I want to use Lavender seed in in vitro study. However, I could not sterilized lavender seed. I had high percentage of contamination even after ı used different methods. Although ı used different concentrations and exposure time of commercial bleach with/without Tween20 and ethonol, it did not solve my problem. Also ı do not want to use mercury chloride during sterilization process. So How can we do seed sterilization of Lavender ?
Thank you sharing your idea.
First, try with higher concentration of chlorine and/or time. Anyway, check your complete desinfection protocol. Desinfection is a routine; however, it must to be conducted in a proper way.
I have two suggestions.
(1) Reduce the burden of your starting material. You can e.g. try to grow the plants making seeds in a semi-sterile environment.
(2) Use more seeds so that you have enough sterile ones to do your experiments. Use multiwell plates for better separation of contaminated seeds from non-contaminated once. Transfer sterile tissue rapidly to fresh plates to avoid cross-contamination.
Unfortunately you don't provide details of what you want to do. Grow sterile plants? Establish callus material?
Ricardo Julian Licea-Moreno thank you your recommendation.
Markus Sack Thank you for your suggestions and for your time. My first goal is to produce contaminations-free plant for next steps. Then, I will focus on direct and indirect organogenesis. I already started to do your second suggestion. However, still I should have a efficient sterilization protocol for lavender.
What type of contamination do you have? Bacteria, Yeast or filamentous fungi?
Markus Sack After 2 days of culturing in MS media, fungal contamination appeared. Most probably my lavender seeds are more dirty than normal but for now ı have only these seeds.
Please tell me how you sterilize the seeds. Can you attach a photo showing the contamination?
Markus Sack I tried different combinations of commercial bleach, ethanol and Tween20. Commercial bleach range was between 10%-%80 with/without 70% ethanol and for sterilization. Tween20 and ethanol application was 1 min and than commercial bleach exposure time range was between 15 - 45 min.
Dear @Mehmet this kind of contamination is normally occured when the plant materials aren't disinfected correctly. Many factors might cause these results. For example, more than percentage of bleach you must check the content of chlorine of bleach. CommerciaI bleaches usually have between 3 to 5% of chlorine. Avoid to use old bleaches. It's preferable to buy a new bottle. A treatment with 1% of chlorine during 30 minutes usually works. However, you can perform a two- step disinfection and/or reduces the pH of chlorine solution (in this case, use adequated protection since toxic gases are released in this process).
Ricardo Julian Licea-Moreno, Firstly thank you for answer and information. I used new bleach and it contains 4.6% chlorine. also, I did not try yet 1% of bleach with 30 minutes exposed time. I will try this one. Thank you again.
Use domestos 10% 10 min only at 300 rpm shaking. If not working, use 0.1% of Mercuric chloride 3 min at 200 rpm.
For that plant, I offer you to use green plant, seed germination may take time and very slow. Sometimes the seeds are not vigor. If your plan is on clonal propagation, you can use stem cuttings ex vitro.
Buhara Yucesan Thank you for your comment. You are right about germination and vigor of lavender. Same time with sterilization process, I planted seed into soil and filter paper. It is thirteenth days now, and still they did not germinate.
That the seeds did not germinate so far in soil, is not such a good sign.
You still have got the plants?
Of course you could use unripe seeds.
As sometimes embryo rescue is used too.
But you could of course to induce tissue culture from leaves in order to get plantlets for further research.
If you have got the mother plant...
S. Wenkart I planted seed into filter paper as ı mentioned before. Today some of seeds are started to germinated but germination rate is so low. It is around 2-3%. Most probably ı will use those germinated plant for future work. Thank you for your comment.
Ok can you try this protocol I used for many seeds and it's work very good
1_ but the seeds in sterilize container.
2_ add the ethanol at concentration 70% (V/V) and be sure all the seeds inside the ethanol With continuous stirring for 1 minute.
3_ add Sodium hypochlorite or Clorox at concentration 50% (V/V) ( NaoCl.5.45%) for 15 minutes With continuous stirring
4- Wash the seeds with sterile distilled water for 15 minutes, with water changing every 5 minutes.
5_ Sterile seeds are planted in Petri dishes with sterilized forceps flame in each cultivation and closed on Petri dishes by paraffilm.
Keep all the culture seeds in incubator with 25C°.
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