Progress 09/01/23 to 08/31/24

Outputs
Target Audience:1) My research was of particular interest to the whole scientific community working in Plant Synthetic Biology and Genetic engineering. Last year I presented my research program at two international conferences: i) ASPB Plant Biology Conference, Savannah 5th to 9th of August 2023; and ii) PAG Plant and Animal Genome conference, San Diego 12th-17th of January 2024. I also presented my research at the University of Tennessee (UT)/ Tennessee State University (TSU) research summit in Nashville on the 1st of March 2024. This is an annual event that these two Universities organized to facilitate collaborative projects. Furthermore, my research activity and expertise were of particular interest to fundamental scientists looking for potential collaborations. For instance, during the last year I have developed two new important collaborations for my scientific career. A collaboration with Prof. Feng Chen in the Dept. of Plant Sciences, University of Tennessee, and Prof. Meng Chen in the Botany and Plant Sciences Dept. University of California Riverside. 2) My research was of particular interest to my institution. In April 2024, I completed my first year of tenure as Assistant Professor, in plant genetic engineering and gene editing at the Department of Plant Sciences at the University of Tennessee. Being the major expert of genome engineering and gene editing at the University of Tennessee, I have delivered important knowledge and expertise within my laboratory (laboratory meetings) and institution (presentations/meetings). Last year I started a new group of research, that at this time is composed of one Master student, one PhD student, one postdoc, and one undergraduate/technician. As a part of my new role, I mentor and supervise my group of research, as well as advise other faculties if they need expertise in genetic engineering and gene editing techniques. 3) My research was also of particular interest to both undergraduate and graduate students. Last year I presented my research during my courses at the University of Tennessee. In particular, I have delivered knowledge on genetic enginnering of chloroplast genome using novel technologies for producing marker-free transplastomic plants. I have been involved in three courses: i) Plant Sciences Seminar (PLSC 504), ii) Plant Science Plant Propagation (PLSC 330), iii) and in a new course that I developed last year focusing on Engineering and Gene Editing of Plant Cells (PLSC 493). 4) Last year I submitted an invention disclosure entitled "Mini-synplastome hybrid vectors for production of marker-free transplastomic plants" to the UT Research Foundation (office in charge of technology transfer and licensing at UTIA). This invention disclosure describes a new set of transformation vectors developed in this research project that can be used for fast and efficient production of full-marker-free transplastomic plants. The UT Research Foundation has started marketing these novel tools and contact with potential collaborators have been activated. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Improving career at the University of Tennessee A very important professional development achieved during the past two years was to obtain a new position of Assistant Professor in Plant Genetic Engineering and Gene Editing. This position is part of the Agri-research plan to improve the Biotech curriculum in Plant Sciences at UTIA, and to develop an outstanding research program il Plant Biotech and Synthetic Biology. At this time, I am the PI of a new laboratory at the University of Tennessee, where I am leading a group of research composed of one Master and one PhD students, a postdoctoral scientist, and an undergraduate technician. During this time, I had the opportunity to strength many important skills, including writing, communication, supervising, and mentoring skills. Success during this period of time is extremely important to put solid foundations on my future career. Collaborations At this time, I am the major expert in plant genetic engineering in my department. My research program and expertise were of particular interest for finding collaborations with other groups of research in my department and in other institutions. During this year, my expertise in plastid genetic enginnering was very important to develop two new collaborations. The first collaboration is focused on production of secondary metabolites in chloroplasts with Prof. Feng Chen in the Dept. of Plant Sciences, University of Tennessee. The second collaboration is focused on improving tuber biofortification with Prof. Meng Chen from the Botany and Plant Sciences Dept. University of California Riverside. Teaching This research program provided an important opportunity in improving my teaching and mentoring skills. In fact, my research program and expertise were of particular interest to develop new courses for both undergraduate and graduate students. These courses are part of the new Biotech curriculum in my Department of Plant Sciences. In particular, my research in genetic enginnering of chloroplast genome using novel technologies for producing marker-free transplastomic plants was delivered in these courses, for which I was the main instructor: i) Plant Sciences Seminar (PLSC 504), in two semesters, ii) Plant Science Plant Propagation, with my session focused on tissue culture and micropropagation (PLSC 330), iii) Engineering and Gene Editing of Plant Cells (PLSC 493), which is a completely