Project Number: 6034-22320-007-04-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2020
End Date: Jun 30, 2021
1. Utilize a gene promotor from Caladium flowers to boost floral fragrance production in cut flowers. 2. Prospect coriander genotypes for novel floral fragrance compounds to increase attraction to biological control agents.
In response to Objective 1: We will clone three kilobases of the Caladium THI4 promoter and will construct a traditional transformation vector with this promoter in tandem to the petunia Phenylalanine Reductase (PAR) coding sequence, which will be codon optimized to eliminate any co-suppression effects. The PAR enzyme functions to produce the 2-phenylethanol (rose oil) volatile molecule from phenylacetaldehyde and has been well characterized in several plant systems.The well-established model system Petunia x hybrida cv ‘Mitchell Diploid’) will then be transformed with the THI4 promoter-PAR coding sequence vector along with a ‘control’ vector containing a constitutive promoter (35S) in tandem to petunia PAR. The hypothesis here is that the Caladium THI4 promoter will be flower specific and many times stronger than even a 35S promoter based on flower specificity and tremendous upregulation demonstrated in Caladium. This work will be a proof of concept, which will give merit to the use of the THI4 promoter sequence in the development of transgenic plants with CRISPR/Cas technology. Any time metabolic engineering is proposed it is always a gamble that the overall metabolism of the plant system will cooperate. In this case, we are very confident for the petunia system because treatments of red wavelengths of light can double the emission of rose oil (Colquhoun et al., 2013), overexpression of a tomato PAR doubles the production of rose oil (Tieman et al., 2007), and current work in our lab has demonstrated that both temperature and red light treatment can triple the production of rose oil. Therefore, the ability to ‘steal’ the shared substrate of phenylalanine has been demonstrated in various ways using the established metabolic model of petunia flowers. The long-term project of using CRISPR/Cas to insert the THI4 promoter sequence in front of the PAR genic sequence in both petunia and rose varieties will be an ongoing process. Admittedly a difficult process, but there are many documented cases in the literature of using CRISPR/Cas for large sequence insertion in a directed manner. Additionally, Prong 2 of this proposal will provide new or novel floral volatiles, which can then be characterized for biosynthesis. The corresponding coding sequences responsible for specific volatile production can then be cloned and inserted into the CRISPR/Cas pipeline for petunia and rose. In response to Objective 2: Because of the apparent importance of floral fragrance in dictating the composition and magnitude of beneficial insect assemblages, identifying the floral fragrance composition of different coriander accessions will be a central focus of the proposed study. As a first step, we will measure the abundance and diversity of beneficial insects on different accessions of coriander at different seasons to identify the accessions that are most promising with respect to attracting beneficial insects.