The efficiency of wheat biolistic transformation systems strongly depends on the bombardment parameters, the condition of the donor plant, and the plant genotype chosen for the transformation process. This paper analyzes the transformation efficiency of the 129 wheat sister lines generically called 'Bobwhite', originally obtained from the cross 'Aurora'//'Kalyan'/'Bluebird 3'/'Woodpecker'. A number of factors influencing the transformation were examined, such as the ability to produce embryogenic callus, regeneration in selection medium, and overall transformation performance. Of the 129 genotypes evaluated, eight demonstrated transformation efficiencies above 60% (60 independent transgenic events per 100 immature embryos bombarded). Among the eight genotypes identified, we studied agronomic characteristics such as earliness to identify the most adaptable line(s) for different lab conditions. 'Bobwhite' SH 98 26 was identified as a super-transformable wheat line.

The use of transgenic plants in durum wheat breeding makes possible the utilization of genes not previously available in the wheat genetic pool. However, to effectively use this technology, it is necessary to be able to continuously produce large numbers of transgenic plants. This paper describes the development of a routine biolistic transformation technology for durum wheat and its adaptation to the mass production of transgenic plants. The method involves subjecting the donor plant to mild drought stress 1 day before the transformation experiment, subsequent bombardment of the immature embryos harvested from these plants with plasmid DNA, and reduced levels of tissue culture manipulation. The in vitro culture response of bombarded immature embryos and the production of transgenic plants were investigated in three durum wheat cultivars: Mexicali, D5c31YN S74, and D5c31YN S48. In all three genotypes, immature embryos (1.0 mm long) were transformed with a plasmid containing a phosphinothricin-resistant gene (bar) under the control of the maize ubiquitin promoter. The transformation frequency was highest in Mexicali, with a range of 0-6.46% transformation efficiency. The data suggest that variations in transformation were frequently influenced by differences in the genotypes and physiological status of the donor plant, rather than by the efficiency of the particle bombardment procedure.

Diseases adversely affect grain yield of crop plants. Leaf rust is a major disease of wheat. As race-specific resistance breaks down, introduction of newer sources of resistance often from older accessions is necessary. Linkage drag from the donor accessions adversely impacts grain yield. As a result, CIMMYT breeding effort has shifted to using durable resistance, also referred to as non-host resistance, which allows slow rusting but maintains grain yield. Two of the three known genes for durable resistance have been isolated. One of these two, Lr67, is absent in the elite CIMMYT lines. We have initiated efforts to convert the susceptible copy of Lr67 into its resistant form directly in elite CIMMYT lines using gene editing. This would eliminate backcrossing and thus save time as well as eliminate linkage drag that would accompany the resistant copy of the gene if it were to be introgressed from an older accession. As first steps, we have isolated and sequenced the genomic copies from each of the A, B, and D genome of the Lr67 gene from three elite CIMMYT lines and an experimental line. Identification of more than 50 single nucleotide polymorphisms (SNPs) in the open reading frames (ORF) among these lines would be useful in designing the guide RNA molecules with precision. Further, we have streamlined genetic transformation of the elite CIMMYT lines, a prerequisite step for gene editing.