Modifying fatty acid profiles through a new cytokinin-based plastid transformation system

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Genetics

Background:

• Antibiotic and herbicide resistance genes are commonly used for transgenic plant selection but face commercialization and public acceptance challenges.
• Alternative selection systems are crucial for advancing plant biotechnology and overcoming limitations of current methods.

Purpose of the Study:

• To evaluate the efficacy of the bacterial ipt gene as a positive selectable marker for plastid transformation in plants.
• To compare the ipt selection system with the traditional spectinomycin-based aadA system.
• To demonstrate the proof of principle by transforming the kasIII gene into tobacco plastids using the ipt system.

Main Methods:

• Utilized the ipt gene from Agrobacterium tumefaciens for positive selection in plastid transformation.
• Compared ipt-mediated selection with spectinomycin-based aadA selection on different media.
• Introduced the kasIII gene into tobacco plastids via Agrobacterium-mediated transformation using the ipt selection system.

Main Results:

• The ipt selection system proved as effective as the spectinomycin-based system for selecting transplastomic plants.
• Transplastomic tobacco plants were successfully generated using the ipt system, exhibiting normal phenotypes despite ipt over-expression.
• Over-expression of the kasIII gene led to significant alterations in fatty acid profiles, specifically increasing 16:0 and decreasing 18:0 and 18:1 levels.

Conclusions:

• The ipt gene provides a novel and effective positive selection system for plastid transformation, independent of transgene expression or protein activity.
• This system shows potential for application in monocots, which are often recalcitrant to antibiotic-based selection.
• The ipt system can be integrated into a two-step selection strategy with a negative marker to achieve homoplasmic plant lines.