Poster # 301
Rong Di, Jun Qin, and Michael A. Lawton
1. Rutgers, the State University of New Jersey, Department of Plant Biology, New Brunswick, NJ
Corresponding Author: Rong Di, rongdi@sebs.rutgers.edu
Di, Rong
We have established the Barley Genetic Engineering Facility for FHB Research Community since 2022. This facility, supported by the USWBSI, provides services to develop tissue culture protocols for client-based barley cultivars and for optimizing transformation efficiency to produce transgenic barley plants with client-provided constructs. After testing different explants, such as mature barley seeds, which save time needed to grow up plants that can provide immature embryos, or germinated seedlings, which provide meristematic tissues, we found that immature scutellum explants proved to be the optimal material for producing stable transgenic barely plants. Significantly, we observed that
different barley cultivars require different hormone regimes at the initial stages of in vitro culture to produce
regenerable embyogenic calli. We have developed tissue culture protocols for the in vitro regeneration of several
barley cultivars, including the two-rowed spring barley “ND Genesis”, the winter cultivar “Thunder”, and the six-rowed cultivar “Morex”. Using these protocols, we generated barley embryogenic calli within 3 weeks that could then be transformed by either gene gun bombardment or Agrobacterium. After 8 weeks, transformed barley calli could be selected and used to regenerate transgenic plantlets. We have also demonstrated that co-bombardment with our transiently expressing barley HvBBM and HvWUS growth regulators, the barley regeneration rates were improved from single to multiple shoots per embryo. Our improved barley transformation and regeneration protocols have allowed us to transform Morex embryogenic calli with our custom integrative CRISPR-gene editing vector and to produce transgenic Morex plants that are phenotypically normal and able to produce seeds. We have also been able to produce transgenic ND Genesis plants that were transformed with our double-knock out (KO) CRISPR-gene editing vector to mutate the HvHRC gene. In addition, we have produced transgenic ND Genesis with client-provided construct over-expression Arabidopsis lipid transfer protein (LTP). Our optimized barley tissue culture and transformation protocols will aid in the production of transgenic and gene-edited barley plants for the FHB Research Community.
ACKNOWLEDGEMENT AND DISCLAIMER
This material is based upon work partially supported by the U.S. Department of Agriculture, under Project ID #FY22-GD-003. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of
the U.S. Department of Agriculture.
© Copyright
2025 by individual authors. All rights reserved. No part of this abstract or paper publication may be reproduced without prior permission from the applicable author(s).