Cotton Genome Editing via Embryogenic Callus and Endonuclease
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Solution Overview
Problem
Current methods for targeted genome modification in plants, such as cotton, are inefficient due to recalcitrance in tissue culture and transformation, limiting precise nucleotide sequence modifications.
Innovation Solution
The use of friable embryogenic callus in cotton plants, maintained under dim light and active carbon conditions, with the introduction of a rare-cleaving endonuclease enzyme to induce double-stranded DNA breaks, allowing for precise modifications like insertion, deletion, or substitution at predefined sites, facilitated by DNA delivery methods like particle bombardment or Agrobacterium-mediated transformation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional transformation methods are used in cotton plants, then the transformation process can be performed, but the efficiency is low due to recalcitrance in tissue culture and transformation
Solution Approach 1:
The invention changes the physical and chemical parameters of the culture medium by adding specific hormones (2,4-D at 1-5 µM, kinetin at 0.1-1 µM), adjusting pH to 5.8, and incorporating activated carbon (0.1-10 g/L) to create optimal conditions for embryogenic callus formation in cotton, thereby overcoming tissue culture recalcitrance and improving transformation efficiency
Solution Approach 2:
The invention uses embryogenic callus as an intermediary stage between the cotton plant tissue and the transformed plant. The callus acts as a highly receptive intermediate tissue that facilitates efficient DNA uptake and integration, solving the recalcitrance problem of direct cotton plant transformation
2Manufacturing precision
If double stranded DNA breaks are induced to increase homologous recombination frequency, then targeted modifications can be achieved, but the process complexity increases
Solution Approach 1:
The invention performs preliminary action by pre-inducing embryogenic callus with specific hormone treatments and activated carbon before introducing the DSBI enzyme. This preparation creates a highly receptive cellular state that enhances subsequent homologous recombination efficiency while simplifying the overall process
Solution Approach 2:
The invention segments the transformation process into distinct stages: callus induction phase (with hormones and activated carbon), DSBI enzyme introduction phase, and regeneration phase. This segmentation allows optimization of each stage independently, achieving high precision targeted modifications without excessive overall complexity
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables efficient targeted genome modifications in cotton plants, achieving precise nucleotide changes at predefined sites with high accuracy and regeneration into viable cotton plants.
Implementation Method 1
inducing a double stranded DNA break in the vicinity or at of said predefined site, said double stranded break being induced by the introduction into said cell of a rare-cleaving endonuclease enzyme which recognizes a recognition sequence in the vicinity of or at said predefined site
Implementation Method 2
said cell is comprised within friable embryogenic callus, wherein said friable embryogenic callus has been maintained under dim light conditions and on medium comprising active carbon
Data Source
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AI summary
Methods and means are provided to modify in a targeted manner the genome of a cotton plant using a double stranded DNA break inducing enzyme and embryogenic callus.