I saw many papers that started with seed germination to achieve the in vitro cultures and from the in vitro raised seedlings they took leaves, petioles and apical buds for further multiplication. I have started with the leaves, petioles and shoot apex directly taken from the field, sterilized them and taken them as explants, are there any technical issues regarding that? I have taken these plants from the alpine region and the plant is mainly propagated by rhizome cuttings and seed germination % is very low in nature. I have tried in vitro seed germination (26% germinated) and used those as explants too. If I want to check genetic stability among the in vitro raised plants, is this procedure OK?
Only concern with the material collected from field is higher contamination rate. If the plant is cross pollinated then genetic variability may be an issue- depending on your end goal of the experiment. If you need more help, contact Dr. Pramod Tandon.
Good to see someone from NEHU. Did my PhD from NEHU in 1987.
Thank you Sir. Thank you so much. Actually, I am working on some high altitude medicinal plants and I took shoot apex, leaf, petiole and coteledonary explant from the donor plant taken from the field. Ya, I had a problem regarding contamination but not so much. Sir, for paper purpose is this protocol is ok?
Sir, good to know Sir, you are also from NEHU. We are proud of you Sir.
I am doing my research work under the supervision of Prof. pramod Tandon and Prof. Suman Kumaria. Sir and Mam always helping me with their valuable comments. I thought, why not to see what other experienced person says.
Yes, for publication purpose this approach is fine. Try to include as many replicates as possible for each treatment. Maintain size and developmental stage uniformity of the explants you are using. What plant you are working on?
Good luck for your research.
tell me the plant habit. This infleuences the behaviour of the explant in the medium.....
also add the aim of your experiment.. Callusing, multiple shoot induction etc.. then will be able to answer
The plant grows @ altitude of 3500-5500 m asl. Its a perennial, rhizomatous herb. I want to induce callus from leaf and petiole and also from the shoot apex, petiole and coteledonary explants.Finally I will check the genetic stability among regenerants.
I understand that you do not want to disclose the name of the plant. If you want help from people you need to be open. At least share family of the plant. Do not worry. Believe me- people will not run away with your plant or jump on your project.
Some work on its distribution and medicinal components have been done from Nepal. It is endangered in many places so better acquire your material through reliable agencies and sources. It is distributed all along western and central Himalayas.
Technically, the procedure is OK. But since you are doing PhD, I would rather suggest to use both, explant materials directly collected from field, as well as seedling derived explants. Obviously, seedling derived explants wont trouble you as far as contamination is concerned. But at the same time you also need to establish a procedure to regenerate and multiply a plant which Is present in open environment. Initially, the contamination may be problem, but over the period with some standardization and due to seasonal effects, you would be able to establish them in in vitro conditions. After all, another dimention of doing PhD is to generate information/data. Then this whole exercise would be helpful. Best wishes.
as per your requirement and the habit of the plant, I suggest that you should start with tender stem explant (which will resist the HgCl2 treatment or any surface sterilisation treatment) for callus induction and then go for other explants as well..
good day
I understand your goal, I made the same application to potatoes and I assure you that the use of the stem as explant is the fastest way to have a organogenic callus _. I have published in this goal. Now to help you better now requires more explanation I give you my email if you want more details: [email protected]
Hi, Biswajit, so, you goal is to get organogenic (embryogenic) callus and thereafger get plants, right?
Do you want to study genetic stability of regenerated plants?
I would suggest you to use young leaf, petioles and stem.
I think that relatively young seedlings will be ab excellent options.
Good luck!
Thanks all.....Yes Sir, my goal is to obtain organogenic callus and direct organogenesis and to check the genetic stability of in vitro raised plants from both direction....
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