BIOLUMINESCENCE OF GERMINATING SEEDS*)

Since the discovery by Gurwitsch of the so-called mitogenetic rays, it has been known that, at least in a number of cases, biological processes are accompanied by the emission of light. It is quite obvious that the study of bioluminescence may prove very useful for investigating the mechanism and features of biological processes themselves and, in particular, the process of development of a living organism—one of the most important problems of modern science. However, because of the extreme weakness of bioluminescence, until recently it remained inaccessible to ordinary methods of direct physical investigation and could be studied only by indirect and comparatively ineffective methods. All the greater interest is presented by the brief communication reviewed here by Italian authors, who used photoelectric multipliers to measure the intensity of bioluminescence of germinating seeds of a number of plants.

Preliminary investigations by the authors showed that RCA-5819 and EMI-6260 photomultipliers possess sufficient sensitivity to be used as counters of individual photons. According to the authors’ data, the sensitivity of these multipliers in the appropriate regime is from 0.03 to 0.06 photoelectrons for each incident photon (in the region of maximum spectral sensitivity).

The minimum light intensity still accessible to measurement is determined by the dark count of the multiplier and amounts to from 30 to 120 photons per second per 1 cm² of the multiplier’s photosensitive surface. The comparatively large surface of the photocathode (about 10 cm²) makes it possible to cover a sufficiently large range of solid angles, i.e., to make effective use of the weak glow of the object under study.

*) L. Colli and U. Facchini, Nuovo-Cimento 12, No. 1, 150 (1954); L. Colli, U. Facchini and A. Rossi, Nuovo-Cimento 11, No. 3, 225 (1954).

FROM CURRENT LITERATURE

The objects under investigation were placed by the authors in a glass cuvette 14 cm in diameter. The photomultiplier was positioned directly above the cuvette and was surrounded by screens coated with magnesium oxide (to increase the coefficient of utilization of light). Cooling of the photomultiplier (to reduce the dark current) was carried out either with water or with a mixture of dry ice and acetone. The cuvette containing the biological material was in a thermostat. To avoid fluorescence, the entire apparatus was placed in a light-tight box. The output of the photomultiplier was connected to a counting device that made it possible to record individual pulses.

As the objects of investigation, seeds of wheat, maize, lentils, and beans were chosen. About 300 seeds with a total weight (fresh) of 60 g were placed in the cuvette and, over the course of 6 days, were regularly wetted with water. During all this time, in order to avoid possible fluorescence, they were kept in darkness. Under these conditions the formation of chlorophyll, and of the well-known red fluorescence of the latter, was practically excluded.

The intensity of bioluminescence, measured after 6 days, is given in Table I (the statistical error was less than 5%).

The results presented clearly indicate the presence of luminescence in germinating seeds; moreover, as established by the authors, the intensity of the luminescence depends substantially on temperature, but does not change if the seeds are subjected to preliminary illumination.

The aseptic conditions noted in the last line of Table I consisted in the seeds being subjected to 20-minute sterilization in a 0.3% solution of HgCl₂. Special experiments established that such sterilization completely freed the seeds from viable bacteria.

Since sterilization did not eliminate the luminescence of the seeds, it could therefore be concluded that the latter cannot be attributed to bacterial luminescence.

Using light filters, the authors established that, in the main, the luminescence occurs in the wavelength interval from 4500 to 6500 Å.

Table I

The next stage of the study consisted in determining the luminescent capacity of individual elements of the germinated seed. For this purpose a seedling was cut into separate parts and made into a pulp, which was then diluted in water, placed in a cuvette, and subjected to examination. The measurement results are given in Table II.

Table II

The measurements showed that absorption of the luminescent radiation in the mass of the luminous material itself is small—no more than 50%. A clearly expressed, slowing decrease in the intensity of the glow over time is observed (over 40 minutes the intensity decreases by about half). Temperature has a substantial effect on the intensity of the glow. When the temperature is raised to 60–70°C, the intensity of the glow rises steadily; a further increase in temperature causes a sharp decrease in the intensity of the glow. The authors see in this evidence of the biological nature of the reactions leading to the glow. A manyfold (up to 10 or more times) increase in the intensity of the glow is obtained when small amounts of H₂O₂ are added to the plant pulp.

On the basis of estimates of the sensitivity of the photomultiplier and the geometrical parameters of the apparatus, the authors find that the intensity of the bioluminescence they observed averages about 100,000 photons per second per 1 g of plant matter.

G. R.