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Abgeschickt von Michael Weinert am 30 Oktober, 2003 um 06:22:09
Encouraged by Bill Steele's posting about his bleaching model, I would like to post some of my former observations on seed treatment with NaOCl which might be helpful for the germination of difficult Cyp species. The results were published 1992 in "Die Orchidee" 43(6), p.287-293 (in German, download 4 MB). My trials at that time were made with seed of Epipactis palustris, but I am pretty sure that the results are applicable to Cyps, too. I used 0.3 % NaOCl and tested the viability by staining the seed with 2,3,5-Triphenyltetrazolium chloride (TTC). Physiologically active embryos turn red, slightly active embryos turn orange, inactive embryos remain yellowish. All trials were made with the same batch of seed (several capsules harvested from one clump of Epipactis palustris and all seeds mixed together).
There were two main results:
1. The ratio between the amount of seed and the
volume of NaOCl is relevant for the intensity of bleaching.
The less seed in a given volume of NaOCl, the more intense the bleaching. This effect is especially visible with a long duration of the treatment. The reason is the slower decrease of the chlorine content in the NaOCl solution (indicated by a iodine test). Please see below the data of 3 repetitions with 48 - 6 mg seed in 15 ml 0.3% NaOCl and a bleaching time of 1 - 5 hours, the numbers showing the percentage of TTC-positive embryos (the Tukey letters of significance must only be compared within the same duration of treatment).
This means that it is possible to determine the best bleaching time for a specific batch of seed before sowing by making small scale trials with a fixed seed/NaOCl ratio at different bleaching times and using the TTC test. After this the quantity of seed and NaOCl is multiplied and the optimal bleaching treatment applied for the major amount of seed. No longer losing precious seed by erroneously using the wrong pre-sowing treatment for the complete batch!
2. Seed of hard-to-germinate orchids can contain two different types
of seed which require different bleaching times.
I was very surprised not to find a normal distribution regarding the bleaching time and percentage of viable (i.e. TTC-positive) embryos. The different symbols show the amount of seed used with 15 ml 0.3 % NaOCl solution: the square means 48 mg seed, the rhombus 24 mg, the triangle 12 mg and the star 6mg.
This contradiction to my expectations could only be solved by differentiating between orange and red stained embryos, i.e. between less and more physiologically active embryos. The figure below shows the results for the orange embryos, which is the expected normal distribution.
The next figure shows the results for the red embryos.
This is very interesting as it proves the existence of a seed type which requires very little bleaching and should be easy to germinate. I assume that it makes sense for the orchid to produce seed with varying germination times as every year is different and sometimes the fast germinating embryos may have an advantage and another year the slow germinating part of the seed may be better off. In the light of these observations it might be worth a try to apply only a light bleaching treatment on seed of hard-to-germinate orchid species in order to preserve the easy germinating group of embryos. Additionally it might be better for these species to use low concentrations of NaOCl and a wide seed/NaOCl ratio. Then the intensity of bleaching is high enough but not killing the embryo immediately after the carapace has been penetrated. The TTC test and the seed/NaOCl ratio makes it possible to determine the optimum bleaching treatment with a small part of the seed before sowing and should make germination more predictable.
Unfortunately the end of my studies at the university put an end to these trials. So I have never applied these findings on sowings of difficult Cyp species. Maybe that one of the fellow propagators is encouraged to try it out and report about his results! Or is there anybody who already has made any experience in this matter? Comments solicited.
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