Javadian Neda, Karimzadeh Ghasem, Sharifi Mohsen, Moieni Ahmad, Behmanesh Mehrdad
Department of Plant Breeding, Faculty of Agriculture, Tarbiat Modares University, P. O. Box 14115-336, Tehran, Iran.
Department of Plant Biology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Planta. 2017 Jun;245(6):1165-1178. doi: 10.1007/s00425-017-2671-2. Epub 2017 Mar 14.
Induction of tetraploidy was performed and podophyllotoxin production increased by upregulating the expression level and enzyme activity of genes related to its biosynthesis in tetraploid compared to diploid Linum album. Linum album is a valuable medicinal plant that produces antiviral and anticancer compounds including podophyllotoxin (PTOX). To achieve homogeneous materials, in vitro diploid clones were established, and their nodal segments were exposed to different concentrations and durations of colchicine. This resulted in successful tetraploidy induction, confirmed by flow cytometry, and is being reported for the first time. The highest efficiency of tetraploid induction (22%) was achieved after 72 h exposure to 2.5-mM colchicine treatment. The stable tetraploids were produced after being subcultured three times, and their ploidy stability was confirmed after each subculture. The effects of autopolyploidy were measured on the morphological and phytochemical characteristics, as well as enzyme activity and the expression levels of some key genes involved in the PTOX biosynthetic pathway, including phenylalanine ammonia-lyase (PAL), cinnamoyl-Coa reductase (CCR), cinnamyl-alcohol dehydrogenase (CAD), and pinoresinol-lariciresinol reductase (PLR). The tetraploid plants had larger leaves and stomata (length and width) and lower density stomata. Increasing the ploidy level from diploid to tetraploid resulted in 1.39- and 1.23-fold enhancement of PTOX production, respectively, in the leaves and stem. The increase in PTOX content was associated with upregulated activities of some enzymes studied related to its biosynthetic pathway and the expression of the corresponding genes. The expression of the PAL gene and PLR enzymatic activity had the most positive correlation with the ploidy level in both leaf and stem tissues. Our results verified that autotetraploid induction is a useful breeding method, remarkably increasing the PTOX content in the leaves and stem of L. album.
与二倍体亚麻相比，通过上调四倍体亚麻中与其生物合成相关基因的表达水平和酶活性，进行了四倍体诱导并提高了鬼臼毒素的产量。亚麻是一种珍贵的药用植物，可产生包括鬼臼毒素（PTOX）在内的抗病毒和抗癌化合物。为了获得同质材料，建立了体外二倍体克隆，并将其节段暴露于不同浓度和持续时间的秋水仙碱中。这导致成功诱导出四倍体，经流式细胞术确认，且首次报道。暴露于2.5 mM秋水仙碱处理72小时后，四倍体诱导效率最高（22%）。经过三次继代培养后产生了稳定的四倍体，并在每次继代培养后确认了其倍性稳定性。测定了同源多倍体对形态和植物化学特征的影响，以及参与PTOX生物合成途径的一些关键基因的酶活性和表达水平，包括苯丙氨酸解氨酶（PAL）、肉桂酰辅酶A还原酶（CCR）、肉桂醇脱氢酶（CAD）和松脂醇-落叶松脂醇还原酶（PLR）。四倍体植物的叶片和气孔（长度和宽度）更大，气孔密度更低。将倍性水平从二倍体提高到四倍体，分别使叶片和茎中PTOX的产量提高了1.39倍和1.23倍。PTOX含量的增加与其生物合成途径中一些研究的酶活性上调以及相应基因的表达有关。在叶片和茎组织中，PAL基因的表达和PLR酶活性与倍性水平的正相关性最强。我们的结果证实，同源四倍体诱导是一种有用的育种方法，可显著提高亚麻叶片和茎中PTOX的含量。
