Probing the Role of RDR and DCL proteins in Virus Induced Gene Editing in Tomato Plants

Genome editing is an important tool for biological research and crop improvement. Tissue culture is the traditional method for genome editing but requires large time investments, extensive experimental procedures, and can have unintended effects on the genome. Virus induced gene editing (VIGE) uses viral vectors to create heritable edits by delivering single-guide RNA to the meristem via direct delivery. Tobacco Rattle Virus (TRV) has successfully created heritable edits in dicotyledonous plants, but many species exclude viruses from the meristem as part of protection. RNA-dependent RNA polymerase (RDR) and Dicer-like (DCL) genes encode key proteins involved in viral protection in plants. This project seeks to evaluate how they affect VIGE efficiency by creating loss of function mutant tomato plant lines using CRISPR Cas9 and tissue and conducting TRV mediated VIGE on these plants using phytoene desaturase (PDS) as a reporter gene. The loss of function mutants were crossed with high Cas9 expressing, high editing efficiency wildtype plants and then VIGE was conducted on the crossed plants. PDS photobleaching symptoms began showing 11 days post editing after which a western blot was performed to evaluate the Cas9 expression in the crossed plants. Positive sequencing results indicate successful creation of loss of function mutants. Western blot results indicate high Cas9 expression in the crossed plants. This work can lay the foundation for future work on developing protocols for more efficient VIGE work and act as progress towards the overall goal of overcoming the barriers of tissue culture-based genome editing.

I have had the pleasure of working in the Van Eck as part of the BTI/ CROPPS Research Experience for Undergraduates (REU). My project involved evaluating the role of RNA-dependent RNA polymerase (RDR) and Dicer-like (DCL) genes in the efficiency of Virus Induced Gene Editing (VIGE). My mentor, Dr. Fan Xia, has helped me gain an in-depth understanding of CRISPR-Cas9 genome editing methods, the genotyping process, experimental design and problem-solving from a scientific mindset. The Van Eck lab is a collaborative and uplifting environment, and it has been an honor work with these individuals these past 10 weeks.

Investigating insect defense systems in groundcherry as a model for related Physalis species

Physalis is a genus of fruit plants native to Central and South America. Goldenberry (Physalis peruviana) has abundant nutritional and medicinal properties that make it desirable for large-scale agricultural production. However, it is susceptible to insect herbivory, which reduces fruit yield and poses a challenge to growers. A close relative of P. peruviana, groundcherry (Physalis grisea), is more resistant to insect herbivores. Its leaves produce specialized metabolites called withanolides that confer insect resistance in other plant species. Withanolide content in P. peruviana may be manipulated to make it more viable for large-scale production. To further explore the application of withanolides as a defense mechanism against insect herbivory, through deterrent or toxic effects, we used CRISPR gene editing to alter withanolide accumulation in P. grisea leaf tissue. We targeted two branch points in the withanolide biosynthetic pathway: BETA-AMYRIN SYNTHASE (PgꞵAS) and STEROL METHYLTRANSFERASE 2 (PgSMT2). Three mutant lines with unique alleles for each gene were recovered. We used these lines in a no-choice insect bioassay in which Trichoplusia ni neonate larvae were placed individually in a petri dish with one leaf disc. The larvae did not exhibit a significant feeding preference for wild type P. grisea or any edited line, and the larvae had similar survival rates on all lines. This may be due to T. ni being a generalist species and having an acute sensitivity to withanolides. Future bioassays will investigate the effect of withanolides on Lema daturphila beetles, which are Physalis specialists and can likely tolerate higher levels of withanolides.

This summer, I had the opportunity to work in Joyce Van Eck’s lab at BTI, using chemical ecology to study insect defense mechanisms in groundcherry as a part of the Physalis Improvement Project. I was closely mentored by Savanah Dale who gave me the freedom to think independently and the support I needed as I delved into a new project. Through my work and the weekly seminar series, I’ve learned about a plethora of plant research projects and their practical applications. Understanding how scientists can use their research to improve crops has enriched my love of plant science and has sparked my interest in plant breeding as a career path.

