Work is under way in three areas to determine the possibilities for producing novel resistance to H. maydis using tissue culture techniques.

1) Expression of T-toxin sensitivity in callus tissue: Our experiments confirm the results of Gengenbach and Green (Crop Sci. 15:645, 1975) and others that callus tissue derived from plants with T cytoplasm (T-callus) is markedly inhibited or even killed by the presence of quite small amounts of T-toxin in the nutrient medium. N-callus grows normally under these conditions. Malate oxidation as measured by DCPIP reduction (Peterson, Flavell and Barratt, Plant Disease Reporter 58:777, 1974) has been examined in mitochondrial preparations from both N and T callus. Despite difficulties in extracting a high mitochondrial malate dehydrogenase activity from callus, we find that oxidation is inhibited in a T-callus preparation and not in an N-callus preparation. That a differential 'cytoplasmic' sensitivity to T-toxin is expressed in callus means that it should be possible to use undifferentiated maize tissue (or a single-cell system) to select for genetic changes, whether nuclear or cytoplasmic, which affect this expression.

2) Selection of T-toxin resistance in T-callus: We are selecting T cytoplasm callus lines resistant to T-toxin without mutagenic treatment. Callus was initially obtained according to the method of Green and Phillips (Crop Sci. 15:417, 1975) in the presence of 2 mg/l 2,4-D. After 3 subcultures on medium containing 2 ml/l of a purified T-toxin extract, some of the T-calluses are showing a remarkable resistance to T-toxin, growing almost as well as N-callus. T-callus not previously exposed to T-toxin normally will not grow at all at this sublethal toxin concentration. These results substantially confirm the findings of Gengenbach and Green (1975, op. cit.). Experiments are under way to determine whether this toxin resistance reflects a permanent genetic change or is the result of a physiological adaptation by the callus to the presence of toxin in the medium.

We have obtained a similar pattern of resistance development in tobacco cultures by the selection of callus on medium containing sublethal levels of cycloheximide or 1-amino-2-nitrocyclopentane carboxylic acid (ANCPA). Further investigations have suggested that the high degree of resistance to these toxins developed in culture is not due to permanent genetic changes.

3) Development of a haploid T-callus cell line: We are working in this area to test the feasibility of selecting for nuclear genetic changes affecting the expression of T-toxin sensitivity in T-callus. Two possible approaches have been considered:

Anther culture. Success in generating haploids of cytoplasmically male sterile material by means of anther culture has been reported for wheat (E. Picard and J. de Buyser, CR Acad. Sci. Ser. D 277:1463, 1973) and tobacco (Tsikov et al., Dokl. Bolg. Akad. Nauk 27:1727, 1974). Systems for maize anther culture are not as well defined, although the Chinese workers are beginning to have some real success (Acta Botanica Sinica 19:89, 1977). We have plated out some 35,000 maize anthers from various maize hybrids and inbreds (including T cms material) on several types of medium. So far we have produced callus only from anthers of maize hybrid MV 201, plated on a modified MS medium containing 12-18% sucrose, 2.5 mg/l 2,4-D, 1.0 mg/l 6-BAP and 0.5 mg/l 1-NAA. One of the calluses obtained in this way has continued to proliferate away from the anther on a modified Green and Phillips medium for three months. The callus is rather reminiscent of callus obtained from small immature embryos and is producing shoots and roots. Experiments are in progress to confirm the pollen origin of this callus.

Spontaneous androgenesis/parthenogenesis. Means are not at our disposal to make use of the ig mutation or 'stock 6' to generate immature haploid embryos for culture. We are, however, trying to initiate callus from some mature haploid seed kindly supplied by Prof. Peter Peterson. Callus has been initiated from mature seed in this laboratory and has been maintained for over a year in culture, although it is generally slow growing and has so far differentiated to produce roots only.

With regard to the initiation of maize callus from immature embryos, our experience stresses the need to use embryos less than 2 mm long to generate callus successfully according to the method of Green and Phillips (Crop Sci. 15:417, 1975). Larger embryos tend to give results similar to those reported by Rafaill (MNL 50:84). We also find "the number of days post-pollination" as a misleading parameter for gauging embryo development. Owing to the vagaries of the British climate, our American seed took 30-35 days from pollination to produce embryos 1.0 mm long!

Dick Brettell and David Ingram

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