new course at the University of Tennessee. Conferences and seminars Many new opportunities have arisen to present my research program during international conferences and other scientific events. In particular last year I have presented my research in form of poster presentation at the ASPB Plant Biology Conference, Savannah 5th to 9th of August 2023, and I have been invited to give a talk at the PAG Plant and Animal Genome conference, San Diego 12th-17th of January 2024. My research was also presented at our annual University of Tennessee (UT)/ Tennessee State University (TSU) research summit in Nashville on the 1st of March 2024. This research summit is organized annually to foster potential collaboration within these two major universities in Tennessee. How have the results been disseminated to communities of interest?Lab meetings My research program was disseminated during lab meetings in the Department of Plant Sciences at UT. Lab meetings are organized weekly, or anytime a lab member needs to discuss the latest results or problems. I also participate in monthly lab meetings with my collaborators at the Center for Agricultural Synthetic Biology (CASB) where research is delivered in the form of scientific presentations. Lab meetings are also organized with my collaborators at the University of Tennessee, Professor Feng Chen, and Professor Jennifer DeBruyn if research updates need to be discussed. Face-to-face meetings In my role of Assistant Professor before, I was involved in training and mentoring students both within my group of research and from other laboratories if my expertise is required. I am a committee member of 5 PhD students at the University of Tennessee. Being a major expert in plant genetic engineering, my role of mentor in this domain of research is very important at the University of Tennessee. Social networks and UT Research Foundation My research achievements and activities are disseminated using my lab social network. Another important aspect in disseminating my research activities is covered by the UT Research Foundation. In particular this organization at the University of Tennessee has already started marketing technologies developed in my laboratories. Smith Global Leadership fellowship I have been a Smith Global Leadership fellow since last year. During this program, we have visited several Federal Grant Institutions in Washington DC, including USDA, NSF, USAID, and the national academy of science. During this program, I had the opportunity to meet and talk about my research program with several project managers. Furthermore, we visited Capitol Hill, where we had meetings with representatives of the state of Tennessee. Conferences My research program was disseminated in two international conferences: i) ASPB Plant Biology Conference, Savannah 5th to 9th of August 2023; and ii) PAG Plant and Animal Genome conference, San Diego 12th-17th of January 2024. My research was also disseminated during our annual University of Tennessee (UT)/ Tennessee State University (TSU) research summit in Nashville on the 1st of March 2024. What do you plan to do during the next reporting period to accomplish the goals?Goal-1.Evaluate and quantify the risks associated with the use of the mini-synplastome technology in agriculture. Objective 1.Phenotypic characterization of the above and below-ground portion of synplastomic and conventional transplastomic plants compared to wild-type control plants. This objective was completed in years 1 and 2. However, every 2-3 months we grow our germplasm in potting mix in a growth chamber for collecting leaf samples for testing plant-to-bacterium transgene flow. In addition, during the last 2 years we have developed an optimized version of the mini-plastome. We are also planning to test these plants for plant-to-bacterium transgene flow, since our preliminary results of biochemical characterization indicate a high copy number of the episome. Objective 2.Evaluation of transgene and other backbone sequences escapes to pollen of synplastomic and traditional transplastomic plants. We will optimize a protocol for genome extraction from pollen collected from flowers of mature plants. In a first step, absence of transgene escape will be investigated by PCR techniques (conventional and qPCR). While it is anticipated that the mini-synplastome will follow the same maternal inheritance of conventional transplastomic lines, next generation sequencing (NGS) of genomic samples will be performed in case transgene sequences will be detected by quantitative PCRs. Objective 3.Evaluation of transgene and other backbone sequences escape from synplastomic and conventional transplastomic plants to soil bacteria. During the second year of this project, we have created a library comprising about 50 bacterial strains isolated from soil samples. These strains have been classified by morphology and sequencing analysis of the 16S RNA ribosomal gene. In the next step, all these bacteria strains will be characterized by their ability to uptake foreign DNA. The same protocol developed for our bacteria model Acinetobacter baylyi (strain ATCC 33305; https:// www.uniprot.org/taxonomy/62977) will be used for these soil bacteria. For this purpose, bacteria at a logarithmic phase of growth (OD: 0.3-0.4) will be incubated with increase amount of pure plasmid (0-1 µg). Natural competency to uptake DNA will be indicated as colony forming units (CFU) of treated bacteria plated in selective media. In parallel, the same bacteria cultures will be