DIMBOA production in tissue culture

Recent work by Williams et al. (Agric. Ecosystems Environ. 18:185) has indicated that resistance to several corn insect pests including the European corn borer can be identified using tissue culture material. Both antibiosis and nonpreference were indicated to be important in determining the resistance. The hydroxamic acid, 2,4dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) has been shown to play an important part in resistance to the first brood ECB. This raises the question of whether or not DIMBOA is produced in tissue culture and if so at what levels.

To try to answer this question 18- to 21-day old embryos were plated out on Murashige and Skoog medium supplemented with 1mg/l 2,4-D, 4mg/l a-naphthaleneacetic acid and 0.05 mg/l 6-(-g,-g-dimethylallylamino-purine. The callus which developed was transferred and divided 4 times at 2 to 4 week intervals before being used. DIMBOA was extracted from the callus using a modification of the methods of Gutierrez et al. (J. Agric. Food Chem. 30:1258). The major breakdown product of DIMBOA, 6-methoxy-2-(3H)-benzoxazolinone (MBOA), co-extracts with DIMBOA. Both DIMBOA and MBOA were identified by HPLC methods and quantitated using standard curves based on peak height.

Both C131A and Oh43 normally produce high levels of DIMBOA in seedlings and maintain high levels long enough to give resistance to the first brood ECB. Under the tissue culture conditions used here Oh43 produced an average of only 2.2mg DIMBOA/g tissue. The C131A calli were quite variable, producing from 0 to 44.8mg DIMBOA/g tissue. The breakdown product, MBOA, was found at levels of 2.9 and 7.0mg/g tissue in C131A and Oh43 respectively. The level of DIMBOA was much lower than that found in seedling tissue where DIMBOA can reach greater than 10 fold higher amounts in seedling tissue. It is noted that most of the calli had far more MBOA than DIMBOA. Under the extraction conditions used this is unexpected. In normal cells DIMBOA exists as a glucoside and MBOA is not found. It may be that what DIMBOA is produced is not sequestered normally and breaks down. A second possibility is that the MBOA comes from cells that died during culture and therefore released their DIMBOA allowing it to break down. In order to get a better picture of DIMBOA production under tissue culture conditions several other corn lines and several other tissue culture conditions will have to be tested.

B.A. Bailey, J. Bussard and R.L. Larson

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