Abstract: 【Objective】 Plant organs are made from multiple cell types, and 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 genotype, and chimeric plants were often produced through grafting in citrus. 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 + Citrus aurantium) is a periclinal chimera consisting of L1 cell layer denoted by Citrus unshiu and L2/L3 cell layers derived from Changshan Huyou (Citrus 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, the tetraploid seedlings generated from chimera is 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 origination of the tetraploid seedling was 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 as control. Following the DNA extraction, 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, respectively. The semi thin sections were used to observe the anatomical structure of leaves, and the thickness 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, respectively. The density of fat granules and starch granules in leaf were compared between the tetraploid seedling and the diploid seedling, respectively, based on the ultramicrotomy and observation under a transmission electron microscope (TEM). The stomatal density and the size of stoma and guard cells in the leaves were compared between the tetraploid seedling and the diploid seedling by observation under a scanning electron microscope (SEM), respectively. The chlorophyll content and carotenoid content were measured by spectrophotometry and compared between the tetraploid seedling and the diploid seedling, respectively. 【Results】 The investigation of embryo number indicated that Hongrou Huyou was a polyembryonic species, and the proportion of monoembryos was 42%, and that of polyembryos was 58%. Flow cytometry (FCM) indicated that one tetraploid seedling of Hongrou Huyou was found among 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 Citrus 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 thickness of upper and lower epidermis, palisade tissue, and sponge tissue were significantly greater in the tetraploid seedling compared with the diploid seedling, respectively. The SEM analysis showed that the tetraploid seedling was significantly reduced in the stomatal density and enlarged in the size of guard cells compared with the diploid seedling, respectively. The TEM observation showed that the density of fat globules was increased but the density of starch granules was decreased in the tetraploid seedling compared to the diploid seedling, respectively. The chlorophyll content and carotenoid content were significantly increased in the tetraploid seedling compared to the diploid seedling, respectively. 【Conclusion】 In this study, one tetraploid seedling was identified from one hundred and twenty seedlings of periclinal chimera Hongrou Huyou. SSR Ploymorphism of the tetraploid seedling was identical to the Citrus aurantium that is the L2 donor to Hongrou Huyou. The tetraploid seedling showcased the typical morphological and anatomical characteristics in leaf shape and thickness, stomatal density and size, and chlorophyll and carotenoid content compared with the contemporary diploid seedling, respectively. This work provides a novel tetraploid germplasm promising the application on triploid breeding in citrus in the future.
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