washed, treated using DNase to eliminate any plastid contamination in the supernatant, and thereafter bacteria incorporating foreign DNA (plasmids) will be characterized for the presence of transgenes by qPCR. In case foreign DNA uptake is confirmed, the same bacterial samples will be used for RNA extraction, cDNA synthesis and qRT-PCR to confirm transgene expression and functionality of plastid regulatory elements in these bacterial strains. Only naturally competent bacterial strains able to uptake pure plasmid will be tested in plant-to-bacterium transgene flow. For this purpose, the same high throughput method developed for testing transgene flow using Acinetobacter baylyi in vitro in 1.5 mL tubes (or 96-well plates) will be used. Leaf tissue samples collected using year 1-2 from conventional integrating lines, mini-synplastomic lines and wild-type control plants will be used. Plant-to-bacterium transgene flow will be expressed as CFU (colony forming unit) per leaf area, or amount (mg) of leaf tissue, or transgene copy number in the samples. The presence of the marker gene into bacteria colonies will be confirmed by PCR analysis and NGS as described in the achievement session. In parallel, the same experimental design will be used to test plant-to-bacterium transgene flow to the environmental microbiome using microcosms containing soil samples. Soil samples have already been collected from our Research Station at East Tennessee (ETREC). For these experiments, an increase in absolute copy number of marker-genes over time (0 to 72 hours) expressed as a proportion of total bacterial 16S rRNA copies in samples incubated with transgenic material vs wild-type controls, will be considered an indicator of potential increased of plant-to-bacterium HGT. The increase in transgene copy number in soil microcosms will be determined by digital droplet PCR (ddPCR) that provides higher sensitivity compared to conventional qPCR methods. Goal-2 To demonstrate a potential path of commercialization, next-gen mini-synplastomes will be developed to generate marker-free transplastomic plants. Objective 1-2.Design of next-gen mini-synplastomes to produce transplastomic marker free lines and phenotypic analysis. During the year 1-2 we have developed an optimized genetic engineering platform for producing transplastomic marker-free lines. Transplastomic plants have been fully characterized by the presence of the transgene integrated at homoplasmy, while the selectable marker gene was completely removed. Marker gene removal was confirmed by PCR, Southern blot and NGS of the whole plastome preparations extracted from isolated chloroplasts. In parallel, marker free transplastomic plants were subjected to phenotypic analysis at anthesis. During year 3, marker free transplastomic plants will be grown to plant maturity at tuber production for phenotypic characterization of the below ground part of the plant. These experiments will be necessary to confirm normal tuber yield in marker free lines, that is a critical aspect for the potential use of this technology for crop improvement. For this purpose, at least 5 plants from down selected lines during year 1-2 along with wild-type controls will be grown in a green house space. At the end of the experiment, the total number of tubers, and total tuber biomass (fresh and dry) will be collected. Moreover, the second vegetative generation of plants germinated from tuber on soil will be collected. These plants will be genotyped by Southern blot and PCR analysis to confirm transgene stability at homoplasmy across generation. The confirmation of transgene stability in the next vegetative generation is a fundamental characteristic that supports potential utilization of this genetic engineering platform in agriculture. Goal-3 I will continue to deliver science-based knowledge to both an academic and non-academic public. I will disseminate and discuss results during lab meetings and seminars at the University of Tennessee. I have already been invited to give a seminar at the Center for Renewable Carbon at the University of Tennessee. I will strengthen my already established collaborations that I have developed during the past two years, and I will foster new collaborative projects. For this purpose, my participation in the Smith Global Leadership Fellowship at UT will help me to develop international collaborative projects. I am planning to participate in the next ASPB Plant Biology conference, where I will present my research in the form of a poster or talk. I will promote my research activity through my social networks and University websites. I will promote marketing of my research invention through the UT research foundation. Goal-4 Education and training of the future generation of scientists in several cutting-edge Synthetic Biology and Genetic Engineering techniques. I am the major expert in genetic engineering of plant cells in my department, and my role of mentor, supervisor and advisor will be very important. I will be the direct supervisor of a Master and a PhD student working in my laboratory. I will be a committee member of five PhD students working within the Department of Plant Science and other departments. I will be the main instructor of two courses for both undergraduate and graduate students: 1) Plant Science Plant Propagation, with my session focused on tissue culture and micropropagation (PLSC 330), 2) Engineering and Gene Editing of Plant Cells (PLSC 493), which is a completely new course at the University of Tennessee.

