(660h) Genetic and Environmental Manipulation of Somatic Embryo Formation for Plant Improvement

We present our progress in developing technology for plant improvement based on transient genetic manipulation of the developmental process of embryo formation as well as the environmental manipulation of the bioreactor in which the tissue culture takes place.

Somatic embryogenesis (SE) is a way to make superior plants with a desired trait. SE allows the reprogramming of a somatic cell to form a new plant. This extremely complex system is tightly controlled by a network of regulating factors in the plant’s signaling mechanism. This network can be tweaked by environmental changes or more recently by the introduction of transcription factors to activate or repress specific pathways. This manipulation can be done through an agrobacterium mediated gene transfer however, very precise timing and control is necessary to prevent abnormal development or the production of transgenic plants. As a result, transient expression, the introduction of a gene into the plant for a short period of time, is a critical tool that can be used to increase productivity of SE. In addition to the modified genetic environment, an optimal culture environment is essential for the successful implementation of this process at a scaled-up or commercial scale. Plant tissue bioreactors provide a way to control this environment but are often prohibitively expensive. A bioreactor system has been fabricated for plant tissue culture propagation which utilizes low-cost plastic bags combined with gravity driven flows as an approach to achieving scale-up and reducing costs. These temporary immersion bioreactors (TIB) provide for complete media-tissue contacting while minimizing boundary layer mass-transfer resistances and water-logging the plant tissue.

Using cacao (chocolate tree) as a plant model, we are mapping the gene profile expression during the process of SE. Using bioinformatics, transcription factors involved in regulation of SE have been identified and their interaction between these genes are being studied with the goal of developing a dynamic model to better understand the genes responsible for the somatic to embryo transition. Preliminary results of these genes overexpressed in Arabidopsis have shown regenerative capacity resulting in the development of new embryo like structures. These new structures developed abnormalities due to the continued expression of these transcription factors. Using a transient expression strategy however, cacao embryo production was doubled when the transcription factor Babyboom was expressed without stable integration into the genome.

Despite the success observed using transcription factors, the process remains labor intensive at a small scale. In order to scale up the production the process must be automated while providing appropriate growing conditions. Bioreactors provide means to dynamically control the environmental conditions as well as incorporate transient gene expression. To minimize the cost of production we are developing a low cost TIB. The rationale behind the design focuses on the unique needs that come from plant propagation. The slow growth rates of plant embryos require much less stringent operating conditions than typical cell culture allowing the use of low cost disposable plastic bags as vessels allowing this control to be economically feasible. The different gas compositions required by the different stages of plant propagation can easily be adjusted by decoupling of gas and liquid flow, something that gravity driven flow makes possible. This low cost design has introduced minor setbacks including an increased risk of contamination and mechanical failure. These problems have been addressed through iterative design improvement. As a result the TIB has already been shown to improve the growth of N. benthamiana roots under elevated oxygen conditions as well as successfully grow watermelon photoautotrophically when supplemented with carbon dioxide.

Due to the growing prevalence of generic bio-products in the market, the future of bioengineering relies in increasing productivity and decreasing the cost of production. Using a transient gene expression approach to increase yield alongside a low cost TIB system provides for a plant tissue culture propagation system designed to meet these requirements.