The spatial resolution enabled by in situ Fourier-transform infrared (FT-IR) microspectroscopy as predicted from our earlier report in Spectroscopy (1) is applied to localized chemical analysis in this vital biological process of seed germination. Germination includes several different biochemical and structural processes. Ultimately, the entire seed is consumed in sustaining the new life that results after sprouting and growth (2–4). Alpha amylase production is the standard evidence for detection of sprouted (germinated) wheat at harvest. Moist preharvest conditions can cause devastating losses and render the harvested wheat unfit for flour production. Dormancy of dry seeds following harvest retards sprouting under proper storage.

This microspectroscopic study is limited to the relationship of the embryonic axis to the surrounding scutellum within the germ upon germination. The chemical microstructures of these botanical parts were characterized extensively in our previous FT-IR microspectroscopic studies, including individual cell probing across the primary root and surrounding tissue (5–7). In all of the previously reported sample preparations, sprouting was avoided deliberately by soaking kernels to be sectioned overnight in 4 °C water before freezing, sectioning, and thaw mounting on BaF₂ windows. For this study, control sections for each variety were prepared in the same manner for comparison with deliberately germinated counterparts. Germination was effected on moist blotter paper in a Petri dish for 36 h at 25 °C before sectioning.

Related prior experimentation with near-IR imaging in the InGaAs region nondestructively revealed subsurface evidence of germination in whole kernels at early stages. In these images, the developing embryo showed contrast from the body of each kernel for a select wavelength and for principal component images (8). Whereas the embryonic axis spectrum has amide I and II bands and very little lipid, the scutellum is rich in 1740-cm^-1 carbonyl bands. The scutellum is the storeroom of readily available nourishment for conversion to protein of the developing embryo. With this in mind, kernels identified by whole seed InGaAs imaging as germinated were sectioned to examine the lipid-to-protein population in their scutella with synchrotron IR microspectroscopy. Ungerminated kernels of the same lot were sectioned and examined for comparison. The resulting maps showed dramatic qualitative indications of differences.

Wheat that under moist conditions sprouts in the field has an economically devastating effect; therefore, identification of breeding lines resistant to early sprouting is desirable. IR microspectroscopy enables in situ investigation of the microchemical structure within frozen sections of wheat kernels. Kernel frozen sections were mapped that were sprouted under controlled conditions for a specific time, terminated at –80 °C, and mounted for sectioning.

Experimental

Specimens

In preliminary experiments, kernels were allowed to germinate under controlled conditions on moist blotter paper in Petri dishes for a specified time period (24 and 36 h) before terminating the process at –80 °C. Kernels exposed to moisture for only 3 h that showed no evidence of germination were chosen as a control and were freeze-dried. Also, kernels that were not exposed to moisture were used as controls in subsequent studies.

Sections containing both germ and endosperm were prepared by cryomicrotomy. Sections 4-μm thick were thaw mounted onto IR-reflecting, "low e" glass microscope slides (Kevley). Alternatively, 6-μm-thick sections were thaw mounted onto 1 mm × 13 mm BaF₂ disks for mid-IR microspectroscopy in a transmission mode. Sections chosen for study were those in which germ meets endosperm and the scutellum is highly exposed.

Instrumentation at Synchrotron

FT-IR microspectroscopy was done on beamlines U10b and U2b at the National Synchrotron Light Source (NSLS) of Brookhaven National Laboratory (BNL) (Upton, New York). Beamline U10b was equipped with a Continμum IR microscope optically interfaced to a Magna 850 FT-IR spectrometer (Nicolet/Thermo, Madison, Wisconsin). Schwarzschild 32X objective and condenser mirror lenses were used on a double pass single mask beam path to project a 10 μm × 10 μm confocally targeted spot. A 50 μm × 50 μm liquid nitrogen–cooled (MCT) detector essentially matched the beam from the image plane mask of the microscope. A resolution of 8 cm^-1 was used with 16 scans co-added unless otherwise stated. Beamline U2b was equipped with a NicPLAN model infrared microscope (Nicolet/Thermo, Madison, Wisconsin).