February 2, 2012
Plants swap chloroplasts via grafts
November 27, 2011
2011 NJ Inventor of the Year
Plastids are semi-autonomous organelles with a relatively small (120-180 kb), highly polyploid genome present in 1,000 to 10,000 copies per cell. The best-known plastids, chloroplasts, convert sunlight into chemical energy. Plastid engineering, in contrast to nuclear engineering, offers higher protein yields, the opportunity to express several genes controlling complex traits, and natural containment that prevents transgene flow via pollen. During the past twenty years we developed protocols for transformation of the tobacco (Nicotiana tabacum) plastid genome, efficient post-transformation excision of the marker genes, and high-level expression of recombinant proteins. Patents based on research from our laboratory cover all aspects of the genetic manipulation of plastid genomes.
Currently, we pursue research in the following areas:
(1) Plastid marker excision using phage recombinases. Marker gene excision is typically accomplished by integration of a recombinase gene in the plant nucleus. The objective of research supported by the National Institute of Food and Agriculture (NIFA) USDA Biotechnology Risk Assessment Grants (BRAG) Program is marker gene excision by recombinases directly exported from Agrobacterium into chloroplasts by Type IV secretion.
(2) Genetic control of plastid inheritance in Medicago truncatula. Plastids in most plants are inherited by the maternal parent only. The genus Medicago is exceptional among flowering plants because some of its species inherit plastids maternally, paternally or biparentally. Currently we are conducting a mode of plastid inheritance survey supported by the NIFA USDA BRAG Program in Medicago truncatula, a diploid species, with the intent to identify the genes that determine maternal, paternal or biparental modes of plastid inheritance. The ultimate goal is to experimentally modify the mode of plastid inheritance to prevent gene flow via pollen. Currently we use plastid DNA polymorphic markers to follow plastid inheritance. Our ongoing efforts on plastid transformation in Medicago truncatula aim at developing a visual marker system to replace PCR-based tracking of plastid DNA in crosses. Plastid transformation in Medicago (alfalfa) will also be useful to provide a vehicle for oral delivery of vaccines.
(3) Plastid engineering in cereal crops. Plastid transformation is routine in tobacco, tomato, potato, soybean and lettuce, but is not reproducible in any of the cereal crops. The objective of this new program is to develop a marker system that enables routine plastome engineering in cereals, particularly maize. In support of this goal we are developing new marker and vector systems, sequence plastid genomes using next generation technologies, study plastid inheritance, and intend to determine the level of containment achievable by plastid localization of transgenes. The latter is particularly important in the wind-pollinated cereal crops, in which pollen is an efficient vehicle for transgene dissemination.
(4) Inter-cellular movements of DNA-containing organelles in plants. Land plants developed highly sophisticated intercellular channels or plasmodesmata, which mediate cell-to-cell movement of nutrients, hormones and information macromolecules. Functional equivalents of plasmodesmata in animal cells are the tunneling nanotubes, which were shown to mediate intercellular trafficking of organelles, including mitochondria. Our objective is to test whether or not organelles move between cells in plants. Our experimental approach is grafting different species of tobacco with plastid, mitochondrial and nuclear markers and selecting for new combinations of the markers to recover rare events.