Wusheng Liu

The overall goal of this project is to develop effective management practices to minimize the biological risks associated with the newly developed genetically engineered (GE) tomato. Tomato is a predominantly self-fertilizing crop and also a facultative outcrossing species with a substantial pollen-mediated gene flow (PMGF) rate in the field. The adoption of genetic engineering significantly contributed to crop improvement. The inclusion of GE tomato into the agricultural landscape carries high risks related to the introduction of transgenes into related agricultural and wild relatives. Thus, effective biological containment technologies are needed to prevent PMGF from GE to non-GE tomato. We proposed to engineer tomato to produce CRISPR pollen which will degrade endosperm development in the non-GE plants but not the GE plants which will express a mutated endosperm lethality gene. It is expected that the engineered CRISPR pollen will not interfere with seed production in tomato. By doing this, the newly-developed GE tomato could be used as the background source for genetic engineering of any traits for sustainability. Our goals are intimately tied with the management practices to minimize environmental risks of the newly developed GE tomato, and fits squarely into the BRAG objectives. We expect our results will help develop regulatory and best management practices for future GE tomato plantings. We also expect we will develop an efficient biological containment strategy that is applicable to other GE crops currently or soon-to-be incorporated into non-GE agricultural settings.

Our goal is to make stable transgenic lines in strawberry (and tomato and lettuce) for gene editing and trait improvement.

There is great interest in further developing hemp as a valuable specialty crop. Hemp is morphologically identical to marijuana; the only difference is that marijuana is grown for its primary psychoactive chemical �����������9-tetrahydrocannabinol (i.e., THC), while hemp is grown for seed/fiber or cannabidiols (CBDs) that are not psychoactive but attenuate the psychoactive effect of THC. Hemp farmers face high risks of destroying their hemp plants from their farms if they accidently grow hemp with THC content higher than the 0.3% legal limit. It is one of the highest priorities for the hemp industry to significantly reduce the hemp THC content. The proposed research aims to genetically enhance industrial Hemp by using a combination of breeding and biotechnological approaches to improve CBD production and reduce THC content. Firstly we will use conventional breeding strategies to develop and assess new tetraploid and triploid cultivars with the intent of developing seedless forms with enhanced CBD production. Secondly we will use biotechnological approaches to develop regeneration and agrobacterium transformation protocols for gene editing approaches to reduce THC content.

The overall goal of this project is to develop bioengineering pipelines for specific landscape crops with high economic value in order to provide the most immediate impacts for crop improvement. These systems will have broad utility and provide a foundation for developing improved crops with enhanced disease and insect resistance, non-invasiveness, unique phenotypes and greater commercial potential. Transgene-free end products will be realized by segregating out transgenes with selective breeding or with the use of DNA-free delivery systems.

Crop gene editing relies on the DNA delivery of Cas9/gRNA via crop transformation, making the end product GMO which is undesirable for marketing. The overall goal of this project is to develop a novel transgene-free method for gene editing to significantly accelerate breeding methods for tomato. Our goal is to develop a pollen-mediated protein delivery system for the Cas9/gRNA delivery into tomato for gene editing. We anticipate that success in the proposed research will contribute greatly to crop improvement, food security, and agricultural sustainability by providing more resilient, diverse, and profitable crops.

Both DNA and protein delivery of the CRISPR-Cas9 system have been used in horticultural crops for gene editing, which are involved in viral sequences (i.e., Agrobacterium) or plant regeneration (i.e., PEG or biolistic bombardment). Chemical delivery using cationic lipids or polymers is a promising alternative due to its advantages of ease of use, low cost and great safety. Magnetic nanoparticles (MNPs) contain magnetic iron oxide cores that are cationic polyethylenimine-coated, can form magnetic lipoplexes with DNA or RNA, and deliver DNA or RNA into cells under the condition of an external magnetic field. To date, MNPs have been used successfully for direct pollen-mediated delivery (i.e., pollen magnetofection) of plasmids into cotton cells for the generation of transgenic seeds without regeneration. However, pollen magnetofection has not been used for Cas9/gRNA delivery into plants yet. Here, we proposed to conduct proof-of-concept research on using MNPs to deliver the DNA, mRNA, or protein version of Cas9 into greenhouse-grown tomato and hemp for gene editing.

Rapid plant growth development after transformation is key to increase the efficiency and reduce time during crop breeding. At NC State University, the Control Environment Research Lab (ceh.cals.ncsu.edu) is focused on maximizing plant growth, morphology, quality, and efficiency by developing crop-specific and growth stage specific environmental strategies. The CE research group is currently working on other environmental strategies to increase plant-based nutraceuticals, propagation of berry crops, leafy green breeding for CE systems, dynamic spectral control, among others. The expertise of the CE research team will be used to improve the production efficiency of ex-plants selected for this project. In collaboration with BASF, the objective of this proposal is to increase the production efficiency (initiation, shoot elongation, root establishment) of specific ex-plants using environmental optimization for each of the growing stages.

We propose to assess the effectiveness of this candidate gene in BW resistance in tomato so that it could be used to increase host resistance. We expect that this candidate gene could provide robust BW resistance in susceptible tomato genotypes. The enhanced host resistance will be healthy for the grower and environmentally friendly by limiting the need for chemicals. It is also economically feasible as no extra treatments are required after a grower buys the seeds or plants. The enhanced host resistance will also allow us to develop effective and viable IPM approaches together with other methods that can eliminate or reduce the initial inoculation, reduce the effectiveness of initial inocula, delay the onset of disease, and/or slow secondary cycles. The candidate gene could also be introduced to elite tomato genotypes through breeding, genetic engineering or gene editing.

Wusheng Liu������������������s lab at North Carolina State University (NCSU) will analyze confidential transcriptome data from BASF and public sources using up to seven (7) publicly available computational tools. The Liu lab at NCSU will map and annotate detected motifs back to their respective promoters and identify overlapping motif regions (OMRs). Pre-approved OMRs will be cloned into an expression vector for activity confirmation using transient expression assay by tobacco leaf agroinfiltration. OMRs that enhance expression of a reporter gene will be subjected to rounds of vector construction followed by expression analysis to identify the minimal motifs that enhances expression. The OMRs and/or motifs will be used individually or in combination for synthetic promoter engineering.

Plant regeneration is highly species- and genotype-dependent, and specialty crop producers have been forced to rely on traditional breeding methods which take 8-10+ years to deliver improved specialty crops. The overall goal of this project is to develop a novel genotype-independent regeneration method to significantly accelerate breeding methods for sweetpotato. Our goal is to dramatically reduce the time required to introduce a new trait or traits into existing cultivars. Shorter breeding times and improved varieties will have a significant impact on a wide range of stakeholders along agricultural supply chain from biotechnology and seed companies to growers to consumers. We anticipate that success in the proposed research will contribute greatly to crop improvement, food security, and agricultural sustainability by providing more resilient, diverse, and profitable crops.