Document Type

Dissertation - Open Access

Award Date

2019

Degree Name

Doctor of Philosophy (PhD)

Department / School

Agronomy, Horticulture, and Plant Science

First Advisor

Xingyou Gu

Keywords

Genetically engineered crop, RNAi, Seed dormancy, Transgene flow, Transgenic mutation, Transgenic rice

Abstract

Transgene flows into wild/weedy relatives may cause ecological and economic problems. Seed dormancy is an adaptive trait that distributes germination over time and promotes persistence of weeds in agroecosystems. Silencing natural genes controlling seed dormancy (SD) could promote germination and reduce weed adaptability. The goal of this project was to develop a transgenic mitigation (TM) technology by linking to a primary transgene with an SD gene-silencing structure, as a tandem construct for transformation, to reduce the risk of gene flow into weed populations. In this research, the Bar (Bialaphos) gene for resistance to the glufosinate herbicide was used as a primary transgene, and inverted repeat sequences (IRS) from the seed dormancy genes SD7-1 or SD12, were used as a mitigating factor to develop the Bar::IRSSD7-1::IRSSD12 construct. The tandem construct was used to transform a white pericarp-colored cultivar that contains non-functional alleles at SD7-1 and SD12. A transgenic T0 plant was crossed with a line of weedy red rice to mimic transgene flow. The weedy rice parent contains functional alleles at SD7-1/Rc and SD12. SD7-1 is identical to Rc for red pericarp and the pleiotropic gene encodes a transcription factor controlling both seed dormancy and red pericarp color. It is expected that the IRSs linked with Bar in the construct could be activated to silence SD7-1/Rc and SD12 by a mechanism of RNA interference (RNAi), resulting in reduced dormancy and change in the pericarp color from red to white in herbicide-resistant plants. Thus, the objectives of this research were: 1) to identify inheritance patterns for the Bar::IRSSD7-1::IRSSD12 transgene and for the traits HR, SD, pericarp color, hull color, awn length/percentage of awned seeds, flowering time and plant height in the F2 population; and 2) to evaluate the silencing efficiency of RNAi on SD7-1/Rc in the advanced (F3 and F4) generations. A population of 288 F2 plants were evaluated for the traits and genotyped with markers distributed on 10 of the 12 chromosomes. Data from the F2 population revealed one copy of the Bar::IRSSD7- 1::IRSSD12 tandem construct segregating in the population and that the phenotypic frequency for red pericarp was greatly reduced, seed dormancy was correlated with the other tested traits except for flowering time, more than two SD (SD7-1 and SD12) genes segregated in the population, the RNAi-mediated silencing effects on both SD7-1 and SD12 were detected, and the silencing efficiency on SD7-1/Rc was about 30%. Seven F3 lines selected from the F2 populations and eight F4 lines selected from two F3 lines were evaluated for seed dormancy and pericarp color to estimate the silencing efficiency on SD7-1/Rc. Data from the F3 and F4 lines confirmed the observation for one copy of the transgene in the generations of segregating populations and demonstrated that the RNAimediated gene silencing efficiency increased to about 90% in the two advanced generations. The silencing efficiency reached 100% in some of the F3 or F4 lines and the results were consistent with transcriptional data of the gene SD7-1/Rc. This research provided evidence that natural genes with a major effect on seed dormancy can be silenced by an RNAi mechanism linked to a primary transgene in hybrids with weeds and the silencing efficiency increased with the generation advancement.

Library of Congress Subject Headings

Transgenic plants.
Seeds -- Dormancy.
Oryza.
Rice.
Weeds.

Format

application/pdf

Number of Pages

168

Publisher

South Dakota State University

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Rights Statement

In Copyright