A study on the induction of polyploidy in red-fleshed apple‘Hongcuitian’

Abstract:【Objective】The method of polyploid induction of red-fleshed apple was established by in vitro regeneration combined with polyploidy induction on the basis of tissue culture of red-fleshed apple‘Hongcuitian’. The expected goals were: (1) to obtain the bigger red-fleshed apple by chromosome doubling; (2) to select the new triploid apple by crossing the new tetraploid apple with the new diploid apple; (3) to evaluate the adaptability of apple rootstocks based on the characteristics and stress resis- tance of the polyploid plants.【Methods】The new shoots of‘Hongcuitian’were used as the explantlet, disinfection treatment, bud induction and subculture protocols were established. The stem segments were shaking cultured in 250 mg · L- 1 colchicine solution for 12, 24 or 48 h. The explants were then transferred to regeneration medium (MS + 6- BA 0.5 mg · L- 1 + NAA 0.05 mg · L- 1). The leaf explants were precultured in 200 mg·L-1, 250 mg·L-1, 350 mg·L-1 or 500 mg·L-1 colchicine solution for 4 days and then transferred to the most suitable medium for regeneration, which was screened from MS medi- um supplemented with different concentrations of TDZ. All the regenerated plants were tested for ploi- dy by flow cytometry. The plant height, leaf length and leaf width of the diploid‘Hongcuitian’and the tetraploid‘Hongcuitian’were measured and the changes of leaf edge sawtooth and leaf color were ob- served at 55d seedling age.【Results】Plant regeneration was affected by hormone types and concentra- tions in the medium. The effect of MS medium with different concentrations of TDZ on adventitious budregenerationwasdifferent.MS+TDZ2.0mg·L-1+NAA0.1mg·L-1 hadthebestinductioneffecton the leaves. The average regeneration frequency of adventitious buds was 76.47%, which was significantly higher than that of the other two mediums. The average number of regenerated buds was 5.3. The ad- ventitious buds could further develop into seedlings by transferring them into the medium of MS+6-BA 0.1 mg·L-1+NAA 0.05 mg·L-1. Polyploidy induction of stem segments showed that the survival rate of stem segments decreased with the extension of shaking culture time, indicating that long-term colchi- cine immersion accelerated the destruction of cells and caused tissue death. The results of ploidy deter- mination showed some mixed ploidy, but no tetraploidy was detected. Polyploidy was induced from leaves at room temperature with colchicine at concentrations up to 500 mg·L-1. Under this high concen- tration leaf explants turned brown on the second day. Under the other three concentrations, the regener- ated plants were identified as mixed and tetraploid. Therefore, 200-350 mg·L-1 colchicine was suitable for chromosome doubling of regenerated plants from the leaves. It was also found that the proportion of mixed-ploidy plants decreased from 31.85% to 5% with the increase of subculture times, and diploidy of plants was restored after the stem regeneration. The tetraploid plants could not be obtained by stem regeneration, but stable tetraploid plants could be obtained by leaf regeneration induced by colchicine solution. The yield of tetraploid plants measured in two trials (2018-10 and 2019-06) was close to 20%. The characters of diploid and tetraploid plants at 55 days seedling age were determined, the height of tetraploid plant was 7.4 cm, which was 30% lower than that of the control; the leaf widths of tetraploid and diploid plants were similar, but the leaf length of tetraploid plants was shorter, which caused the leaf shape index to reduce from 2.05 to 1.42, and leave color changed from light green to dark green.【Conclusion】The method of chromosome doubling by leaf induction was more appliable than by stem induction, and chromosome doubling caused significant changes in plant height, leaf length, leaf color and leaf edge compared with the control diploidy plants.