【作者】 张敏；

【导师】 邓秀新；

【作者基本信息】 华中农业大学 ， 果树学， 2006， 博士

【摘要】 芽变选种是柑橘育种的一种重要方法。柑橘果实扇形嵌合体和嫁接嵌合体隶属于广义的芽变范畴，可以用其作为柑橘品种改良的资源。本实验旨在研究自然界中柑橘果实扇形嵌合体发生的频率，通过组织培养技术分离纯化其中的变异，为选育新品种积累候选材料；并对2组嫁接嵌合体进行评价，探明了它们的嵌合模式。主要研究结果如下：1．2002—2006连续5年，对湖北秭归的柑橘果实嵌合体发生频率进行了调查，调查品种有‘纽荷尔’脐橙(Citrus sinensis Osbeck)，‘长红’脐橙(C．sinensis Osbeck)，‘红肉’脐橙(C．sinensis Osbeck)，脐血橙(C．sinensis Osbeck)，桠柑(C．reticulata Blanco)，‘卡特’夏橙(C．sinensis Osbeck)。2003—2006连续4年，对江西寻乌的柑橘果实嵌合体发生频率进行了调查，调查品种有‘纽荷尔’脐橙，‘萘维林娜’脐橙(C．sinensis Osbeck)，‘朋娜’脐橙(C．sinensis Osbeck)。2004—2006连续3年，对湖北兴山的柑橘果实嵌合体发生频率进行了调查，调查品种有‘纽荷尔’脐橙，‘锦橙’(C．sinensis Osbeck)。调查结果表明，9个品种中果实扇形嵌合体的发生频率为0.02％到3.97％。其中‘红肉’脐橙的嵌合体发生频率为最高(3.97％)，‘桠柑’最低(0.02％)。果实扇形嵌合体的发生频率受品种基因型，环境条件和栽培管理方式等因素的影响。嵌合体以果皮颜色的变异最普遍，类型有绿扇，深红扇，深橙扇，黄扇，棕扇，粉红扇；还有一种常见的变异是果皮凸出或凹陷。2．收集了上述9个品种的180个嵌合体果实，取其败育胚珠或种子进行组织培养。共接种5000颗败育胚珠和30粒饱满种子，再生了2078棵苗。其中有1508棵来源于嵌合部分对应的种子或败育胚珠，另外的570棵来源于正常部分对应的种子或败育胚珠。从50个果皮凸出类型的果实扇形嵌合体再生的600棵苗中，经流式细胞仪检测到有‘纽荷尔’脐橙四倍体3棵，‘萘维林娜’脐橙四倍体4棵和‘纽荷尔’脐橙混倍体植株2棵。目前，已从嵌合体分离再生的小苗中选择了1000棵移入温室和大田。这些植株为我国柑橘新品种的选育提供了材料。3．收集到两个柑橘嫁接嵌合体，暂定名为‘早红’脐橙(Citrus sinensis Osbeck+Citrus unshiu Marc．)和‘红肉桃叶橙’(Citrus sinensis Osbeck+Citrus unshiu Marc．)。‘早红’脐橙产生于‘罗伯逊’脐橙(C．sinensis Osbeck)高接于温州蜜柑(C．unshiu Marc．)的树上，‘红肉桃叶橙’产生于‘桃叶橙’(C．sinensis Osbeck)高接于温州蜜柑的树上。采用形态学，细胞学，孢粉学，生理生化和分子标记等手段对两组嫁接嵌合体及其亲本进行了评价。发现‘早红’脐橙和‘红肉桃叶橙’中由L1层细胞决定的性状，如汁囊，气孔长度等都与温州蜜柑相似，而叶形指数，果实形状，脐，花粉的形态和育性，种子的数量，果皮的颜色和气味等由L2层细胞决定的性状分别与‘罗伯逊’脐橙和‘桃叶橙’相同。高效液相色谱(HPLC)分析果肉所含类胡萝卜素表明，‘早红’脐橙，‘红肉桃叶橙’和温州密柑的果肉具有相同的类胡萝卜素指纹图谱，主要类胡萝卜素为β—隐黄质(β-cryptoxanthin)。利用引物TAA15，TAA27和引物SPCC1分别进行SSR和cpSSR分析表明，‘早红’脐橙和‘红肉桃叶橙’的核基因组和叶绿体基因组分别同时具有‘罗伯逊’脐橙和温州蜜柑，‘桃叶橙’和温州蜜柑所有的带形。根据这些结果，可以认为‘早红’脐橙是由‘罗伯逊’脐橙和温州蜜柑构成的周缘嵌合体，其L1层来源于温州蜜柑，L2／L3层来源于‘罗伯逊’脐橙；而‘红肉桃叶橙’是由‘桃叶橙’和温州蜜柑构成的周缘嵌合体，其L1层来源于温州蜜柑，L2／L3层来源于‘桃叶橙’。两个嫁接嵌合体分别具备了各自亲本的有利性状，而且性状稳定，有可能成为新的鲜食品种。4．采用引物TAA27对‘早红’脐橙败育胚珠再生苗和‘红肉桃叶橙’的实生苗进行SSR分析，结果表明，‘早红’脐橙败育胚珠再生苗的核基因组和其L2亲本‘罗伯逊’脐橙具有相同的带形，而‘红肉桃叶橙’实生苗的核基因组和其L2亲本‘桃叶橙’具有相同的带形。验证了珠心胚由L2层细胞发育而来。更多还原

