E. L. Sorensen

Direct embryogenesis from single mesophyll protoplasts in alfalfa (Medicago sativa L.).

SummaryWe used a tetraploid clone derived from an anther culture operation of ‘Ladak’ alfalfa to study the pathway of direct embryogenesis from leaf-mesophyll protoplasts. About 72% of the protoplasts divided, and 7% of those produced proembryos. Approximately 38% of the proembryos developed into green embryos, and 33% initiated calluses. Other proembryos dedifferentiated into calluses which later redifferentiated embryos. Sixteen percent of the embryos developed directly into plants, whereas 81% produced plants indirectly via secondary embryos. The remaining 3% of the primary embryos failed to develop into plants. The lowest plating efficiency for direct embryogenesis was 0.3%. The high percentage of direct embryogenesis observed was related to the genetic nature of the clone, low density of liquid medium, low protoplast culture density, and the composition of culture media.

Regeneration of plants from Alfalfa (Medicago sativa L.) Protoplasts by Direct Embryogenesis

Alfalfa plants are usually regenerated from protoplasts with an intervening callus stage (Johnson et al. 1981; Arcioni et al. 1982; Atanassov and Brown 1984; Pezzotti et al. 1984). Because considerable somaclonal variation could occur during callus development and growth (Bingham et al. 1988), direct embryogenesis, whereby the callus stage is bypassed or maintained for a very short period, appears to be advantageous in preserving genetic identity and reducing the time required for the operation, especially for protoplast fusion, microinjection, and direct transfer of specific genes to produce transgenic plants.