Institute for Biological Research 'Siniša Stanković', University of Belgrade, Bulevar despota Stefana 142, Belgrade, Serbia.
USDA-ARS, Molecular Plant Pathology Laboratory, 10300 Baltimore Avenue, Beltsville, MD, USA.
Tree Physiol. 2019 May 1;39(5):888-905. doi: 10.1093/treephys/tpz006.
Allelopathic plants exploit their chemical 'weapons' to prevail over the competition, suppress neighboring plants and consequently use the available resources more efficiently. However, the investigation of plant allelopathic interactions in rhizosphere is difficult to perform because of its high complexity due to interactions of biotic and abiotic factors. Thus, autonomous, aseptic root cultures of apple (Malus × domestica Borkh.) could facilitate allelopathic studies. We report on the successful genetic transformation of apple cultivars Melrose, Golden Delicious, Čadel and Gloster using Agrobacterium rhizogenes (Riker et al. 1930) Conn 1942 strain 15834 and for the first time the establishment of apple autonomous and permanent in vitro hairy root cultures that could be used as a new tool for apple allelopathic assays. Molecular characterization of transgenic hairy root lines was conducted to elucidate the possible relationship between expression of T-DNA genes and root growth characteristics that include branching. Similar content of phenolic acids (chlorogenic, caffeic, syringic, p-coumaric and ferulic), glycosilated flavonoids (rutin, quercitrin, isoquercitrin, kaempferol-3-glucoside) and flavonoid aglycones (quercetin and naringenin), and dihydrochalcone phloridzin, was detected in untransformed and transgenic apple root tissue by ultra high-performance liquid chromatography with mass spectrometry (UHPLC/(+/-)HESI-MS/MS) analyses, confirming that genetic transformation did not disturb secondary metabolite production in apple. Chlorogenic and caffeic acids and dihydrochalcones phloridzin and phloretin were detected as putative allelochemicals exuded into the growth medium in which transgenic roots were maintained for 4 weeks. Apple hairy root exudates significantly affected shoot and root development and growth of test plant Arabidopsis thaliana (L.) Heynh. seedlings after 5 or 10 days of treatment. Additionally, core cell-cycle genes CDKA1;1, CDKB2;1, CYCA3;1 and CYCB2;4 were down regulated in Arabidopsis shoots suggesting, in part, their role in inhibition of shoot growth. The present work highlighted an autonomous and permanent in vitro hairy root culture system as a valuable tool for studying allelopathic potential of apple, offering new perspective for allelopathy background elucidation in this important fruit species.
自毒植物利用其化学“武器”战胜竞争，抑制邻近植物，从而更有效地利用可用资源。然而，由于生物和非生物因素的相互作用，根际植物化感相互作用的研究具有很高的复杂性，因此难以进行。因此，苹果（Malus × domestica Borkh.）的自主、无菌根培养可以促进化感研究。我们报告了利用根癌农杆菌（Riker et al. 1930）Conn 1942 菌株 15834 成功转化苹果品种 Melrose、Golden Delicious、Čadel 和 Gloster，并首次建立了苹果自主和永久性的体外毛状根培养，可以作为苹果化感测定的新工具。对转基因毛状根系进行了分子特征分析，以阐明 T-DNA 基因表达与包括分枝在内的根生长特征之间的可能关系。超高效液相色谱-串联质谱（UHPLC/(+/-)HESI-MS/MS）分析表明，未转化和转化的苹果根组织中检测到相同含量的酚酸（绿原酸、咖啡酸、丁香酸、对香豆酸和阿魏酸）、糖基化类黄酮（芦丁、槲皮苷、异槲皮苷、山奈酚-3-葡萄糖苷）和类黄酮苷元（槲皮素和柚皮素），以及二氢查耳酮根皮苷，证实遗传转化没有干扰苹果次生代谢产物的产生。在转基因根维持 4 周的生长培养基中，检测到绿原酸和咖啡酸以及二氢查耳酮根皮苷和根皮素作为可能的化感物质渗出。苹果毛状根分泌物显著影响拟南芥（L.）幼苗的芽和根发育和生长，处理后 5 或 10 天。此外，拟南芥芽中核心细胞周期基因 CDKA1;1、CDKB2;1、CYCA3;1 和 CYCB2;4 的表达下调表明，它们在部分程度上抑制了芽的生长。本研究强调了自主和永久性的体外毛状根培养系统作为研究苹果化感潜力的有价值工具，为该重要水果物种的化感背景阐明提供了新的视角。
