Li Mingzhuo, Poonam Aditi Dey, Cui Qirui, Hsieh Tzungfu, Jagadeesan Sumeetha, Xu Jin, Bruce Wesley B, Vogel Jonathan T, Sessions Allen, Cabrera Antonio, Saville Amanda C, Ristaino Jean B, Paul Rajesh, Wei Qingshan
Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, USA.
Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, USA.
Plant Biotechnol J. 2025 Jun;23(6):2317-2329. doi: 10.1111/pbi.70055. Epub 2025 Mar 19.
Crop breeding plays an essential role in addressing food security by enhancing crop yield, disease resistance and nutritional value. However, the current crop breeding process faces multiple challenges and limitations, especially in genotypic evaluations. Traditional methods for seed genotyping remain labour-intensive, time-consuming and cost-prohibitive outside of large-scale breeding programs. Here, we present a handheld microneedle (MN)-based seed DNA extraction platform for rapid, non-destructive and in-field DNA isolation from crop seeds for instant marker analysis. Using soybean seeds as a case study, we demonstrated the use of polyvinyl alcohol (PVA) MN patches for the successful extraction of DNA from softened soybean seeds. This extraction technology maintained high seed viability, showing germination rates of 82% and 79%, respectively, before and after MN sampling. The quality of MN-extracted DNA was sufficient for various genomic analyses, including PCR, LAMP and whole-genome sequencing. Importantly, this MN patch method also allowed for the identification of specific genetic differences between soybean varieties. Additionally, we designed a 3D-printed extraction device, which enabled multiplexed seed DNA extraction in a microplate format. In the future, this method could be applied at scale and in-field for crop seed DNA extraction and genotyping analysis.
作物育种在通过提高作物产量、抗病性和营养价值来解决粮食安全问题方面发挥着至关重要的作用。然而，当前的作物育种过程面临多重挑战和限制，尤其是在基因型评估方面。传统的种子基因分型方法仍然劳动强度大、耗时且在大规模育种计划之外成本过高。在此，我们展示了一种基于手持微针（MN）的种子DNA提取平台，用于从作物种子中快速、无损且在田间进行DNA分离，以便即时进行标记分析。以大豆种子为例，我们证明了使用聚乙烯醇（PVA）微针贴片可成功从软化的大豆种子中提取DNA。这种提取技术保持了较高的种子活力，微针采样前后的发芽率分别为82%和79%。微针提取的DNA质量足以用于各种基因组分析，包括PCR、环介导等温扩增（LAMP）和全基因组测序。重要的是，这种微针贴片方法还能够鉴定大豆品种之间的特定遗传差异。此外，我们设计了一种3D打印的提取装置，其能够以微孔板形式进行多重种子DNA提取。未来，这种方法可大规模应用于田间作物种子DNA提取和基因分型分析。
