Determination of the Optimal Dosage of Physical and Chemical Mutagenesis Agents in Callus Tissue of Kiwifruit Leaf

Induced mutations have been used in a wide range of horticultural crops for more than half a century. This approach has opened up great opportunities for biotechnologists and plant breeders to produce new varieties with several new traits including resistance to disease, and tolerance to adverse environmental conditions or even produce new horticultural characters in some crops. The optimization of dosage of the physical and chemical mutagenic agents for inducing mutations is the first step in using this technique in plant breeding. This study was thus conducted to determine the optimum dosage of two mutagenic agents including gamma radiation as a physical agent and ethyl methanesulphonate (EMS) as a chemical agent in kiwifruit callus in in-vitro conditions. To that end, simple sequence repeat (SSR) markers were used to detect the mutations in the regenerated plantlets.

Having induced the callus in tissue culture media containing naphthaleneacetic acid, 6-benzylamino purine and meta-Topolin, the produced calli were exposed to different dosage of gamma radiation (0, 10, 20, 30, 40, 50 Gy) or different EMS concentrations (0, 0.4, 0.6, 0.8, 1, 1.2 % for 60 min). Then, the callis were transferred to the shoot regeneration media. The number of dead calli, and the size of live calli as well as the number of shoots generated in live calli were then recorded. Afterwards, the regenerated shoots were transferred to elongation media, and the DNA was extracted. The SSR markers analysis was followed on the randomly selected plantlets to detect the SSR markers using a Genetic Analyzer.

As expected, with increasing the dosage of gamma radiation, the number of dead calli increased and the callus growth was thus retarded. The lowest number of dead calli was, indeed, detected in the control and the 50 Gy dose resulted in the highest number of dead calli. The morphology of shoots generated in the treated calli and the SSR marker analysis showed that 50 Gy dose could be used as the optimum radiation dose to produce an acceptable mutation rate in kiwifruit callus. In turn, EMS as a chemical mutagenic agent showed a different pattern in increasing the number of shoots regenerated with increasing dosage, however the same trend happened with gamma radiation in the number of dead calli and live callus size. Considering the effect of EMS on callus and the number of shoots regenerated per callus, a treatment with 1% EMS for 60 min can be used to produce a high density mutant population in kiwifruit. The highest mutation rate (12.5 %) was observed in the 50 Gy gamma ray treatment with changes in 12 pairs of SSR markers among 25 loci. For EMS, this value was 2.6 % in 1% concentration treatment.

To detect the induced mutations using Gamma ray and EMS, the SSR markers can be applied. However, for the EMS mutation detection, more studies are needed to evaluate the efficiency of other techniques such as single nucleotide polymorphism (SNPs).