An Efficient Protocol for Root Studies in the Common Sunflower Using Composite Plants

Sunflower, Helianthus annuus, is one of the most important oilseed crop in the world. Oil from the seeds is prized for its\u27 exceptional quality and flavor. Despite this, sunflowers have been forced onto marginal lands often on semi-arid and non-fertile soil, due to disease pressures and economics making it critical to produce more resilient lines and varieties to withstand these new stresses. The recent availability of the sunflower genome can allow genome-wide characterization of gene families. Stable transformation protocols, which can be used for characterization studies, have been developed for H. annuus using Agrobacterium tumefaciens, but they produce low efficiency and are time-consuming. For this reason, very few functional studies have been done in the sunflower. Composite plants, produced using Agrobacterium rhizogenes, are plants with transgenic \u27hairy\u27 roots and wild type shoots. Composite plants offer benefits over creating fully transgenic plants, namely time and cost. With plant transformation usually being the rate limiting step for gene functional studies of non-model plants, composite plants can alleviate this bottleneck. Originally produced in vitro, the method developed here outlines a less expensive ex vitro alternative to produce composite sunflowers for study. The critical steps and parameters are outlined and discussed further herein. The testing of more than a dozen genotypes validates the utility and efficiency of this protocol. We also study the performance of three constitutive promoters for the expression of genes of interest. The functional characterization of a root specific transcription factor HaLBD16, via overexpression and RNAi silencing, further reinforce the utility of the system