甜橙与酸橙体细胞杂种核质组成鉴定(英文)

采用流式细胞术(flow cytometry, FCM)、简单重复序列(simple sequence repeat, SSR)和酶切扩增多型性序列(cleaved amplified polymorphic sequence, CAPS)等技术分析酸橙(Citrus aurantium L. )叶肉原生质体和甜橙(C. sinenis Osbeck cv. Shamouti)胚性愈伤组织原生质体电融合再生的体细胞杂种。FCM研究结果表明,所有的体细胞杂种植株荧光强度是二倍体对照的2倍,说明所分析的植株为四倍体。用SSR和CAPS分析了体细胞杂种的核质遗传组成,在试验的4对SSR引物中,有2对能区分开融合亲本。在2对引物中,体细胞杂种植株包含双亲的全部特异带,表明它们为异核杂种。通用引物扩增结合限制性内切酶酶切能鉴别融合亲本,在具有多型性的引物/酶组合中,所有体细胞杂种的线粒体和叶绿体DNA带型与胚性亲本(甜橙)完全一样。结果表明体细胞杂种核基因组来自双亲,而胞质基因组来自悬浮系亲本。讨论了所用技术的特点、柑橘四倍体体细胞杂种核质遗传规律及本组合体细胞杂种的应用。     

甜橙与酸橙体细胞杂种核质组成鉴定

采用流式细胞术(flow cytometry,FCM)、简单重复序列(simple sequence repeat,SSR)和酶切扩增多型性序列(cleaved amplifiedpolymorphic sequence,CAPS)等技术分析酸橙(Citrus aurantium L.)叶肉原生质体和甜橙(C.sinenis Osbeck cv.Shamouti)胚性愈伤组织原生质体电融合再生的体细胞杂种.FCM研究结果表明,所有的体细胞杂种植株荧光强度是二倍体对照的2倍,说明所分析的植株为四倍体.用SSR和CAPS分析了体细胞杂种的核质遗传组成,在试验的4对SSR引物中,有2对能区分开融合亲本.在2对引物中,体细胞杂种植株包含双亲的全部特异带,表明它们为异核杂种.通用引物扩增结合限制性内切酶酶切能鉴别融合亲本,在具有多型性的引物/酶组合中,所有体细胞杂种的线粒体和叶绿体DNA带型与胚性亲本(甜橙)完全一样.结果表明体细胞杂种核基因组来自双亲,而胞质基因组来自悬浮系亲本.讨论了所用技术的特点、柑橘四倍体体细胞杂种核质遗传规律及本组合体细胞杂种的应用.     

Hormonal and Nutritional Changes in the Flavedo Regulating Rind Color Development in Sweet Orange [Citrus sinensis (L.) Osb.]

The objective of this research was to determine the changes in the levels of endogenous gibberellins GA₁ and GA₄, abscisic acid (ABA), and ethylene during fruit coloring of on-tree fruits of sweet orange. The time course of carbohydrates and nitrogen content in the flavedo prior to fruit color break and during peel ripening were also studied. To identify nutritional and hormonal changes in the fruit, 45?days before fruit color break the peduncles of 15?C30 fruits per tree of ??Washington?? navel, ??Navelate,?? and ??Valencia Delta Seedless?? sweet orange, located in single-fruited shoots, were girdled to intercept phloem transport. A set of 15?C30 fruits per tree remained intact on the peduncle for control. Girdling significantly delayed fruit coloration for more than 2?months; the delay paralleled higher GA₁ and GA₄ concentrations in the flavedo and retarded the rise of ABA concentration prior to color break. Girdling also reduced carbohydrate concentrations and increased N concentrations in the flavedo compared to control fruits; no ethylene production was detected. Therefore, in sweet orange, fruit changes color by reducing active gibberellin concentrations in the flavedo, which are involved in regulating sugars and ABA accumulation and in reducing N fraction concentration as rind color develops. This was demonstrated in vivo without removing the fruit from the tree. Comparable results were obtained with experiments carried out over four consecutive years in two countries (Spain and Uruguay).     

Iron Chlorosis in Grafted Sweet Orange (Citrus sinensis L.) Plants: Physiological and Biochemical Responses

Fe deficiency was imposed in Citrus sinensis L. cultivars Valencia and New Hall grafted on C. aurantium and Swingle citrumelo rootstocks by the absence of Fe (-Fe) or by the presence of bicarbonate in the Hoagland nutrient solution. In Fe-deprived leaves total and active Fe concentration, and peroxidase and catalase activities were decreased while the ratios carotenoids/chlorophylls, P/Fe, and K/Ca were increased. Fe(III) chelate reductase activity was induced in (-Fe)-treated roots whereas it was depressed in bicarbonate-treated roots.     

