Non Technical Summary
Natural Rubber (cis-1,4-polyisoprene; NR) is a vital commodity that is used in the production of tens of thousands of consumer products, with applications in industries ranging from medical devices to shoes and tires. Synthetic rubber (SR) is also used in the manufacturing of many products, however, it is not renewable like NR and it is synthesized from petroleum oil byproducts. SR is inferior to NR in many chemical and mechanical properties. It cannot replace NR in most applications, including the majority of tires. An overly simplified way to view it is that the bigger the tire, the higher the percentage of natural rubber used in compounding and formulation. Therefore, the transportation, construction, mining, agricultural and defense sectors of the economy are all impacted by NR production. The market is also strong for NR in other applications; approximately 20% of NR is sold into non-tire markets in the US, often at substantially higher cost (i.e., vs the tire market) for the raw material. Currently, the main worldwide source of NR comes from Hevea brasiliensis, the para rubber tree. It is being produced in a relatively small climatic region in Southeast Asia and there are many biological, economic, and social threats facing today's NR supplies.Kultevat, Inc. aims to produce natural rubber from Taraxacum kok-saghyz (TKS) at scale to achieve high quality product at a price point that is attractive to customers, in particular to US based companies. To date, the economics of growing TKS and extracting rubber from its roots at a price point that is competitive with Hevea rubber has not been demonstrated. During Phase I research, an RNAi gene silencing strategy was used to reduce the activity of genetic repressors that control plant flowering, with the goal to delay flowering of TKS. The technology, referred to as Biomass Enhancement Technology (BET), was developed by Performance Plants Inc. and was soley licensed to Kultevat, Inc. for use in TKS in North America. A similar technical approach resulted in a 1-4 fold increase of biomass in crop plants in which it has been applied. The immediate goal of this Phase II project is to scale up confirmed T0 TKS-BET lines by clonal propagation to use for T1 seed production. Cellular, molecular and biochemical characterization will be done to verify (1) an increase in cell/biomass growth and (2) total rubber content per plant, of ensuing T1 plant lines. The best T1 lines identified will be used in pollen-crossing to produce pre-commercialization T2 varieties. Success will lead to future R&D to develop a commercial variety for the US, spawning a new US-based industry that will supply markets for natural rubber and inulin.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
511	2249	1030	100%

Knowledge Area
511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
2249 - Rubber, gum, and resin plants, general/other;

Field Of Science
1030 - Cellular biology;

Goals / Objectives
Work in Phase I led to the development of 4transgenic TKS T0 lines (founder lines) that exhibit the predicted molecular phenotype. Twoof the lines were identified in earlier transformation eventsand two additional lines were identified after the Phase I award period ended. An additional 3 T0 lines have since been confirmed for the desired gene expression level. The first twoconfirmed lines have been clonally propagated for the purpose of developing seeds by cross pollination between the T0 plant lines (i.e., T01 x T02) and between T0 lines and maintenance non-transgenic plants (recall that TKS plants are self-incompatible; thus outcrossing to other TKS plants is required). With that in mind, the technical objectives of the proposed research are as follows:(1) To produce a small number of T1 seeds by hand pollinating/crossing select transgenic T0 TKS FVE-RNAi plants under greenhouse conditions as described; the technical goal is to produce a small population of seeds to maintain the transgene inserts.(2) To grow a small number of replicate clones of each initial T0 line in greenhouse conditions to:a. determine mature root dry weight and percent dry weight rubber content,b. carry out microscopy studies on mature TKS rootsThe goal is to test the hypothesis that knockdown expression of TKS FVE will cause an increase in plant (root) biomass and thus an overall increase in total rubber yield per plant, and that the effect can be measured in T0 plants.