Date of Award

Spring 2019

Degree Type

Honors Project

School

College of Liberal Arts

First Advisor

Irina Makarevitch

Abstract

Most of today’s maize is cultivated outside its original climate zone, where yields are constrained by the changes in climate. Maize is especially vulnerable to high temperatures and drought stress, both of which negatively affect corn yields. An important strategy to combat this is early sowing, which avoids the effects of summer droughts and high temperatures in many places around the globe. However, maize is a cold sensitive species (Sanghera et al., 2011), making improvement to cold stress crucial for its adaption. The relatively new system CRISPR (Clustered regularly-interspaced short palindromic repeats)/Cas9 offers the potential to study cold-stress related genes through targeted mutagenesis. Using an enzyme called Cas9 and guide RNA, scientists can target a specific region in the genome and make a double stranded break so that any DNA can then be added or removed through DNA repair mechanisms (Jiang, Yang, & Weeks, 2014). Our research project aimed at investigating cold response in maize through development of an application that can be used to analyze the function of plant genes. We designed and implemented CRISPR/Cas9 technology on a model organism Arabidopsis thaliana (rockcrest) to knock out eleven plant genes (from the website database arabidopsis.org) that would produce easily distinguishable phenotypic traits once mutated, or were homologs to potential stress candidates in maize. Using published CRISPR/Cas9 protocols (Čermák et al., 2017) we selected appropriate gRNA regions to create approximately 200 bp out of frame deletions in coding parts of the genes and constructed transformation vectors, using golden gate cloning technology. The vectors at each of the cloning steps were analyzed by restriction digests, colony PCR, and sequencing, demonstrating the success of vector assembly. The T0 plants were transformed with T-DNA transformation vectors for select genes and T1 seeds were harvested and screened for transformants. Results showed the success of using CRISPR/Cas9 to create transgenic plants. Further investigation of mutant response to cold stress conditions is necessary to investigate the involvement of maize candidate genes in controlling cold tolerance in maize.

dc_type

text

dc_publisher

DigitalCommons@Hamline

dc_format

application/pdf

dc_source

Departmental Honors Projects

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