Identification and evaluation of new mixoploid variants in Citrus reticulata‘Orah’

Abstract: 【Objective】Orah (Citrus reticulata) is an important late-maturing citrus variety. Previously, putative polyploid bud mutation materials were discovered in Orah. In this study, the material was identified and evaluated in terms of ploidy, morphological characteristics, fruit quality and leaf microstructure to provide a theoretical basis for its utilization.【Methods】Flow cytometry was used for ploidy identification, the paraffin sections were used for leaf microstructure identification, scanning electron microscopy was used for stomatal morphology, size and density identification, the morphological characteristics of leaves, flowers, seeds and fruits were compared, and the conventional methods were used to determine fruit quality parameters such as soluble solids content, titratable acid content, vitamin C content, edible rate and juice yield.【Results】Orah was diploid, while the variant material was a mixo-ploid of diploid and tetraploid (2x+4x), with a cell ratio of 2x∶4x = 39.1∶60.9. There were differences in leaf morphology between the mixoploid and diploid. The mixoploid leaves were ovate with short acuminate tips and dark green color, while the diploid leaves were broad-lanceolate with gradually acuminate tips. The length, width, perimeter and area of the mixoploid leaves were 101.68 mm, 54.29 mm, 266.02 mm and 3 598.92 mm2 , respectively, which were 14.75%, 31.01%, 19.38% and 46.09% higher than those of the diploid, respectively. The leaf shape index was 1.87, which was 12.61% lower. Both had bisexual flowers with similar petal numbers, petal colors, filament cohesion states and style states. The petal length and width of the mixoploid were 14.48 mm and 8.22 mm, respectively, which were significantly larger than those of the diploid, increasing by 34.32% and 49.18%, respectively. The seeds were both monoembryonic, and the mixoploid had higher seed weight, transverse diameter and longitudinal diameter than the diploid, increasing by 30.77%, 8.12% and 26.64%, respectively. The single fruit weight of the mixoploid was 206.16 g, the longitudinal diameter of fruit was 63.37 mm, the transverse diameter was 77.90 mm, the fruit shape index was 0.81, the peel thickness was 4.26 mm, and the fruit firmness was 6.47 kg· cm-2 , which were 29.67%, 13.38%, 8.57%, 3.85%, 38.31% and 16.37% higher than those of the diploid, respectively. However, the number of seeds per fruit was 8.2, which was 61.68% lower than that of the diploid. There were no significant differences in soluble solids content (15.10%) and titratable acid content (0.58%) between the mixoploid and diploid. The edible rate decreased by 8.09%, the juice yield increased by 4.34%, and the vitamin C content increased by 39.70%. The cellular arrangement of the transverse section tissue structure of the mixoploid leaves was basically the same as that of the diploid. The thicknesses of the leaf, upper epidermis, lower epidermis, palisade tissue and spongy tissue were 310.96 μm, 12.26 μm, 72.89 μm and 227.94 μm, respectively, which were 19.75%, 4.15%, 10.10%, 9.38% and 43.92% higher than those of the diploid, respectively. The stomatal length and density were 7.35 μm and 520.40 per mm2 , respectively, which were 7.36% and 15.76% lower, respectively. The stomatal width was 3.33 μm, which was 5.09% higher.【Conclusion】In this study, a new mixoploid germplasm of Orah with diploid and tetraploid was identified. There were significant differences between the mixoploid and diploid in leaf, flower, seed, fruit morphology, fruit quality and leaf microstructure. Overall, the mixoploid showed larger and thicker leaves, increased petal length and width, increased seed weight, transverse and longitudinal diameters, but reduced number of seeds per fruit. The thicknesses of the leaf, upper and lower epidermis, palisade tissue and spongy tissue increased, while the stomatal length and density decreased and the stomatal width increased. Additionally, there were increases in single fruit weight, fruit transverse and longitudinal diameters and vitamin C content. This mixoploid can be used as a new type of single-embryo type of small-nucleus and largefruit Orah, and can also be further used as a new germplasm resource for breeding program.