Genome Wide Characterization of Short Tandem Repeat Markers in Sweet Orange (Citrus sinensis)

Sweet orange (Citrus sinensis) is one of the major cultivated and most-consumed citrus species. With the goal of enhancing the genomic resources in citrus, we surveyed, developed and characterized microsatellite markers in the ≈347 Mb sequence assembly of the sweet orange genome. A total of 50,846 SSRs were identified with a frequency of 146.4 SSRs/Mbp. Dinucleotide repeats are the most frequent repeat class and the highest density of SSRs was found in chromosome 4. SSRs are non-randomly distributed in the genome and most of the SSRs (62.02%) are located in the intergenic regions. We found that AT-rich SSRs are more frequent than GC-rich SSRs. A total number of 21,248 SSR primers were successfully developed, which represents 89 SSR markers per Mb of the genome. A subset of 950 developed SSR primer pairs were synthesized and tested by wet lab experiments on a set of 16 citrus accessions. In total we identified 534 (56.21%) polymorphic SSR markers that will be useful in citrus improvement. The number of amplified alleles ranges from 2 to 12 with an average of 4 alleles per marker and an average PIC value of 0.75. The newly developed sweet orange primer sequences, their in silico PCR products, exact position in the genome assembly and putative function are made publicly available. We present the largest number of SSR markers ever developed for a citrus species. Almost two thirds of the markers are transferable to 16 citrus relatives and may be used for constructing a high density linkage map. In addition, they are valuable for marker-assisted selection studies, population structure analyses and comparative genomic studies of C. sinensis with other citrus related species. Altogether, these markers provide a significant contribution to the citrus research community.     

哈姆林甜橙与粗柠檬体细胞杂种的育性

对异源四倍体柑桔体细胞杂种“哈姆林甜橙+粗柠檬”及其亲本的花粉活力、花器官发育、花器官形态发生与花粉母细胞减数分裂四分体阶段进行了观测和统计.结果发现“哈姆林甜橙+粗柠檬”的花粉染色活力、萌发率、每花药中花粉粒数均居于其双亲之间,花器官发育及其形态发生具有双亲的特点.但小花粉及花粉母细胞减数分裂过程中形成的不正常四分体比率远远高于其双亲.以体细胞杂种“哈姆林甜橙+粗柠檬”为花粉亲本,与二倍体单胚类型宜昌橙与华农本地早的有性后代杂交,获得了110棵有性后代植株,其中三倍体82棵,二倍体和其它倍性的植株28棵.     

甜高梁离体再生体系的建立和组织结构变化的观察

以甜高粱成熟种子为外植体,调节不同生长调节物质配比建立甜高梁离体再生体系。结果表明在MS＋2．5mg．L^-12,4．D＋0．3mg·L^-1KT培养基上愈伤组织的诱导率可达77．26％;比较不同浓度6-BA或TDZ与NAA配合诱导愈伤组织分化和苗形成的情况,TDZ的作用优于6-BA。观察培养组织的结构变化发现,甜高粱离体再生过程中除了体细胞胚发生途径之外,还伴随有器官发生途径。     

电融合获得用于选择抗CTV砧木的酸橙与甜橙体细胞杂种植株

在已知参数条件下,通过电场诱导酸橙(Citrus aurantium L.)叶肉原生质体和沙漠蒂甜橙(C.sinensis Osbeck cv.Shamouti)的胚性愈伤组织原生质体融合,融合产物经培养再生出40棵植株.染色体检查表明所得到的植株具有36条染色体,为四倍体植株.再生植株具有翼叶,叶片厚,表现出多倍体的特征.采用2个10-碱基随机引物鉴别再生植株的杂种特性.在2个引物的扩增带型中,再生植株的随机扩增带图里出现了融合亲本的特征带.对再生植株染色体计数和RAPD分析的结果表明它们是酸橙和甜橙种间异源四倍体体细胞杂种植株.这些体细胞杂种植株的获得为选择具有酸橙优良性状、同时抗CTV的新型砧木提供了好的试材.     

Growth Responses of Sweet Orange Seedlings to Shoot and Root Pruning

Following combined shoot- and root-pruning treatments the growthof Citrus sinensis (L.) Osbeck seedlings was analysed into responsesto post-pruning size and to the part pruned. The treatmentswere shoots pruned by removing O, , or of their estimated weightin all combinations with roots pruned by removing O, , or oftheir estimated weight. Responses were measured a year laterand are discussed in terms of the mechanisms controlling increment.Total increment was linearly related to initial pruned weightboth within and between treatments and there were no interactionsbetween shoot and root pruning in increment of leaf, new stem,old stem, thorn, fibre-root, or tap-root. Root pruning, pergramme of pruned material, reduced increment more than did shootpruning. With increasing shoot pruning the ratio of new stem to old stemincreased while with increasing root pruning the ratio of fibre-rootto tap-root increased. Following shoot pruning the new-stemincrement was disproportionately large while following rootpruning the increment of top growth was proportionately largerthan that of roots. The results suggest that pruning citrus trees to a constantsize at planting could lead to a uniform tree size in the subsequentorchard but that maximum growth would occur on un-pruned trees.     