Impacts
What was accomplished under these goals? Goal-1 The overarching aim of this session is to evaluate the risks associated with the use of conventional plastid genetic engineering strategies and the mini-synplastome in agriculture. Phenotypic characterization The second generation of transplastomic lines produced with conventional integrating vectors, along with mini-synplastomic lines described by Occhialini et al 2022 have been subjected to phenotypic analysis. As described for the first generation, phenotypic analysis was performed in comparison with wild-type potato plants. The phenotypic analysis was performed at two different developmental stages: 1) in a vegetative stage at appearing of the first floral bolt (anthesis); and 2) at the end of plant life cycle to evaluate tuber production. Each experiment included 5 plants per genotype, and data were compared using ANOVA with Dunnett's test (p<0.05) statistical analysis using wild-type plants as reference group. The following parameters were collected. For the above ground: i) plant height; total biomass (fresh and dry), iv) foliar biomass (fresh and dry) per unit of leaf area; v) leaf chlorophyll content (CCI, chlorophyll content index). For the below-ground: i) total number of tubers, ii) tuber biomass (dry and fresh). As was shown for the first generation of plants, mini-synplastomic lines have identical phenotypic parameters (above and below-ground) compared to wild-type plants. On the contrary, conventional integrating lines showed a reduced amount of chlorophyll per leaf area. Evaluation of transgene flow in vitro Leaf samples from 4-5-week-old plants grown on potting in a growth chamber were collected for testing plant-to-bacterium transgene flow. Samples were collected from conventional and mini-synplastomic lines. Wild-type potato plants of the same edge and kept at the same environmental condition were used as controls. In a first step, plant-to-bacterium transgene flow was performed in vitro using the bacteria modelAcinetobacter baylyi. For this purpose, one independent line per each genotype (transplastomic, synplastomic and wild-type controls) and at least 3 plants (biological replicates) per each genotype were used. Compared to previous publications a high throughput method for testing transgene flow in small scale (2 mL tubes or plates) was developed. For this purpose, 50 mg of leaf samples were collected in tubes (or plates) and then samples were surface sterilized. This sterilization step was fundamental to eliminate any bacteria contamination. Leaf samples were grinded in liquid nitrogen and then incubated with 1 mL of Acinetobacter baylyicultures at logarithmic phase of growth (OD: 0.3-0.4) for 1.5 hours at 37°C. Counting of bacterial cells was performed to obtain the number of cell x OD. In parallel, a negative control (only water) and positive control (plasmid) were used as comparison. After this incubation time, each sample was plated in 5 petri dishes containing selective (spectinomycin) solid media. Plant-to-bacterium gene flow data were expressed the number of bacteria cells incubated per mg of fresh tissue necessary to produce 1 CFU. Our preliminary results data indicate very low risk, as to obtain 1 CFU using conventional integrating lines we need to incubate 2.2x105 bacteria cells x 1 mg of tissue, while for mini-synplastomic lines we need 1.9x105 bacteria cells x 1 mg of tissue. The presence of full-length transgene (aadA) in colonies was confirmed by colony PCR. However, the transgene was very fast removed starting from the first bacteria subculture. While the transgene was present, no transgene expression was detected (qRT-PCR). In parallel, PCR analysis using oligonucleotides designed on the CRISPR locus indicates no transgene integration in this portion of the bacterial genome. These results suggest the ability of bacterial cells to uptake few transgene copies from the environment, and that synplastomic lines do not have an increased risk compared to conventional transplastomic lines. Biochemical analysis indicates that transgenes are very fast degraded from bacterial cells, without specific integration into the CRISPR locus. It is likely that bacterial cells use foreign DNA as source of energy, with no risk of potential integration into the genome. Evaluation of transgene flow to bacteria strains extracted from soil microbiome. In order to expand the number of bacteria strains that will be tested for transgene flow in vitro, an optimized method for isolation of soil bacterial clones was developed during this second-year project. Soil samples were collected from the top layer (10-15 cm) of a soybean field at the East Tennessee Education Center (ETREC). For microbiome extraction soil samples were homogenized and treated in sodium chloride saline solution. Several serial dilutions were made and plated in conventional R2A media for isolation of cultivable bacteria colonies. Colonies were selected by morphology, trying to get one clone per morphological type. A total of 50 bacterial strains were collected from single colonies and organized in a database in form of glycerol stocks at -80°C. In the first step, bacteria were characterized by their morphology, and then in parallel, the same bacteria were subjected to genotypic characterization by sequencing of the 16S Ribosomal RNA. This database represents a snapshot of the whole cultivable bacterial population present in soil samples. At this time, we have already started to test all these bacterial strains for their ability to uptake foreign DNA contained in transgenic plants using the same protocol that we have developed for the bacteria model Acinetobacter baylyi. Goal-2 The overarching aim of this session is to demonstrate a potential path for commercialization, by designing an optimized engineering platform for producing marker-free transplastomic lines. Characterization of marker-free lines Marker-free transplastomic lines (19 independent lines) have been fully genotypically and phenotypically characterized. For phenotypic characterization all marker-free lines along with wild-type controls have been grown in pots in a growth chamber until the appearance of the first floral bolt (anthesis). At this developmental stage, phenotypic data were collected from the above ground portion of the plant. As described before, the plant height, leaf fresh and dry weight per unit of foliar area, together with the total biomass (fresh and dry) and the leaf chlorophyll content index (CCI) have been measured. While a phenotypic variation was observed across independent transformation events, down selected lines can reach the same phenotypic parameters of wild-type plants. In particular phenotypic variation was observed in a reduced chlorophyll content. This particular phenotypic penalty is often associated with transgene integration into the trnG/trnfM integration site, commonly used in plastid genetic engineering. To mitigate this problem of plastid engineering, alternative integration sites that do not show any phenotypes have been tested in the laboratory. The complete removal of the selectable marker-gene and backbone sequences have been already confirmed by Southern blot and PCR analysis. However, in this second year the complete removal of all transgene copies was confirmed by Next Gen sequencing using chloroplast genome preparations extracted from transgenic plants and wild-type controls. For this purpose, 5 down selected lines covering a range of phenotypic variations from wild-type phenotype to reduced size and chlorophyll content have been used. Next generation sequencing confirmed again removal of all transgene copies as well as correct transgene integration in all marker-free lines. Taken together these experiments confirmed functionality of our strategy to produce potato marker-free transplastomic plants.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2024 Citation: Occhialini Alessandro, Reed Andrew C., Harbison Stacee A., Sichterman Megan J., Baumann Aaron, Pfotenhauer Alexander C., Li Li, King Gabriella, Vincent Aaron G., Wise-Mitchell Ashley D., Stewart C. Neal Jr, and Lenaghan Scott C. (2024) Utilization of episomes for marker-free chloroplast engineering. The Plant Journal. Submitted.
