Mutagenic effects of laser radiation and 6-mercaptopurine on seedlings
--V. K. Burilkov, V. M. Paschenko and V. N. Lysikov

Mutagenic effects of acridine orange (AO) and ethidium bromide (EB) on prokaryotes and eukaryotes have been studied (Burilkov and Krochik,in Laser in the Life Sciences, pp. 253-274, 1988; Dragan and Khrapunov, Cytol. Genet. 26:32-35, 1992). We have compared cytogenetic effects of the known sensitizers, AO and EB, and of ones not used previously, such as 6-mercaptopurine (6-MP) and cloroxine (CX), each used in combination with laser radiation (LR).

Seedlings of A-346 grown on media containing the above sensitizers at a concentration of 0.00001 M each, were exposed to LR for 1 min (l=337.1 nm, l = 70 MWt/m.m.sec).  Counts of chromosome aberrations were made in temporary preparations during mitotic anaphase and telophase of maize (Gostimsky, Practical Guide on Cytogenetics, 1974).

The studies have shown that the highest rate of chromosome aberrations occurred when 6-MP was used as a sensitizer (Fig. 1). This much exceeded the rates of chromosome aberrations resulting from exposure to EB+LR, CX+LR, and AO+LR.

One possible cause of chromosome aberrations induced by LR and sensitizers may be the formation of one- and two-strand breaks due to laser radiation energy which is transferred from the sensitizer molecule to certain DNA sites. The resultant one- and two-strand breaks may be repaired during mitosis, or they may eventually turn into chromosome aberrations. To test this hypothesis, we studied maize genomic DNA by the gel electrophoresis technique. Electrophoretic patterns and break counts from densitograms (Zhizina et al., Radiobiology 23:783-786, 1983) have suggested that the hypothesis is not implausible (Fig. 2, Table 1).
 

Table 1. The relative number of breaks in total DNA of maize resulting from exposure to sensitizers and LR.
 
LR 6-MP+LR EB+LR AO+LR CX+LR
3.87 64.50 24.94 12.47 14.62

The induction of chromosome aberrations in maize plants with the aid of 6-MP and LR has a number of advantages over the conventional techniques: (1) it shows relative selectivity--interaction is primarily with DNA; (2) it enables the molecular mechanisms of mutation and recombination to be more precisely identified; and (3) it produces no cytotoxic effects.

A high level of mutations of various types, inherited in M2 and M3, were discovered following the treatment of maize seedlings of the A-346 line. The analysis is in progress.

Figure 1. The rate of chromosome aberrations during mitotic anaphase-telophase in maize rootlets resulting from exposure to sensitizers and LR. 1 - control; 2 - LR; 3 - AO; 4 - AO+LR; 5 - CX; 6 - CX+LR; 7 - 6-MP; 8 - 6-MP+LR; 9 - EB; 10 - EB+LR.

Figure 2. Electrophoretic patterns (non-denaturating) for maize DNA and DNA+sensitizer complex. Lanes: 1 - DNA; 2 - DNA+LR; 3 - DNA+6-MP; 4 - DNA+6-MP+LR; 5 - DNA+AO; 6 - DNA+AO+LR; 7 - DNA+CX; 8 - DNA+CX+LR; 9 - DNA+EB; 10 - DNA+EB+LR.


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