Strategies to obtain stable transgenic plants from non-embryogenic lines: complementation of the nn(1) mutation of the NORK gene in Medicago sativa MN1008

Strategies to introduce genes into non-embryogenic plants for complementation of a mutation are described and tested on tetraploid alfalfa (Medicago sativa). Genes conditioning embryogenic potential, a mutant phenotype, and a gene to complement the mutation can be combined using several different crossing and selection steps. In the successful strategy used here, the M. sativa genotype MnNC-1008(NN) carrying the recessive non-nodulating mutant allele nn(1) was crossed with the highly embryogenic alfalfa line Regen S and embryogenic hybrid individuals were identified from the F1 progeny. After transformation of these hybrids with the wild-type gene (NORK), an F2 generation segregating for the mutation and transgene were produced. Plants homozygous for the mutant allele and carrying the wild-type NORK transgene could form root nodules after inoculation with Sinorhizobium meliloti demonstrating successful complementation of the nn(1) mutation.

Incidence of Stagonospora meliloti and Acrocalymma medicaginis in Lucerne crowns and roots in eastern Australia, and their comparative aggressiveness to Lucerne and inheritance of reaction to S. meliloti in lucerne

The research presented indicates that lucerne crown and root rot caused by Stagonospora meliloti is prevalent in southern New South Wales, whereas Acrocalymma medicaginis is the more commonly observed pathogen in Queensland. Although both pathogens cause reddening of internal root and crown tissue of lucerne, they can be distinguished by symptomatology. S. meliloti causes a diffuse red blotching of the internal tissue accompanied by the presence of an external lesion, whereas A. medicaginis causes red streaking at the extremity of wedge-shaped, dry-rotted tissue. Inoculation of propagules of a susceptible lucerne clone indicated that S. meliloti was the more aggressive pathogen. Although A. medicaginis does not cause leaf disease, there was a strong relationship between the leaf and root reaction of clones to S. meliloti. Inheritance of resistance to S. meliloti in lucerne appeared to be conditioned by a single dominant gene, based on segregations observed in S-1 and F-1 populations, but not in a backcross population from the same family where an excess of susceptible individuals (74% v. expected of 50%) was obtained in a cross of a resistant F-1 individual to the susceptible parent. Resistance appears to be highly heritable, however, and amenable to population improvement by breeding. A conclusion of the research is that breeding for resistance to S. meliloti for lucernes to be grown in southern Australia would appear to be a worthwhile objective. Presently, no highly resistant cultivars exist anywhere in the world.