Colchicine-induced tetraploidy in Dendrobium cariniferum and its effect on plantlet morphology, anatomy and genome size

Abstract

Dendrobium cariniferum Rchb. f. is an important crossing parent that is primarily used to produce aromatic Dendrobium orchid cultivars due to its beautiful flowers and pleasant orange fragrance. However, there are no offspring within the limited progenies that fully inherit this odor trait. Thus, converting diploid D. cariniferum into tetraploids for interploid hybridization is another promising approach to creating a new orange aroma germplasm. This study reports a novel in vitro colchicine-induced chromosome doubling protocol that would effectively convert diploid D. cariniferum protocorms into tetraploids. A total of 120 tetraploid genotypes were identified by flow cytometry. The tetraploid induction rate was significantly affected by interaction between the colchicine concentration and exposure time. Treatment with 0.05% colchicine for 24 h produced the optimal results, yielding 54% surviving regenerated plantlets with a 33% tetraploid induction rate. The tetraploid plantlets displayed obvious morphological variations (broader, thicker leaves and greater stem and root diameters) than the diploid plantlets. Tetraploidization also caused a significant change in the leaf anatomical structure, including larger stomata with lower densities, more chloroplasts per stomata, thicker spongy tissues, larger leaf veins and decreased adaxial epidermal cell and trichome densities. We were the first to observe that there are one to five branched trichomes in the leaf sheath in both diploids and tetraploids, and we propose that the leaf adaxial epidermal cell density can be used to identify tetraploids. Moreover, tetraploids exhibited a 1.75-fold greater genome size than diploids. Our study lays the foundation for breeding triploid aromatic Dendrobium orchids. Autotetraploidy causes morphological, anatomical and genome size variations. This is the first report of trichome types in the leaf sheath. Leaf adaxial epidermal cell density is useful for identifying tetraploids.