Page 268 - Plant Canada 2024 Proceeding

PLANT CANADA 2024

[P130] OPTIMIZATION OF A RAPID, SENSITIVE AND HIGH THROUGHPUT ASSAY TO MEASURE
CANOLA PROTOPLAST RESPIRATORY METABOLISM AS A MEANS OF SCREENING
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NANOMATERIAL CYTOTOXICITY. Zhila Osmani , Muhammad Amirul Islam , Feng Wang , and
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Marianna Kulka . Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada; and Quantum
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1,2 1
and Nanotechnologies Research Centre, National Research Council Canada, Edmonton, Alberta,
Canada
Correspondence to: marianna.kulka@nrc-cnrc.gc.ca

Introduction:
Plant genetic engineering using nanomaterials is a versatile method of developing new cultivars for food
production. However, a key challenge is that many nanomaterials are reactive and toxic to cells at
concentrations needed for efficient transformation. Protoplasts, plant cells lacking a cell wall, are often
used in nanomaterial-mediated genetic engineering but due to their fragility, protoplasts are particularly
sensitive to nanomaterial treatment. Therefore, it is necessary to utilize a rapid, sensitive, and high
throughput method to measure the cytotoxicity of various nanomaterials on protoplasts. Conventional
methods of measuring protoplast viability are technically challenging, require specialized instrumentation
and are labour intensive. We have developed a rapid, cost-effective and high throughput assay to
measure respiratory metabolism of protoplasts using the reduction of resazurin, a non-toxic dye that is
converted to highly fluorescent resorufin during cell respiration. Although resazurin has been used
extensively to study cell viability in mammalian cells, its applicability to protoplasts has not been tested.

Methods and Results:
Initial optimization revealed that both glucose and mannitol directly reduce resazurin, and thus W5 buffer
lacking glucose was chosen as the ideal buffer for this assay. Next, we determined whether protoplasts
could reduce resazurin as a sign of their metabolic activity. We isolated protoplasts from hypocotyl canola
(Brassica napus L.) using cellulase (15 mg/ml) and macerozyme (6 mg/ml) for 16 h at 22 C. We
o
incubated healthy protoplasts with resazurin at different cell densities, for varying times and at different
temperatures and determined that the most sensitive parameters for the detection of protoplast viability
occurred when 20,000 cells were incubated with 40 uM of resazurin at room temperature for 3 hours.
Next, we determined the applicability of our assay to nanomaterial cytotoxicity measurement. We
incubated protoplasts with silver nanospheres, silica nanospheres, a cholesteryl-butyrate nanoemulsion,
and lipid nanoparticles and measured protoplast viability using our optimized resazurin protocol. Silver
and silica nanospheres had no effect on protoplast viability even at the highest concentration of 500 ng/µl.
However, the cholesteryl-butyrate nanoemulsion and lipid nanoparticles were toxic to the protoplasts at
500 ng/µl.

Conclusions:
In conclusion, the resazurin assay offers a precise, rapid, and high throughput method for assessing
canola protoplast viability, enabling more accurate analysis of nanomaterial effects on protoplasts. The
versatility of this approach and its scalability make it a valuable tool for nanomaterial cytotoxicity
screening prior to the use of nanomaterials for genetic engineering of protoplasts.

[P131] DEVELOPMENT OF A MOBILE, HIGH-THROUGHPUT, AND LOW-COST IMAGE-BASED
PLANT GROWTH PHENOTYPING SYSTEM. Li’ang Yu , Hayley Sussman , Olga Khmelnitsky , Maryam
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1
1
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Rahmati Ishka , Aparna Srinivasan , Andrew D L Nelson , and Magdalena M Julkowska . Boyce
1 1
Thompson Institute, Cornell University, Ithaca, NY 14850, USA
Correspondence to: an425@cornell.edu; mmj55@cornell.edu

Nondestructive plant phenotyping forms a key technique for unraveling molecular processes underlying
plant development and response to the environment. While the emergence of high-throughput
phenotyping facilities can further our understanding of plant development and stress responses, their high
costs greatly hinder scientific progress. To democratize high-throughput plant phenotyping, we developed
sets of low-cost image- and weight-based devices to monitor plant shoot growth and evapotranspiration.
We paired these devices to a suite of computational pipelines for integrated and straightforward data
analysis. The developed tools were validated for their suitability for large genetic screens by evaluating a
cowpea (Vigna unguiculata) diversity panel for responses to drought stress. The observed natural
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