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Propagation -- Colchicine Treatment and Toxicity
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Introduction:
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 the Darlingtonia 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.
Results:
The results from the colchicine treatment indicates that there is a high degree of inter-genera susceptibility to colchicine as applied in this dosing experiment (Table 1). None of the Drosera species showed any visible effect from the colchicine; the germination was comparable to the control group and the seedlings looked normal. These plants, due to their short life cycle, were monitored until some of the plants started blooming. Almost none of the plants looked different from “normal” plants of the same species except that a few of the D. capensis looked a little more washed-out in terms of color, but this is qualitative at best. Two of the D. binata plants looked larger than the control, so they were isolated and they will be evaluated for differences in growth rate. If these plants seem to be larger in the long run, then chromosome counts will be conducted on them to verify if polyploidy were generated.
On the other extreme, Byblis liniflora showed a very high degree of susceptibility to colchicine. The germination and survival rate among seedlings was very low in the treated group. Furthermore, the seedlings were clearly stunted and most were not viable (Figure 1). Some of the colchicine seedlings developed a few stunted leaves but then died. These plants were probably polyploid, but the lack of plant viability prevented testing for chromosome counts. Any future attempts at making polyploid Byblis liniflora will need to use lower concentrations of colchicine.
The Sarracenia leucophylla showed no observable effect from the colchicine treatment. The treated group had effectively the same germination rate as the controls and the seedlings appeared to be normal. Future experiments to induce polyploidy will need to use higher concentrations of colchicine.
The Darlingtonia seedlings germinated and where then scored in the following spring approximately 6 months after germination, thus these scores represent both germination as early survival. The data suggests that colchicine may have reduced the germination/seedling viability, but the rather small sample set (57 control seeds and 57 treated seeds) makes any comparison rather tenuous.
Lastly, the Dionaea showed an effect from the colchicine treatment, but not as dramatic as the Byblis. The germination rate of both of the treated groups was lower than the control group by almost a factor of two. The higher colchicine treatment also started to generate clearly stunted plantlets that were comparable to the Byblis plantlets. It would appear that 5100 ppm colchicine would be a good dosing concentration for future experiments since germination was still reasonable (albeit ½ of normal) and some of the plants were clearly affected. Whether any of these plants will develop into anything of cultural interest remains to be seen. If any of the plants are visibly different, then they will be tested for polyploidy.
The ultimate objective of this experiment is to generate polyploid plants with desirable characteristics. Since chromosome counts require considerable effort, only plants that appear to be different will be tested in the future. The long development times for some species (e.g. Sarracenia, Dionaea) means that some species may take considerable time to show effects. Although some plants in this test were abnormal (Byblis, Dionaea, Drosera), none of them have been confirmed as polypoid and abnormal Byblis were not viable. Until some of the plants are confirmed as polyploid, the results of this experiment are best used as a “range-finding” experiment that determines what concentrations of colchicine are needed to produce effects without causing excessive seedling mortality. Byblis needed dosing concentrations below 500 ppm while Darlingtonia and Dionaea produce biological effects at 500 ppm dosing concentration. Drosera and Sarracinea require dosing concentrations of greater than 1000 ppm or a longer dosing exposure time.
Additional experiments may be conducted in the future to add more species to the colchicine susceptibility table and to generate new plants with different characteristics (hopefully positive!). If such experiments are conducted in the same fashion again, they will be added to the table presented here.
Table 1. Germination success (%) for several species of carnivorous plants exposed to different concentrations of colchicine (in ppm) for 96 hours. The number of seeds per each treatment group is given after the species name.
Species (number of seeds in each treatment) Germination success (%) by
colchicine concentration in ppm
0 (control) 102 510 2550 5100
Byblis liniflora (n = 150)
20.6
---
1.3 a
---
---
Darlingtonia californica (n = 57)
33.3 (b)
17.5 b
Dionaea muscipula (n = 96)
38.5
---
22.3
---
18.2 (a)
Drosera binata (n = 150)
35.3
---
34.7
---
---
Drosera capensis ‘red’ (n = 300)
67.5
74.0
69.7
65.0
---
Drosera filiformis “Florida red” (n = 150)
54.0
---
50.0
---
---
Sarracinea leucophylla (n = 150)
48.7
---
50
---
---
(a) some germinating seedlings were clearly stunted and were not viable.
(b) The seedling success was scored in the following spring approximately 6 months after germination.
Thomas Cahill
Arizona State University
