In updating our previous report (Sheridan, Neuffer and Bendbow, MNL 52:88-90) on the search for auxotrophs by culturing immature embryos of lethal, defective kernel mutants we have two points of interest.
First, we have determined that mutant E1121 and Coe's A342 mutant are allelic to the pro-1 mutant of Gavazzi et al. (Theor. Appl. Genet. 46:339-346, 1975). Furthermore, pro-E1121 and pro-A342 respond identically to immature embryo culturing. They die on basal medium, are rescued on basal medium plus amino acids and basal medium plus proline, but are not rescued by basal medium plus ornithine.
In a recent report from Gavazzi's laboratory, it was suggested that the genetic block in proline synthesis in the pro-1 mutant occurs between D' pyrroline-5-carboxylic acid (P5C) and proline (Racchi, Gavazzi, et al., Pl. Sci. Lett. 13:357 364, 1978). This conclusion is based on the assumption that proline biosynthesis proceeds from glutamate via glutamic d-semialdehyde and D '-pyrroline-5-carboxylic acid and that ornithine is connected to this pathway by conversion to glutamic d -semialdehyde. It has been recently reported, however, that the actual route of proline synthesis in plants may be by conversion of ornithine to proline via a-keto-d-aminovaleric acid and D '-pyrroline-2-carboxylic acid (P2C) (Mestichelli et al., J. Biol. Chem. 254:640-647, 1979).
The lack of rescue of the pro-1 mutant by including P5C in the culture medium (Racchi, Gavazzi et al., 1978) is in agreement with the suggested pathway of Mestichelli et al. (1979); however, since Gavazzi and coworkers also observed that a-amino-d-hydroxyvaleric acid would not rescue the mutant, then the block must occur either between this compound and P2C, or between P2C and proline if, indeed, Mestichelli et al. are correct. It is apparent, therefore, that the critical test will be to attempt to rescue the pro-1 mutant with P2C.
We will be grateful for assistance in locating a source of P2C since it is not commercially available.
Second, among the 102 EMS induced mutants tested to date by embryo culturing, 19 mutants have displayed a superior shoot growth on enriched medium compared to that on basal medium. These mutants are listed in the accompanying table. Among these mutants, there were 11 mutants with a mean shoot growth value on enriched medium of greater than 100 mg. and two mutants displayed that amount of growth on ammonium-free enriched medium. The table also includes the data for the pro-1 mutant and similar results have been obtained for the pro-A342 mutant (not shown). Among these mutants the E1121 mutant has been shown to be proline requiring (see above) and the other mutants are promising mutants for future study.
Promising mutants with greater than 150% growth on
enriched medium compared to basal medium*
|Enriched 1977||Enriched 1978||NH4-free enriched 1978|
|Mean Wt.-mg E/B||Number Shoots E/B||E#||Wt. E/B %||Mean Wt.-mg E/B||Number Shoots E/B||E#||Wt. E/B %||Mean Wt.-mg E/B||Number Shoots E/B|
*The mean fresh weights of the shoots is shown with the value to the right of the diagonal line indicating the weight for shoots grown on basal (B) medium and the value to the left of the diagonal line indicating the weight for shoots grown on enriched (E) medium. The ratio of these weights is presented in the percent columns.
The number of plants harvested and used to determine the fresh weight of shoots is shown in the column labeled "E/B" with the number to the right of the diagonal line indicating the number for basal (B) medium and the number to the left of the diagonal line indicating the number from the enriched (E) medium tested (enriched or NH4-free enriched).
All of the above mutants are lethal and fail to germinate when tested as mature kernels except for 948A, which is uncertain, and E1431 which remains to be tested. The chromosome arm locations are E744, 9L; E873, 9S; E948A, 1L; E1024A, 2L; E1121a, 8; E1308A, 1S; E1417, 10L and pro-1, 8; while the other mutants, although tested, were not uncovered by the 18 B-A translocation stocks used in the test cross.
William F. Sheridan and M. G. Neuffer
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