Cell Penetrating Peptides for Citrus Genetic Improvement – 752

Cell Penetrating Peptides for Citrus Genetic Improvement - 752

Report Date: 02/05/2016
Project: 572   Year: 2015
Category: Horticultural & Management
Author: Gloria Moore
Sponsor: Citrus Research and Development Foundation

This quarter, using Cas9m4, with conjugated activation or repression domains, we intended to modify the expression of citrus proteins responsible for regulating flowering, namely TERMINAL FLOWER-1 (TFL), in order to reduce juvenility. TFL is a repressor of flowering and has been shown to inhibit flowering when overexpressed and to increase flowering when enhanced in Arabidopsis thaliana. We want to down-regulate TFL transiently, so we intend to decrease maturation times and do so without the use of transgenic insertion that is deemed unfavorable. For this quarter, we have run two different time course experiments, one with Agrobacterium and the other using cell penetrating peptides (CPPs). The time course experiments were performed on three different citrus varieties: ‘Duncan’ grapefruit for the Agrobacterium experiment and ‘Pineapple’ sweet orange and a trifoliate cultivar ‘812’ for the CPP experiment. For both experiments, they plants were microinjected with the Cas9 repressor of and a sgRNA construct target the 5′ UTR of TFL. Control solutions were included. Sample leaves that had been treated with the experimental or control treatment were removed for a period of up to five days. After which, the leaves were harvested for their RNA and cDNA preparations were made. The real-time analysis run on these samples has been performed and the results are being investigated for accuracy. For the next quarter, we hope to have good, clear results of the experiments above. While perfecting the real-time analysis of the data more experiments will also be run to further test our CRISPR/Cas9 transient expression system in citrus.

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