Alpizar E, Dechamp E, Lapeyre-Montes F, Guilhaumon C, Bertrand B, Jourdan C, Lashermes P, Etienne H
Centre de Coopération Internationale en Recherche Agronomique pour le Développement-Département des Systèmes Biologiques (CIRAD-BIOS), UMR-RPB, 911 Avenue Agropolis, BP 64501, 34394 Montpellier, France.
Ann Bot. 2008 May;101(7):929-40. doi: 10.1093/aob/mcn027. Epub 2008 Mar 2.
The aims of this study were to set up proliferation conditions for hairy roots of Coffea arabica regenerated after transformation by Agrobacterium rhizogenes strain A4-RS, and to carry out the morphological and molecular characterization of hairy root clones maintained over the long term.
Auxin supply, light conditions and sucrose concentration were modified with the aim of establishing efficient root proliferation conditions. The morphological variability among 62 established hairy root clones was phenotyped by scanning the roots and analysing the images using 'whinRHIZO' software procedures. PCR analysis of integration in transformed root cells of rol and aux oncogenes from the T-DNA of the Ri plasmid was used to study the molecular variability among clones.
Auxin supply was necessary to obtain and stimulate growth and branching, and IBA applied at 0.5 microm was the most efficient auxin. Significant differences were shown among the 62 clones for total root length and for the percentage of fine roots. These variables were stable across subcultures and could hence be used for efficient characterization of hairy root clones. The majority of hairy root clones (86 %) exhibited non-significant phenotype differences with non-transformed roots. Eight clones were significantly different from the non-transformed controls in that they possessed a low proportion of fine roots. Two other hairy root clones grew significantly faster than the other clones. The PCR analysis revealed a low variability in the integration of rol and aux oncogenes in transformed root cells. The T(R)-DNA was never integrated as aux1 and aux2 genes were not found, although rolB and rolC genes from the T(L)-DNA were always present.
The discovery of low morphological variability among coffee hairy roots together with the identification of morphological variables allowing easy identification of phenotypically altered clones represent two important results. They make hairy roots a possible, and efficient, tool for functional-genomic studies of coffee root genes.
本研究的目的是为发根农杆菌A4-RS转化后再生的阿拉伯咖啡毛状根建立增殖条件，并对长期保存的毛状根克隆进行形态学和分子特征分析。
通过改变生长素供应、光照条件和蔗糖浓度来建立有效的根增殖条件。利用“whinRHIZO”软件程序扫描根系并分析图像，对62个已建立的毛状根克隆的形态变异性进行表型分析。通过对Ri质粒T-DNA中rol和aux致癌基因在转化根细胞中的整合进行PCR分析，研究克隆间的分子变异性。
生长素供应对于获得和刺激生长及分支是必要的，0.5微摩尔的吲哚丁酸（IBA）是最有效的生长素。62个克隆在总根长和细根百分比方面存在显著差异。这些变量在继代培养中保持稳定，因此可用于高效鉴定毛状根克隆。大多数毛状根克隆（86%）与未转化根表现出不显著的表型差异。八个克隆与未转化对照有显著差异，因为它们的细根比例较低。另外两个毛状根克隆的生长速度明显快于其他克隆。PCR分析显示转化根细胞中rol和aux致癌基因的整合变异性较低。由于未发现aux1和aux2基因，T(R)-DNA从未整合，尽管T(L)-DNA中的rolB和rolC基因总是存在。
咖啡毛状根形态变异性低的发现以及易于鉴定表型改变克隆的形态变量的确定代表了两个重要结果。它们使毛状根成为咖啡根基因功能基因组学研究的一种可能且有效的工具。
