Project summary

FloraForge is a startup developing a modular genome engineering platform to generate new aesthetic traits in ornamental plants. Right now, the startup is focused on establishing and validating a reusable petunia host line. This host line can be used for the site-specific cassette exchange of new genetic modules, instead of having to generate and compare random insertion events for every single new design.

Using a reusable host can aid in systematic testing of novel traits. One of the main problems with random insertion in plant engineering is reproducibility. Independent transformation events are expected to end up at different genomic sites and express at different levels. In order to take full advantage of a library of landing-pad lines, FloraForge first selects a well-characterized host from the library. New payloads can then be tested for expression at the same genomic site as opposed to having to perform another random insertion for every new design. The selection system is intended to make random donor integrations less likely to be selected. The ability to easily compare between experiments, and test out new designs will allow FloraForge to move beyond making individual engineered plants and instead test color and pattern designs in the same host. If successful, this could make the development of new ornamental traits more reproducible and easier to compare by allowing new payloads to be tested at the same characterized genomic site instead of relying on a new random insertion site for every design.

The work is organized around two separate workflows at NC State. The first workflow covers the biological work in the NC State Phytotron. In the Phytotron I established controlled plant growth, acclimation, line tracking, Agrobacterium infiltration, fluorescence testing, vegetative cloning and initial leaf-disc tissue culture and regeneration.

The second workflow uses shared laboratory facilities at NC State for DNA fabrication and plant characterization. In the shared laboratory space I established plasmid construction and iteration, genomic DNA extraction, PCR, amplicon-sequencing preparation and analysis, fluorescence imaging and preparation for genomic qPCR copy-number testing.

PBROC at Michigan State University completed the first stable transformation campaign and produced 41 transgenic lines of Petunia hybrida cv. Mitchell. The lines were shipped to North Carolina and placed into soil alongside wild-type controls for acclimation. Genomic DNA was extracted from all 41 lines and PCR was run using various primers to test for the presence of the expected sequences. PCR products from candidate lines were then sent for amplicon sequencing. In total, 18 of the candidate lines were sequenced. Broad or full coverage of the tested region was obtained for most of the lines. Three of the lines, however, had small gaps at the 5' end of the PCR products. Multiple stable lines also show strong, uniform GFP fluorescence across flower petals.

I also created a working transient-expression system for Mitchell petunia as a secondary goal for Round 1. A transient-expression system allows for rapid testing of designs before committing them to stable transformation and long-term characterization.

The work is part of the landing-pad program. Round 1 focused on a vein-pattern rescue. The vein-pattern goal has not changed. Only the order of the work has. Once the stable lines arrived from PBROC at Michigan State University, I determined that the first order of business would be to identify the best landing-pad host for repeat testing of designs for new aesthetic and functional plant traits. Only after establishing a suitable host would I focus on the completion of the first design’s pigment swap.

Images and sequencing summary: www.floraforgelabs.com/manifund

What are this project's goals? How will you achieve them?

The petunia campaign generated 41 stable transgenic lines. For future work, I really only need to maintain 1-2 healthy lines that express GFP consistently, contain the expected sequence, and can be maintained by cloning and/or tissue culture. I am comparing the strongest lines using GFP expression, plant health, PCR, amplicon sequencing, and qPCR. I have made backup clones of the 5 strongest lines before another round of transformation.

A preliminary experiment to assess the ability to regenerate plants from leaf-discs is currently underway to determine which elements of the transformation process can be carried out in-house and which would be better to outsource to a specialist provider. The following parameters will be monitored for each experiment: contamination, callus formation, shoot formation, survival.

The next major event in the lab will be the first cassette exchange. A donor for the first cassette is in production and is designed to replace the GFP marked cassette at the same genomic site. Therefore, the first step in this process will be to select the lead host lines. The selected lines will be carried forward using the existing backup clones of the 5 strongest lines, and I will receive the verified donor construct for the first cassette. The first exchange attempt will then use the leaf-disc regeneration workflow that is currently in progress. This will help determine whether I can complete the necessary steps for petunia transformation internally or whether it would be better to have an outside provider complete some or all of the steps for me. Initially, I will look for loss of the GFP signal as an initial indication that the exchange may have occurred. This will then be followed by PCR and sequencing to confirm that the desired donor cassette was inserted into the genome in the correct orientation. The first exchange will test whether the cassette exchange system works in the selected host, while later exchanges with different payloads will test its reusability.

