Performing Department
Agr & Env Sci Deans Office
Non Technical Summary
Genome editing is a revolutionary technology that allow one to make precise changes in a plant's genetic blueprint for agriculture. However, its application to many minor crops like alfalfa, grapevines, lemons, limes and oranges, is limited. The limitation in using gene-editing techniques in these crops is mainly due to the inability of stem cells of these plants to reform whole plants in tissue culture. There are only a very few varieties of alfalfa, oranges, limes, lemons and grapevines for which we are capable of forming whole plants from cells in tissue culture and the few varieties that we have been successful with do so at a very low frequency.We recently discovered that a gene sequence encoding a chimeric protein that comprises a GROWTH REGULATING FACTOR (GRF) and its cofactor GRF INTERACTING FACTOR (GIF) (henceforth GRF-GIF) dramatically increased the ability of wheat stem cells to reform whole plants in tissue culture. When combined with the gene editing system CRISPR-Cas9, we found that this technology also significantly increases the total number of gene-edited plants we can produce using tissue culture. A novel feature of this technology is that although the chimeric gene enhances regeneration in young cells, the chimeric gene is turned off in older tissue so that the plant grows normally to maturity. In wheat, the technology also allows us to make gene edits in plants without the use of a plant selectable marker gene. The GRF-GIF system stimulates the cells that contain it to reform plants on tissue culture plates that lack certain growth factors normally added to the culture medium and which are required for the cells to reform plants in a petri dish. Plant selectable marker genes are used to allow plant cells to grow on compound on which they normally cannot grow. These selective agents allows only cells containing the new gene edit along with the plant selectable marker gene to reform whole plants in the presence of the selective agent. Plant selectable marker genes currently used are often those that confer resistant to antibiotics or herbicides. Although regulatory agencies have approved genetically modified plants containing these genes for commercialization, the public would prefer not to have these genes in their food supply.Promising preliminary results in citrus suggest that this technology may be applicable to multiple plant species. Therefore the overall goal of this proposal is to use this technology to expand the benefits of gene editing to minor crops by increase the efficiency with which plant cells reform plants across multiple varieties of alfalfa, citrus and Vitis vinifera. In addition, we want to use the ability of the GRF-GIF technology to replace antibiotic and herbicide plant cell selection system. Lastly, we want to combine GRF-GIF technology with Genome Editing for each of the crop to enhance the production of gene-edited plants. This project will open the door to multiple minor crops to benefit from the power of genome editing technologies
Animal Health Component
30%
Research Effort Categories
Basic
70%
Applied
30%
Developmental
0%
Goals / Objectives
The overall goal of this proposal is to expand the efficient GRF-GIF regeneration technology from wheat to other minor crops to improve their transformation efficiencies so they can benefit from gene editing technologies. The ability to generate large number of transgenic events combined with genome editing will be a significant improvement in the breeding and research approaches used in these species. For this project, we selected an economically important and diverse group of minor crops that includes alfalfa, citrus and grape. Regeneration frequencies are low and highly genotype dependent for each of these minor crops.Alfalfa (Medicago sativa) is a cross-pollinated, polyploid, perennial legume that is distributed throughout the entire United States, and provides high quality hay and grazing. It is a heterozygous, continually segregating population in which every seedling is genetically different. Therefore, an alfalfa cultivar is not a uniform population but instead a random, interbreeding population of plants. Transformation of alfalfa like wheat relies on somatic embryogenesis. The transformation process is strongly limited by genotype with non-dormant genotypes of alfalfa particularly difficult to transform due to their inability to undergo somatic embryogenesis. Even for alfalfa cultivars that are competent to regenerate, only a very limited number of seedlings within the population exhibit efficient regeneration. Therefore, one needs to identify specific individual seedlings within the segregating population that are competent to regenerate, establish a clone of that seedling in vitro and maintain the clone as a tissue culture stock plant. This limitation significantly complicates the development of transgenic cultivars, since the breeding population must be recreated by incorporating the transgenic allele back into the segregating population. If the GRF-GIF technology allows transformation of more expanded germplasm, more individuals within the segregation