Use of internal reflection spectroscopy for maize (Zea mays L.) grain diagnosis

Infrared (IR) spectroscopy and Raman spectroscopy (RS) are modern methods on the basis of which biotechnological approaches are being successfully developed that allow genetic and functional analysis of individual plant organs and tissues at the molecular level. In the present work, using surface internal reflection spectroscopy (SIR), which is a modification of IR spectroscopy, differences in the content and conformation of biomolecules in grain homogenates and its components (endosperm, pericarp, germ) of ZP 735 maize hybrids were first recorded and revealed. Our goal was to develop a methodology for surface SIR for the identification of organic molecules, their content and conformation in corn seed and its tissues. The grains of the corn hybrid ( Zea mays L.) ZP 735 (originator Maize Research Institute, Zemun Polje, Belgrade, Serbia) served as the object of the study. Thirty grains were selected by random sampling. To obtain SIR spectra, samples of grains and their parts (endosperm, pericarp, and embryo) were crushed, homogenized, and placed in a special cell of the device. A spectrometer (FT-IR spectrophotometer, Thermo Scientific, USA) with diamond (diamond ATR crystal, Thermo Scientific, USA) was used to record the SIR spectra, and a software package (Thermo Scientific™ ) was used to analyze the spectra. The SIR spectra were compared in the data library of the Advanced ATR correction Algorithm program. The SIR spectroscopy method is based on the reflection of a light beam at the interface between two phases: the phase of a crystal with a high refractive index, which is part of the SIR device, and the phase of the sample under study with a lower refractive index. During the measurement, the light beam penetrates to a small depth into the phase of the sample, where it is partially absorbed. During subsequent irradiation of the sample, this phenomenon is repeated, and as a result, the SIR spectrum is recorded. It has been proven that in the range from 400 cm-1 to 4000 cm-1 ATR spectra of whole grain, endosperm, pericarp and embryo of the ZP 735 hybrid are similar to the previously obtained IR spectra of grain, which indicates the possibility of using a new method for molecular breeding technologies. The location of the bands of the SIR spectrum characterizes various forms of vibrations of the valence bonds of the functional groups of the organic molecules of the seed, which makes it possible to identify not only the presence of certain molecules, but also their conformation. It has been proved that by analyzing the amplitude of the SIR spectrum bands (maximum intensity amplitude and high intensity amplitude), it is possible to control changes in the content of a number of organic compounds in seed tissues: proteins, lipids, sugars, esters, amides, ketones, aldehydes, carboxylic acids, simple ethers, phenols, alcohols, aromatic hydrocarbons, acyclic compounds, alkenes, alkanes and alkynes. The obtained results are important for testing the presence of the necessary organic compounds in the grain or changes in their conformation during the selection process. The important advantages of SIR spectroscopy compared to IR spectroscopy include, on the one hand, the preservation of the nativeness of the object (the possibility of conducting research without fixing or staining the object) and simple sample preparation, on the other hand, an effective assessment of the content and conformation of molecules with high sensitivity (resolution about 1.0 cm-1). The implementation of the developed methodology for the formation of molecular breeding technology will increase the profitability of cultivation and the efficiency of breeding not only corn, but also other agricultural plants.