homogenized to prevent inter-packet variation.  The seed was counted into equal number of seeds for each treatment.  For each treatment, the seed was divided into three equal portions which were treated separately.  The one exception was the Dionaea seed where each treatment was dosed together.

            The dosing procedure involved creating a concentrated colchicine solution by adding 0.51 g of colchicine into 100 mL of purified (HPLC-grade) water.  This created a “stock” solution of 5100 ppm colchicine by weight.  For each treatment, the stock solution was diluted to achieve the desired concentration of colchicine.  For example, the 510 ppm solution involved diluting 1 mL of the stock solution by adding 9 mL of purified water in a test tube.  The control group just had 10 mL of pure water added to it.  The seeds were added to the test tubes and they were allowed to soak in the solution for 96 hour.  The test tubes were wrapped in aluminum foil to protect the solutions from light since colchicine breaks down in light.  They were stored at room temperature for the 96 hours.  The solutions were shaken each day to make sure that the seeds were in contact with the solution since many of the seeds initially floated on the solution.  The Sarracenia seed were stratified in a refrigerator for 4 weeks before being dosed with colchicine.

            The seeds were planted into 4” (~10 cm) square pots that were filled with a mixture of 50 sand and 50% peat (“CP mix”) except for theDarlingtonia that were planted on pure sphagnum.  The pots were individually placed in plastic bags and then the solution with seeds in it was poured into the pots as to avoid coming in contact with the toxic solution.  The test tubes were rinsed with clean water and poured into the pots to wash out any remaining seeds into the pots.  The plastic bags were sealed and the pots were placed in a greenhouse or sunny windowsill for germination. 

            The seed germination was monitored weekly.  Once the number of new germinations in the control group ceased, then a final count of the number of seedlings was conducted.  The germination counts did include plants that were probably not viable in the long run.  These stunted seedlings were noted.  For example, Figure 1 shows the normal and stunted seedlings for Byblis liniflora.  Any seedling that died before the final count was not counted as a “germination”.  The pots were counted twice and the two counts were averaged to get the final number of seedlings.  The Darlingtonia pots were the exception in that they were scored the following spring about 6 months after germination.  The Darlingtonia score does not include plants that died within the 6 month period.

Figure 1.  Panel A shows the control Byblis liniflora plants 9 days after planting.  Panel B shows the 510 ppm colchicine treated group, which are clearly stunted by the treatment.  None of the plants picture here were viable.

http://cancerres.aacrjournals.org/content/amjcancer/38/2/169.full.pdf

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