Home › Microbiology › Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes
Microbiology JoVE (Open Access) Citable · DOI

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

DOI: 10.3791/60782-v
What you'll learn
  • ✓ Perform Agrobacterium-mediated transformation of recalcitrant maize inbred lines
  • ✓ Optimize immature embryo dissection and co-cultivation protocols
  • ✓ Apply morphogenic genes to improve transformation efficiency
  • ✓ Regenerate and select transformed maize plants through heat treatment
Protocol

Plant morphogenic genes can be used to improve genetic transformation of recalcitrant genotypes. Described here is an Agrobacterium-mediated genetic transformation (QuickCorn) protocol for three important public maize inbred lines.

Difficulty
advanced
Total time
~3–4 months (donor plant growth ~4–6 weeks, transformation & regeneration ~8–10 weeks)
Model organism
Maize (Zea mays) inbred lines
Biosafety
BSL-1

Steps

1
Grow donor plants and harvest immature ears

Cultivate maize inbred lines and collect immature ears at the appropriate developmental stage for embryo extraction. Timing is critical for embryo viability.

▶ 01:01
2
Dissect embryos and perform Agrobacterium infection

Isolate immature embryos from harvested ears, expose them to Agrobacterium tumefaciens, and initiate co-cultivation to allow gene transfer into plant cells.

▶ 02:40
3
Select transformed tissue and regenerate plantlets

Culture infected embryos on selective medium under heat treatment to eliminate non-transformed cells and promote morphogenic gene-driven shoot and root regeneration.

▶ 05:54
4
Transplant regenerated plants and produce T1 seed

Transfer regenerated transgenic plantlets to greenhouse conditions, allow plants to mature, and collect T1 seed from self-pollinated transgenic lines for validation.

▶ 07:32
5
Verify transformation results and analyze data

Assess representative outcomes from Agrobacterium-mediated immature embryo transformation, including transformation frequency and regeneration success rates across maize inbred lines.

▶ 08:08
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