1a.Objectives (from AD-416):
Evaluate potential of myb1A gene constructs (from Vitis vinifera) for use in producing anthocyanin in citrus. If effective, the citrus gene equivalent will be extracted and utilized as a cis-genic selectable marker.
1b.Approach (from AD-416):
Use two constructs obtained from the collaborator in Agrobacterium mediated transformation of citrus and tobacco. Regenerate shoots and assess for presence of myb1A and evaluate gene expression. If promising plants will be grown out for further assessment.
This project is related to Objective 1: Create new genetic combinations of citrus, Objective 2: Screen germplasm for important traits and select superior individuals, Sub objective 1 D: Create new scions and rootstocks with potential resistance to huanglongbing (HLB) and citrus bacterial canker (CBC) by genetic transformation.
Anthocyanin production genes were inserted into citrus and expressed. Transformation was compared with and without kanamycin in the shoot regeneration medium. In sum over ten separate experiments 1000 explants each of Carrizo and Hamlin were treated. In all cases, 6-10X more shoots resulted when kanamycin was excluded from the medium, since there was no negative selection against non-transformed shoots, but no shoots with red pigmentation were recovered. When kanamycin was included, 26% of resulting Hamlin shoots (5 out of 19) and 54% of Carrizo shoots (156/289) displayed anthocyanins. Phenotypes recovered included plants with blotchy reddish leaves and plants with cupped leaves. Pigmented Hamlin shoots were very weak and subsequently died. Several deep red Carrizo transformants resulted with potential as research tools and ornamentals. The initial report has been submitted, but further work is underway.