Phytohormone Changes and Carbohydrate Status in Sweet Orange Fruit from Huanglongbing-infected Trees

Huanglongbing (HLB) infection alters citrus fruit growth and development, resulting in small, misshapen, and poorly colored fruit containing aborted or partially developed seeds. Typically, symptomatic fruit have delayed maturation and abscise prematurely. We studied carbohydrate and phytohormone changes in HLB-affected fruit to explain symptom development because (1) carbohydrate shortage has been linked to fruit growth arrest and eventually abscission and (2) hormonal signals regulate, at least partially, fruit set and development. Symptomatic fruit (S), asymptomatic fruit (AS) from symptomatic trees, and healthy fruit (H) from asymptomatic trees were harvested from ‘Valencia’ sweet orange trees [Citrus sinensis (L.) Osbeck] infected with the HLB pathogen or not, as verified by PCR. Mature S weighed less, had lower °Brix, were smaller, had more aborted seeds, and were greener than AS or H. Starch and sucrose contents were lower in mature S flavedo compared with that of H and AS. S and AS harvested 7 and 12 months after full bloom produced significantly less ethylene than H. Indole-3-acetic acid (IAA) and abscisic acid (ABA) contents in flavedo removed from the stylar end, middle section, or stem end of fruit generally were higher in S flavedo than in AS and H. ABA content was fourfold higher in flavedo from the middle section of S than in AS and H. Flavedo excised from the large shoulder of misshapen S had significantly higher IAA content when compared with the normal-sized area of the same fruit on the opposite side. This increase corresponded to an increase in hypodermal cell area in S flavedo. Overall, these data reveal an imbalance of carbohydrate and phytohormone status in fruit from HLB-infected trees and suggest a role of such changes in fruit symptom development.     

原生质体融合获得柑桔种间体细胞杂种

粗柠檬(Citrus jambhiri Lush)叶肉原生质体与哈姆林甜橙(C.sinensis L.Osbeck)胚性悬浮细胞系原生质体经PEG诱导融合,培养7天时原生质体恢复分裂。再生的胚状体在含有GA_3的培养基中萌发出茎芽。茎芽经生根诱导成为完整植株。对首批再生的5棵植株进行染色体检查,结果表明,全为四倍体,2n=4x=36。淀粉胶电泳分析过氧化物酶同工酶,结果显示这5棵植株为体细咆杂种。粗柠檬和哈姆林甜橙在该位点上均为同质结合,基因型分别是MM和FF。体细胞杂种含有双亲的酶带,基因型为MMFF。杂种植株生长旺盛,根系发达,叶片及植株形态介于双亲之间。本文对其作为砧木品种的可能性等问题作了讨论。     

An HPLC-MS Characterization of the Changes in Sweet Orange Leaf Metabolite Profile following Infection by the Bacterial Pathogen Candidatus Liberibacter asiaticus

Huanglongbing (HLB) presumably caused by Candidatus Liberibacter asiaticus (CLas) threatens the commercial U.S. citrus crop of an annual value of $3 billion. The earliest shift in metabolite profiles of leaves from greenhouse-grown sweet orange trees infected with Clas, and of healthy leaves, was characterized by HPLC-MS concurrently with PCR testing for the presence of Clas bacteria and observation of disease symptoms. Twenty, 8-month-old ‘Valencia’ and ‘Hamlin’ trees were grafted with budwood from PCR-positive HLB source trees. Five graft-inoculated trees of each variety and three control trees were sampled biweekly and analyzed by HPLC-MS and PCR. Thirteen weeks after inoculation, Clas was detected in newly growing flushes in 33% and 55% of the inoculated ‘Hamlin’ and ‘Valencia’ trees, respectively. Inoculated trees remained asymptomatic in the first 20 weeks, but developed symptoms 30 weeks after grafting. No significant differences in the leaf metabolite profiles were detected in Clas-infected trees 23 weeks after inoculation. However, 27 weeks after inoculation, differences in metabolite profiles between control leaves and those of Clas-infected trees were evident. Affected compounds were identified with authentic standards or structurally classified by their UV and mass spectra. Included among these compounds are flavonoid glycosides, polymethoxylated flavones, and hydroxycinnamates. Four structurally related hydroxycinnamate compounds increased more than 10-fold in leaves from ‘Hamlin’ and ‘Valencia’ sweet orange trees in response to Clas infection. Possible roles of these hydroxycinnamates as plant defense compounds against the Clas infection are discussed.     

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Research into the design and utilization of brain-implanted microdevices, such as microelectrode arrays, aims to produce clinically relevant devices that interface chronically with surrounding brain tissue. Tissue surrounding these implants is thought to react to the presence of the devices over time, which includes the formation of an insulating "glial scar" around the devices. However, histological analysis of these tissue changes is typically performed after explanting the device, in a process that can disrupt the morphology of the tissue of interest.Here we demonstrate a protocol in which cortical-implanted devices are collected intact in surrounding rodent brain tissue. We describe how, once perfused with fixative, brains are removed and sliced in such a way as to avoid explanting devices. We outline fluorescent antibody labeling and optical clearing methods useful for producing an informative, yet thick tissue section. Finally, we demonstrate the mounting and imaging of these tissue sections in order to investigate the biological interface around brain-implanted devices.