Robust Agrobacterium-Mediated Transient Expression in Two Duckweed Species (Lemnaceae) Directed by Non-replicating, Replicating, and Cell-to-Cell Spreading Vectors.

Plant-based transient expression systems have recognized potential for use as rapid and cost-effective alternatives to expression systems based on bacteria, yeast, insect, or mammalian cells. The free-floating aquatic plants of the Lemnaceae family (duckweed) have compact architecture and can be vegetatively propagated on low-cost nutrient solutions in aseptic conditions. These features provide an economically feasible opportunity for duckweed-based production of high-value products via transient expression of recombinant products in fully contained, controlled, aseptic and bio-safe conditions in accordance with the requirements for pharmaceutical manufacturing and environmental biosafety. Here, we demonstrated Agrobacterium-mediated high-yield transient expression of a reporter green fluorescent protein using deconstructed vectors based on potato virus X and sweet potato leaf curl virus, as well as conventional binary vectors, in two representatives of the Lemnaceae (Spirodela polyrhiza and Landoltia punctata). Aseptically cultivated duckweed populations yielded reporter protein accumulation of >1 mg/g fresh biomass, when the protein was expressed from a deconstructed potato virus X-based vector, which is capable of replication and cell-to-cell movement of the replicons in duckweed. The expression efficiency demonstrated here places duckweed among the most efficient host organisms for plant-based transient expression systems, with the additional benefits of easy scale-up and full containment.

Production of cybrids in Brassicaceae species.

This chapter describes a method of cytoplasm transfer within the brassicaceae family through Ca-PEG-mediated protoplast fusion. The method includes a protocol of nonmutagenic albinism induction based on spectinomycin-induced plastid ribosome deficiency (PRD). The proposed application of spectinomycin-mediated albinism allows speeding up creation of albino lines, as well as of hybrid production with substituted cytoplasm. According to described method cybrids between Orychophragmus violaceus and Brassica napus, O. violaceus and Lesquerella fendleri have been produced. Methods of further molecular analysis are also presented. The time-scale and reliability of described methods are indicated.