Rapid separation of developing Arabidopsis seeds from siliques for RNA or metabolite analysis

Abstract

BackgroundProtein, starch and oil produced in plant seeds are major renewable sources of food, chemicals and biofuels. Developing Arabidopsis thaliana seeds are commonly utilized as a model for seed crop research. However, due to the very small size of Arabidopsis seeds efficient collection of large amounts of tissue for gene expression or metabolite analysis is very difficult and time consuming.Results/conclusionsHere we describe a method that allows very rapid separation and collection of large amounts of developing Arabidopsis seeds from their encapsulating silique tissue after flash freezing whole siliques in liquid nitrogen. The efficient popping open of the frozen siliques on dry ice and filtering the seeds away from the silique tissue with liquid nitrogen cooled funnels and sieves allows large amounts of developing seeds to be quickly isolated while remaining frozen. This method increases the speed of developing seed collection approximately 10 fold over methods which dissect individual siliques one at a time.

Fatty acid synthesis is inhibited by inefficient utilization of unusual fatty acids for glycerolipid assembly

“…RNA Expression Analysis. Frozen developing seeds of 7-8, 9-10, and 11-12 days after flowering (DAF) were collected from liquid N 2 frozen siliques (47). Approximately 100 mg of frozen seed samples (three biological replicates for each plant line/developmental stage) were ground to a fine power, RNA extracted (48), and DNA removed (DNA-Free RNA Kit, Zymo Research).…”

“…Acyl-CoA and acyl-ACP were extracted from developing seeds of 9-12 DAF that were collected from liquid N 2 frozen siliques (47). Acyl-CoA were exacted as in ref.…”

Identification of Arabidopsis GPAT9 (At5g60620) as an Essential Gene Involved in Triacylglycerol Biosynthesis

“…Transcript levels were analyzed by quantitative PCR using Platinum SYBR Green qPCR SuperMix-UDG (Life Technologies) and the Stratagene Mx3005P Quantitative PCR system (Agilent Technologies). For developing seed transcript analysis, frozen developing seeds (9-11 DAF) were collected from liquid N 2 frozen siliques (Bates et al, 2013a). Approximately 50 to 100 mg of frozen seeds was mechanically pulverized to a fine powder with steel beads (TissueLyser LT; Qiagen), RNA was extracted (Suzuki et al, 2004), and DNA was removed (DNA-Free RNA Kit; Zymo Research; www.…”

Castor LPCAT and PDAT1A Act in Concert to Promote Transacylation of Hydroxy-Fatty Acid onto Triacylglycerol

“…We harvested developing Arabidopsis siliques at ten DAF and flash froze them in liquid nitrogen. Seeds were released from siliques as described (Bates et al, 2013). RNA was extracted from seeds with the RNeasy Plant Mini Kit (Qiagen) and the quality and quantity was determined by nanophotomer (Implen).…”

“…Counting continued until each independent replicate accounted for 100 siliques at 8, 10, 12, and 14 DAF. We extracted seeds by immersing siliques in liquid nitrogen followed by rapid thawing to break open the wall (Bates et al, 2013). We extracted lipids from the seeds and separated TAG and PC using thin-layer chromatography with a single development in a solvent system of chloroform:methanol:acetic acid (75:25:8, v/v/v).…”