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Gunadi, Andika [1], Van Eck, Joyce [2], Van Eck, Joyce [2], Li, Fay-Wei [3].

Improved tissue culture medium for accelerating gametophyte growth of model hornwort Anthoceros agrestis.

Represented by the model species Anthoceros agrestis, hornworts are an understudied lineage of bryophytes having critical traits for elucidating the evolution of land plants. The thalloid hornwort gametophyte phase has a remarkable ability to recover and proliferate following mechanical wounding and fragmentation, making it a suitable starting material for both in vitro propagation and genetic modifications. Recently, A. agrestis gametophyte tissues have been successfully transformed using Agrobacterium- and Biolistics-mediated transformation. However, efficiencies of recovering stably transformed plants remain low in large part due to poor tissue recovery. In this study, we evaluated the contributions of several culture medium addenda on the axenic growth of A. agrestis (Oxford strain) gametophytes to identify external factors that influence the gametophyte development and to augment its growth. A streamlined growth assay using time-lapse image analysis was developed for rapidly quantifying and comparing tissue development spanning four weeks of culture on solidified medium. For each medium variant, tissue growth was estimated based on averaging the two-dimensional area of green tissue (thalli) following trainable image segmentation over four petri dishes (replicates) containing equal amounts of starting material. Significant phenotypic effects including gametophyte tissue survival, tissue color, patterns and speed of thalli growth, as well as frequency of rhizoid formation were identified following addition of activated charcoal, ammonium nitrogen source, sucrose, 2-(N-morpholino)ethanesulfonic acid (MES) buffering, and growth regulators (6-benzylaminopurine, 2,4-dichlorophenoxyacetic acid, and thidiazuron) to the culture medium. Based on these findings, an improved medium composition and growth regimen for A. agrestis gametophyte tissue was formulated, which increased the average area of thalli by more than five times following three weeks of culture. Notably, this amount of thalli growth was more than double the amount after four weeks of culture on various baseline solidified media (Hatcher's, BCD and KNOP) commonly used for hornwort in vitro culture. The improvements reported here for the development of vigorous starting material and accelerated tissue recovery serve as important foundation for advancing studies relating to gene functional characterization and genome editing in hornworts.

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1 - Boyce Thompson Institute, Boyce Thompson Institute, 533 Tower Rd, Ithaca, New York, 14853, United States
2 - Boyce Thompson Institute, 533 Tower Road, The Boyce Thompson Institute, Ithaca, NY, 14853, United States
3 - Boyce Thompson Institute, 533 Tower Rd, Ithaca, NY, 14853, United States

Tissue Culture
Image Segmentation.

Presentation Type: Oral Paper
Session: PHYS, Physiology
Location: /
Date: Wednesday, July 21st, 2021
Time: 10:30 AM(EDT)
Number: PHYS003
Abstract ID:253
Candidate for Awards:None

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