In vivo tetraploid induction of mono-embryonic citrus genotypes by colchicine treatment

Abstract

Polyploid holds great significance in citrus breeding, to produce dwarf tetraploid rootstocks with wide adaptability and triploid scion varieties bearing seedless fruits. In citrus, tetraploids have been generated through somatic hybridization and autonomous chromosome doubling of nucellar embryos. However, most tetraploids were recovered from poly-embryonic (apomictic) citrus, while tetraploids of mono-embryonic (non-apomictic) citrus were seldomly reported. Herein, we developed an in vivo tetraploid induction method by colchicine treatment, which is especially high-efficient in mono-embryonic citrus. The epicotyl of citrus seedling was decapitated and incubated in dark for different time durations (0–15 d) to induce callus formation on the epicotyl wound. The callus on wound was then immersed in different concentrations (0.0125 %-0.100 %) of colchicine for 2 h, enabling regeneration of tetraploid shoots. We achieved high tetraploid induction rate (20.69 %) but low mixoploid induction rate (1.72 %), when 0.025 % colchicine was applied on wound cultured in dark for 9 d By using this method, 147 tetraploids were recovered from mono-embryonic mandarin, tangerine, sweet orange, pummelo and citron. Our method exhibited high tetraploid induction efficiency (14.28 %-20.69 %), low rate of mixoploid (1.72 %-5.30 %), short recovery period (45 d), and no need of tissue culture. The majority (75 %-100 %) of the recovered tetraploids maintained stable ploidy during subsequent growth, and the mono-embryonic nature of the tetraploids was identified by InDel marker analysis. The mono-embryonic tetraploids obtained herein enriched polyploid germplasm pool for citrus breeding.