A novel method for rapid in vivo induction of homogeneous polyploids via calluses in a woody fruit tree (Ziziphus jujuba Mill.)

Abstract

Conventional approaches to the polyploid breeding of fruit trees are severely limited by the unavoidable problem of mixoploidy. This is caused because the traditional approach to inducing polyploidization is the application of anti-mitotic agents like colchicines to organs, such as apical or axillary buds, which prevents the doubling of chromosomes in some cells of the organs. To overcome this challenge, we first developed a mixoploid-free in vivo autopolyploid induction system in the Chinese jujube tree (Ziziphus jujuba Mill.) by integrating in vivo bud regeneration via calluses with polyploid induction. The novel system included three steps: field callus induction, callus cell polyploidization, and shoot regeneration from a chromosome-duplicated callus cell. We used the following optimized approach. First, strong branches of 15–25 mm in diameter were chosen and clipped. Second, the cambium of the cut sections was treated with 4 mg/L thidiazuron +2 mg/L AgNO₃ for 14 h to induce calluses and buds, and the cut sections were then covered with humid mud and plastic film. Third, 3 days later, the calluses newly initiated from the cambium were treated with 0.05 % colchicine dissolved in 1 % dimethylsulphoxide. Fourth, the coverings were removed after regenerated shoots reached a length of 2 cm. Finally, the ploidy and purity of the new shoots were checked with flow cytometry. On the tetraploid shoots, leaves were thicker, broader and darker in appearance compared to diploid leaves. However, leaf size became much smaller on octoploid shoots. The size of stoma increased and their densities decreased in response to the increase in ploidy. In addition, we established a simple, stepwise approach for morphological polyploid determination that was able to distinguish among diploid, tetraploid, octoploid and mixoploid individuals with high accuracy. Based on morphological and anatomical observations, the buds regenerated from calluses can be classified into somatic embryo-derived buds and adventitious buds, with adventitious buds being the main route of regeneration.