【摘要】：柑橘是我國(guó)南方乃至世界上最重要的經(jīng)濟(jì)果樹(shù)之一。雖然我國(guó)柑橘品種多樣性豐富,但地方良種普遍多籽,影響了其商品價(jià)值,因而缺乏市場(chǎng)競(jìng)爭(zhēng)力。原生質(zhì)體融合能夠較好地克服柑橘中普遍存在的多胚、雌/雄性器官敗育、遠(yuǎn)緣雜交不親和等生殖障礙,因而是創(chuàng)造新型種質(zhì)并培育優(yōu)良接穗及砧木的有效手段。目前通過(guò)細(xì)胞融合創(chuàng)制新種質(zhì)應(yīng)用較多的策略是用二倍體愈傷原生質(zhì)體與二倍體葉肉原生質(zhì)體對(duì)稱融合的模式,用這種方法獲得的四倍體植株作為親本與二倍體進(jìn)行倍性雜交培育三倍體,育種年限較長(zhǎng);相比之下,通過(guò)單倍體與二倍體融合能夠直接獲得三倍體,可大大縮短育種年限,提高育種效率。本研究擬借助前人建立的柑橘原生質(zhì)體電融合技術(shù)體系,旨在通過(guò)單倍體-二倍體體細(xì)胞融合直接培育三倍體;另一方面通過(guò)常規(guī)的二倍體愈傷原生質(zhì)體與二倍體葉肉原生質(zhì)體的融合模式創(chuàng)造四倍體;此外,還對(duì)‘早金’甜橙單倍體和二倍體愈傷原生質(zhì)體直徑進(jìn)行了統(tǒng)計(jì)比較。主要研究結(jié)果如下:1、單倍體愈傷原生質(zhì)體與二倍體葉肉原生質(zhì)體融合。以‘早金’甜橙單倍體為愈傷親本,分別與‘雞尾’葡萄柚、‘城固冰糖’橘、‘默科特’橘橙為葉肉親本進(jìn)行融合實(shí)驗(yàn),其中‘早金’單倍體+‘默科特’橘橙再生出細(xì)胞團(tuán),而當(dāng)雙親均為愈傷親本時(shí),成功再生出白色愈傷組織。通過(guò)流式細(xì)胞儀檢測(cè)倍性,結(jié)果表明:‘早金’甜橙單倍體+‘默科特’橘橙再生愈傷的倍性較復(fù)雜,是一個(gè)混倍體,包含單倍體、二倍體、三倍體和四倍體。2、二倍體愈傷原生質(zhì)體與二倍體葉肉原生質(zhì)體融合創(chuàng)造四倍體體細(xì)胞雜種。以‘默科特’橘橙為愈傷親本,分別與‘雞尾’葡萄柚、‘桃葉’橙、‘南充本地’甜橙葉肉原生質(zhì)體進(jìn)行細(xì)胞融合,其中,‘默科特’橘橙+‘雞尾’葡萄柚再生出愈傷組織。通過(guò)流式細(xì)胞儀對(duì)其倍性進(jìn)行檢測(cè),結(jié)果表明,再生愈傷組織的倍性為二倍體與四倍體的混倍體。3、‘早金’甜橙單倍體與其二倍體的愈傷組織原生質(zhì)體大小比較。分別統(tǒng)計(jì)了200個(gè)‘早金’甜橙二倍體及其單倍體的原生質(zhì)體直徑,結(jié)果表明:‘早金’甜橙二倍體懸浮系原生質(zhì)體直徑平均值為16.3μm,標(biāo)準(zhǔn)偏差為2.6;單倍體懸浮系原生質(zhì)體直徑平均值為14.6μm,標(biāo)準(zhǔn)偏差為2.8,表明二倍體體積較單倍體大且細(xì)胞長(zhǎng)勢(shì)更趨于一致。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S666

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