Production of nucellar seedlings and exploration of tetraploid from Satsuma mandarin Guoqing No. 1

Abstract:【Objective】Satsuma mandarin (Citrus unshiu Marc.) is a polyembryonic cultivar with typical cytoplasmic male sterile (CMS) trait, and it is widely grown in China because of its seedlessness and excellent fruit quality. Due to the CMS characteristic, the Satsuma mandarin has been used as female parent in sexual hybridization and contributed to the production of many excellent seedless cultivars such as Kiyomi tangor, Shiranui mandarin, Harumi mandarin and Setoka tangor. Satsuma mandarin Guoqing No. 1 (hereafter abbreviated as G1) is an early maturing variety selected from Satsuma mandarin Kamei via exploring spontaneous bud mutation by Huazhong Agricultural University. G1 fruit theoretically appears seedless, but it may produce a small number of seeds when it encounters high temperature during flowering period, indicating that its fertility may be partially restored under high temperature conditions. Citrus seedling production is usually based on asexual propagation, like grafting, which can keep the excellent characteristics of the original cultivar. However, long-term asexual propagation might result in the infection of viruses, viroids and bacterial diseases in citrus trees, leading to a certain degree of variety degeneration, and gradually show the decline of vigor, yield, quality and other phe-nomena. It is urgent to carry out purification, rejuvenation and variety improvement. Most virus diseases cannot be transmitted by seed, and citrus seedlings regenerated from the nucellar embryo can eliminate most virus diseases and usually show more vigorous growth than their parents, so as to achieve the purpose of purification and rejuvenation. In addition, selection breeding based on the variation of nucellar seedling is also an important approach in citrus breeding, and many new citrus varieties with different ripening stages, fruit types and peel colors were selected. The G1 trees were grafted on Poncirus trifoliata and planted in the citrus breeding orchard in Huazhong Agricultural University for more than ten years. In January 2021, due to the low temperature and freezing injury, G1 fruits were frozen and inedible, thus all the fruits were picked for seed collection. Because polyembryonic citrus has variable number of embryos and the nucellar embryos are usually more vigorous than zygotic embryo, mature seeds of G1 may regenerate more nucellar seedlings, which can be used as the valuable germplasm for tetraploid exploration. Here, we used G1 as the material, explored their nucellar seedlings for producing virus-free G1 and screened tetraploid germplasm for interploid breeding. It can be a model for the utilization of other Satsuma mandarin cultivars.【Methods】Following G1 fruits being picked, seeds were extracted and divided into developed and undeveloped types. By stripping the exopleura of all seeds, under aseptic conditions, the seeds were soaked and disinfected in 3% (ρ) NaClO solution for 15 min. In order to make the seeds germinate neatly, the endopleura was removed and then the seeds were sown in vitro on the germination medium. When the seedlings grew with 3-5 leaves, using flow cytometry and shoot tip chromosome counting to determine their ploidy level. Using a known diploid as a control, the fluorescence intensity of its leaves was set at 50, and then the peak at 100 indicated that the sample was tetraploid. In addition, because the basic chromosome number of citrus was 9, thus 18 and 36 chromosomes meant diploid and tetraploid, respectively. Whereafter, the seedlings were transplanted into plastic pots filled with nutrient soil and placed in a growth chamber. When the seedlings grew with 7- 8 leaves, they were transferred to the greenhouse, and normal fertilizer and water management were guaranteed during this period. The genetic origin of the seedlings obtained in this study were further analyzed by automatic capillary electrophoresis system using four simple sequence repeat (SSR) markers, which were screened from the reported work.【Results】A total of 106 seeds were obtained from about 12 500 G1 fruits, with a seed setting rate of 0.85%. Among them, the numbers of developed and undeveloped seeds were 80 and 26, respectively, and the former was about three times as many as the latter. All seeds were sown in vitro on the germination medium. Due to the contamination of some developed seeds and the failure of germination of undeveloped seeds, only 41 developed seeds finally germinated, with a germination rate being 38.7%. From the germinated seeds, 62 seedlings were obtained, with an average of 1.5 plants regenerated per seed. By analyzing the ploidy level of 62 seedlings, one tetraploid plant was obtained, with the tetraploid occurrence rate of 1.61%. And the remaining 61 seedlings was proven to be diploid seedlings. These results determined by flow cytometry was also confirmed by roottip chromosome counting. Four SSR markers were used to analyze the genetic origins of the tetraploid and 38 randomly selected diploid seedlings. The result showed that the bands of the tetraploid seedlings and 35 diploid seedlings were identical to that of G1, indicating that the tetraploid seedling might originate from the chromosome doubling of nucellar cells of G1 and all 35 diploid seedlings were the nucellar seedlings of G1. And the remaining three diploids showed some bands that G1 did not possess, indicating they might originate from the sexual hybridization of G1 with unknown pollen parent. In total, the seedlings derived from the nucellar embryo accounted for 92.11% of the 38 randomly selected diploid seedlings, indicating that nucellar embryos might be more vigorous than zygotic embryo.【Conclu-sion】From the results above, it showed that in the seeds of polyembryonic citrus, certain proportion survival seeds was derived from zygotic embryos, and SSR molecular marker analysis can be an efficient tool to distinguish between nucellar and zygotic seedlings. The diploid and tetraploid nucellar seedlings obtained here may hold great potential for genetic improvement, polyploid breeding and related fundamental researches of Satsuma mandarin Guoqing No. 1