Improved cotton transformation protocol mediated by Agrobacterium and biolistic combined-methods

Area of Science:

• Plant Biotechnology
• Agricultural Science
• Molecular Biology

Background:

• Cotton (Gossypium spp.) is a vital global crop for textile fiber production.
• Significant crop losses occur due to insect pests, pathogens, and abiotic stresses.
• Genetically modified (GM) cotton varieties offer a solution to enhance crop resilience.

Purpose of the Study:

• To describe an improved, straightforward, and cost-effective protocol for genetic transformation of cotton embryo axes.
• To merge Agrobacterium-mediated transformation with biolistic methods for enhanced DNA delivery.
• To overcome limitations in cotton genetic modification, particularly recalcitrance in certain genotypes.

Main Methods:

• A combined protocol involving Agrobacterium preparation, seed sterilization, embryo excision, biolistic bombardment, Agrobacterium transformation, co-culture, and in vitro regeneration.
• Utilized cotton embryo axes, leveraging their high regenerative capacity and meristem cell potential.
• Employed tungsten particle bombardment for enhanced DNA delivery into shoot cells.

Main Results:

• Achieved an average plant regeneration rate of 5.5% and a transformation efficiency of 60% within 4-10 weeks.
• Demonstrated stable transgene inheritance with most transgenic plants possessing a single transgene copy.
• Confirmed stable transgene expression across generations, with high protein levels in T2 progeny, showing no phenotypic or productivity differences from wild-type.

Conclusions:

• The combined Agrobacterium and biolistic approach provides a highly efficient protocol for producing transgenic cotton.
• This method successfully overcomes recalcitrance issues in cotton genotypes, enabling efficient transgenic plant recovery.
• The developed protocol is crucial for advancing global agribusiness through the creation of GM cotton with desirable traits.