Quantification of leghaemoglobin content in pea nodules based on near-infrared hyperspectral imaging spectroscopy and chemometrics

Leghemoglobin content in nodules is closely related with the amount of nitrogen fixed by the legume-rhizobium symbiosis. It is therefore commonly measured in order to assess the effect of growth-promoting parameters such as fertilization on the symbiotic nitrogen fixation efficiency of legumes. The cyanmethemoglobin method is a reference method in leghemoglobin content quantification, but this method is time-consuming, requires accurate and careful technical operations and uses cyanide, a toxic reagent.
As a quicker, simpler and nondestructive alternative, a method based on near-infrared (NIR) hyperspectral imaging was tested to quantify leghemoglobin in dried nodules. Two approaches were evaluated: (i) the PLS approach was applied to the full spectrum acquired with the hyperspectral device, and (ii) the potential of multispectral imaging was also tested through the preselection of the most relevant wavelengths and the building of a multiple linear regression model. The PLS approach was tested on mean spectra acquired from samples containing several nodules and acquired separately from individual nodules. Peas (Pisum sativum L.) were cultivated in the greenhouse. The nodules were harvested on four different dates in order to obtain variations in leghemoglobin content.
The leghemoglobin content measured with the cyanmethemoglobin method in fresh nodules ranged between 1.4 and 4.2 mg leghemoglobin.g-1 fresh nodule. A partial least square (PLS) regression model was calibrated on leghemoglobin content measured with the reference method and mean NIR spectra of dried nodules acquired with a hyperspectral imaging device. On a validation dataset, the PLS model predicted well the leghemoglobin content in nodule samples (R² = 0.90, RMSEP = 0.26). The multispectral approach showed similar performance. Applied to individual nodules, the PLS model highlighted a wide variability of leghemoglobin content in nodules harvested from the same plant. These results show that NIR hyperspectral imaging could be used as a rapid and safe method to quantify leghemoglobin in pea nodules.