Regenerating cultures of the important New York State inbred W182BN in three different cytoplasms (N, C and S) have been established and maintained since March, 1981. Immature embryos (ca. 1 mm long) were cultured on Murashige-Skoog medium containing 5 mg/l 2,4-D. Scutellar callus from some of these embryos has been subcultured repeatedly on the same medium, either liquid or agar-solidified; the tissue forms small superficial organized areas. Reduction or elimination of the 2,4-D leads to development of compact white structures. Continued growth into plantlets is promoted by increase of the sucrose level to 10%. Morphogenesis appears to be via an embryogenic pathway. Further growth of leaves and roots proceeds on media containing decreased levels of salts and sucrose.
Several dozen plants from each cytoplasm have been transferred from culture to vermiculite and then to soil, and grown to maturity under greenhouse conditions. A wide range of morphology has been observed, from essentially normal phenotype to greatly reduced form with only a few leaves before development of a terminal ear shoot. Tassels that developed usually contained some silks as well. No shifts from male-sterile to male-fertile tassels (as have been seen in some plants regenerated from T cytoplasm scutellar cultures) were seen in the plants regenerated from W182BN cms-C and cms-S cultures, but the generally poor tassel development makes ratings difficult. Mitochondrial DNA from leaves of plants regenerated from S cytoplasm embryos is being analyzed to see whether the characteristic S-1 and S-2 plasmids are still present.
Many of the plants regenerated from N, C and S cultures have successfully been pollinated with pollen from seed-grown W182BN plants. Study of the progeny of regenerated material is in progress; it should help clarify whether morphological changes seen in the plants obtained directly from long-term cultures are ephemeral physiological ones induced by culture conditions, or true nuclear or cytoplasmic mutations.
Elizabeth D. Earle
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