The objective of this activity is to develop herbicide tolerant lines in dry bean via genome editing technology. Genome editing can be used to modify endogenous genes to improve traits, including resistance to the herbicide imazethapyr (Pursuit) as part of the current study. Genome editing technology consists of three major steps: 1) establishment of cell culture and plant regeneration system, 2) development of plant transformation system, and 3) implementation of genome editing.
We have previously screened a large number of dry bean varieties grown in Canada and identified those with high capacity of regeneration from cell culture. We have successfully developed regeneration in three dry bean varieties including two pinto bean varieties (AAC Burdett and Olathe) and one red kidney bean variety (Montcalm). High levels of regeneration of some other varieties are also possible with method optimization. We are currently testing regeneration efficiency of Apex navy bean.
We have tested different factors and conditions for dry bean plant genetic transformation using previously published protocols as guidelines. Firstly, we are testing a stable transformation system in cotyledon tissues of AAC Burdett pinto bean variety. To date, we have used several hundred explants in transformation trials with Agrobacterium tumefaciens EHA105 strain containing pCAMBIA3301 vector conferring resistance to ammonium glufosinate (also known under the trade names Liberty or Basta), but have not yet identified transformants or tissues showing positive GUS (β-glucuronidase reporter gene) expression. We are working to improve the efficiency of AAC Burdett transformation through protocol optimization. As an example, we have optimized the selection parameters using glufosinate ammonium at a high concentration of 1 mg/L. Secondly, as a proof-of-concept we have performed transformation in AAC Burdett hairy root system (using Agrobacterium rhizogenes K599 strain with pCAMBIA3301) and have shown positive GUS expression. Since the hairy root transformation efficiency is currently low, we are optimizing the method in order to improve this efficiency.
We have analyzed dry bean ALS genes and identified locations within the genes which, if mutated, would cause resistance to herbicide Pursuit. Using this information, we developed genome editing constructs via cloning the ALS1 expression cassette into pMDC99 plant expression vector. We are currently assembling the ALS2 construct. We will be delivering these constructs into cell culture for editing ALS genes to confer herbicide resistant trait. Once the transformation efficiency is improved, we will be transforming the explants using the developed genome editing constructs targeting ALS1 or ALS2 via CRISPR/Cas9 platform.
In a separate mutagenesis approach, we have developed chemically mutagenized (using ethyl methanesulfonate or EMS) populations of 150,000 M2 Apex navy bean seeds to screen for herbicide resistance. We will be testing another mutagen (N-nitroso-N-methylurea or NMU) as it appears to result in slightly higher rates of the desired ALS mutation. Apex seeds will be mutagenized using NMU by first testing this protocol in a greenhouse experiment followed by field testing. Field conditions for herbicide selection have been identified at eight times the spray rate of Pursuit compared to recommendation for soybean providing high efficiency in identifying herbicide resistant lines.
Project lead: Dr. Frederic Marsolais (519) 457-1470 Ext 311 firstname.lastname@example.org