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Botany JoVE (Open Access) Citable · DOI

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

DOI: 10.3791/2633-v
What you'll learn
  • ✓ Prepare Medicago truncatula seedlings for hairy root transformation
  • ✓ Generate composite plants via Agrobacterium rhizogenes transformation
  • ✓ Inoculate transgenic roots with rhizobia for nodulation assays
  • ✓ Identify transgenic roots using a visual reporter marker
Protocol

Biopharma Insights We demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species Medicago truncatula.

Difficulty
intermediate
Total time
~6-8 weeks
Model organism
Medicago truncatula
Biosafety
BSL-1

Steps

1
Understand hairy root composite plant system

Introduce the hairy root composite plant approach for studying plant-rhizobium interactions and nodulation in hard-to-transform Medicago truncatula.

▶ 00:05
2
Prepare Medicago truncatula plant materials

Sterilize, germinate, and grow Medicago truncatula seedlings under controlled conditions before transformation.

▶ 02:39
3
Generate hairy root composite plants

Inoculate seedling explants with Agrobacterium rhizogenes to induce transgenic hairy roots, then select composite plants.

▶ 06:09
4
Infect roots with symbiotic rhizobia

Apply compatible rhizobia to transgenic roots to initiate symbiotic infection and nodule formation.

▶ 09:39
5
Identify transgenic roots visually

Use a fluorescent or visual reporter marker to distinguish transgenic hairy roots from non-transgenic roots.

▶ 11:10
6
Analyze nodulation results

Summarize the workflow and interpret nodulation phenotypes in composite plants.

▶ 12:00
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