Alfalfa was one of the first major crop plants to be regenerated from tissue culture. Alfalfa plants have been regenerated from long-term callus cultures,suspension cultures, protoplast cultures and from fused protoplasts. In most cases the mode of rege neration is somatic embryogenesis.
Somatic embryos are bipolar structures arising from sporophytic cells that have no vascular connection with the maternal tissue. In vitro somatic embryogenesis can occur from different types of explants and can be induced by the auxin analogue 2,4 -D in many different species.
The ability of alfalfa cultures to regenerate plants is genetically controlled and occurs with a frequency 0f 1-10% in most cultivars. The variable response within a cultivar reflects the fact that alfalfa is an open-pollinated species and each cultivar is actually a heterogeneous mixture of genotypes. Somatic embryogenesis in tetraploid alfalfa is a qualitative trait under the control of two dominant genes with complementary effects.
Somatic embryogenesis is a potentially useful trait in alfalfa because it could be used to obtain large quantities of parental material for the production of limited synthetic or hybrid seed. Vegetative propagation through somatic embryogenesis would cir cumvent self-incompatibility and inbreeding depression that have hinderd traditional alfalfa improvement. A prereqisite for this approach is a method for recognizing embryogenic genotypes in alfalfa breeding populations. A simple tissue culture test tha t utilizes a single cotyledon is an effective method for screening a population for embryogenic plants but the utilization of a molecular marker that is tightly linked to the trait would simplify and accelerate the identification process. Furthermore, mo lecular markers for each of the two genes would enable plant breeders to track their segregation, individually, in populations without resorting to test crosses. We have identified a molecular marker associated with one of the genes (Yu and Pauls, 1993, Plant Mol. Biol. and 22:269-277)
We are also interested in understanding the molecular events that are
involved in the initiation of somatic embryogenesis. From a cDNA library
differential screen three clones (ASET1, ASET2, ASET3) were shown to be
derived from transcripts that are abund ant at the early stages of somatic
embryogenesis. Two of these clones have been sequenced (EMBL accession
#'s 452525 and 250800). Plant transformation studies with various parts
of these genes are in progress to determine their function in embryo development.