• Type: Book Chapters Status: Submitted Year Published: 2024 Citation: Occhialini Alessandro and Lenaghan Scott. Chapter 12: Plastid Genetic Engineering. In Book: Stewart, C.N., Jr. (Ed.) Plant Biotechnology and Genetics: Principles, Techniques and Applications, John Wiley and Sons, Hoboken, New Jersey, New Third Edition.
• Type: Book Chapters Status: Submitted Year Published: 2024 Citation: Occhialini Alessandro. The genetic engineering toolbox for transformation of higher plant plastids Chloroplast. In book, Gene Expression - Stress Signaling, Biotechnology and Regulation. Tessa Burch-Smith (Ed.). Springer Nature. Submitted.

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:The first year of this research project, my research was of particular interest to the scientific community working in chloroplast genetic engineering and plant synthetic biology ofSolanum tuberosum(potato). During this period of time, I have delivered important knowledge within the research group working at the Center for Agricultural Synthetic Biology (CASB) at the University of Tennessee. At that time, the CASB was counting about 30 people, including grad and undergrad students as well as Post-Docs and technical staff. Until April 2023, my role of Research Assistant Professor also included co-advising and mentoring students, as well as supervising Post-Doc and technical staff. Starting from April 2023, I have got a position as Assistant Professor Tenure, in plant genetic engineering and gene editing at the Department of Plant Sciences at the University of Tennessee. At this time, I am the expert of genetic engineering of chloroplasts in all University. I am running a new lab of genetic engineering and synthetic biology of plant cells and part of my actual job is to advise in these novel research domains a large group of scientists within my research institute. My research was also of particular interest for students. In fact, starting in January 2023, I have also been involved in teaching a special course (Plant Synthetic Biology and Genetic Engineering) within the Department of Food Science. During several lectures, I have delivered knowledge regarding the genetic enginnering of chloroplasts using the mini-synplastome and utilization of this novel technology for producing marker-free transplastomic plants. This year I have also been invited to give a guest lecture (webinar) in Italy at CNR-IBBA (Institute of Agricultural Biology and Biotechnology). During these research lectures, I have delivered my research focus to both scientists and students, and many potential collaborations will be started this year. This year, an invention disclosure entitled "Mini-synplastome hybrid vectors for production of marker-free transplastomic plants" was submitted to the UT Research Foundation (office in charge of technology transfer and licensing at UTIA). This novel genetic engineering tool developed during this first year of my research project consists of a new mini-synplastome-hybrid transformation vector that has been designed to produce marker-free transplastomic plants. This technology will be particularly important for improving biosafety of genetic engineered plants in agriculture. The UT Research Foundation has already started marketing this novel tool, contributing to broadening the target audience to all public community interested in this novel technology. Changes/Problems:This first year has been quite challenging to recruit students in a time frame compatible with the beginning of this progect. At this time I have recruited master students that will start to work in my lab from fall 2023 (August 2023). What opportunities for training and professional development has the project provided?I have started this research project as Research Assistant Professor at the Center for Agriculture Synthetic Biology (CASB) at UT. During this period of time (September 2022 - April 2023), I got the opportunity to strength many important, writing, communication, supervising, and mentoring skills. My role included several activities: i) co-advising and mentoring students; ii) supervising Post-Doc and technical staff; iii) performing research within my several research projects. During this period of time, I have delivered knowledge within the group of research (lab meetings), and at the same time, I have supported my supervisors in leading research and technical staff. During this period of time, I have also reinforced my writing skills that are particularly important for being successful in grant applications. Another very important professional development acquired during this first year consists in teaching experience and reinforcing my communication skills to students (under- and graduate students). In fact, an activity accomplished during this period of time is that, starting from January 2023, I am in charge of a special course "Plant Synthetic Biology and Plastid Genetic Engineering". During this special course, I have dedicated three lectures and one journal club on plastid genetic engineering. Aside from the description of conventional methods of chloroplast engineering, during these lectures I have also delivered information about novel technologies that will be developed during this research project (marker-free transformation