Abstract:Liquidambar is an important forestry species used to generate many commercial wood products, such as plywood. Inducing artificial polyploidy is an effective method to encourage genetic enhancements in forestry breeding. This report presents the first in vitro protocol for the induction of genus Liquidambar tetraploids based on the established in vitro regeneration system of hybrid sweetgum (Liquidambar styraciflua × Liquidambar formosana). The leaves and petioles from three genotypes were pre-cultured in woody plant medium (WPM) supplemented with 0.1 mg/L thidiazuron (TDZ), 0.8 mg/L benzyladenine (BA), and 0.1 mg/L α-naphthalene acetic acid (NAA) for a variable number of days (4, 6 or 8 days), and exposed to varying concentrations of colchicine (120, 160, 200 mg/L) for 3, 4 or 5 days; the four factors were investigated using an orthogonal experimental design. Adventitious shoots were rooted in 1/2 WPM medium supplemented with 2.0 mg/L indole butyric acid (IBA) and 0.1 mg/L NAA. The ploidy level was assessed using flow cytometry and chromosome counting. Four tetraploids and nine mixoploids were obtained from the leaves. Pre-treatment of the leaves for 8 days and exposure to 200 mg/L colchicine for 3 days led to the most efficient tetraploid induction. Producing 11 tetraploids and five mixoploids from petioles, the best tetraploid induction treatment for petioles was almost the same as that with the leaves, except that pre-culturing was required for only 6 days. In total, 15 tetraploids were obtained with these treatments. This study described a technique for the induction of tetraploid sweetgum from the leaves or petioles of parental material. Based on the success of polyploid breeding in other tree species, the production of hybrid sweetgum allotetraploids constitutes a promising strategy for the promotion of future forestry breeding.

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Liquidambar formosana (Hamamelidaceae) is a relatively fast-growing deciduous tree of high ornamental value that is indigenous to China. However, few molecular markers are available for the species or its close relatives; this has hindered genomic and genetic studies. Here, we develop a series of transferable expressed sequence tag-simple sequence repeats (EST-SSRs) for genomic analysis of L. formosana. We downloaded the sequence of the L. formosana transcriptome from the National Center of Biotechnology Information Database and identified SSR loci in the Unigene library. We found 3284 EST-SSRs by mining 34,491 assembled unigenes. We synthesized 100 random primer pairs for validation of eight L. formosana individuals; of the 100 pairs, 32 were polymorphic. We successfully transferred 12 EST-SSR markers across three related Liquidambar species; the markers exhibited excellent cross-species transferability and will facilitate genetic studies and breeding of Liquidambar. A total of 72 clones of three Liquidambar species were uniquely divided into three main clusters; principal coordinate analysis (PCoA) supported this division. Additionally, a set of 20 SSR markers that did not exhibit nonspecific amplification were used to genotype more than 53 L. formosana trees. The mean number of alleles (Na) was 5.75 and the average polymorphism information content (PIC) was 0.578, which was higher than that of the natural L. formosana population (0.390). In other words, the genetic diversity of the plus L. formosana population increased, but excellent phenotypic features were maintained. The primers will be valuable for genomic mapping, germplasm characterization, gene tagging, and further genetic studies. Analyses of genetic diversity in L. formosana will provide a basis for efficient application of genetic materials and rational management of L. formosana breeding programs.

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