Development and Optimization of Nicotiana benthamiana Seed Production for Biopharmaceutical Applications

Thursday, July 31, 2014: 3:00 PM
Salon 8 (Rosen Plaza Hotel)
Joey Norikane, PhD , Fraunhofer USA, Newark, DE
Rebecca Snow , Fraunhofer USA - Center for Molecular Biotechnology, Newark, DE
David Wienner , Fraunhofer USA - Center for Molecular Biotechnology, Newark, DE
Ryan Dayton , Fraunhofer USA - Center for Moecular Biotechnology, Newark, DE
Audio File Recorded Presentation

Central to the plant-based pharmaceutical production are the plants, and more specifically the plants seeds.  Food and Drug Administration (FDA) regulators have multiple questions, e.g., what is the source of the seeds, how are the seeds multiplied, what testing is conducted to ensure the seeds are viable and virus-free, among other questions.  To address these concerns during the scale-up process from the laboratory bench to the pilot plant capacity, Nicotiana benthamiana (USDA PI555478) seeds were acquired from the USDA Nicotiana Germplasm Collection (Raleigh, North Carolina USA).  These seeds were multiplied in a controlled environment, but seed yields and germination rates were low.  The early seed production efforts were resource limited, i.e., space and environment, which impacted seed yield and quality.  Later, a dedicated seed production area was developed with metal halide lighting (500 – 700 µmol m-2 s-1) and temperature control (26 - 28¢ªC).  Nutrient content and irrigation methods were optimized in the seed production area which increased average seed yields from 5.0 ± 1.8 g plant-1 to 48.1 ± 11.6 g plant-1.  Maximum single plant seed harvests increased from 9.4 g plant-1 to 74.6 g plant-1.  Post-harvest processing protocols were developed to ensure seed quality, consistency, and viability, which are particularly important at large scale with automated equipment.  Harvested seeds were sieved and germination tests were conducted according to size.  As the environmental conditions and cultivation methods were optimized, seed size distribution trended towards larger seeds, where the 500, 425, and 355 µm sizes shifted from 3.6, 62.5, and 32.0% to 45.8, 48.4, and 4.6%, respectively.  Larger seed sizes had higher germination rates where seeds from the 500 and 425 µm sieve sizes germinated at a rate of 99.6 and 99.2%, respectively.  Seeds from the 355 µm sieve size germinated at an 84.2% rate and the germination rates for smaller seeds (< 355 µm) were less.  The improvements in seed production environment and cultivation methods have yielded higher quality seeds with satisfactory germination rates.  These are two critical aspects for seeds supplying the manufacturing of a plant-based biopharmaceutical product.

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