Understanding Tomato Regeneration and Maize Transformation Through Bioengineering

Tomato and maize are major crops and genetic models for studying gene function. Different genotypes exhibit varying responses to transformation. In plant bioengineering, some genotypes are not amenable to transformation and regeneration, while others are. Improving the bioengineering of tomato and maize is crucial. In this study, we aim to enhance transformation efficiency in genotypes that show a low response to transformation. We intend to generate overexpression constructs for genes A, B, and C, identified from previous GWAS studies, linked to plant regeneration in tomatoes. In another study, we tested different Ancymidol concentrations to increase maize leaf transformation in genotypes LH287, LH245, and 90DJD28, which showed a low response at lower Ancymidol concentrations. Genes A and C were amplified using cDNA from cotyledons and roots. Gene A was cloned into a construct with the 35S promoter and Nos terminator using Nimble Cloning. Cloning was confirmed by colony PCR, and construct sequencing will confirm cloning. In maize transformation, higher Ancymidol concentrations were tested on six genotypes at 0, 3.4 mg/L (1X), and 6.8 mg/L (2X). GFP expression was observed after two days. Higher GFP expression in 2X Ancymidol may improve transformation, especially in genotypes LH287, 90DJD28, and LH245, showing that 2X Ancymidol enhances transformation frequency in maize leaf tissues. In the future, we aim to generate overexpression constructs and transform low-regenerative genotypes of tomatoes. Additionally, we will further explore maize leaf transformation to determine how increased Ancymidol concentrations can improve the stable transformation of maize plants.

During this REU experience, I have learned the importance of interdisciplinary collaboration. Plant science is often intersecting and complexly interrelated to other STEM fields such as bioinformatics, robotics, physics, and many others. This integrated work taught me the importance of science communication at every education and interest level. Being able to simplify and condense complex research is a crucial skill that I will carry forward into my future science career. The research that I was able to conduct at BTI set me up to have a foundation in bioengineering principles and a deeper understanding of fundamental laboratory skills such as PCR, RNA isolation, and data analysis. BTI’s holistic approach to this internship allowed me to gain a comprehensive understanding of computational strategies, interdisciplinary collaboration, and real-world applications.

Examining Lipid Nanoparticles as an effective method for pDNA delivery into tomato plants

The need for viable means of plant gene delivery is more crucial at this moment in time than it possibly ever has been. Plant genetic engineering in its current form uses the process of agrobacterium-mediated delivery along with particle bombardment of the target cells within a plant. These existing methods of tissue culture prove to face many different limitations in plant gene delivery such as a limited yield to said biological target, extended time consumption, risk of gene damage, along with a limitation of applicability to a broad variance of species. This summer, with the resources provided by the Alabi, and Van Eck Labs respectively, We have studied the use of Lipid Nanoparticles to deliver transgenic DNA into plant cells. Lipid Nanoparticle technology has recently been used in the delivery of the Moderna mRNA vaccine into mammalian cells. Our objective this summer is to utilize LNP technology to achieve transport of plasmid DNA into the nucleus of the cells of tomato plants. The complications with a transport delivery vehicle such as an LNP is hypothesized to have to fit a size exclusion limit (SEL). A plant cell’s SEL begins with the rigid cell wall (5-20 nm) and then its semipermeable membrane (~500 nm), compared to just the membrane of a mammalian cell. The LNPs that will be formulated in the Alabi Lab will be 100-200 nm in size to form soft fusogenic cells. The most successful composition of LNPs include a PEG-lipid, phospholipid, ionizable lipid, and cholesterol.

My ten weeks spent at the Boyce Thompson Institute have been pivotal in the development of myself not only as in my career goals, but as a person as well. I would like to thank Megan and Delanie for their continued grace throughout this summer. My experience in the Van Eck, and Alabi labs respectively, has been nothing short of amazing. I will miss the camaraderie, and overall cohesiveness that these labs have displayed to me. Participating in this project has allowed me to learn new techniques, as well as increasing my confidence to conduct meaningful research. I plan to take my experiences back home with me to share the knowledge I have accumulated throughout this program. I am excited to encourage more Tuskegee students to join this program as a token of my gratitude to BTI for this life-changing experience.