I also plan to make one more purple infiltration attempt in order to obtain a visible result, but this will remain secondary to the main aims of selecting a suitable host line, continuing regeneration, and attempting the first exchange.

How will this funding be used?

$15,000 (minimum requested) will be sufficient to allow selection of best lines, qPCR copy-number testing on the best lines, and any remaining sequencing needed for the best lines. Funding will also be used to continue the leaf-disc regeneration protocol, to pay for the donor that has already been ordered, to maintain the plants and to set up the first exchange attempt.

If funded the full $75,000, I would have the funds to test out several different host lines and different donor conditions for transformation. I would also be able to have the initial transformation done by an outside service provider while building up the internal capacity for the pigment and color experiments that follow the first exchange. With this amount, I would also be able to get assistance from a technician for the leaf-disc regeneration work and repeat the first experiment if necessary.

Proposed full-budget allocation:

• Outside transformation and repeat-attempt reserve: $22,000
• Donor costs, pigment/color follow-up constructs, and sequence verification: $8,000
• Phytotron, shared laboratory access, and plant maintenance: $10,000
• Tissue-culture media, plastics, and regeneration supplies: $8,000
• qPCR, amplicon sequencing, and follow-up molecular testing: $5,000
• Part-time tissue-culture or plant support: $8,000
• Founder time: $10,000
• Shipping, permits, replacement materials, and contingency: $4,000

Total: $75,000

This budget can change based on the provider’s final quote for the experiment as well as results from the first exchange attempt. In general, a larger budget gives me more ways to run the experiment and more room to respond to the results.

Who is on your team? What's your track record on similar projects?

I am the sole founder of FloraForge and design and run the experiments myself. I also perform the molecular testing to verify results. This includes other administrative tasks needed to establish the company, such as contacting vendors, obtaining permits, and fundraising.

My background is in genome and cell engineering. I began in Douglas H. Smith’s lab at the University of Pennsylvania, where I worked with patterned cortical neuron cultures and used live calcium imaging to study responses after axonal injury. In Century Therapeutics’ iPSC engineering group, I designed and ran experiments to engineer and characterize iPSCs.

When I was at Colossal Biosciences, I worked on the thylacine and dire wolf programs. One of the single cell clones I engineered was later used in the dire wolf program. I designed edits and built donor constructs. I then recovered and screened clones to see which ones had the desired edit.

I started FloraForge in Spring 2025 with construct designs and a plan for a reusable plant platform. That work has since produced 41 living transgenic petunia lines. These include several sequencing-supported landing-pad candidates, and multiple stable lines show strong GFP expression across the flower petals.

At the NC State Phytotron, I set up the plant workflow for receiving lines, planting them in soil, tracking the plants, running infiltrations, measuring fluorescence, making backup clones, and preparing plant material for tissue culture.

I use shared laboratory space for the DNA work, including plasmids, PCR, sequencing, imaging, and qPCR preparation. The plant work, including Agrobacterium infiltration, growth in soil and measurement of expression, is done at the NC State Phytotron. This allows me to take a design into plants and follow the result myself rather than having to hand off the project at the end of each step.

What are the most likely causes and outcomes if this project fails?

Many things can go wrong at the first exchange step, e.g. a bright GFP line can make a very poor host for subsequent transformations; weak recombination in certain tissue types; poor selection; low regeneration; or cultures contaminated with fungi/ bacteria. In the worst case, a whole run could be lost. Even if an attempt fails, I will still learn more about the potential host lines, how the lines compare by copy number, about the quality of the sequence of the introduced DNA, about the quality of the leaf-disc regeneration work, and about potential problems in the plant engineering workflow, including the host, donor, delivery, selection, or regeneration. If one of these steps fails, I should be able to narrow down the failure point and what needs to be changed before the next attempt. The most important thing to test for cassette exchange is whether any of the selected lines can be used for this kind of manipulation.

How much money have you raised in the last 12 months, and from where?

$5,000 from Manifund Round 1. I have also put approximately $17,000 of my own money into FloraForge.

This money has been spent on construct synthesis, transformation work through PBROC, plant growth, lab space at NC State, permits to ship materials, shipping, reagents, imaging, etc. and initial rounds of molecular testing and tissue culture to set up a working plant engineering system. This funding has enabled me to build out a working plant engineering setup for FloraForge, and I now have 41 lines of plants stably transformed with the first generation of constructs. Amplicon sequencing has been completed for candidate lines and GFP images have been taken. Round 2 funding will be used for the first attempt at a cassette exchange in FloraForge plants.