I am interested to induce a mutation in some lilium species because of very low variation and susceptibility to soil conditions. I want to use Sodium Azide as a mutagen but don't have any experience with effective dosage of this mutagen. I will use seeds, callus and scales as explants and after treatment with sodium azide, I will culture them in MS medium for better germination and higher success.
In addition to the clear description by Kornelia Polok, I suggest not to use tap water for seed imbibition but reverse osmosis water as Chlorine or Chloramine in tap water can be very stable and react with the later added NaN3. Best to establish the suggested 'kill curve' using RO water all the way through. After the NaN3 treatment and before sowing the seeds, make sure that you thoroughly wash the treated seeds. Go for at least 8-10 washing steps using plenty of tap water. Here the tap water is better than RO as you want to get rid of any active NaN3.
Thank you very much Kornelia , Klaus and Pg
this is my first experience with mutagenic agents , i only have experience with using Surflan (instead of Oryzalin) herbicide as chromosome doubling agent in lilium scales and callus with some good results. I have to use scale , callus and seeds because i want to use cut flower lilies and also Lilium species and use of seeds from cut flower variaties is not ideal because lilium is self incompatible , for seed production i have to use two different varieties that can product lots of variation result of sexual production and i don't want sexual variation because i have to use original variety for compartion with mutated plants.Seed trial : there is only one form of Lilium Ledebourii and of plants look similar in flower color and form and i can use seeds and seedlings from this species and other species and later when they flower i can select mutated plants if there is any change flower form or color .
Is NaN3 water soluble ? what is best material to dissolve Sodium Azide and is there any other necessary ingredient to be mixed with Sodium Azide for best results ? there is no one here to have experience with Sodium Azide as mutation agent .Merck's Sodium azide tablets (for the preservation of milk samples) is available here is it possible to use it instead of Pure Sodium azide ? I have to wait some month if i want to order from Sigma because their agent in Iran deliver orders after some month .
Thank you Pg for this article , it is interesting
Thank you Kornelia for your help .
please what do you mean of double treatment ?
what kind of self protection i need to use sodium azide ? it is very toxic for breath .I do Oryzalin Treatment in home but don't know if sodium azide can be used easy .
Sodium azide solution is freshly prepared in 0.1 M phosphate buffer ( pH 3 adjusted with phosphoric acid ), fully imbibed seeds ( presoaked in distilled water for 6 h in our case) were put in mutagenic solution , with intermittent shaking , for 4 h then treatment terminated by washing in tap water for 6 h. You can try either 0.2 to 1.o mM concentration range or 1.0 to 10 mM range to determine LD 50 in your plant sample using reduction in germination percentage under lab ( use Petri dishes ) . I have worked on following plant species in the past.
Trigonella foenum 0.8/1.65/2.5 mM, in Vigna unguiculata 3mM, Foeniculum vulgare 1 and 3 mM , Coriadrum sativum 1 mM and Eruca sativa 1,3 and 5 mM
For further query, if any, please mail at [email protected]
The treatments were followed at room temperature in our experiments i.e. 28-30 degree Celsius . Further for preperation of 0.1 M buffer we used HCl instead of phosphoric acid . You may use HCl as of ours.
Thank you for all of your answers , my friend Mr Arthur Sale Reccomend me to not use Sodium Azide for now and try to product some polyploid plants for comparison with original plants to see if polyploid plants can resist botrytis attack or no , he mentioned than percent of positive mutations are very low and this mutagenic agent is highly dangerous .thicker leafs (in polyploid lilies) resist botrytis attack better than thin leafs . i have to read and learn more about mutation and also i don't think that i can't have safety equipments now.I will continue my researches about mutation untill i can have the safety equipments that i need to use sodium azide .
Thank you again
Hello Mr Mohammad. Yes any chemical mutagen must be handled with safety. Sodium azide is a respiratory poison and should not be inhaled. However, SA is very easy to handle. Its higly water soluble and works at very low concentration,i.e. below 5mM. As regards its effeciency, we have been getting encouraging results as compared to other chemical mutagens like EMS/MMS/ HA etc. There is nothing like safety equipments required for treatment accept that it should be handles carefully and disposed at a proper place. But one should use the pure form of NaN3 . The suggestions given by Dr Kornelia Polok are very useful ones.
How about the uses of SA on fresh tissues? how long the explants should be exposed to SA and what the range of concentration should be used. thanks
Hello Mr Aziz, I have worked only on seeds and not on fresh explants. However one way is to treat the seeds with SA, germinate it under aseptic conditions and then use its fresh leaves as explants. Somatic mutations may be arrested , if any, this way. Thanks
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