Production of a doubled haploid from a haploid pummelo using colchicine treatment of axillary shoot buds

Abstract

To produce the homozygous strain of a haploid plant derived from small seed-derived seedlings of 'Banpeiyu' pummelo (Citrus grandis Osbeck), we carried out colchicine treatment to axillary shoot buds of the haploid. Many shoots with cytochimeras (X+2X and 2X+4X) arose from the colchicine-treated axillary buds. When cytochimeric buds of 2X+4X were top-grafted onto trifoliate orange [Poncirus trifoliata (L.) Raf.], a complete diploid shoot with 18 chromosomes was obtained from the cytochimera. This diploid strain showed vigorous growth compared with the original haploid. The leaf weight per unit area and the stomata size in this diploid were significantly larger than those of the original haploid plant, and were almost equal to those of 'Banpeiyu' pummelo. The diploid strain was confirmed to be a doubled haploid of a haploid from 'Banpeiyu' pummelo, based on random amplified polymorphic DNA (RAPD) analysis and chromosome composition analysis by chromomycin A3 (CMA) staining.