Investigating the Impact of 1-Methylcyclopropene and Gene Editing on Physalis grisea Abscission

Fruit abscission is a necessary process for many plants, and describes the shedding of fruit from the stem. This process spreads seeds, which is important for advancing the next generation of that species. Sometimes, fruit will abscise before they are ready to be harvested, which can be a detriment to crop harvest, especially in large-scale agricultural settings. This premature fruit abscission is especially prevalent in Physalis grisea, also known as groundcherry, which hinders its adoption as a widely-grown agricultural crop. Two separate strategies were explored to investigate solutions to groundcherry fruit abscission: applying 1-Methylcyclopropene (1-MCP), a synthetic growth inhibitor, and editing the plant genome for genes that cause fruit abscission. Applying 1-MCP to fruit is known to delay ripening, and studies have shown that there is an association between abscission and fruit ripening in plants like tomatoes. We hypothesized that 1-MCP application would result in slowed groundcherry ripening and thus decrease rates of abscission. Our experiment consisted of three treatments: an untreated control, a treated control, and a 1-MCP treatment group. The 1-MCP treatment consisted of 1-MCP powder dissolved in distilled water and surfactant solvent, while our treated control group received the solvent without 1-MCP. After treating the fruit, we recorded the total number of fruit that dropped approximately every other day for 21 days. The second experiment observed the effects of editing the groundcherry genome. INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) and JOINTLESS are two groundcherry genes that are known to be involved in abscission zone (AZ) development and cell separation, which play an important role in the detachment of groundcherry fruit. When these genes were edited in groundcherry via CRISPR/Cas9 , we expected reduced AZ development and a drop in fruit abscission rates. To test this hypothesis, wild type groundcherry, ida mutants, and jointless mutants were randomly assorted in the greenhouse and flowers were tagged weekly. After four weeks of tagging, a force meter was used to measure the force required to remove the fruit of varying developmental stages from the plants. A necessary step in improving the quality of groundcherry fruit harvest is determining the best method to reduce abscission. These experiments serve as an early assessment of the effectiveness of current methods of groundcherry abscission reduction, and similar experiments at a larger scale can be derived to optimize groundcherry cultivation.

When I first heard about CRISPR technology, I was confused but very excited by the potential to make something new. I saw myself developing a grass that my mom could plant on our lawn to outgrow the ever-persistent weeds. Before making this plant, though, I had to learn about the technology, so I reached out to Joyce Van Eck and asked to join her research. She graciously welcomed me to the lab, despite my lack of experience, and I worked with Elise Tomaszewski throughout the beginning of 2023. Elise taught me every step of transformation, and in return, I provided some very gnarly-looking contaminated plates. I am so grateful for the members of the Van Eck lab, who all showed me patience and kindness and made the experience a ton of fun. During the REU program, I got to meet students from around the country who were even more excited than I was about plant science. I’ve made some very good friends who I know are going to change the world.

“Understanding the Roles of Auxin and Ethylene in Physalis grisea Fruit Drop”

Project Summary:

Physalis grisea (groundcherry) is a fruit crop native to North America and is an underutilized species. Groundcherry easily drops its fruit through a process known as abscission, allowing it to disperse seed and shed injured organs. This process occurs at the abscission zone (AZ) on the pedicel, which is the stalk that attaches fruit to the plant stem. However, large amounts of fruit abscission result in a loss of profit for farmers. To develop groundcherry varieties with reduced fruit drop, we first need to understand the mechanisms behind P. grisea fruit abscission.

Auxin and ethylene are plant hormones known to be implicated in organ abscission in other species. However, this has not been confirmed in any Physalis species. My goal is to understand the role of auxin and ethylene in P. grisea fruit abscission. I hypothesized that applying auxin to groundcherry pedicels will delay fruit abscission and ethylene will accelerate abscission. Furthermore, auxin applied distally will delay abscission more than proximally.

To accomplish this, I applied auxin distally and proximally to the AZ to groundcherry pedicels and applied ethylene to the entire pedicel. Data were collected twice a day by counting the number of abscised pedicels and fruit. Data analysis confirmed that applying auxin to groundcherry pedicels delayed abscission, especially when applied distally. Also, ethylene application accelerated abscission. These results confirm that auxin and ethylene have similar effects on fruit abscission as in other species. This experiment will be useful in the future to determine if these hormones affect abscission differently in abscission-edited groundcherry lines.

My Experience:

Over the summer, I gained an incredible amount of professional and lab experience and made lifelong friends. I had the opportunity to do CRISPR editing, work with an underutilized crop species, and perform my own experiment under a wonderful mentor. I also learned about professional development through workshops on communication and social science. There were also seminars from PIs at Cornell and BTI where I could learn about other types of research in the field. Finally, I enjoyed learning about graduate school at Cornell and thinking about what I want out of my career in the future.

