Cas9/gRNA Transfection of Grapevine Protoplasts for Low-Stress Regeneration
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Solution Overview
Problem
Existing methods for transfecting protoplasts from grapevine plants and regenerating edited grapevine plants face challenges in reducing stress on protoplasts and improving the likelihood of successful plant regeneration, while maintaining the identity and organoleptic profile of grapevine fruits.
Innovation Solution
A method involving the preparation of a Cas9:gRNA complex with specific molar ratios, dark incubation, and the use of PEG and saline solutions to transfect protoplasts, followed by minimal centrifugation and immediate cultivation, reduces stress and enhances the regeneration efficiency of edited grapevine plants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional transfection methods are used to introduce CRISPR/Cas9 complex into protoplasts, then genetic modification can be achieved, but the stress on protoplasts increases and regeneration efficiency decreases
Solution Approach 1:
The patent optimizes multiple parameters of the transfection protocol including PEG concentration (40%), incubation time (20 min), and temperature (25°C) to minimize protoplast stress while maintaining transfection efficiency. These parameter adjustments resolve the contradiction by finding the optimal balance between introducing the CRISPR/Cas9 complex and preserving protoplast viability for regeneration.
Solution Approach 2:
The patent performs preliminary optimization of the transfection conditions before actual experimentation, establishing a standardized protocol that minimizes stress on protoplasts. This preliminary action ensures that subsequent regeneration processes start with healthy, low-stress protoplasts, thereby improving regeneration efficiency while reducing harmful stress effects.
2Reliability
If extended incubation periods are used during transfection steps, then transfection efficiency may improve, but protoplast stress increases and regeneration capability decreases
Solution Approach 1:
The patent applies partial action by using a moderate incubation period (20 minutes) rather than extended incubation, which is sufficient to achieve adequate transfection efficiency while avoiding the excessive stress that would result from longer exposure to transfection reagents. This resolves the contradiction by finding the optimal midpoint between insufficient and excessive incubation.
Solution Approach 2:
The patent rapidly proceeds through the transfection process with minimized incubation times, quickly moving from PEG addition to washing and regeneration steps. This rushing through the necessary steps achieves adequate transfection without allowing excessive stress to accumulate, thereby preserving protoplast regeneration capability.
3Adaptability or versatility
If CRISPR/Cas9 complex is introduced into protoplasts for genome editing, then disease resistance can be achieved, but the complexity of the regeneration process increases
Solution Approach 1:
The patent uses a universal transfection protocol that can accommodate different CRISPR/Cas9 target sequences and gRNA designs without requiring separate optimization for each editing target. This multi-functional approach allows disease resistance editing while maintaining a standardized, manageable regeneration process, resolving the contradiction between versatility and complexity.
Solution Approach 2:
The patent divides the complex regeneration process into distinct, manageable stages: transfection, washing, initial culture, and regeneration. Each stage is optimized independently with specific parameters, making the overall complex process more controllable and reproducible while achieving the desired disease resistance through CRISPR/Cas9 editing.
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
The method achieves a high regeneration efficiency of edited grapevine plants by minimizing stress on protoplasts, allowing for successful plant cultivation and maintaining the grapevine's identity and organoleptic qualities.
Implementation Method 1
addition of a PEG 3000 - PEG 5000 solution, preferably PEG 4000, to the mixture obtained in step (II) and incubation
Implementation Method 2
centrifugation of the transfected protoplasts washed in the previous steps preferably for 2 - 4 minutes at 80 - 120×g
Data Source
Figure 1A~2B
Figure 3A~5
Figure 6~7
AI summary
The invention relates to a method for transfection of protoplasts from grapevine plants with the Cas9:gRNA system which includes the addition of a PEG solution and incubation in the dark, followed by washing with saline solutions, centrifugation of the mixture and cultivation of the protoplasts preferably without further incubations. An improved protocol is also described for culturing the transfected protoplasts which specifies details regarding the addition of the culture medium. The effectiveness of the system has been demonstrated, verifying mutations in the genome of the edited plants cultivated from the corresponding transfected protoplasts. The stress-reducing effect of incubating in the dark could be shown.