Study of rice pollen grains by multispectral imaging microscopy

Cellular images obtained by light microscopy have been analyzed qualitatively, but there is still a lack of quantitative information about the variations in cellular metabolism of selective substances. A new approach using the multispectral imaging microscope (MIM) to observe rice (Oryza sativa L.) pollen grains is reported. A liquid crystal tunable filter device was used for wavelength selection from 400 to 720 nm and a cooled two-dimensional monochrome charge coupled device for image detection. Rice pollen were stained respectively by acetocarmine, Coomassie blue, or iodine potassium-iodine, and then imaged by MIM. The images were processed by the WuDa Image Analysis System 2003 (computer software), and the transmittance spectra for pollen grain images were obtained. The statistical analysis of the transmittance data showed that the macromolecular amount (nucleic acid, protein, starch) of male-sterile line (MSL) pollen grains was less than those of the fertility-maintaining line (FML). For instance there was a significantly lower nucleic acid content in the MSL than in the FML pollen. The results revealed that pollen abortion was directly related to the diminution of intracellular substances for metabolism. Consequently, we have established a quantitative criterion to determine pollen sterility. Comparing the spectra features of the FML with the MSL, we found that certain spectra features can be used to identify various types of abortion pollens and the deficient cytoplasm of male-sterile rice. Our experimental results offer the first quantitative understanding for evaluating cell morphological structure correlated with cellular physiological status.