Institute of Genetics, Bulgarian Academy of Sciences
AgroBio Institute
Screening of herbicide resistance in maize calli in vitro --Nedev, T, Dimitrov, B, Kruleva, M, Krapchev, B, Nencheva, D Immature maize embryos from several genotypes were screened for in vitro development and regeneration, and the A619 genotype was reported to have the highest regeneration ability (Todorova, L. et al., MNL 72:76, 1998). This genotype would allow in vitro selection for herbicide resistance. Vigorous weeds have been one of the main causes of crop losses ever since humans started to cultivate plants. Overall, more than one third of all agricultural production is still lost in the same proportion as a century ago (although, of course, the total production is much larger than it was then) (May, R.M., Nature 361:593-594, 1993). Stomp 330 (a product of BASF) is a dinitroanilin herbicide (active substance pendimetalin-N-(1-ethyl-propyl) 3-4 dimethyl-2, 6-dinitroanilin) with a wide range of action, mainly against annual cereals and some dicotyledonous weeds of many crops, vegetables and fruits and vines. A positive effect of this herbicide was observed on the following major crops: maize, wheat, barley, cotton, soy bean, beans, sunflower etc. The mechanism of the action of pendimetalin includes inhibition of the proliferation of root and stem meristemic tissue cells, as well as their elongation. For early selection of herbicide resistance, a laboratory method is highly desirable. The larger number of calli that can be screened, and the limited space that is needed, make the application of in vitro selection an attractive approach. There have been several attempts to create bioassays in which resistance at the tissue culture level can be used as an indicator for resistance of whole plants (Dryanova and Dimitrov, Cytologia 65:17-23, 2000; Kosturkova et al., Proceedings of 4th European Conference on Grain Legumes, pp. 158-159, 2001; Vasic et al., Helia 25(36):145-152, 2002). A correlation between the response of the explants of the same genotypes, when grown on medium to which the herbicide was added, and field resistance of genotypes was found.

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 herbicide treatment.
Genotype Concentration of Stomp 330 (%) Total number of embryos inoculated Undeveloped embryos (%) Fresh calli (%) Organogenic calli (%)
A619 0% (Control) 332 12.33±1.48 60.86±1.17 26.77±1.16
  0.01 % 300 72.00±1.48* 28.00±1.70* 0
  0.05 % 324 100.00 0 0
  0.10 % 312 100.00 0 0
  0.26% 340 100.00 0 0
*Significant at P>99.90%.

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 weeds.

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