Recipient Organization
NAPIGEN, INC.
200 POWDER MILL ROAD, EXPERIMENTAL STATION E400/3431
WILMINGTON,DE 19803
Performing Department
(N/A)
Non Technical Summary
The goal of this project is to produce rice plants with stable and long-lived herbicide resistance traits. Weeds can gain herbicide resistance by receiving pollen from crop plants and acquiring resistance genes directly. Such weeds are tremendous problems, especially for rice farmers in the southern US states as well as in Asia, Latin America and Europe. This weedy rice problem is eroding the efficacy of herbicides resulting in significant gain yield losses. Consequently, it is highly desirable to produce crop plants that cannot transmit herbicide resistance through pollen. Such crop plants are not available currently; however, the new technology we develop at NAPIGEN will provide a means for their development. We previously have developed technologies for gene editing of plant mitochondrial DNA, a goal that had eluded plant scientists for decades. Therefore, we can introduce an herbicide resistance gene into the mitochondrial genome. Since mitochondria are maternally inherited, the mitochondrial herbicide resistance gene will not be transmitted by outcrossing to weed plants. Unlike nuclear DNA, plant cells can have hundreds of copies of mitochondrial DNA. A current challenge is ensuring that engineered mitochondrial DNA is the dominant form in a transformed plant cell. In this project, we propose selection methods to increase the proportion of engineered mitochondrial DNA having an herbicide resistance gene derived from rice itself. The resulting plants will not have any exogenous genes from other species, accommodating public preference regarding gene edited products. The technology obtained from this project can be used to develop other novel mitochondrial traits in crop plants such as cytoplasmic male sterility for hybrid seed production, improved nitrogen use efficiency, and enhanced drought tolerance, each of which would increase crop yields under normal as well as stressful conditions worsen by climate change.
Animal Health Component
30%
Research Effort Categories
Basic
0%
Applied
30%
Developmental
70%
Goals / Objectives
The goal of this project is to produce crop plants with stable and long-lived herbicide resistance traits. Weeds can gain herbicide resistance by receiving pollen from crop plants and acquiring resistance genes directly. Such weeds are tremendous problems, especially for rice farmers in the southern US states as well as in Asia, Latin America and Europe. This weedy rice problem is eroding the efficacy of herbicides resulting in significant gain yield losses. Consequently, it is highly desirable to produce crop plants that cannot transmit herbicide resistance through pollen. Such crop plants are not available currently; however, the new technology we develop at NAPIGEN will provide a means for their development. Specific objectives of this Phase I project are the following: (1) Achieve a level of engineered mitochondrial DNA in plant tissue culture cells to allow for stable inheritance of an herbicide resistance gene. We have previously made a breakthrough by developing the first selectable marker genes for plant mitochondrial transformation. We will pursue various strategies to enrich for the proportion of transformed mitochondrial DNA. (2) Confirm herbicide resistance in T0 plants, i.e., plants regenerated from transformed tissue culture cells. The resulting plants contain an herbicide-resistance gene derived from rice itself. They will not have any exogenous genes from other species, accommodating public preference regarding gene edited products.
Project Methods
Over the last several years, we have successfully developed the technology to transform plant mitochondria with exogenous DNA. This was a first in plant science. Part of the development was supported by a USDA SBIR grant (Award No. 2020-33610-31806; Title: Development of mitochondrial transformation in plants toward creating elite hybrid wheat). Since the conclusion of the SBIR project, we have been identifying trait genes of interest to incorporate into the mitochondrial DNA. One such gene is an herbicide-resistant ALS gene, which has enabled us to explore the use of resistance harbored in mitochondria for the development of a new product at NAPIGEN. Our first objective derives from the special features of plant mitochondria. There are several hundred mitochondria present in each cell on average. Many of them appear to carry multiple copies of mtDNA. The main hurdle in mitochondria engineering is to propagate engineered mtDNA that contains a new trait gene to become predominant within the entire mtDNA population in the transformed cells. To achieve the homoplasmic state of engineered mtDNA in transformed plant cells, we have developed two independent strategies. These two strategies are not mutually exclusive, i.e., they could be combined to maximize their effects on homoplasmy. In the first strategy, we will co-transform the mitochondria with a separate gene as a selectable marker. In a second strategy, we will use an enzyme that will modify mitochondrial DNA to faciliate the replication of mitochondrial DNA with a trait gene. After implementation of these two strategies, their effectiveness of enrichment will be assayed by determining the percentage of transformed mitochondrial DNA in the cell, e.g., by use of DNA fragment amplification and deep sequencing. Upon achieving this goal, plants will be regenerated from the tissue culture cells and assayed for their degree of herbicide resistance. The herbicide-resistant ALS gene in our constructs confers a broad spectrum resistance to all five classes of ALS inhibitor herbicides. The extent of resistance to each class of ALS inhibitor herbicides will be determined. The goal will be herbicide resistance comparable to or exceeding that for commercial lines having the herbicide-resistant ALS gene in the nuclear genome.