Ahmed Zaheer, Fu Yong-Bi
Plant Gene Resources of Canada, Saskatoon Research and Development Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2 Canada.
Plant Methods. 2015 Dec 21;11:56. doi: 10.1186/s13007-015-0099-x. eCollection 2015.
Mitochondria perform a principal role in eukaryotic cells. Mutations in mtDNA can cause mitochondrial dysfunction and are frequently associated with various abnormalities during plant development. Extraction of plant mitochondria and mtDNA is the basic requirement for the characterization of mtDNA mutations and other molecular studies. However, currently available methods for mitochondria isolation are either tissue specific or species specific. Extracted mtDNA may contain substantial chloroplast DNA (cpDNA) and nuclear DNA (nDNA) and its end use efficiency can be reduced. Clearly, an effective mitochondria isolation method is warranted with wider applicability and with minimum contamination from cpDNA and nDNA.
Here we reported an improved method for isolating mitochondria from dry wheat seeds and its extension to dead seeds, viable seeds, etiolated leaf tissue and several other plant species: oat, Arabidopsis, flax, and yellow mustard. The isolated mitochondria were successfully used to extract mtDNA with QIAamp DNA mini kit (Qiagen). The extracted mtDNA from the assayed samples of these species was intact in large quantity and showed little contamination from nDNA, cpDNA, RNA, and proteins. The mtDNA extracted from dead wheat seeds was also substantial, but more degraded and less intact when compared to those from viable seeds and other tissues.
The improved method was successfully applied to isolate mitochondria and extract mtDNA from several different tissues and plant species. The major advance in the improvement lies in its wider application with the same mitochondria extraction medium to different tissues and species. The improvement is significant, as it helps to widen the scope of future plant mitochondria research.
线粒体在真核细胞中发挥着主要作用。线粒体DNA（mtDNA）的突变可导致线粒体功能障碍，并常常与植物发育过程中的各种异常情况相关。植物线粒体和mtDNA的提取是表征mtDNA突变及其他分子研究的基本要求。然而，目前可用的线粒体分离方法要么是组织特异性的，要么是物种特异性的。提取的mtDNA可能含有大量的叶绿体DNA（cpDNA）和核DNA（nDNA），其最终使用效率可能会降低。显然，需要一种有效的线粒体分离方法，具有更广泛的适用性，并且来自cpDNA和nDNA的污染最小。
在此，我们报道了一种从干燥小麦种子中分离线粒体的改进方法，并将其扩展应用于死种子、活种子、黄化叶组织以及其他几种植物物种：燕麦、拟南芥、亚麻和黄芥。使用QIAamp DNA mini试剂盒（Qiagen）成功地从分离的线粒体中提取了mtDNA。从这些物种的测定样品中提取的mtDNA大量完整，并且显示出来自nDNA、cpDNA、RNA和蛋白质的污染很少。与从活种子和其他组织中提取的mtDNA相比，从死小麦种子中提取的mtDNA数量也很多，但降解程度更高，完整性更低。
该改进方法成功地应用于从几种不同的组织和植物物种中分离线粒体并提取mtDNA。改进的主要进展在于使用相同的线粒体提取培养基对不同组织和物种具有更广泛的适用性。这一改进意义重大，因为它有助于拓宽未来植物线粒体研究的范围。
