Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system

Area of Science:

• Plant Biotechnology
• Genetics and Molecular Biology
• Agricultural Science

Background:

• Maize (Zea mays) transformation is crucial for crop improvement.
• Standard Agrobacterium tumefaciens binary vectors are widely used but can be inefficient for maize.
• Developing robust and efficient transformation protocols is essential for genetic studies and breeding.

Purpose of the Study:

• To establish a routine maize transformation protocol using a standard Agrobacterium tumefaciens binary vector system.
• To evaluate the effect of L-cysteine on transformation efficiency and embryo response.
• To confirm the stable integration, expression, and inheritance of transgenes in transgenic maize.

Main Methods:

• Immature zygotic embryos of maize hybrid line Hi II were infected with Agrobacterium tumefaciens strain EHA101 carrying a standard binary vector.
• Cocultivation was performed in the presence of L-cysteine (400 mg/L).
• Stable transformation efficiency was assessed by bialaphos resistance, and transgene integration/expression was confirmed using Southern-blot and progeny analysis.

Main Results:

• Inclusion of L-cysteine improved transient beta-glucuronidase expression and significantly increased stable transformation efficiency to an average of 5.5%.
• L-cysteine supplementation decreased embryo response post-cocultivation.
• Transgene integration, expression, and inheritance (bar and gus) were confirmed across R0, R1, and R2 generations.

Conclusions:

• A routine and efficient method for producing fertile, stable transgenic maize using a standard Agrobacterium tumefaciens binary vector system has been developed.
• L-cysteine is a key component for enhancing transformation efficiency in this maize system.
• This protocol provides a valuable tool for genetic manipulation and breeding of maize.