Identification and characterization of tetraploids from seedlings of Citrus junos ‘Quzhou xiangcheng’

Abstract: 【Objective】In this study, we aimed to obtain a batch of tetraploid germplasm from the seed-lings of C. junos‘Quzhou xiangcheng’, and analyze the characteristics of tetraploid seedlings of C. ju-nos‘Quzhou xiangcheng’by evaluating morphology, stomata and leaf anatomical structure to provide a reference for screenin polyploid germplasm.【Methods】The seeds of C. junos‘Quzhou xiangcheng’were collected from the ripe fruits in autumn, and sown in the soil germination. Four hundred seedlings of 4 months old were firstly used for selecting tetraploids according to the morphological characteristics of leaf size, stem thickness, root thickness, and so on. And then the flow cytometry and the chromo-some compression method of root tip were employed to further identify the chromosome ploidy of the cells of the seedlings primarily screened based on the morphological characteristics. The plant height,leaf width, root diameter and leaf length of the seedlings of both tetraploid and diploid were deter-mined. Meanwhile the oil gland density of the epidermis of the plants was also detected through count-ing the number of the oil gland within 1 cm2 with a light irradiate Additionally, the stoma density and size on the leaf epidermis were determined with scanning electron microscope. Moreover, upper epider-mis, under epidermis, palisade tissue, sponge tissue, and total leaf thickness of plants were measured us-ing Digimizer 4 software, and then the ratio of palisade cell and spongy parenchyma were caculated.【Results】According to preliminary morphological observations, a total of 13 suspected tetraploid seed-lings were screened from 400 seedlings. A peak at 50 of elative content of DNA was observed in the plants of the diploid C. junos‘Quzhou xiangcheng’, and a peak at 100 in the plants of the tetraploid C. junos‘Quzhou xiangcheng’by flow cytometry. The number of chromosomes of the diploid of C. ju-nos‘Quzhou xiangcheng’was 18, and the number of chromosomes of the tetraploid was 36. 3 tetra-ploid plants were finally identified and the natural occurrence rate of the tetraploid was 0.75%. Charac-teristic research showed that the tetraploid plant had a thicker stem, lower height compared with the dip-loid plants. The tetraploid plants had a thicker main root a, fewer lateral root branches and darker and larger leaves. The average lengths of the leaves of the diploid and tetraploid plants were 7.27 cm and 4.18 cm, and the average widths were 1.69 cm and 2.23 cm, respectively. The oil gland density of the tetraploid plants was significantly smaller (only 50% of that of diploid) than that of the diploid plants. The length and width of the stomata of the leaves of the tetraploid plants was significantly larger than that of the diploid plants. The main leaf veins of the tetraploid leaf was more prominent than the dip-loid. The xylem diameter of the tetraploid was significantly larger than that of the diploid xylem and the number of xylem cells was about twice as much as that of the diploid. The area of parenchyma cells of the tetraploid was obviously larger than that of diploid. The leaf thickness, upper epidermis, under epi-dermis, palisade tissue and sponge tissue thickness of tetraploid were significantly larger than those of the diploid. In addition, compared with diploids, the ratio of palisade cell and spongy parenchyma and leaf structure compactness of the tetraploid were extremely significantly increased. The leaf structure porosity of the body of the tetraploid was significantly less than that of the diploid.【Conclusion】The combination of preliminary morphological screening and ploidy identification technology was an effec-tive way for the selection of tetraploid from the seedlings of C. junos‘Quzhou xiangcheng’. There were significant difference of several morphological characteristics and anatomical structures between the tetraploid and diploid plant in C. junos‘Quzhou xiangcheng’.