- Vegetables: greens (lettuces)
- Crop Production: crop improvement and selection, plant breeding and genetics, heat stress resistance, biotechnology
In the coming decades, the demands on global agriculture are expected to escalate from the increase in population and other challenges. The rapid decrease in arable land due to urban expansion and land degradation, and the adverse effects of climate change aggravate worldwide food shortages. Climate change imposes severe crop production limitations, especially for cool-season crops like lettuce in the Southern States. Lettuce quality is extremely susceptible to high temperatures, which restrict their growing areas to only California and Arizona in the U.S. This limitation poses a high risk to the lettuce supply chain as climate change continues to threaten lettuce production. By providing strategies to improve heat tolerance in lettuce, growers can expand lettuce growing seasons and areas, which will alleviate the burden and risk of its supply chain from two western states. Development of heat-tolerant lettuce is highly hampered by the unavailability of ideal breeding materials; therefore we propose the following strategies to enhance sustainable production of lettuce under the changing demographic and climatic situations: 1) suppress bolting under high temperature with foliar delivery of small interfering RNAs (siRNAs) particles, which is a NON-GMO approach; 2) develop heat-tolerant lettuce variety through consumer-friendly biotechnology. The outcome may increase lettuce production to provide more people with higher quality lettuce, reduce greenhouse gas, and reduce water consumption.
Project objectives from proposal:
Objective 1. Evaluate foliar delivery of siRNA particles for suppressing bolting under high temperatures
RNA interference (RNAi) is a commonly used biotechnology tool to inactivate gene function by degrading their transcripts, thus leading to an expected phenotype. Direct foliar application of naked double-stranded RNA (dsRNA) may efficiently penetrate the plant cells and activate RNAi. A recent report indicated a substantial increase in the effectiveness of gene silencing when spraying plants directly with methylated dsRNA-derived small interfering RNA (siRNA) . The external application of siRNA on the plant’s surface is short-lived and temporarily changes the plant’s internal mechanisms for protection against specific stimuli within a certain period. Successful application of the siRNA system may extend lettuce production time and increase productivity in many southern states. Thus, we will be evaluating a delivery platform utilizing siRNAs methylated at 3’ ends for systemic silencing of miR172 in lettuce to prevent bolting. It is expected that in vitro-synthesized siRNAs to be rapid and reproducible resulting in stable particles that can be applied via canopy spraying in ambient conditions. The miR172-siRNA will be sprayed on Salinas lettuce leaves grown in the field using a hand-held sprayer and bolting time will be recorded. Additionally, lettuce yield will also be measured to determine the effects of miR172-siRNA on lettuce production.
Objective 2. Develop heat tolerant lettuce through consumer-friendly CRISPR genome editing
We will utilize CRISPR/Cas9 genome editing technology to induce double-stranded breaks and create knockout mutations within the miR172 gene which disrupt their functions. Gene-edited lettuce plants will be generated from genetic transformation and plant tissue culture regeneration. The optimized genome editing system in our lab has gene editing efficiency of up to 40 percent with 40-50 transformed plants in the first generation . Thus, it is expected that lines carrying a mutation in miR172 will be identified in T0 plants through sequencing. Segregated homozygous gene-edited lines without Cas9 will be grown in the greenhouse and the seeds will be collected for high-temperature treatments. We expected that the miR172 lettuce mutant will display increased tolerance to high temperature when compared to the wild type. MiR172 mutant and wild-type Salinas seedlings will be subjected to high day/night temperature and measurements such as ion leakage, chlorophyll fluorescence (Fv/Fm), and reactive oxygen species (ROS) will be analyzed to determine heat tolerance between the mutants and the wild type. Additionally, fresh/dry weight and bolting time will also be recorded and compared between the gene-edited plants and wild-type Salinas. Lines that showed high potential for heat tolerance will be propagated for future characterization and field trials.