populations could be transformed accelerating the reestablishment of the breeding populations.Grapevine (Vitis vinifera): A limited number of grapevine clones have been used for many decades to produce high quality wine. These clones are maintained by vegetative propagation to preserve the intrinsic quality of this material. Therefore, the introduction of new traits into existing Vitis cultivars without altering their essential characters and identity is crucial. Transformation, coupled with genome editing is a powerful method to achieve this goal, but is currently limited by the low transformation efficiency in this species. Table grapes and rootstock are more amenable to transformation, but wine grapes have proven particularly difficult to transform. A major bottleneck in transformation of wine grape is the regeneration of embryos and shoots from transgenic tissue. Indeed, inoculation of wine grape genotypes with an Agrobacterium-engineered to express the DsRed marker, gives high frequencies of transgenic tissues. However, the success in converting this transgenic tissue into embryos and plants has been limited. The transformation process in this species is further complicated because, pro-embryogenic callus must be generated from anther filaments of immature flowers collected in the spring of each year. The frequency of filaments that produce embryogenic callus is extremely variable, ranging from 8.9% in some rootstocks to a 0.7 % for Cabernet and Chardonnay. Even lower percentages are observed for other wine grapes such as Zinfandel and Pinot noir. Furthermore, the transformation process is slow and very laborious requiring 8 to 12 months for generating transgenic plant lines. Currently, there are only a few facilities in the US capable of grape transformation. Like wheat, grape transformation process utilizes embryogenesis. If the GRF4-GIF1 chimera allows for higher regeneration frequencies from callus of wine grapes, it would allow for genetic engineering of these important genotypes. If GRF4-GIF1 transgenic lines could be created for these wine grapes, this would also facilitate the identification of more accessible tissue (e.g. leaves and internodes) to be used as sources for Agrobacterium-mediated transformation.Citrus: Citrus farmers and industry are currently threatened by the devastating effects of the Citrus Greening or Huanglongbing (HLB) disease. Most of the commercial citrus trees in Florida are currently infected, and the insect vector of the disease is now present in California. Therefore, one of the main motivations for the selection of this species is to improve the efficiency and range of citrus genotypes amenable to transformation to accelerate research and development of HLB resistant plants. Currently the most efficient citrus transformation uses juvenile epicotyl tissue from seedlings generated from nucellar embryos. Although plants generated from nucellar embryos generate plants with the identical genotype, those plants require an extended time to fruiting. Low transformation efficiencies have made it difficult to evaluate the efficacy of transgenes against HLB since a large number of independent insertion events must be evaluated to assess the efficacy of each construct.The specific objectives of this proposal are to:1. Increase regeneration efficiency in multiple cultivars of alfalfa, citrus and Vitis vinifera (grape) usingthe GRF-GIF technology.2. Establish a positive selection method that does not require plant pest sequences.3. Combine GRF-GIF technology with Genome Editing systems for each of the three species toenhance the efficiency of recovery of gene-edited plants.This project will open the door to multiple minor crops to benefit from the power of genome editing technologies.
Project Methods
Methods to increase regeneration efficiency in multiple cultivars of alfalfa, citrus and Vitis vinifera using the GRF-GIF technology.GRF-GIF chimeras for each species will be generated by gene synthesis of the closest homologs to wheat GRF4 and GIF1 identified in the phylogenetic analysis . The resulting synthetic genes will be cloned in binary vectors under the control of Arabidopsis UBIQUITIN promoters. In these initial vectors, antibiotic-based selection markers will be included in the same T-DNA. In addition to the regular GRF-GIF chimeras, we are going to generate GRF-GIF versions with silent mutations in the miR396 target site that abolish the miR396-mediated repression, which will be referred hereafter as resistant GRF-GIF or simply rGRF-GIF. The presence of this mutation interferes with the cleavage of the GRF-GIF chimera in tissues with high levels of miR396. Since miR396 levels are particularly high in fully developed tissues, the rGRF-GIF chimera will particularly useful for transformation protocols involving developed tissues (like leaf, root and epicotyl explants). For all three species, we will test the ability of different GRF-GIF chimeras (regular and rGRF-GIF) to improve regeneration efficiency in standard Agrobacterium-based transformation protocols, which are routinely used at the UC Davis Plant Transformation Facility. In all species, regeneration efficiencies (the number