vector). This year I have also been invited for a guest lecture (webinar) at the CNR-IBBA (Institute of Agricultural Biology and Biotechnology) in Milan, Italy. During this research seminar, I have delivered my research interest to both scientists and students, and many potential collaborations will be started this year. With regard to starting potential collaborations with this strategic area, this year I have applied for the Smith Global Leadership Fellows at the University of Tennessee. This program at UT has been designed to provide new faculty with knowledge, experience, and networking opportunities in global food, agriculture, natural resources, family and consumer sciences, and veterinary medicine. Another very important professional development achieved during this research project is that I have obtained a tenure position of Assistant Professor in the Department of Plant Sciences at the University of Tennessee (start date April 2023). At this time, I am running a new laboratory at UT focusing on genetic engineering and synthetic biology of plant cells. I have been hired during a new strategic Agri-research plan to improve both the biotech curriculum at UT Institute of Agriculture, and to develop an outstanding research program in cutting edge research domains focused on novel genetic engineering strategies to improve crops. How have the results been disseminated to communities of interest?The results have been disseminated as following: Lab meetings within my novel group of research, the group of research at the Center for Agricultural Synthetic Biology (CASB), and in the Department of Plant Sciences at UT. I have lab meetings to discuss results and general problems with my collaborators multiple times a week if necessary. Lab meetings at the CASB are organized ones a month. During these meetings, research is delivered in the form of scientific presentations. Face-to-face meetings. In my role of Research Assistant Professor before, and Assistant Professor tenure now, I am involved in training and mentoring students (under- and graduated) and technical staff. I always adopt an open-door policy, communicating with my collaborators every day or anytime is necessary. I communicate results through my social networks and through the UT Research Foundation. For this purpose, the UT Research Foundation has already started marketing my invention disclosure "Mini-synplastome hybrid vectors for production of marker-free transplastomic plants" that will reach a broader audience in academia and private sector. I am active in finding potential collaborations nationally and internationally. Recently I have been invited to give a webinar at the Institute of the Agricultural Biology and Biotechnology Institute (IBBA-CNR) in Milan, Italy. The Smith Global Leadership Fellows will also give me all tools necessary to develop novel collaborations. I will disseminate my scientific results during conferences, such as the next ASPB Plant Biology meeting 2023 that will be in Savannah, Georgia (USA). I have already submitted two poster presentations for this conference. What do you plan to do during the next reporting period to accomplish the goals?Goal-1.Evaluate and quantify the risks associated with the use of the mini-synplastome technology in agriculture. Objective 1.Phenotypic characterization (above and below-ground) of synplastomic and conventional transplastomic plants compared to wild-type control plants. All transgenic plants, including i) conventional transplastomic lines produced with homologous recombination vectors and ii) mini-synplastomic lines (produced with the Gen1 and Gen2 mini-synplastomes) are already available in the laboratory from a previous research project (Occhialini et al., Plant Biotechnol J. 2022 doi: 10.1111/pbi.13717). The phenotypic parameters of these lines have been collected during the first year of this research project. During the second year, a phenotypic analysis will be conducted on the second generation of these plants as follows. Phenotypic analysis of transgenic plants (conventional and mini-synplastomic) along with wild-type controls will be performed in controlled environment facilities, using at least 5 plants per genotype. Phenotypic data will be compared using ANOVA with post-hoc (p<0.05) statistical analysis. Both the above and below-ground part of the plant will be characterized at plant maturity: For the above-ground part of the plant, the following data will be collected: i) plant height, ii) number of nodes, iii) foliar and total (fresh and dry) weight of the entire plant, iv) foliar biomass (fresh and dry) per unit of leaf area; v) leaf chlorophyll content (CCI, chlorophyll content index); vi) photosynthetic performances expressed as CO2assimilation (A) per unit of leaf area. For the below-ground part of the plant, the following data will be collected: i) total number of tubers, ii) tube size, iii) tuber biomass (dry and fresh). Objective 2.Evaluation of transgene and other backbone sequences escapes to pollen of synplastomic and traditional transplastomic plants. For this purpose, pollen will be collected from flowers of mature plants described in objective-1 and will be subjected to next generation sequencing (NGS) and data analysis to identify any remaining