I am trying to generate wheat callus from mature wheat, by using standard protocol, which includes MS Medium PT102 plant tissues culture medium manufactured by Himedia with 2 mg/l,3mg/l, 4mg/l different concentration 2-4,D. After 15 days I am not able to identify the proper callus growth.
Can anyone please explain what the possible reasons are and provide suggestions to implement in my protocol?
First of all which type of explant whether mature embryo or immature embryo of wheat is not well defined.
If you are using mature embryo, then the protocol which you can try is : MS salts (g/l)-4.43; MS vitamins(1000X)(ml/l)-1; sucrose(g/l)-20; Dicamba(mg/l)-12; IAA(mg/l)-0.5 ; casein-hydrolyzate(g/l)-0.2 ;MES hydrate(g/l)-1.95; and culture period is three weeks.
Dear Saurabh
The monocot plants such as wheat need to high amounts of auxin for callus and regeneration. Please apply more concentrations.
Good Luck
Your culture may be high concentration of 2,4-D and 15 days may be not enough. Please wait, gave long time and low concentration of 2,4-D.
Usually when working with growth media, it is advisable to optimize using different levels of the hormone. In your case, the concentrations of 2,4-D may need to be varied further. I would try a lower concentration. Moreover, response to culture media varies among plant species, even though, for most plants 15 days is long enough to produce the callus.
Hello Saurabh,
regenerating plant tissue might be troublesome. Actually I am working on ornamentals but I had some discussions with people who work on crops for their income. Whoever I asked, I was told that the main reasons for not responding cultivars are the cultivar itself or the overall fitness of the donor plants.
Did you ever succeed in generating callus in your lab? If so, was it the same cultivar? If not, it might be suitable to try one of the published cultivars to get the right feeling.
For the overall fitness of your plants: it doesn't hurt do give them optimal growth conditions.
Try with the media defined in beakdong barley. The paper is available on Journal of Nationa Science Foundation Sri Lanka. Authot WTPSK senarath. That helps
Sir, may be U have not injured the mature embryo properly. it should be horizontally injured while its isolation and kept with scutellum region up on culture medium. I have used wheat mature embryos for inducing callus. Callus was induced with in almost 4-5 days or a week in different wheat genotypes and at 1mg/L 2,4-D concentration. U should also taken care about the himedia MS salts concentration. Because this company gives MS salts in MACRO and MICRO nutrients pack. For MS, both are needed. Check the conc. of these salts to be used for preparation of culture medium (written on pack).
If mature embryo is cut longitudinally, regeneration take place rather than callus induction. This problem occur at low rate in immature embryos, because cells are meristmatic in immature while in mature most cells are differentiated. Even Genotypic dependent differences occur for callus induction, u can also use higher concentration of 2,4-D, but it may be toxic because its hydrolysis is slow in medium. The concentrations U have used, these are enough to induce callus.
Hi.., you can get relevant information for your problem in this article DOI number of the article:10.5402/2012/367851. You can standardize using different concentration of components of media. Good luck...
Hi, you can use medium published in this paper: http://dx.doi.org/10.1016/S0176-1617(99)80119-9, Under optimal conditions you also can got good callus formation form mature embryos of many wheat and barley cultivars. Good Luck.
First of all you have to check the minimum requirement for callus induction, please check ph of the medium, temperature of the incubator, incubation in dark and be careful from sterilization because it sometimes kill your embryo? if everything is ok i suggest you to soak the sterilized seed in sterilized water overnight and excise the embryo without injuring using 2.5 mg 2,4,D or follow Neha steps i think it will work good with you.
Hi Guys
Where can I get some Wheat seeds that can grow in hot tropical climate like Singapore for experimentation. Drop me a line at [email protected]
Dear Sourabh,
Basically tissue culture is a trial and error method where you need a lot of patience, try using 2, 4-D along with MS media for better results. as it helps in callogenesis.
Hi, Saurabh, which kind of materail have you used? What do you as "mature wheat"? Do you mean mature embryos or mature leaf? In cerelas cells very rapidly undergo irreversible differentiation and you can not get cells re-programming back to callus. Only certain tissues were able to produce callus. You can use mature embryos (but not scutelluar tissue, what is irreversble differentiated). or young leaf base (2-3 mm from apical meristem). All other tissue most probably wiil not give you any callus. Good luck!
Hi, Omneya, 2,4-D is a strong hormone, but it have effect only on cells which are competent for cell re-programming/ cell division. If cells are not competent (fully, irreversibly differentiate, with high ploidy level with condense chromatin), even 2,4-D will not help to re-programm these cells. As an example we can consider cereal (wheat, barley) leaf except 3-5 mm of the leaf base, which are close to meristem.
Am trying to do the same thing and am getting the same problem.However, there is a possibility that your embryos get contaminated by bacteria in the process of their removal and this render them in-viable. You can try using the whole seed instead of isolating the embryo, that has worked for me.
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