Home › Genetics / Genomics › Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii
Genetics / Genomics JoVE (Open Access) Citable · DOI

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

DOI: 10.3791/59971-v
What you'll learn
  • ✓ Perform Agrobacterium tumefaciens-mediated stable transformation in Parasponia andersonii
  • ✓ Design and validate CRISPR/Cas9-based genome edits in tropical tree species
  • ✓ Establish nodulation phenotyping protocols for nitrogen-fixing symbiosis assessment
Protocol

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae) and can form nitrogen-fixing root nodules in association with the rhizobium. Here, we describe a detailed protocol for reverse genetic analyses in P. andersonii based on Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing.

Difficulty
advanced
Total time
~8–12 weeks (transformation to stable line generation and nodulation assessment)
Model organism
Parasponia andersonii
Biosafety
BSL-1

Steps

1
Grow Parasponia andersonii trees from seed

Establish healthy P. andersonii seedlings under controlled conditions to serve as transformation donors or control plants. Seedlings are typically grown for several weeks before use.

▶ 01:08
2
Perform stable transformation via Agrobacterium tumefaciens

Inoculate P. andersonii explants with Agrobacterium carrying CRISPR/Cas9 or transgene constructs, culture on selection media, and recover stable transformants. Selection and regeneration typically require multiple weeks.

▶ 03:14
3
Prepare rooted transformant plantlets for nodulation

Establish well-developed root systems in transgenic plantlets prior to inoculation with rhizobium. Roots must be sufficiently mature to support nodule formation.

▶ 06:40
4
Inoculate plantlets with rhizobium and assess nodulation

Introduce nitrogen-fixing rhizobium symbionts to rooted P. andersonii plantlets in sterile pots and monitor nodule formation and phenotypic traits. Nodulation typically develops over 2–4 weeks post-inoculation.

▶ 07:31
5
Validate stable transformation and phenotypic outcomes

Confirm transgene integration via molecular methods (PCR, sequencing) and quantify nodulation phenotypes (nodule number, biomass, nitrogen fixation markers). Results validate genome editing or overexpression effects.

▶ 09:48
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