Date of Award

Fall 12-16-2022

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Master of Science (MS)

Department

Botany and Plant Pathology

Advisor

Ek Han Tan

Second Committee Member

Yongjiang Zhang

Third Committee Member

Benjamin King

Abstract

The production of haploids through crossing was first discovered from instances of interspecies crosses in Nicotiana spp. in 1924. Since then, haploid induction crosses has been used to improve plant breeding programs and commercially utilized in a number of crop industries. The generation of doubled haploid instantaneously creates a pure homozygous line, therefore eliminating the need for several generations of inbreeding. There are several pathways to induce haploids in plants: of these methods, centromere-mediated genome elimination pathway engenders the highest haploid induction rate (HIR) with up to 45% in Arabidopsis compared to 15% through phospholipase-mediated haploid induction in maize. Centromere-mediated genome elimination operates through the manipulation of CENH3, a histone H3 variant that is associated with the formation of centromere on chromosomes. Although centromere-mediated genome elimination is highly efficient in inducing haploids, the best haploid inducer in the Arabidopsis system is stunted in its growth and is partially male sterile. In the work presented here, we were able to induce haploids in Arabidopsis using lines that were vigorous and can be crossed as either a male or female. This was achieved using four mutant allele combinations based on two recessive cenh3 alleles: cenh3-1, a null allele and cenh3-2, a missense allele. Our results demonstrated that we could induce haploids while balancing the trade-off between the efficacy to induce haploids and haploid inducer vigour. As CENH3 can be found across all plant species, centromere-mediated genome elimination pathway can be employed by other plants as well. On top of that, the concept that haploids can be induced without the introduction of transgene is attractive for crop industries as it can eliminate the need to go through regulatory bodies for plant breeding programs or crop improvement efforts. Meanwhile, detailed molecular characterization of events that govern haploid induction via centromere-mediated genome elimination is still largely not known. Here, we also provide a framework and potential protocol that would eventually allow expression profiling of early Arabidopsis embryos undergoing centromere-mediated genome elimination.

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