【Abstract】 Selection of bud mutation is one of the most important methods for Citrus breeding. Generally, citrus fruit sector chimeras and graft chimeras were considered to belong to bud mutation and could be used as genetic resources for cultivar improvement. In the present research, a 5-year investigation was carried out to study the frequency of citrus fruit sector chimeras. And some chimeras were separated and purified by in vitro technique as the candidates for selecting new cultivars. Two graft chimeras were genetically evaluated, their layer constructions were studied. The main results are as follows:1. During the years of 2002-2006, frequency of citrus fruit sector chimeras were investigated in 6 citrus cultivars in an orchard of Zigui county, Hubei province. The cultivars included’Newhall’ navel orange(Citrus sinensis Osbeck),’Changhong’ navel orange(C. sinensis Osbeck),’Cara Cara’ navel orange(C. sinensis Osbeck), navel blood orange(C. sinensis Osbeck),’Carter’ orange(C. sinensis Osbeck) and’Ponkan’(C. reticulata Blanco). During the years of 2003-2006, same work was conducted in 3 citrus cultivars in the orchards of Xunwu county, Jiangxi province. The cultivars included’Newhall’ navel orange,’Navelina’ navel orange(C. sinensis Osbeck) and’Bonanza’ navel orange(C. sinensis Osbeck). During the years of 2004-2006, same investigation of 2 citrus cultivars in the orchards of Xingshan county, Hubei province was carried out. The cultivars included’Newhall’ navel orange and’Jincheng’ sweet orange(C. sinensis Osbeck). The results indicated that the frequency of the fruit sector chimeras of nine citrus cultivars’ varied from 0.02% to 3.97%. The frequency in’Cara Cara’ navel orange(3.97%) is the highest and that in’Ponkan’ is the lowest(0.02%). Frequency varied with genotypes, environment and cultivation methods. Different types of sector mutations were observed in chimeric citrus fruits, out of them alterations of sector rind color were predominatedly observed, as shown by brown, green, yellow, dark red, dark orange, pink sectors. Another common mutation was the thicker or thinner rind sector than the normal.2. A total of 180 chimeric fruits were collected from 9 cultivars described above. 30 seeds and 5000 aborted ovules from the fruit chimeras were cultured and 2078 plantlets were recovered from them. Among them, 1508 plantlets were recovered from the seeds and aborted ovules beneath the chimeric part of each fruit and 570 plantlets were from the normal sectors. Among 600 plantlets which were recovered from 50 fruit chimeras with thicker rind, 3 tetraploid plants of’Newhall’ navel orange, 4 of’Navelina’ navel orange and 2 mixploid plants from’Newhall’ navel orange were obtained after flow cytometry confirmation. In total, 1000 plantlets were transferred to the greenhouse and field and which will be the candidates for new cultivars selections.3. Two new strains of citrus from graft chimeras were collected as were named’Zaohong’ navel orange(Citrus sinensis Osbeck+Citrus unshiu Marc.) and’Hongrou Taoye’ sweet orange(Citrus sinensis Osbeck+Citrus unshiu Marc.) respectively.’Zaohong’ navel orange arose at the junction area where a’Robertson’ navel orange(C. sinensis Osbeck) scion was top-worked onto Satsuma mandarin(C. unshiu Marc).’Hongrou Taoye’ sweet orange was from the junction where a’Taoye’ sweet orange(C. sinensis Osbeck) scion was top-worked onto Satsuma mandarin. These two graft chimeras and their donors were evaluated by morphological, cytological, biochemical and molecular markers. Characters of the’Zaohong’ navel orange and’Hongrou Taoye’ sweet orange, such as juice sacs of fruits, stoma length, which were determined by L1 cell layer, were similar to Satsuma mandarin. While other traits, including leaf index, fruit shape, navel, pollen morphology and fertility, the number of seeds, the color and aroma of the rind determined by L2 cell layer, were the same as’Robertson’ navel orange and’Taoye’ sweet orange respectively. The carotenoid extract of the flesh of’Zaohong’ navel orange,’Hongrou Taoye’ sweet orange and their donor plants was studied by using high performance liquid chromatography(HPLC). The results indicated that’Zaohong’ navel orange and’Hongrou Taoye’ sweet orange had the same carotenoids profile of Satsuma mandarin, in whichβ-cryptoxanthin accumulated predominantly in the juice sacs in mature fruit. SSR and cpSSR analysis showed that both nuclear and chloroplast genomes of’Zaohong’ navel orange and’Hongrou Taoye’ sweet orange were composed from both of their donor plants respectively. Based on these facts,’Zaohong’ navel orange was a periclinal chimera consisting of L1 derived from Satsuma mandarin and L2/L3 from’Robertson’ navel orange.’Hongrou Taoye’ sweet orange was a periclinal chimera consisting of L1 derived from Satsuma mandarin and L2/L3 from’Taoye’ sweet orange. They combined the elite traits of their donor plants and genetically stable, they could be commercially acceptable chimeric cultivars for the fresh market of citrus.4. SSR primer TAA27 was used to verify the seedlings derived from the nucellar embryo of’Zaohong’ navel orange and’Hongrou Taoye’ sweet orange. The results showed that the band model of the plants were the same as the L2 donors. And it is concluded that the nucellar embryo in citrus is derived from the cell in L2 layer.更多还原