The First Steps in the Genetic Improvement of Goji (Lycium barbarum)

Goji (Lycium barbarum) is a member of the Solanaceae family alongside tomato, potato, and pepper. Goji berries possess a wide range of reported health benefits in Chinese traditional medicine (including antioxidants and antidiabetic effects). As such, it has become regarded in the United States as a “superfruit.” However, the delivery of fresh goji berries to the United States has been hampered by growth and harvesting issues that diminish the economic and agricultural viability of L. barbarum, namely its 3-year vegetative period before fruit are produced and its tall, drooping height that requires pruning and other management. With the expertise and materials available in the Van Eck lab, we aim to improve these issues. We generated an in vitro regeneration protocol by measuring “shooty” callus development on 3 sets of media containing different concentrations of 6-benzyladenine (BA, a synthetic cytokinin) and α-napthaleneacetic acid (NAA, a synthetic auxin). We determined that in vitro regeneration occurs most efficiently with hypocotyl sections on media containing 2 mg/L BA and 1 mg/L NAA. After this finding, we began the first steps in genetic improvement of L. barbarum using Agrobacterium-mediated transformation. CRISPR-Cas9 constructs were designed to knockout SELF PRUNING (SP) and SELF-PRUNING 5G (SP5G), two homologs partially responsible for plant architecture and day-length sensitivity, respectively.

My Experience

Spending the summer living in Ithaca and working in the Van Eck lab has been an incredibly transformative experience. I expanded my scientific knowledge greatly beyond my previous classroom experiences. Even with no background in plant science, I found myself comfortable and excited learning about the background of both my project and my new friends’ projects. I learned invaluable molecular and tissue culture techniques that I will surely carry forward into my future. Moreover, I learned what it means and feels like to be a true scientist, working full time on a committed, valuable project.
Outside the laboratory, my time spent in Ithaca has been one of the best summers of my life. I have found myself surrounded by an incredibly diverse and friendly group that has made my time much more fun than I could have imagined. Hiking, eating, and dancing with this group has formed bonds that I hope to carry far into the future. I highly recommend any interested student to apply to the BTI program with no hesitation.

“Approaches for the genetic improvement of Physalis”

About me:

My name is Kyle Keating and I come from a town called Centereach in the middle of Long Island, New York. Earlier in life I always knew I wanted to work with plants. I started my botanical education with the Master Gardener program through Cornell Cooperative Extension. When I finished the program, I knew I had to take my knowledge and abilities to the next level. That’s when I applied to the plant science program at SUNY Cobleskill. It was there I enhanced my skills and became interested in the field of plant breeding. Now, after almost 4 years at SUNY Cobleskill, I was accepted as an intern at Boyce Thompson Institute where I learned  applicable lab and research techniques as part of the Physalis project.

Project Summary:

The Physalis research project required several different approaches for acquiring information. Overall, the purpose of this project is to make Physalis a new specialty crop for New York state. To do that, we need to determine the most needed and desirable traits. We are currently in the process of talking with farmers about their experience growing Physalis. We also made a catalog of all existing germplasm to better understand the lines we already had available to us. By applying lab techniques like ovule, ovary-slice, and tissue culture we hope to overcome interspecific barriers and create a plant that is more desirable to the public and farmers for future agriculture production.

My Experience:

My time here at BTI has been a beautiful and rewarding experience. When I began my internship, I didn’t know what to expect but after only a few days I felt right at home. My mentors guided me in new directions, helped me improve my lab techniques, and built my overall confidence in the field. I’m glad I had the opportunity to be exposed to this type of work and look forward to applying my new-found techniques in the near future.

Factors that affect Agrobacterium tumefaciens-mediated transformation of Solanum prinophyllum

Project Summary

The human population is predicted to reach 9.6 billion and the food demand is predicted to increase 70 to 100 percent by 2050. Arable land is being lost to urbanization, water supply is slowly dissipating, and the climate is gradually changing. Genetic engineering and crop improvement will play a crucial role to meet the demands and alleviate crop damage from both biotic and abiotic stresses. One major plant family is Solanaceae, which contains approximately 2,700 species including many important food crops.  Solanum prinophyllum, a member of the Solanaceae, was the focus of this study.  The purpose of the experiment was to investigate factors that can affect the transformation of S. prinophyllum.  Factors included different Agrobacterium tumefaciens strains (AGL1 and LBA4404), two explant types (cotyledons and mature leaves) and selective plant regeneration medium that contained two different concentrations of the antibiotic kanamycin (75 mg/l and 100 mg/l). Tissue culture and biotechnology techniques were used throughout this study. To confirm the recovery of transgenic lines, a β-Glucuronidase (GUS) assay and Polymerase Chain Reaction (PCR) will be used. The ultimate goal of this study was to develop the first successful transformation methodology for S. prinophyllum. S. prinophyllum can then be used as a model plant system for a larger NSF funded project in the Van Eck lab to investigate the genes and networks involved in meristem maturation and transition from vegetative growth to reproductive growth in the Solanaceae.

