Botany JoVE (Open Access) Citable · DOI

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Name: Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale
Uploaded: 2026-06-17
Duration: 8 min 56 s
Description: The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

DOI: 10.3791/58169-v

What you'll learn

✓ Prepare and perform vacuum infiltration of Agrobacterium suspensions into N. benthamiana leaves
✓ Execute preparative-scale triterpene extraction using pressurized solvent methods
✓ Apply ion exchange chromatography to remove chlorophyll contaminants from plant extracts

Protocol

Transient heterologous expression of biosynthetic enzymes in Nicotiana benthamiana leaves can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products. Herein is described a detailed protocol for rapid (5 days) preparative-scale production of triterpenes and analogs utilizing this powerful plant-based platform.

Difficulty

intermediate

Total time

~5 days (includes 5-day growth period post-infiltration)

Model organism

Nicotiana benthamiana

Biosafety

BSL-1

Steps

Prepare Agrobacterium infiltration suspension

Culture and prepare Agrobacterium tumefaciens suspension containing desired triterpene biosynthetic genes. Adjust optical density and resuspend in infiltration medium to optimal concentration for transient plant expression.

▶ 00:33

Perform vacuum infiltration of plant leaves

Subject intact N. benthamiana plants to vacuum pressure while submerged in Agrobacterium suspension to facilitate rapid uptake of expression constructs into leaf tissue.

▶ 02:11

Grow infiltrated plants and harvest tissue

Maintain infiltrated plants for 5 days to allow protein expression and triterpene biosynthesis. Harvest leaves and perform pressurized solvent extraction to recover triterpene products.

▶ 03:56

Treat extract with ion exchange resin

Pass crude plant extract through ion exchange resin columns to remove chlorophyll and other polar contaminants, yielding purified triterpene product.

▶ 05:38

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