Plant regeneration from root segments of Anthurium andraeanum and assessment of genetic fidelity of in vitro regenerates

Abstract

A highly efficient regeneration system of Anthurium andraeanum Linden was established using root segments as explants of four genotypes, Arizona, SWD, OYH, and Mississippi. The root explants were firstly incubated on callus induction media containing NH4NO3 level of 206 mg L−1 or 825 mg L−1, and the calluses were formed from both ends of the root segments after 1 mo of culture. Root-derived calluses were then transferred to shoot regeneration media with NH4NO3 level of 412 mg L−1 or 1650 mg L−1, and numbers of shoot were successfully produced from the surface of calluses after 2 mo of culture. Medium containing 206 mg L−1 NH4NO3 induced more callus compared to 825 mg L−1. In contrast, the number of shoots per callus was the same between the above two shoot regeneration media indicating that NH4NO3 level had little effect on shoot regeneration. Long-termin vitro maintenance of the callus-bearing shoot initials was possible without compromising shoot regeneration and subsequent rooting. Histological analysis showed that early cell division was initiated from the inner cortical parenchyma cells near the vascular tissue and from the vascular parenchyma cells adjacent to the xylem vessel. The primary callus mainly comprised inner parenchyma cells with scattered procambial-like cells and xylem elements and two different meristematic zones beneath epidermal layers. Transfer of callus to the regenerative medium caused meristemoid formation and subsequent shoot primordia production. Calcium oxalate crystals and tannin-like deposits were visible on the outer callus layer or within the cotyledon-like structure of shoot primordium throughout callus development. Starch grains were abundant in root explants, but disappeared in the profusely growing primary callus, and were mobilized in the shoot primordium, indicating energy requirements of these two developmental stages. Flow cytometry analysis demonstrated that ploidy levels were maintained during in vitro cultures, including long-term regenerated plants. The inter-simple sequence repeat analysis indicated genomic DNA stability of Arizona and OYH but occasional genetic variation in primary regenerates of SWD and Mississippi.