the protocol is very easy. you can find information about it on my paper "Production of novel allelic variation for genes involved in starch biosynthesis through mutagenesis" pubished in Molecular Breeding journal. Before to start, you have to find the right concentration of mutagen (EMS). I can advise to mutagenize 100 seeds with different EMS % (0.1%, 0.3% 0.5% 0.7% 0.9% 1.1% 1.3%). when you increase EMS %, you observe a decrease in plant germination. For a good mutagenised population, you have to have a germination percentuage of 60%.
The suggestion of Francesco is very good. Additionally you also should variate treatment time (1 hour, 2 hours, overnight). It depends how fast the seeds start to germinate.
Thank you very much for your help. The idea is not to do TILLING but to obtain herbicide-resistant mutants (IMI f.ex.) that can be selected in field or greenhouse by appliyig the herbicide. Another idea is to obtain cold and drougth tolerant plants (is this method the best for it?) Dear Carlos, my email is [email protected] or [email protected]
Thanks n very very sorry i do not know to much about on protocols for mutating seeds with EMS...........i am also specialist of plant tissue culture protocol...............................
When you do Mutagenesis with seeds, you will obtain useful mutants if only the mutated cells happen to be at the meristematic regions or at the layer that will develop into generative tissues (flowers). That way, the mutant gene will be transmitted into the next generation. In such case, grow up the seeds after EMS treatment several generations (by selfing) and collect as many seeds (progenies) as possible. Evaluate the presence of mutant phenotypes you are looking for among the harvested seeds (progenies). I do not recommend you to evaluate the mutant phenotype directly to the EMS treated seeds since there might be a chimera or the mutant cell genotypes are in a heterozygous state. In the case of recessive mutant, you will not see the mutant phenotype if the genotype is heterozygous. Best of luck...
We have developed and commercialised a sulfonylurea and imidazolinone tolerant mutant (cultivar "Angel") of Medicago littoralis using EMS. Reference: Heap, J. (2000). Increasing Medicago resistance to soil residues of ALS-inhibiting herbicides. PhD thesis, University of Adelaide. This is now being used extensively in our medic breeding program to cross into other species (eg M. truncatula, M. tornata).
Dear juan we are also working on mutation breeding on medicinal plants using ems and des.you first presoak the seeds for 5-8 hours after that you willmake the concentrations of ems based on ld 50 of your seeds,and then put 100 seeds in marked test tubes and put different concentrations of ems as i recomend u 0.1% to 1.5% concentration for 24 hours................(rest it depends on the seed coat)..............................................................cheers
Dear Juan. I have experience on physical mutagens i.e. gamma rays. Regarding chemical mutagens, the suggestions from Mohsin and Francesco looks better. If you are not interested in TILLING then physical mutagens will be better for you.
First imbibe seeds in sterile distilled water overnight wash the imbibed seeds without damaging the seed coat if you are treating small samples cut a whatman filter paper toa size of a petri dish (glass) arrange imbibed seeds uniformly in each petridish arrange petridishes for different fractions of the EMS dissolved in D.W. for eg. o.1, o.2. o.3., o.4.,o.5 and 0.01 to 0.09 minimum three fractions for diffrent durations 4,8,12,16,24. Hours after treatment is over wash the treated seeds in tap water or steriled tap water for 15 to 30 minutes trasfer the treated seeds immediately to nursery beds with agronomic inputs equal number of untreated seeds also have to be sown besides the treated seeds to compare the spontaneous and in born genotoxic mortality and mutations before all this dance you have to identify % of germination of seeds from the seed stock the whole process again depends on changes in environmental factors repeat the experiment for your satisfaction and standardise the suitable dose which works for 50% of the seeds to germinate in a dose where optimum mutation spectra is observed with reference to changes in enzyme activity changes in Gibberellin biosynthesis, cell mortality chngrs in spindle behaviour microspore behaviour seed sterility ,chlorophyll mutations and much more contact me on my E-mail:[email protected]
Hi Use Jayaramiahs method but only add tween 20 or some pH balanced soap drop which acts as a surfactant. I have used this method for tissue culture as well. However concentration and duration depends on the species and the hardseededness of the seed. Be careful to use gloves and eye goggles as and dont dispose liquids in the drain as EMS can be toxic to humans as well as fish etc in waterways. do not damaage the environment.
Hiii Mr Juan, do your prelime reseach first to to determine the target dose....so you can find the suitable dose of EMS ...and do the real research next....good luck
Thank you very much people! I have calculated the DL50, and mutated several kilograms of seeds. I´m going to start the selection in M2 seed, that will be avaliable in months.
Digitaria eriantha is such a diverse species of grass which can be propagated vegetatively using stolon or seeds. Mutating seeds with EMS is more preferred since it is much easier to handle. However, it is highly carcinogenic and volatile therefore treatment must be performed with all required precautions. For good uptake of EMS by treated seeds, it is a common procedure to first imbibe the seeds in water between 12-24 hrs in darkness. Effective of treatment depends on concentration and duration. As an indication for effective EMS treatment, M1 should show reduced seed germination and seedling growth, sterility and different types of colour mutants in the leaf (albino).