Induction of polyploid Malus prunifolia and analysis of its salt tolerance

The apple rootstock Malus prunifolia (Willd.) Borkh. is widely used for apple production. Because polyploid plants are often more tolerant to abiotic stress than diploids, we wondered whether polyploidy induction in M. prunifolia might improve its stress tolerance, particularly to high salinity. We used a combination of colchicine and dimethyl sulfoxide (DMSO) to induce chromosome doubling in M. prunifolia and identified the resulting polyploids by stomatal observations and flow cytometry. We found the best way to induce polyploidy in M. prunifolia was to use 2% DMSO and 0.05% colchicine for 2 days for leaves or 0.02% colchicine for stem segments. The results of hydroponic salt treatment showed that polyploid plants were more salt tolerant and had greater photosynthetic efficiency, thicker leaf epidermis and palisade tissues, and shorter but denser root systems than diploids. During salt stress, the polyploid leaves and roots accumulated less Na+, showed upregulated expression of three salt overly sensitive (SOS) pathway genes, and produced fewer reactive oxygen species. The polyploid plants also had considerably higher ABA and jasmonic acid levels than diploid plants under salt stress. Under normal growth conditions, gibberellins (GAs) levels were much lower in polyploid leaves than in diploid leaves; however, after salt treatment, polyploid leaves showed upregulation of essential GAs synthesis genes. In summary, we developed a system for the induction of polyploidy in M. prunifolia and response to salt stress of the resulting polyploids, as reflected in leaf and root morphology, changes in Na+ accumulation, antioxidant capacity and plant hormone levels.