In vitro tetraploid induction from multigenotype protocorms and tetraploid regeneration in Dendrobium officinale

Abstract

In vitro mitotic polyploidization using anti-microtubule agents has been commonly used for polyploid production. The present study was an attempt to develop an in vitro oryzalin chromosome doubling and stable tetraploid regenerating protocol for Dendrobium officinale. In this study, we successfully induced tetraploids by treating protocorms developed from the seeds of multiple genotypes. The highest frequency of polyploidy was 37.40%, achieved with 14.4 µM oryzalin treatment for 24 h. Among the obtained polyploid seedlings, 72 solid tetraploid, 33 mixoploid and 3 octoploid genotypes were identified via screening using flow cytometry (FCM). Three peaks were observed in the histograms of the diploid, tetraploid, and octoploid leaves, but four peaks were observed only in mixoploid leaves with FCM, indicating the existence of endopolyploid cells and the occurrence of conventional endoreduplication in the leaves of all ploidy levels. Recurrent ploidy identification of various tetraploid genotype regenerated plantlets obtained via protocorm-like bodies (PLBs) derived from axenic stem-node segments maintained stable tetraploid levels. Comparisons of phenotypic characteristics revealed that relative to the diploid plantlets, the tetraploid plantlets exhibited increased stem diameter, root diameter, labellum width and gynostemium width. Furthermore, the tetraploid plantlets showed lower plant height, leaf length and root length than the diploid plantlets. This efficient polyploid induction and ploidy-stable regeneration protocol can be used for the mass production of tetraploid D. officinale. The tetraploid genotypes regenerated in this study might be useful for nobile Dendrobium breeding in the future.