(3) To produce T1 seeds from clones of T0 plants that exhibit the desired molecular phenotype; the technical goal is to use clones of T0 lines to produce large amounts of seeds via insects. This will be conducted by Kultevat's current seed producers, Priority Seeds (Yuma AZ; see technical objective (5) below) using;a. equal numbers of wild-type plants (to serve as maintainer lines) and clones of transgenic T0 plants (mixed assortment of clones of different T0 plants of known lineage) in a netted outdoor cage(4) To establish a genetic marker or 'DNA foot-print' for each of the independent T0 'parent' plants;a. DNA foot-printing will be used to identify parentage of T1 progeny.(5) To grow T1 seeds generated through (3) for the following:a. genetic phenotyping using DNA foot-printing method,b. anatomical phenotyping using microscopy,c. biochemical phenotyping through determination of the percent dry weight rubber and concomitant increases in total rubber yield(6) To cross the 'best' T1 plants from (5) to produce T2 pre-commercialization seeds;a. Through the development and characterization studies laid out in objectives (1)-(5), it is possible and anticipated that a population of T2 seeds will be produced by the end of the Phase II award period.The objectives stated here are feasible in the 3 year award period proposed. The Kultevat laboratory is set up for some of the initial genotyping and rubber extraction work that is required during the award period. Kultevat's greenhouse space is located within the Donald Danforth Plant Science Center (DDPSC) plant growth buildings (Field 10). Additional small scale field growth of the best lines may be done if necessary, at a time and location TBD. For larger scale genetic characterization, Kultevat will use a local molecular diagnostics company, MOgene LC, on a contract basis. Additionally, the imaging and microscopy work proposed will be completed at the DDPSC Imaging Core Facility under a fee-for-service agreement.

Project Methods
Kultevat's use of the BET technology during the Phase I award term led to the generation of several T0 parent lines. Technical objective (1) of the proposed research herein is to produce a small number of T1 seeds by hand pollination.A. Controlled hand crossing of T0 plants/clonesAs TKS is an obligate out-crosser, a mixed population of clones of T0 plants and non-transgenic parental lines from which the T0 plants were developed will be grown to 6-8 weeks of age. The young plants will be moved to a vernalization chamber for 4-6 weeks, set to 6°C, 14 hour day length. After vernalization, the plants will be moved to the Kultevat greenhouse and monitored for stem and flower development. Crossing will be done as described in Iaffaldano et al. (2018), with optimization to be tested and determined.Some of the replicate T0 clones will be grown in greenhouse conditions until at least 6 months of age to determine mature root dry weight and percent dry weight rubber content to test the hypothesis that suppression of TKS FVE will cause an increase in plant biomass and total rubber yield per plant (Technical objective (2)).B. Plant growing conditionsAll plants in soil will be grown in the Kultevat-designated (leased) greenhouse located at the Danforth Plant Science Center. Plant growth conditions in the greenhouse are set for 16 hour day length (06:00-22:00), with a day temperature of 24ºC, a night temperature of 20ºC and 40% relative humidity.C. Accelerated Solvent Extraction of rubber from TKS root tissueA Dionex Thermo Scientific ASE 350 Model (Sunnyvale, CA) is the standard technology to extract rubber from TKS and is used routinely in the Kultevat laboratory for that purpose. The protocol is designed to first extract organic contaminants (including resins) followed by extraction of natural rubber from TKS dry root tissue. Details of the methods and the protocol for quantitating the amount in a sample are described in the Phase I proposal.Technical objective (3) involves scaling up clonal propagation through tissue culture of selected T0 TKS-BET plants that show reduced TKS FVE expression to generate T1 seeds. A larger set of clones will be needed in a cross pollination program using bees. An equal mix of wildtype ('maintainer lines') and a mixed assortment of transgenic T0 plants will be netted in an outdoor cage. Kultevat's contract seed production company, Priority Seed Productions (Salinas, CA), will grow the plants and collect and grade the seeds.D. Scaling tissue culture clonal propagationYoung (4-5 inches in length) transgenic T0 TKS leaves are harvested and surface sterilized by standard methods. Small leaf pieces are placed onto shooting medium, followed by shoot elongation, followed by rooting medium, all of which contain Kanamycin to avoid development of chimeric plants. The cloning process takes approximately 12 weeks from collecting leaves to planting rooted plantlets in soil. Plantlets are planted in soil in the greenhouse or growth chamber, set for 16 hour day length (06:00-22:00), day temperature of 24ºC, night temperature of 20ºC, 40% relative