Mutagenesis—A Potential Approach for Crop Improvement - Crop Improvement: New Approaches and Modern Techniques - page 162

genic treatment of seeds with chemical mutagens are: pre-soaking in distilled water,
pre-treatment rinsing in tap water, treatment with the mutagen, post-treatment rins-
ing in tap water and drying (if necessary) on a filter paper. All steps of mutagenic
treatment should be done using glass beakers to avoid any interaction of chemical
mutagens with even trace quantities of metallic cataions or other active reagents.
Seeds for each dose of mutagenic treatment (M 0 generation) and for the untreated
control, usually the parent variety, are put into beakers that are visibly labeled with
the applied concentration of mutagen (Roychowdhury 2011 ).
As dry seeds are usually used for treatment, pre-soaking in distilled water should
be applied to activate seeds physiologically before treatment with mutagen. The
amount of water used in pre-soaking should be at least 2-3 times the volume of dry
seeds. The beakers with pre-soaked seeds should be gently shaken a few times to
remove air bubbles, which can block access of mutagen to embryos. Duration of
pre-soaking depends on the biology of germination of a particular crop species. For
example, in barley and other major cereals, 8-10 h of pre-soaking in room tempera-
ture (20-24 °C) is usually applied. Pre-soaking significantly reduces the somatic
effect of chemical mutagen (Roychowdhury and Tah 2011a ). Short washing, 2-3
times in room-temperature tap water should be applied after soaking to remove
water-soluble substances leaching from the seed. Such prepared seeds are ready
for mutagenic treatment. It is advisable to use three doses of mutagen for a large-
scale field experiment. This is especially desired for regions with very variable
and unpredictable weather conditions during the growing period of mutagenetically
treated material. Drought, cold and heat can significantly modify the somatic ef-
fect of a mutagen and influence the final effect of treatment. The concentration of
mutagen, its duration and temperature of treatment are understood under the term
'dose' in chemical mutagenesis. A temperature of mutagenic solution of 22-24°C is
most often applied for the seed treatment of various crop species. The use of other
temperatures is also possible. However, it should be noted that the increased tem-
perature will significantly shorten the half-life of chemical mutagen and generate
products of hydrolysis that can increase undesired somatic effect of a mutagen. This
is especially relevant to treatment with mutagens such as diethyl sulfate (DES) or
ethyl methane sulphonate (EMS). To obtain equal penetration of a mutagen through
the cells of a seed embryo, it is necessary to treat seeds in a water solution of the
mutagen for 3-5 h. Similar to the pre-soaking, the treatment should be done with
a significant surplus of mutagenic solution, some 2-3 times the volume of the dry
seeds. In cereals, about 1-1.5 ml of mutagenic solution is applied per seed. The
concentration of the mutagen should be considered, together with duration of the
treatment. A shorter treatment time with higher concentration of mutagen can in-
crease somatic effects and could be insufficient to penetrate equally all cells in the
plant material. A gentler treatment requires a lower concentration but longer period
of application.
Extensive post-treatment rinsing several times in room-temperature tap water is
necessary to stop action of the mutagen and to remove its residues from the surface
of the seeds. To facilitate sowing, the treated seeds can be dried on filter paper
under a fume hood. However, too intensive drying, especially with increased air