国庆1号温州蜜柑珠心胚苗培育及四倍体发掘

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

【目的】温州蜜柑一般表现为雄性不育、果实无核,收集国庆1号温州蜜柑(Citrus unshiu Marc.,简称G1)种子,用于培育珠心胚苗并发掘多倍体新种质,为其提纯复壮和珠心胚变异育种提供基础材料。【方法】从G1成熟果实剥取种子,浸泡消毒后离体播种至播种培养基;待幼苗长至有3~5枚真叶时,采用流式细胞仪分析倍性和SSR分子标记鉴定遗传来源。【结果】从约12 500个果实中剥取获得106粒种子,离体播种后41粒种子萌发,经培养获得62株实生幼苗;对所有幼苗进行倍性检测,获得四倍体1株,其余61株均为二倍体;用4对多态性SSR引物对获得的四倍体和随机筛选的38株二倍体植株进行分子鉴定,表明发掘的四倍体和35株二倍体的带型与亲本G1完全一致,推测其均为珠心胚来源,且四倍体为G1珠心细胞自然加倍而形成;其余3株二倍体在部分SSR位点与G1带型不一致,推测由G1与周边其他品种杂交而来。【结论】这些珠心胚苗二倍体及四倍体种质为国庆1号温州蜜柑提纯复壮、珠心胚变异育种及相关基础研究提供了珍贵的种质资源。

【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 phenomena. 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 7-8leaves, 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 about12 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.【Conclusion】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.