柑橘单倍体—二倍体原生质体融合及其再生愈伤的倍性分析

发布时间：2018-08-27 12:54

【摘要】：柑橘是我国南方乃至世界上最重要的经济果树之一。虽然我国柑橘品种多样性丰富,但地方良种普遍多籽,影响了其商品价值,因而缺乏市场竞争力。原生质体融合能够较好地克服柑橘中普遍存在的多胚、雌/雄性器官败育、远缘杂交不亲和等生殖障碍,因而是创造新型种质并培育优良接穗及砧木的有效手段。目前通过细胞融合创制新种质应用较多的策略是用二倍体愈伤原生质体与二倍体叶肉原生质体对称融合的模式,用这种方法获得的四倍体植株作为亲本与二倍体进行倍性杂交培育三倍体,育种年限较长;相比之下,通过单倍体与二倍体融合能够直接获得三倍体,可大大缩短育种年限,提高育种效率。本研究拟借助前人建立的柑橘原生质体电融合技术体系,旨在通过单倍体-二倍体体细胞融合直接培育三倍体;另一方面通过常规的二倍体愈伤原生质体与二倍体叶肉原生质体的融合模式创造四倍体;此外,还对‘早金’甜橙单倍体和二倍体愈伤原生质体直径进行了统计比较。主要研究结果如下:1、单倍体愈伤原生质体与二倍体叶肉原生质体融合。以‘早金’甜橙单倍体为愈伤亲本,分别与‘鸡尾’葡萄柚、‘城固冰糖’橘、‘默科特’橘橙为叶肉亲本进行融合实验,其中‘早金’单倍体+‘默科特’橘橙再生出细胞团,而当双亲均为愈伤亲本时,成功再生出白色愈伤组织。通过流式细胞仪检测倍性,结果表明:‘早金’甜橙单倍体+‘默科特’橘橙再生愈伤的倍性较复杂,是一个混倍体,包含单倍体、二倍体、三倍体和四倍体。2、二倍体愈伤原生质体与二倍体叶肉原生质体融合创造四倍体体细胞杂种。以‘默科特’橘橙为愈伤亲本,分别与‘鸡尾’葡萄柚、‘桃叶’橙、‘南充本地’甜橙叶肉原生质体进行细胞融合,其中,‘默科特’橘橙+‘鸡尾’葡萄柚再生出愈伤组织。通过流式细胞仪对其倍性进行检测,结果表明,再生愈伤组织的倍性为二倍体与四倍体的混倍体。3、‘早金’甜橙单倍体与其二倍体的愈伤组织原生质体大小比较。分别统计了200个‘早金’甜橙二倍体及其单倍体的原生质体直径,结果表明:‘早金’甜橙二倍体悬浮系原生质体直径平均值为16.3μm,标准偏差为2.6;单倍体悬浮系原生质体直径平均值为14.6μm,标准偏差为2.8,表明二倍体体积较单倍体大且细胞长势更趋于一致。
[Abstract]:Citrus is one of the most important economic fruit trees in the south of China and even in the world. Although the variety of citrus varieties in China is rich, the local improved varieties are generally multi-seeded, which affects their commodity value and therefore lacks market competitiveness. Protoplast fusion is an effective method to create new germplasm and cultivate excellent scions and rootstocks because it can overcome the reproductive obstacles such as polyembryogenesis female / male organ abortion distant hybridization incompatibility and so on. At present, the strategy of using diploid callus protoplast and diploid mesophyll protoplast symmetrically fused with diploid mesophyll protoplasts is widely used to create new germplasm by cell fusion. The tetraploid plants obtained by this method were used as parents to cross with diploid to breed triploid, and the breeding time was longer, in contrast, triploid could be obtained directly by haploid and diploid fusion, and the breeding life could be shortened greatly. Improve breeding efficiency. The aim of this study was to cultivate triploid directly by haploid-diploid somatic fusion with the help of protoplast electrofusion system established by predecessors. On the other hand, tetraploid was created by conventional fusion model between diploid callus protoplasts and diploid mesophyll protoplasts, and the diploid and haploid diploid callus protoplasts were statistically compared. The main results were as follows: 1. The fusion of haploid callus protoplasts with diploid mesophyll protoplasts. The haploid of 'Zaojin' sweet orange was used as the callus parent, and the fusion experiment was carried out with the 'chicken tail' grapefruit 'Cenggu' Orange 'Orange' Merkett 'orange as the parent of mesophyll. 'Zaojin' haploid 'Merkett' orange regenerated cell mass, and white callus was successfully regenerated when both parents were callus parents. The ploidy was detected by flow cytometry. The results showed that the ploidy of regenerated calli of the haploid 'Merkett' orange of 'Zaojin' sweet orange was more complex, and it was a mixed ploidy, including haploid and diploid. Diploid callus protoplasts fused with diploid mesophyll protoplasts to create tetraploid somatic hybrids. The callus of 'Mercutt' orange was fused with the mesophyll protoplast of 'chicken tail' grapefruit 'peach leaf' orange 'Nanchong' sweet orange. Morcott, orange, chicken tail, grapefruit regenerate callus. The ploidy of regenerated callus was detected by flow cytometry. The results showed that the ploidy of regenerated callus was mixed ploidy of diploid and tetraploid. The size of protoplast of haploid and diploid callus was compared with that of haploid and diploid of sweet orange. The protoplast diploid and haploid diameter of 200 'Zaojin' sweet orange were calculated. The results showed that the average diameter of protoplast of the diploid suspension line was 16.3 渭 m, the standard deviation was 2.6 渭 m, and the average diameter of protoplast of haploid suspension was 14.6 渭 m, the standard deviation was 2.8, which indicated that the diploid was larger than haploid. Cell growth tends to be more consistent.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S666

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