of explants regenerating divided by the number of explants plated) will be compared with a similar vector without the GRF-GIF chimera and with the heterologous GRF-GIF chimeras from the other species. Alfalfa transformation will be performed on trifoliate leaflets harvested from in vitro grown individual seedlings of the non-dormant cultivar Highline and the breeding line UC2705. We will collect ten trifoliate leaves from ten independent seedlings and co-cultivate with Agrobacterium strain EHA105 containing alfalfa-specific GRF-GIF and rGRF-GIF chimeras. The empty vector and the heterologous GRF-GIF chimeras from wheat, grape and citrus will be used as controls. The frequency of leaflet regenerating shoots per plant and the frequency of transformable plants will be scored for the GRF-GIF chimeras and controls. For citrus transformation, seeds of the rootstock cultivar "Carrizo", a hybrid of Poncirus trifoliata L. will be surface sterilized and germinated in vitro. Epicotyls from 2-5 week-old etiolated seedlings will be collected, sectioned into internodal pieces and inoculated with Agrobacterium containing a citrus-specific GRF-GIF chimera (regular and rGRF-GIF). Additionally, new experiments will be performed with the citrus-specific chimeras using Citrus sinensis cv.Valencia seeds. Furthermore, we will determine if expressing the GRF-GIF chimeric genes allows regeneration from non-epicotyl tissue. Transgenic "Carrizo" plants expressing the GRF-GIF chimera will be maintained in vitro in large SteriConTM vessels, and leaves and stems of these plants will be tested as explants sources for regeneration. If we observed positive results we will test transformation with the GRF-GIF chimeras using mature tissue from "Carrizo", Valencia orange and navel orange addition. If regeneration from mature tissue is successful, clones of the GRF-GIF plants will be acclimated to soil in order to provide a source of mature explants for citrus transformation in the future. Grape transformation is performed on embryogenic callus that are induced from anther filaments collected from immature flowers which are only available during a small 3 week window in the spring. We hypothesize that a GRF-GIF chimera might increase regeneration efficiency from other easier-to-obtain tissues. We already have transgenic embryos of Thompson Seedless developing from embryogenic callus inoculated with the grape-specific GRF-GIF chimeric, rGRF-GIF chimera and an empty vector as control. This material will allow us to evaluate the ability of the GRF-GIF chimeras to promote regeneration in our standard transformation protocol. Moreover, to test if the GRF-GIF chimeras allow regeneration from other tissues, we will collect root, leaf, internodes and apices tissues from the T0 GRF-GIF and rGRF-GIF plants and compare their regeneration capacity with the same tissues collected from the corresponding controls transformed with an empty vector. If we see increase regeneration efficiency, we will start transformation of most regenerative tissues from Thompson Seedless and wine cultivars Merlot and Chardonnay as well as the grape rootstock Freedom.2. Methods to establish a positive selection protocol that does not require plant pest sequences.Once we have established efficient transformation protocols, we will test the ability of the GR-FGIF and rGRF-GIF chimeras to regenerate transgenic shoots in the absence cytokinins. Transformation experiments will be set varying the concentration of cytokinins in the culture medium as previously done in wheat. If we observe shoot regeneration in the absence of cytokinins, we will select the shoots and transfer them to rooting media. If we do not see regeneration in the absence of cytokinins, we will test increasing levels of cytokinins that are suboptimal for regeneration in the absence of the GRF-GIF chimera. If we see an increased production of shoots in the tissues transformed with the GRF-GIF chimera, we will transfer them to rooting media and determine the proportion of regenerated plants that are transgenic. If we obtain a very low number of false positives, we will repeat the experiment using a similar vector but without the selectable markers.3. Methods to combine GRF-GIF technology with Genome Editing systems to enhance the efficiency of recovery of gene-edited plants.As previously done in wheat, we will combine in the same T-DNA the best GRF-GIF chimera version for each species and the CRISPR-Cas9 cassettes. First, we will test a guide RNA (gRNAs) targeting the PDS (phytoene desaturase) gene in each of the three species. Mutations in this gene generate albino plants, a phenotype that is easy to score. We will also test two additional gRNAs that have been described in the literature for editing citrus and alfalfa. The generated vectors will be transformed by Agrobacterium and the frequency of regenerated shoots will be scored. Then, the independent regenerated T0 shoots will be analyzed for the frequency of editing by scoring the photo-bleaching phenotype of leaves. In the additional gRNA for citrus and alfalfa, we will test the frequency of editing events by using the disruption of restriction sites in the edited region.