Production of autotetraploid plants by in vitro chromosome engineering in Allium hirtifolium

Persian shallot has been propagated vegetatively through daughter bulbs and has a narrow genetic base. In the present study, to create genetic diversity in terms of ploidy level, the effects of in vitro polyploidization were investigated on morphological, physiological and phytochemical traits. Different colchicine treatments (0, 0.3%, 0.5% and 0.7% concentrations) for 24, 36 and 48 h and oryzalin treatments (0, 0.001%, 0.002% and 0.004% concentrations) for 4, 6 and 8 h were used. A sterile basal plate of bulbs was used for the induction of polyploidy in liquid and solid media. After obtaining plantlets, root tip chromosomes were counted for the determination of ploidy levels. Flow cytometry was performed to confirm the chromosome counting results. The regenerated diploid and tetraploid plantlets were transferred to ex vitro conditions. Colchicine and oryzalin were both successful in inducing polyploids and the polyploids induced were tetraploids and mixoploids. The highest induction of polyploidy was obtained in solid media using 0.5% (w/v) colchicine for 36 h (35.0% polyploidy induction) and in 0.001% (w/v) Oryzalin for 8 h (45.5% polyploidy induction). Differences in plantlet height and weight, bulblet weight, density, stomatal width and length, and chlorophyll content were observed between tetraploid and diploid plants. Chromosome duplication, as a result of tetraploid in-duction, significantly influenced the antioxidant content and enzyme activities. The results showed that total phenolic content, allicin, and antioxidant capacity were significantly higher in the tetraploid plantlets than that in the diploid plantlets. SRAP loci polymorphisms indicated that the 12 autotetraploid plants (with high allicin content) had different genotypes from the parental diploid plant. Besides, in vitro polyploidy induction not only duplicated chromosomes but also altered the DNA sequence in Allium hirtifolium. In vitro induction of tetraploids in A. hirtifolium can be a reliable way to obtain suitable plant material for breeding programs to generate new genotypic variations.