plasmid backbone and transgenes. Sequencing facilities at the Center for Agricultural Synthetic Biology (CASB) at UTIA will be used. Objective 3.Evaluation of transgene and other backbone sequences escape from synplastomic and conventional transplastomic plants to soil bacteria. Leaf tissue has already been collected from conventional, mini-synplastomic and wild-type plants characterized during the first-year project. The frequency of transgene escape will be expressed as a function of the following as parameters: i) leaf area, ii) leaf fresh weight, iii) copy number of selectable-marker genes determined by qPCR. During the next step of this research project plant-to-bacterium horizontal gene transfer (HGT) will be performed in the laboratory using the bacteria modelAcinetobacter baylyi(strain ATCC 33305; https:// www.uniprot.org/taxonomy/62977). For this purpose, one independent line per each genotype (transplastomic, synplastomic, marker free and wild type) and at least 3 plants (biological replicates) per each line will be used. Different ODs of naturally competent bacteria (109cells) will be incubated for 1.5 hours with leaf homogenates (0-5 g) using 96-well plates. A negative control (only water) and positive controls (0-1 µg, of purified marker gene cassette) will be used as comparison. After this incubation time, these bacteria cells will be grown in selective solid media. Plant-to-bacterium gene transfer will be expressed as CFU (colony forming unit) per leaf area, or amount (g) of leaf tissue, or transgene copy number in the samples. The presence of the marker gene into bacteria colonies will be confirmed by PCR analysis and NGS. In parallel the same experimental design and plant samples will be used to test transgene flow to the environmental microbiome using microcosms containing soil samples. For this purpose, an increase in absolute copy number of marker-genes over time (0 to 72 hours) expressed as a proportion of total bacterial 16S rRNA copies in samples incubated with transgenic material vs wild-type controls, will be considered an indicator of potential increased of plant-to-bacterium HGT. The increase in transgene copy number in soil microcosms will be determined by digital droplet PCR (ddPCR) that provides higher sensitivity compared to conventional qPCR methods. After this step, we will attempt to isolate bacteria colonies that will be tested for the presence of transgene integrated by Next Gen sequencing. Goal-2 To demonstrate a potential path of commercialization, next-gen mini-synplastomes will be developed to generate marker-free transplastomic plants. Objective 1.Design of next-gen mini-synplastomes to produce transplastomic marker free lines. As I have described in the accomplishments, this year we have developed the mini-synplastome to produce marker-free transplastomic plants. At this time, we have produced 19 marker-free plants in tissue culture. The complete removal of the selectable marker-gene and backbone sequences have been already confirmed by Southern blot and PCR analysis. In the next step, the complete removal of all transgene copies will be confirmed by Next Gen sequencing using chloroplast genome preparations extracted from transgenic plants and wild-type controls. Along with the aforementioned molecular characterization, marker-free plants will be grown on potting mix and full phenotypic analysis of both above and below ground portions of the plant will be performed. As described before, for the above ground portion of the plant, the plant height, leaf fresh and dry weight per unit of foliar area, together with the total biomass (fresh and dry) will be measured. In parallel, the photosynthetic performance expressed as CO2 assimilation (A) per unit of leaf area and leaf chlorophyll content index (CCI) will be determined using a LICOR gas-exchange device and a chlorophyll content meter, respectively. For the below ground part of the plants, marker-free lines along with wild-type controls will be grown to maturity in a green house space. For this purpose, the total number, the size, and the dry and fresh weights of tubers will be determined. We anticipate that marker-free transplastomic lines will have very similar phenotypic paraments compared to wild-type control plants. Goal-3 Deliver science-based knowledge to both academic and non-academic people. I will disseminate and discuss results during lab meetings and seminars. I will be invited to give a webinar and seminars, and I will participate in the Smith Global Leadership Fellows. I will disseminate my results during my lectures at UT. I will participate to the ASPB Plant Biology conference next August 2023 in Savannah, Georgia (USA). I will promote my research activity through my social networks and University websites. I will promote my research activity through the UT research foundation. Goal-4 Education and training of future generation of scientists in several cutting-edge molecular biology and microbiology techniques. I will continue to co-advise and mentor under and graduate students at the University of Tennessee. I will continue to be involved in teaching my new course "Plant Synthetic Biology and Chloroplast Genetic Engineering". I will hire students (Master and PhD) for my new laboratory.

