Degree Type

Thesis

Date of Award

2016

Degree Name

Master of Science

Department

Agronomy

Major

Genetics and Genomics

First Advisor

Kan Wang

Abstract

With the need for increased food production by 70% by 2050, plant genetic transformation plays a key role in the development of crops more tolerant to stress, pests, and disease. Legumes are immensely important in terms of nutritional and economic value in addition to nitrogen fixation capabilities. However, legumes have often proved difficult to transform with genetic engineering. This thesis evaluates conditions affecting genetic transformation of soybean (Glycine max L.) and common bean (Phaseolus vulgaris L.).

Transformation of soybean is now routine but requires much skill and labor. To reduce the labor required for the transformation of soybean, it is important to maximize the transformation efficiency. An important factor for genetic transformation of any crop is the selectable marker system that allows the identification of transformed cells and tissue. The choice of promoters driving selectable marker genes is important for maximizing transformation frequency in Agrobacterium tumefaciens-mediated plant transformation. The cauliflower mosaic virus 35S (CaMV 35S) and nopaline synthase (NOS) promoter and one regulator, tobacco etch viral (TEV) translational enhancer, were tested in this study. The NOS promoter with enhancer resulted in a significantly higher transformation efficiency than NOS without the enhancer and both CaMV 35S constructs. No significant differences were identified between constructs in the average copy number of the bar gene.

Common bean is one of most important sources of dietary protein for human consumption, especially for small landholder farmers in many tropical countries. Transformation of common bean has proved more challenging than other legumes. The development of a robust and reproducible method of common bean transformation is important to allow crop improvements to be made more efficiently than can be achieved with conventional breeding. Ninety-three (93) common bean cultivars were screened for the ability of each cultivar to turn green and continue shoot development using a half seed transformation method. Twenty cultivars with high regeneration frequency were further evaluated for their responses to infection using two Agrobacterium tumefaciens strains using a transient GUS assay. Transient GUS expression varied between cultivars comparing EHA101 and LBA4404, however higher regeneration resulted from EHA101. Eight cultivars were further used for optimizing regeneration conditions. Overall, tissue culture on MS basal media resulted in healthier growth compared to culture on B5 media. Across eight cultivars, cytokinin 6-benzylaminopurine (5 mg/L) was determined to be optimal for shoot induction, gibberellic acid was important for shoot elongation, and auxin indole-3-butyric acid was important for root induction.

Copyright Owner

Amber Lynn Testroet

Language

en

File Format

application/pdf

File Size

279 pages

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