Recovery and characterization of homozygous lines from two sweet orange cultivars via anther culture

Abstract

Homozygous genotypes are valuable for breeding and genomic studies in higher plants. The production of haploids and DHs through gametic embryogenesis allows a single-step development of complete homozygous lines from heterozygous parents, requiring much less time than the conventional selfing method. Here, we report the regeneration of haploid and double haploid lines of citrus species through anther culture. The anthers of seven citrus cultivars at the uninucleate stage were cultured and induced using four previously reported mediums. Ten haploid lines (2n = x = 9), six DH lines (2n = 2x = 18), two tetraploid lines (2n = 4x = 36) of ‘Early Gold’ sweet orange, and one haploid line of ‘Rohde Red’ Valencia sweet orange were obtained, as identified by ploidy, karyotype and simple sequence repeats (SSRs) analysis. All of them were confirmed to be fully homozygous by SSR analysis using 31 primer pairs that are distributed evenly on each of the chromosomes. Among them, plants regenerated from two DH lines of ‘Early Gold’ sweet orange grew vigorously in the greenhouse. To our knowledge, this is the first report on sweet orange anther culture with successful DH plant regeneration. The haploid, DH and tetraploid lines reported here hold great potential for future citrus genome resequencing in genetic studies and seedless breeding via somatic fusion.