Development of a chlorophyll fluorescence imaging system for the quality assessment of fruits and vegetables

Abstract

Chlorophyll fluorescence has been used for a long time as a powerful tool to investigate the functioning of the photosynthetic apparatus of photosynthetic organisms. It is an extremely sensitive technique, though such sensitivity often makes its results very difficult to decipher. One of its main advantages is that it is a non-destructive technique, which makes it ideally suited for fast screening of large collections of samples. Traditionally, chlorophyll fluorescence is measured by specially designed fluorimeters, which perform point measurements on the sample surface. In the last decade a new approach, chlorophyll fluorescence imaging, has been developed in order to measure the distribution of chlorophyll fluorescence emission in two dimensions. Research work in this area is still at an early stage, and a few research groups developed different approaches to the problem. The present thesis deals with the development of a chlorophyll fluorescence imaging system carried out at the Laboratory of Botany of the Limburgs Universitair Centrum and its application in the field of fruit quality assessment, in the study of plant heavy metal stress, on the effects of disturbances of the endogenous phytohormone balance and as a diagnostic method for pre-symptomatic detection of viral infections. The system is composed of an excitation unit, an imaging unit and a control unit. Chlorophyll fluorescence is detected by a CCD camera fitted with a red cut-off filter, upon excitation with xenon lamps filtered with a solution of copper sulphate, which provides a blue cut-off low-pass filter. A detailed description of the system is presented and relevant technical issues are discussed. In particular, advantages and drawbacks implied by the technical solutions adopted are addressed. The mainstream application of the chlorophyll fluorescence imaging system is in the field of apple quality assessment. After a description of the typical behavior of an apple in terms of fluorescence emission as measured by the fluorescence imaging system, results from various experiments with apple material are reported. The suitability of the technique as a predictor for development of storage diseases is discussed on the basis of an example of successful prediction. In experiments with apples grown with or without extra nitrogen supply, the system proved to be able to detect differences in fluorescence emission related to the nitrogen treatment. New perspectives opened by such result are discussed. Progress made in using advanced analysis tools like Artificial Neural Networks is also addressed. The system was used also in an experiment with apples grown in orchards treated with different water regimes. In this case, unfortunately, the system could not detect any remarkable differences related to the irrigation regimes. The fluorescence imaging systems, though designed with special attention to its application in fruit quality control, can be applied also to other fields of research. A gallery of three examples of an alternative application is presented. In particular, results are shown from experiments made on heavy metal treated bean plants, on virus-inoculated Nicotiana benthamiana and on cytokinin-overexpressing transgenic Pssu-ipt tobaccо. Heavy metal treated plants showed a variation of fluorescence emission in time during the metal treatment. Not only the intensity of fluorescence emission changed, but also its distribution on the leaf, opening the way to speculations about the possible correlation of the difference observed and the distribution of metal ions within the leaf. The inoculation of Nicotiana benthamiana plants with pepper mild mottle virus caused an alteration of the fluorescence emission pattern a few days after the inoculation in otherwise asymptomatic leaves. A peculiar pattern could be observed, which might be related to the spreading of the virus from the site of inoculation to other parts of the plant. Transgenic Pssu-ipt tobacco, characterized by an elevated content of endogenous cytokinin, also showed a peculiar pattern of fluorescence emission, with altered kinetics in mesophyll areas at a distance from the main veins. Possible explanations of such phenomenon are attempted. In the appendices, the software tools used in this research work are presented. In particular, a description is given of the following programs: Grabix: Developed by the author, this is the software that controls the whole fluorescence imaging system. KhorosPro 2001, Student edition: This is a very flexible multi-purpose software suite for image analysis, used to process fluorescence images after being captured with Grabix. R: This is the programming language for statistical data analysis used to extract statistical information from the images. Stuttgart Neural Network Simulator (SNNS): It is the simulator used to analyze fluorescence images by means of artificial neural networks.