My Experience

Being able to participate in the Plant Genome Research Project at the Boyce Thompson Institute for Plant Research has been the opportunity of a lifetime. Working with my mentors Cynthia Du and Sarika Gupta provided me with insight and hands on experience in the field I dream of working in. My advisor, Joyce Van Eck, was very welcoming and she taught me so much. I learned many new lab skills and techniques such as plant tissue culture. This internship has given me confidence for stepping into the graduate school world. I now know what I need to do to prepare for the next step in my career path. I had an amazing time this summer in Ithaca, meeting other summer interns and lab members. Thank you for this amazing opportunity BTI!

Comparison of effectiveness in gene silencing of phytoene desaturase (PDS) gene through RNAi and CRISPR techniques

Project Summary

Targeted genome editing approach- CRISPR/Cas (Clustered Regularly Interspaced short palindromic repeats/CRISPR-associated) is relatively new, and reported to be highly specific and effective in genome modification in a variety of living organisms. These systems are a bacterial defense against invading foreign nucleic acids.They use an array of small CRISPR RNAs (crRNAs) consisting of repetitive sequences flanking unique spacers to recognize their targets, and conserved Cas proteins to mediate target degradation. This project examined the differences in effectiveness between CRISPR and RNA interference (RNAi) in silencing Phytoene Desaturase (PDS) gene in tomato leaves. Constructs for both CRISPR and RNAi with PDS sequence were designed and infiltrated into the tomato leaves via agrobacterium infiltration method. Tissues from yellow patches, which appeared yellow because of the CRISPR and RNAi effect, were collected and subjected to further analyses. DNA was extracted from yellow tissue from leaves transfected with CRISPR constructs and subjected to DNA sequencing. Transcript levels were examined and found reduced in case of RNAi when compared to wild type tomato. In comparison, yellow patches were more prominent in case of CRISPR than in plants infiltrated with RNAi constructs. Together, these findings indicate that CRISPR/Cas method is more promising compared to RNAi.

My Experience

Participating in the PGRP Internship program at BTI has taught me so many great things about plant genetic research. My mentor, Sarika Gupta, as well as other people in Dr. Van Eck’s lab truly guided me and provided me with so much knowledge and exposure to plant gene silencing techniques as well as plant tissue culture techniques, both of which I didn’t have much experience before. The weekly science seminars and many professional development activities at BTI also enhanced my knowledge about plant science, future career and graduate school pathways. This internship definitely has helped me to determine what I want to do after graduating from college. I am so glad to have this opportunity to meet so many other summer interns and people at BTI who are passionate about plant biology research.  Outside of work, I enjoyed hiking around the Cornell Campus, exploring around the finger lake area, and seeing so many wonderful waterfalls around Ithaca!

Study of the germination and a literature review of the nutritional and medicinal values of the Physalis genus

Many species in the Physalis genus have been used in traditional medicine for treating stomachaches, headaches and even certain diseases such as malaria. We were interested in finding more information on what else these plants can be used for in medicine to help stop or prevent other serious illnesses such as cancer.  I studied the growth of 12 Physalis species for the experimental portion of my project, as well as reviewed the nutritional benefits and medicinal uses of the plants.  For the experimental portion, I collected data on the date the radicles appeared on each of the 30 seeds for each species, the date when the first cotyledons developed, the height of the cotyledons, the contamination type, if there was any, the percent of the seeds that germinated, and the date when the first full leaf developed. This information, along with images collected, will be helpful in another study related to the branching, rooting and flowering habits of other plants in the Solanaceae family to increase agricultural productivity.

My Experience

This internship has been a great experience for me. It has given me the opportunity to improve my research and writing skills, and experience working in a lab. I learned how to use different kinds of lab equipment and lab techniques such as plant tissue culture. I was also able to explore science careers, which will help me decide what I want to do in the future. I have really enjoyed learning about the different ways plants have been used in medicine in different cultures, as well as the research that is being done on how these plants can be used in medicine. I am excited to apply the knowledge I have gained from this internship in school and in future careers.