Impacts
What was accomplished under these goals? Goal-1 The first goal of this research project is to evaluate and quantify the risks associated with the use of the mini-synplastome technology in agriculture. The risk associated with this novel tool will also be compared with conventional techniques used to produce transplastomic plants. The risks that will be evaluated during this research project are: i) mini-synplastome backbone and transgene escape to pollen; ii) mini-synplastome and transgene escape through horizontal gene transfer (HGT) to soil environmental bacteria. To achieve this goal, conventional transplastomic lines (produced using homologous recombination integrating vectors), mini-synplastomic lines (produced using the non-integrating mini-synplastome), and wild-type controls have been grown on potting mix to perform the following analysis: i) phenotypic characterization. Both the above and below ground phenotype have been characterized. Per each genotype at least five plants (biological replicates) have been characterized. For the above ground characterization plants have been grown in controlled environment until anthesis. For this purpose, the plant height, leaf fresh and dry weight per unit of foliar area as well as the total biomass (fresh and dry) of the above ground plant have been measured. The photosynthetic performance expressed as CO2 assimilation (A) per unit of leaf area and leaf chlorophyll content index (CCI) have been determined using a LICOR gas-exchange device and a chlorophyll content meter, respectively. For the below ground characterization, plants have been grown until maturity in a green house space. For this purpose, the total number, the size, and the dry and fresh weights of tubers has been determined. Conventional integrating lines appeared statistically identical to wild-type plants for plant height, biomass per unit of leaf area, photosynthetic performances (A) and below ground phenotype (fresh and dry tuber yield, and tuber number). These lines are reduced in chlorophyll content and with the same height, conventional transplastomic lines are reduced in total biomass. On the contrary, mini-synplastomic lines have statistically identical phenotypic parameters compared to wild-type plants, supporting a potential use in agriculture. ii) Tissue collection to test transgene-flow using the soil bacterial model Acinetobacter baylyi and whole soil microbiome. During the phenotypic analysis, leaf tissue and entire plants have been collected at both anthesis and maturity for transgene flow experiments. At this time, tissue ready to be used (starting from the second year of this project) has been collected and stored at -80C. For each sample, the fresh weigh, foliar area, and transgene copy number (obtained by qPCR) was determined. For this purpose, the frequency of transgene flow (CFU: colony forming unit) will be expressed as frequency per unit of fresh weight, leaf area, and transgene copy number. Goal-2 To demonstrate a potential path of commercialization, the mini-synplastome will be re-designed to produce transplastomic marker-free potato lines with an improved biosafety in agriculture. We anticipate that this novel technology will be associated with lower risks compared to both conventional homologous recombination vectors and mini-synplastome technology. Regarding this particular goal, during this period of time, we have re-designed the mini-synplastome transformation vector to produce selectable marker-free Solanum tuberosum (potato) lines. Two different architectures of transformation vectors have been assembled. These two vectors differ for the following features: i) chloroplast-specific regulatory elements used to express the selectable marker (aadA) that allow different levels of transgene expression; ii) architecture of the chloroplast origin of replication of the mini-synplastome backbone that modulate copy number and stability overtime. Both parameters have been tested to modulate the ability to remove the selectable marker-gene in the resulting transplastomic lines. These two vectors have been delivered in potato leaf disks using nano-particle delivery system (gene gun). At this time, transplastomic marker-free lines have been produced in tissue culture using both vectors. Southern blot and PCR analysis has been performed, confirming homoplasmy (all copy of the chloroplast genome with the transgene of interest integrated) and complete removal of the selectable marker gene. It should be noted that a single vegetative generation in tissue culture was enough to produce homoplasmic lines, since a single integration site located in the large single copy region (LSC) of plastome was used. This is of particular interest, because homoplasmy of transgene integrated will provide genetic stability over time. Along with a proper re-design of the mini-synplastome, the production of transplastomic marker-free lines in tissue culture was achieved by modifying the conventional tissue culture method for producing transplastomic plants. After leaf disks transformation and green callus induction, shooting induction was performed in media progressively depleted of the selectable agent spectinomycin. The complete removal of the selectable agent allowed complete removal of the episome-containing aadA, leaving only the transgene of interest (GFP) integrated at homoplasmy in the chloroplast genome. It should be noted that this procedure does not introduce and extra steps respecting the same time frame of producing conventional transplastomic lines with the marker-gene integrated. A total of 19 marker-free independent lines have been produced. These lines will be subjected to full phenotypic and molecular analysis starting from the second year of this research project. The same phenotypic parameters listed in Goal-1 will be collected.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Occhialini, A., Lenaghan, S.C. Plastid engineering using episomal DNA. Plant Cell Rep 42, 11251132 (2023). https://doi.org/10.1007/s00299-023-03020-x