# Workflow for Gene Overexpression and Phenotypic Characterisation in Taraxacum kok-saghyz

**Authors:** Loredana Lopez, Michele Antonio Savoia, Loretta Daddiego, Paolo Facella, Elio Fantini, Linda Bianco, Simone Maci, Francesco Panara

PMC · DOI: 10.3390/mps9010017 · Methods and Protocols · 2026-01-24

## TL;DR

This paper outlines a workflow for overexpressing genes in Taraxacum kok-saghyz to study their role in natural rubber production.

## Contribution

The study introduces a complete workflow for gene overexpression and phenotypic analysis in Taraxacum kok-saghyz.

## Key findings

- A workflow for generating transgenic Tks lines overexpressing target genes is described.
- A reliable method for quantifying natural rubber content in transformed plants is presented.

## Abstract

Taraxacum kok-saghyz (Tks) is a promising plant species for natural rubber (NR) production and represents a model for studying NR biosynthesis in the Asteraceae family. The generation of transgenic plants overexpressing a gene of interest is a well-established strategy to investigate gene function and potential interactions. Here, we present a comprehensive workflow—from the construction of an overexpression vector to the generation, identification, and propagation of stable transgenic Tks lines. In addition, we describe a rapid and reliable method for quantifying NR content in transformed plants, providing essential phenotypic characterisation in this species.

## Linked entities

- **Chemicals:** natural rubber (PubChem CID 6557)
- **Species:** Taraxacum kok-saghyz (taxon 333970)

## Full-text entities

- **Genes:** NOS2 (nitric oxide synthase 2) [NCBI Gene 4843] {aka HEP-NOS, INOS, NOS, NOS2A}
- **Diseases:** Infection (MESH:D007239), Tks (MESH:C538136), necrotic (MESH:D009336), injury to (MESH:D014947), root rot (MESH:D005535), SALB (MESH:C535551), American Leaf Blight (MESH:D006478)
- **Chemicals:** Sephadex G-50 (MESH:C025614), Hexane (MESH:D006586), Glycerol (MESH:D005990), 1-Naphtalenacetic acid (-), aluminium (MESH:D000535), Sepharose CL-6B (MESH:C047264), Rifampicin (MESH:D012293), Acetone (MESH:D000096), carbohydrate (MESH:D002241), CR (MESH:D002857), sucrose (MESH:D013395), Agarose (MESH:D012685), Resin (MESH:D012116), fructose (MESH:D005632), glucose (MESH:D005947), Auxins (MESH:D007210), H2SO4 (MESH:C033158), Tween-20 (MESH:D011136), cellulose (MESH:D002482), IAA (MESH:C030737), salts (MESH:D012492), NH3 (MESH:D000641), Trans-Zeatin (MESH:D015026), PTFE (MESH:D011138), Parafilm (MESH:D010232), agar (MESH:D000362), Amoxicillin/Clavulanic acid (MESH:D019980), nitrogen (MESH:D009584), free fatty acids (MESH:D005230), 1-naphthaleneacetic Acid (MESH:C034182), Water (MESH:D014867), ethanol (MESH:D000431), polyethylene (MESH:D020959), Kanamycin (MESH:D007612), Inulin (MESH:D007444), HCl (MESH:D006851)
- **Species:** Homo sapiens (human, species) [taxon 9606], Agrobacterium tumefaciens (species) [taxon 358], Martinezella rhizogenes (species) [taxon 359], Hevea brasiliensis (jebe, species) [taxon 3981], Escherichia coli (E. coli, species) [taxon 562], H. brasiliensis [taxon 312095], Taraxacum brevicorniculatum (species) [taxon 1262544], Taraxacum kok-saghyz (species) [taxon 333970]
- **Mutations:** C with a 16, M0491L, R3156L, R0145L, C with 200
- **Cell lines:** DH5 alpha — Drosophila hydei (Fruit fly), Spontaneously immortalized cell line (CVCL_Z531), pBI121 — Homo sapiens (Human), Ovarian carcinosarcoma, Cancer cell line (CVCL_G294)

## Full text

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## Figures

8 figures with captions in the complete paper: https://tomesphere.com/paper/PMC12922061/full.md

## References

27 references — full list in the complete paper: https://tomesphere.com/paper/PMC12922061/full.md

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Source: https://tomesphere.com/paper/PMC12922061