During this past summer, we undertook anther culture studies in Columbia, Missouri. During the course of the summer, we plated about 40,000 anthers from 28 different maize genotypes on 45 variations of media. The results described below pertain to the 20,000 anthers evaluated in North Dakota.
The three basic types of media tested were the N6, the Yu Pei, and Murashige and Skoog (M+S) media. There were 6 variations of M+S, 14 variations of Yu Pei, and 25 variations of the N6 media. All of the media contained 0.5% activated charcoal, and all of the media contained 3% sucrose except for 11 variations of the N6 medium which contained 12% sucrose. This modification turned out to be important since all but one of the 15 plates yielding embryos or callus contained one of the variations of N6 medium containing 12% sucrose. The hormone content of the medium did not appear crucial since 7 of the 11 variations of N6 media containing 12% sucrose were effective including media supplemented only with 0.1 mg of TIBA, with 2,4-D and TIBA, with 2,4-D only, with TIBA and 6 benzylaminopurine, or with kinetin only.
There was a degree of genotype specificity for responding to the media. Among the 14 genotypes tested on N6 media containing 12% sucrose, success was obtained with 7 of them. These were Black Mexican Sweet, 1 dish; Illinois Hi Oil x Black Mexican Sweet (F1), 2 dishes; W23 x Black Mexican Sweet (F1), 1 dish; Illinois Hi Oil x Longfellow flint (F1), 3 dishes; Golden 113, 3 dishes; Lai Pin Pai x Golden 113 (F1), 3 dishes; (Lai Pin Pai x Golden 113, F1) x Golden 113 (backcross), 2 dishes. It should be noted that the Golden 113 and Lai Pin Pai are genotypes that were found to be well suited for anther culture by the Chinese workers.
Both callus and embryos emerged from inside the cultured anthers after 30 to 45 days in culture. These continued to develop for a while on the 12% sucrose containing medium but were usually transferred to a 3% sucrose containing medium. When this was done, development ceased except in the case of the embryo produced on the kinetin supplemented medium. This embryo germinated and grew into a plant. Pictures of the anther cultures showing callus, embryos and the young plant are shown in Figure 1.
Our success rate of 15 dishes producing embryo or callus out of about 450 dishes total or about 20 anthers responding out of about 20,000 plated may appear discouraging because of the low frequency of success. Actually, we are pleased and encouraged inasmuch as only about one-fourth, or 5,000 anthers were plated on media containing 12% sucrose, an important feature for success. Furthermore, among these 5,000 anthers, about half were from genotypes that are apparently not responsive to anther culturing on the N6 medium, a result in complete agreement with the genotype specificity observed in China. Finally, among the 2,500 anthers plated from responsive genotypes, about one-third were plated on media that produced no positive response. Therefore, a success rate of 20 anthers out of 1,700 or so is close to a 1% success rate which is comparable to that obtained in the Chinese laboratories.
William F. Sheridan, Colette Nitsch and M. G. Neuffer
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