We have proposed construction of several fusion genes and test them individually or in combination for their effects on improvement of transient expression or stable incorporation of T-DNA genes in citrus tissues. We have shown that the use of some genes we constructed can lead to 2- to 4-fold increases in transient expression activity of T-DNA genes or stable transformation efficiency using juvenile and mature citrus tissues as explants. However, our results are not always consistent in mature shoot tissues of Washington Navel and Valencia oranges. We believe that physiological conditions of explants may play a key role for the observed variations and inconsistence. We have also combined some of these genes and tested their effects. We did not see significant synergistic effects on transient expression or stable transformation efficiencies in mature citrus tissues of Washington Navel and Valencia oranges. We also used a Kn1 gene along with other genes in mature tissues of Washington Navel and Valencia oranges. Little synergistic effects in promoting transformation efficiencies have been observed. Some of the genes we identified may be of useful in improving transformation efficiencies of mature citrus tissues.
We have used root specific auxin biosynthetic gene, root specific-CKX fusion gene (cytokinin oxidase gene), a combination of both the root-specific iaaM and root-specific CKX genes, and other genes produce transgenic Carrizo citrange and HLB-tolerant rootstock US-897. We have produced a large number of independent transgenic citrus plant lines, respectively. Some of these plants have significant increases in root number and root length compared to the WT plants, consistent with our tobacco plant results. We have observed that adventitious root initiation of some of these transgenic citrus plants is faster and enhanced. Our results have also showed that the auxin overproducing plants have improved success rates of grafting. Enhanced root growth and biomass of rootstock may enhance citrus trees’ tolerance to abiotic and biotic stresses. Improved grafting success is desirable for commercial citrus planting because most citrus trees used for fruit production require grafting. We are currently using transgene free genome editing technologies to create the same traits in citrus rootstock.
We have tested a large number of chemical regulators on transient gene expression in mature citrus tissues. Among these chemicals and their combinations, we have identified that some can enhance Agrobacterium-mediated transient gene expression levels. Enhanced transient gene expression activity is important for producing non-transgenic gene-edited mutant citrus plants from mature explants. We have developed a method to produce non-transgenic genome edited mutants without going through sexual reproduction (Chen et al., 2018). We have also observed that that some chemical manipulations can promote shoot regeneration and stable transformation efficiencies of mature citrus tissues.
We have published two articles and two more are under preparation.