Exploration and morphological characteristics evaluation of a tetraploid seedling from periclinal graft chimera Hongrou Huyou (Citrus unshiu + C. aurantium)

[Objective] Plant organs are composed of multiple cell types, and organs of dicotyledonous plants normally have three distinct layers of cells, L1, L2 and L3. Layer L1 is the single layer of cells making up the epidermis, and layer L2 is the single cell for sub-epidermal layer, and layer L3 constitutes the rest of the internal cells. Chimera refers to an organism made of the cell layers with different genotypes, and chimeric plants in citrus were often produced through grafting. Polyembryony hampers the efficiency of sexual hybridization in citrus, and seedlings from citrus chimera can additionally exhibit characteristics closer to the features of the donor of cell layer contributed to the gamete origination. Hongrou Huyou (Citrus unshiu + C. aurantium) is a periclinal chimera consisting of L1 cell layer denoted by C. unshiu and L2/L3 cell layers derived from Changshan Huyou (C. aurantium). Tetraploids are important for rootstock application in citrus production, and for enhanced tolerance or resistance to biotic stress and abiotic stress, and for seedless triploid varieties breeding. Autotetraploid seedlings have been reported in some citrus species with a variable frequency of occurrence. However, tetraploid seedlings generated from chimera are rarely reported in citrus. In this study, one tetraploid seedling of Hongrou Huyou was identified from a population of seedlings transplanted successfully. The ploidy level and genetic origin of the tetraploid seedling were analyzed, and the morphological characteristics of the tetraploid seedling were explored using the diploid seedlings as control, suggesting an application of seedlings of peripheral chimera in citrus. [Methods] In this experiment, the embryo number per seed of Hongrou Huyou was check and identified based on the investigation of one hundred seeds selected randomly. Flow cytometry (FCM) was used to determine the ploidy level of the seedlings of Hongrou Huyou using a diploid species Hongrou Huyou as control. Following DNAextraction, the genetic identification of the seedlings was analyzed using 28 pairs of Simple Sequence Repeat (SSR) primers distributed in 9 chromosomes of citrus species. Morphological differences including leaf length, leaf width, internodal distance, leaf thickness, and leaf shape index were compared between the tetraploid seedling and the diploid seedling of Hongrou Huyou. The semi thin sections were used to observe the anatomical structure of leaves, and the thicknesses of the upper epidermis, lower epidermis, palisade tissue, and sponge tissue were compared between the tetraploid seedling and the diploid seedling under an optical microscope. The density of lipid granules and starch granules in the chloroplast were compared between the tetraploid and the diploid seedlings based on the ultramicrotomic observation under a transmission electron microscope (TEM). The stomatal density and the size of stoma and guard cells in the leaves were also observed under a scanning electron microscope (SEM). The contents of chlorophylls were measured with spectrophotometry. [Results] The results indicated that Hongrou Huyou is a polyembryonic species. The proportion of monoembryos was 42%, and polyembryos 58%. FCM identified one tetraploid seedling of Hongrou Huyou among the 120 seedlings transplanted successfully. The results of SSR molecular markers amplified with 28 pairs of primers showed that the tetraploid seedling had identical genotype to C. aurantium, which was the Layer 2 (L2) cell donor of Hongrou Huyou. Morphological comparison showed that the leaf length, the internodal distance, and the leaf shape index were significantly decreased in the tetraploid seedling compared with the diploid seedling. Semi thin sections showed that the leaf thickness, the thicknesses of the upper and lower epidermis, palisade tissue, and sponge tissue were significantly greater in the tetraploid seedling. The SEM analysis showed that the tetraploid seedling had a significantly reduced in the stomatal density and an enlarged size of guard cells compared with the diploid seedling. The TEM observation showed that the density of lipid globules increased but the density of starch granules decreased in the chloroplasts of the tetraploid seedling compared to the diploid seedling. The chlorophyll and carotenoid contents were significantly increased in the tetraploid seedling. [Conclusion] In this study, one tetraploid seedling was identified from 120 seedlings of periclinal chimera Hongrou Huyou. SSR ploymorphism analysis showed that the tetraploid seedling was identical to C. aurantium, the L2 donor to Hongrou Huyou. The tetraploid seedling displayed the typical morphological and anatomical characteristics in leaf shape and thickness, stomatal density and size, and chlorophyll and carotenoid content of polyploid plants. This work provides a novel tetraploid germplasm for the application of triploid breeding in citrus in the future.