Optimization of polyploidy induction system in dragon fruit

Abstract:【Objective】Dragon fruit (pitaya, pitahaya) is a new and highly commercial tropical fruit spe- cies in China. However, the less variety diversity, self-incompatibility and small fruit size have limited the development of dragon fruit industry. Polyploid breeding, an effective method for fruit crop breeding，has been successfully applied in a lot of plants but little has been done about Dragon fruit. In the ex- periment, the dragon fruit polyploid induction system was optimized, which would be helpful to enlarg- ing the genetic background and innovating new polyploidy germplasm.【Methods】Two dragon fruit cul- tivars‘Dahong’(2n=2X=22) and‘Baishuijing’(2n=2X=22) were used as the materials. Seeds and plant- lets were soaked in colchicine or co-cultivated with colchicine. Germinated seeds were soaked in differ- ent concentrations of colchicines (2.5、5、10 g·L-1) for different times(12, 24, 36 h). In addition, aseptic seedlings were co-cultivated with different concentrations of colchicines (25, 50, 100 mg·L-1) for differ- ent times (3, 5, 7 d). Then germinated seeds and aseptic seedlings were washed with clean water after treated with colchicine, and the mutation rate and death rate after 7 and 30 days were evaluated and sta- tistically analyzed. Stomatal density, length and width of guard cells, chromosomes counting and Ploidy Analyser were used to identified the ploidy of the variants.【Results】The variation of dragon fruit plants showed the following features that longer and wider leaves and wider hypocotyledonary axis ap- peared, and the mutation and death rates increased with increase of colchicine concentrations and treat- ment time. The germinated seeds soaked in 5 g · L- 1 colchicine for 24- 48 h had better induction effect, and the mutation and death rates after 7 d were 13.83%-27.75% and 3.53%-29.28%, respectively, while the mutation and death rates after 30 d were 11.87%-15.08% and 37.56%-52.42%, respectively. Lower and higher concentrations of colchicines were less effective. The hypocotyl enlargement rates at lower concentration of colchicine (2.5 g·L-1) were much lower (3.12%-22.83%), and the mutation rate after 30 d was lower than 5%. The higher concentration of colchicine (10 g·L-1) had higher hypocotyl enlarge- ment rate, but the death rate was very high, and the mutation rates after 30 d were only 3.16%-7.10%. Aseptic seedling co-cultured with 50 mg·L-1 colchicine for 3-5 d had better induction effect. The muta- tion and death rates after 7 d were 26.17%-44.97% and 10.06%-14.15%, respectively, while the muta- tion and death rates after 30 d were 20.36%-26.32% and 22.51%-45.50%, respectively. The lower or higher concentrations of colchicine were less effective. Lower concentration of colchicine (25 mg · L- 1) had lower hypocotyl enlargement rate (7.73%-34.44%) and mutation rate after 30 d (3.66%-6.59%), and higher concentration of colchicines (100 mg · L- 1) had higher hypocotyl enlargement rate (30.47%- 67.55%), but the death rates after 7 d (17.16%-26.56%) and 30 d (31.20%-66.88%) were very high, and the mutation rates after 30 d were very low (8.13%-17.46%). The stomatal density decreased, and the guard cells became larger in variation plants. The stomatal density and size were 81% and 115% of the control group respectively. Ploidy analyser was used to identify the ploidy levels of the variants, which indicated that more than half of the variants were diploid, the polyploid/variation rates of variants in- duced by seed-soaked method were 0.00%-47.00%, and the Polyploid/Variation rates of variants in- duced by co-cultured method were 3.85%-37.31%. Results from chromosome counting showed the chromosome numbers of variants were unstable (2n=44, 56, 66), while that of diploids was 2n=22. The results with Ploidy analyser showed the variants contained tetraploids (2n=44), hexaploid (2n=66) and mixoploid or chimera (2n=22, 2n=44). Autotetraploid/polyploidy rates in co- cultured trial (0.00%- 28.57%) were higher than seed-soaked trial (0.00%-28.57%). The germinated seeds soaked in 5 g·L-1colchicine for 24 h had better induction effect, and the polyploidy and homolog tetraploid induction effi- ciency were 4.04%-5.51% and 1.15%-1.38%, respectively. Aseptic seedling co-cultured with 50 mg · L-1colchicine for 5 d had better induction effect, the polyploidy and homolog tetraploid induction efficien- cy were 8.25%-8.68% and 1.92%-2.43%, respectively. And the polyploidy and homolog tetraploid induction efficiency with co-culture method were higher than soaked method.【Conclusion】Dragon fruit aseptic seedling cocultured with 50 mg · L- 1 colchicine for 5 d was the best combination for tetraploid induction, which had higher mutation and tetraploid rates. Co-culture method was more suitable for dragon fruit polyploidy induction than seed-soaked method, but the tissue culture technique was needed. Although the induce efficiency of seed-soaked method was lower, but it may be a better choice for the breeder without tissue culture condition. Ploidy Analyser is a simple and rapid method to identify the ploidy levels of variants, especially to distinguish tetraploid, mixoploid and chimera. This result will be helpful to germplasm innovation and breeding of dragon fruits.