Evaluating the Development and Potential Ecological Impact of Genetically Engineered Taraxacum kok-saghyz DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Brian J. Iaffaldano Graduate Program in Horticulture and Crop Science The Ohio State University 2016 Dissertation Committee: Professor Katrina Cornish, Advisor Professor John Cardina Professor David Francis Professor Allison Snow Copyrighted by Brian J. Iaffaldano 2016 Abstract Natural rubber is a biopolymer with irreplaceable properties, necessary in tires, medical devices and many other applications. Nearly all natural rubber production is dependent on a single species, Hevea brasiliensis. Hevea has several disadvantages, including a long life cycle, epidemic diseases, and rising production costs which have led to interest in developing new sources of rubber with similar quality to Hevea. One species that meets this criterion is Taraxacum kok-saghyz (TK), a widely adapted species of dandelion that can produce substantial amounts of rubber in its roots in an annual growing period. Shortcomings of TK include an inability to compete with many weeds, resulting in poor establishment and yields. In addition, there is variability in the amount of rubber produced, plant vigor, and seed establishment. In order to address these shortcomings, genetic engineering or breeding may be used to introduce herbicide resistance and allocate more resources to rubber production. We have demonstrated stable transformation in Taraxacum species using Agrobacterium rhizogenes to introduce genes of interest as well has hairy root phenotypes. Inoculated roots were subjected to selection by kanamycin and glufosinate and allowed to regenerate into plantlets without any hormonal treatments or additional manipulations. Transformed plants were reproductively viable and genes of interest ii segregated independently. The natural ability of dandelions to regenerate entire plants from root fragments allows this method to be faster and simpler than methods applied to many commonly transformed species. This method was then leveraged to implement CRISPR/Cas genome editing to potentially improve rubber production. The potential release of improved TK, with novel traits raises questions of potential gene flow into the ubiquitous weedy relative of TK, the Common Dandelion, T. officinale (TO). In order to evaluate this risk, genomic resources were developed to discriminate the nuclear and chloroplast genomes of TK and TO. Furthermore, assemblies of chloroplast genomes of TK and TO were generated. These resources were used to conduct gene flow studies and may also be used to improve and understand TK genetics. In order to understand the potential for gene flow between TK and TO, Flow Cytometry Seed Screening (FCSS) was used to characterize the genome sizes of a global collection of TO. As diploid TO plants are outcrossing sexually reproducing and triploid TO plants are obligate apomicts, genome size estimates can be used to inform the potential for hybridization. All TO screened in North America proved to be apomictic triploids, while diploids where detected as a minority in regions of Central Europe, where they have previously been described. Diploid TO was receptive to TK pollen and readily produced viable, self-incompatible progeny. Triploid TO exhibited obligate apomixis, where clonal seed is produced regardless of pollination, and this phenomenon may preclude pollination by TK. iii While apomixis may limit pollen mediated gene flow, TK may still be receptive to pollen produced by TO. In controlled crosses, TK pollinated by triploid TO produced low seed set; however, 23% of this seed was a result of hybridization. Some of these hybrids demonstrated the inheritance of apomixis and were able to produce viable seed, while those that were not apomictic were sterile. In fitness experiments hybrids produced seed more readily than TO, but had lower biomass after overwintering. In outdoor seed production areas heavily contaminated with TO, seed produced was screened for hybrids over a three year period using phenotypic screening followed by genetic verification. Hybridization of TK by TO was only observed in these areas during one of these years, where bees were introduced, using bee hives, to augment pollination. The estimated frequency of hybridization during this period was 0.001%. Overall, TK can be pollinated by triploid TO, which is the most abundant cytotype. This hybridization may be rare under field conditions due to factors such as interspecific pollen competition. Diploid TO can readily be pollinated by TK, indicating that the potential for gene flow and introgression will be greater in areas with diploid TO, such as Central Europe, than in areas with only triploid TO, such as North America. While apomixis may greatly limit or entirely preclude pollen mediated gene flow between genetically engineered TK and apomictic TO, tools are needed in order to quantify this potential. Accordingly, TK germplasm possessing readily scorable transgenic traits was created for this purpose. Traits such as kanamycin resistance, glufosinate resistance and the expression of florescent proteins and the transcription iv factor IbMYB that colors tissue purple, were introduced into TK, in order to determine if rare pollen mediated hybridization events between TK and TO may occur. Collectively, this research provides a toolkit to develop genetically engineered TK and also understand the potential ecological impact of biotech TK. While the potential for hybridization exists, the ability of novel traits to fully introgress within TO may be limited. However, more work is needed to fully understand the longevity of apomictic hybrid lineages in natural settings. While genetic engineering may contribute novel traits to Taraxacum species, new tools such as CRISPR/Cas offer the opportunity to accelerate the domestication of new crops, such as TK and may be used to further minimize ecological impact. v Acknowledgments I would like to thank my ever enthusiastic advisor, Katrina Cornish, as well as the rest of my committee, John Cardina, David Francis and Allison Snow. I would also like to thank my lab members who contributed to an environment where this research could be conducted. This work wouldn’t have been possible without significant contributions from lab members: Wenshuang Xie, Xiaofeng Zhuang and Yingxiao Zhang. I also appreciate biotechnology advice and bacterial strains which were provided from the labs of John Finer and Chris Taylor. I am grateful to the faculty and staff of MCIC who facilitated many aspects of this work. Lastly, I am appreciative of CAPS and PENRA for financially supporting this research. vi Vita 2007………………………………………..Connetquot High School 2011………………………………………..B.S. Biology, Stony Brook University 2011 to Present…………………………….Graduate Fellow Department of Horticulture and Crop Science The Ohio State University Publications Zhang, Y.1, Iaffaldano, B.J.1, Xie, W., Blakeslee, J.J., and Cornish, K. (2015). Rapid and hormone-free Agrobacterium rhizogenes-mediated transformation in rubber producing dandelions Taraxacum kok-saghyz and T. brevicorniculatum. Industrial Crops and Products 66, 110–118.1 These authors contributed equally to this work. Fields of Study Major Field: Horticulture and Crop Science vii Table of Contents Abstract ............................................................................................................................... ii Acknowledgments.............................................................................................................. vi Vita .................................................................................................................................... vii Publications ....................................................................................................................... vii Table of Contents ............................................................................................................. viii List of Tables ...................................................................................................................... x List of Figures .................................................................................................................... xi Chapter 1: Introduction ....................................................................................................... 1 1.1 Natural Rubber .......................................................................................................... 1 1.2 Taraxacum kok-saghyz .............................................................................................. 2 1.3 Potential hybridization between TK and wild TO .................................................... 3 1.4 Research outline ........................................................................................................ 6 Chapter 2: Development of Efficient and Accessible Genetic Engineering Protocols for T. kok-saghyz ........................................................................................................................... 8 viii 2.1 Rapid and hormone-free Agrobacterium rhizogenes-mediated transformation in rubber producing dandelions Taraxacum kok-saghyz and T. brevicorniculatum ......... 10 2.2 CRISPR/Cas9 genome editing of rubbr producing dandelion Taraxacum kok- saghyz ...........................................................................................................................