In this paper, we describe a method for in vitro screening for resistance to herbicide Stomp 330 by culture of immature maize embryos. Maize inbred genotype A619 was used in our investigations. For phytotoxicity studies, maize control calli were aseptically grown on N6 medium without Stomp 330. Stomp 330 was supplemented to the medium for calli induction after filter sterilization. Treated calli were grown on N6 medium containing different concentrations (0.26%, 0.10%, 0.05% and 0.01%) of Stomp 330. The highest concentration was comparable with that applied in field use. The effect of herbicide on calli development was estimated visually by morphogenetic response after 21 days.
Table 1. Frequency of callus induction and regenerable callus after
|Genotype||Concentration of Stomp 330 (%)||Total number of embryos inoculated||Undeveloped embryos (%)||Fresh calli (%)||Organogenic calli (%)|
The experimental materials were separated into three groups: organogenic calli — with clear formation of green structures, and leaf-like shoots; fresh calli - without evidence of organ - embryogenesis; undeveloped embryos - immature embryos without any kind of growth. Each experimental treatment variant included at least 150 embryos and was repeated twice. The statistical significance of differences between the data obtained from the treated embryos and the controls was made by Least Squares analysis (programmes LSML90, Harvey, 1990, Users Guide for LMSLMW Computer Program, PS2 version, Ohio, Modified model for unbalanced data according to Nencheva D., 2001, Induction of Genetic Variability in Chrysanthemum marifolium Ram., Through Radiation Mutagenesis and In Vitro Techniques, Ph. D., Sofia).
Primary choice of Stomp 330 concentrations was made based on the research of Dryanova and Dimitrov (2000) which was done with triticale callus cultures. In our study, concentrations between 0.26%, 0.10% and 0.05% exerted a negative effect on tested genotype parameters. These explants did not grow, and died after 21 days. In the Dryanova and Dimitrov (2000) study, the concentration of 0.01% of Stomp 330 was efficient for some triticale genotypes with high ploidy levels. The high sensitivity of the maize A619 genotype to the above-mentioned concentrations was evidenced by the percentage of callus induction and the reduced in vitro growth rates. The loss of the ability to form regenerative calli can be explained cytologically by a lack of actively proliferating cells, and histologically by a lack of shoot meristems. Our attempt to control these events by different manipulations including treatment with 2, 4-D levels failed.
Maize calli from inbred line A619, resistant to herbicide Stomp 330,
were obtained by using the method of in vitro selection of mutant cells.
The resistance was proven at a concentration of the herbicide of 0.01%.
The present study seems to be applicable as a preliminary step in the production
of resistant plants. The results obtained will also be useful for investigations
on environmental protection, and for more information about control of
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