Garneau Matthew G, Parchuri Prasad, Zander Nora, Bates Philip D
Institute of Biological Chemistry, Washington State University, Pullman, Washington, 99164, USA.
Plant Methods. 2025 May 22;21(1):67. doi: 10.1186/s13007-025-01388-3.
Seed oils are widely used in the food, biofuel, and industrial feedstock industries, with their utility and value determined by total oil content and fatty acid composition. Current high throughput seed oil analysis methods either lack accuracy in total fatty acid profiling or require extensive labor for lipid extraction prior to derivatization to fatty acid methyl esters (FAME) and quantification by gas chromatography (GC). Alternatively, direct whole seed FAME production methods have been developed for the very small seeds in the model species Arabidopsis thaliana but these have generally not been adapted to larger seeds of most oilseed crops.
High-throughput direct whole seed FAME production methods were optimized for seeds up to 5 mg each utilizing acid-catalyzed esterification. For the oilseed species Camelina sativa, Thlaspi avernse (pennycress), Cuphea viscosissima, and Brassica napus (var. Canola), the total seed fatty acid content and composition from direct seed esterification to FAME matched that of lipid extract derivatization demonstrating the accuracy of the methods. In combination with seed phenotyping using GridFree, this approach enabled the development of a rapid pipeline for simultaneous seed weight, count, size/shape phenotyping, and oil analysis. For the larger and tougher seeds produced by Limnanthes alba (Meadowfoam) and Cannabis sativa L. (hemp) the whole seed acid-based method proved insufficient, and prior laborious homogenization of seeds was required. Therefore, a rapid one-tube bead homogenization and base catalyzed-esterification method was developed. Base-derived fatty acid esterification cannot derivatize free fatty acids leading to slightly lower total seed fatty acid than acid-catalyzed methods, however the seed oil content and fatty acid composition that is valuable for screening large numbers of samples in research populations was accurately measured.
New rapid whole seed fatty acid esterification and phenotyping protocols were developed to accurately assess oilseed lipid content. These methods are particularly valuable in oilseed research, breeding, and engineering applications where efficient analysis of large numbers of samples and accurate oil fatty acid profiling is essential. While having been developed for current and emerging oilseed crops, these methods also provide a foundation from which protocols might be established for new and emerging crop species.
种子油广泛应用于食品、生物燃料和工业原料行业，其用途和价值由总油含量和脂肪酸组成决定。当前的高通量种子油分析方法要么在总脂肪酸谱分析中缺乏准确性，要么在衍生化为脂肪酸甲酯（FAME）并通过气相色谱（GC）定量之前需要大量劳动力进行脂质提取。另外，已经为模式植物拟南芥中非常小的种子开发了直接全种子FAME生产方法，但这些方法通常未适用于大多数油料作物的较大种子。
利用酸催化酯化反应，针对每粒重达5毫克的种子优化了高通量直接全种子FAME生产方法。对于油料作物亚麻荠、遏蓝菜（便士草）、粘毛萼距花和甘蓝型油菜（油菜变种），从直接种子酯化到FAME的总种子脂肪酸含量和组成与脂质提取物衍生化的结果相匹配，证明了这些方法的准确性。结合使用GridFree进行种子表型分析，这种方法能够开发出一种快速流程，用于同时进行种子重量、计数、大小/形状表型分析和油分析。对于白星海芋（泡沫花）和大麻生产的更大、更硬的种子，全种子酸基方法被证明是不够的，需要事先对种子进行费力的匀浆处理。因此，开发了一种快速的单管珠磨匀浆和碱催化酯化方法。碱衍生的脂肪酸酯化不能使游离脂肪酸衍生化，导致总种子脂肪酸略低于酸催化方法，然而，对于在研究群体中筛选大量样品有价值的种子油含量和脂肪酸组成进行了准确测量。
开发了新的快速全种子脂肪酸酯化和表型分析方案，以准确评估油料作物的脂质含量。这些方法在油料作物研究、育种和工程应用中特别有价值，在这些应用中，对大量样品进行高效分析和准确的油脂肪酸谱分析至关重要。虽然这些方法是为当前和新兴的油料作物开发的，但它们也为可能为新出现的作物物种建立方案提供了基础。