humidity. Day 1 in soil is considered day 1 of plant life. Wildtype and transgenic plants will be grown and monitored under the same conditions.A genetic marker or "DNA foot-print" will be established for each independent parent T0 plant such that lineage of the T1 seeds can be ascertained (Technical objective (4)). It is anticipated that each T0 will have a unique 'DNA signature' that can be established as a unique DNA fragment distinguished by its size. Kultevat will use contract services of a local molecular genetics/diagnostics company, MOgene LC (St. Louis, MO), for established DNA signature. MOgene will provide unique primer sets for PCR based amplification of a DNA fragment for each of the T0 lines. While MOgene will conduct the initial diagnostics, and conduct high throughput methods to analyze initial T1 progeny, it is expected that further work will be conducted at Kultevat for cost-savings.T1 seeds obtained through crossing of T0 parent lines, and crosses with non-transgenic plants will first be screened by plating on MS media with Kanamycin to eliminate non-transgenic seeds. Surviving seedlings will be grown in either greenhouse or field conditions (TBD, based on space requirements) to determine genetic lineage through the DNA foot-printing method as above (conducted by MOgene). Individuals that contain one or more alleles (crossing of T0 x T0 will produce T1 plants with two alleles of the transgene) will be grown to near maturity (6-9 months) for further studies. These will include to perform anatomical phenotyping through microscopy methods and to determine the percent dry weight rubber and concomitant increases in total rubber yield (Technical objective (5)).These studies will be critical to determining the value of the BET technology for Kultevat. For example: (1) will one or more transgenic lines have delayed flowering compared with non-transgenic plants; (2) will one or more T0 lines exhibit changes in root anatomy and rubber accumulation when compared with non-transgenic plants; (3) do observed changes occur when one copy of the transgene is present, or are two copies of the transgene required for sufficient suppression of FVE RNA to produce the desired phenotype described in (1) and (2)? Item (3) will subsequently be part of the decision making process that will lead to initial field trials of the transgenic lines.The 'best' T1 plants identified through the efforts aforementioned will be used to produce a population of T2 pre-commercialization seeds; this goal is expected to be reached by the end of the proposed Phase II period (Technical objective (6)).E. Genomic DNA extraction and genotypingFor in-house genotyping/foot-printing, genomic DNA will be extracted from leaf tissue from transgenic lines according to QIAGEN's DNeasy Plant Pro Kit Core Kit (QIAGEN, Valencia, CA). The genomic DNA will be used as template in a PCR reaction using sense and antisense primers specific to the identified T0 transgene inserts.Plant size and morphology will be observed and compared throughout the period of growth in all aforementioned studies to compare transgenic TKS-BET plants and wildtype plants. If expression of the target gene has the anticipated effect, we expect to see significant variation in the size of the stems and roots of plants that have low levels of TKS FVE in T1 and/or T2 seed generations. Statistical analyses will be done on all measurements, with focus on plants derived from T1 and T2 seeds. If the hypothesis is correct, we anticipate to record significant differences in plant size and tissue sizes (i.e., increased stem and root diameter and larger body mass) as described above, in TKS lines with reduced FVE RNA. These differences must exhibit a positive correlation between a decrease in FVE gene expression and the target phenotype.All work will be done either in the Kultevat laboratory or in the Donald Danforth Plant Science Center (DDPSC) Plant Transformation Facility or Plant Growth Facility. Thesespaces includetissue culture hoods, and a small number of incubators and chambers that Kultevat uses regularly for some of the cloning work. The Kultevat laboratory has the necessary molecular biology equipment, and the ASE 350, and a GPC system to analyze high molecular weight polymers; the latter is used to assess the quality of rubber produced by TKS. The DDPSC Imaging facility, Priority Seeds, and MOgene will be used on a contract basis for any necessary microscopy, seed production, and DNA foot-printing work, respectively.With a team including 2 PhD level scientists and 1-2 research technicians, Kultevat sees no obstacles to completing the project and strongly believes it to be technically feasible within the proposed 3 year term length of this SBIR Phase II proposal.