Grape Protoplast Regeneration for Non-Chimeric Gene Editing

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Solution Overview

Problem

Existing methods for introducing genetic modifications into clonally propagated plants like grapevines and regenerating whole plants from protoplasts have not been successful, particularly due to challenges in achieving non-chimeric gene-edited plants and maintaining the fidelity of clonal germplasm.

Innovation Solution

A method involving encapsulating grape protoplasts in a gel matrix, culturing them with osmotically conditioned media and antioxidants or polyamines, forming callus colonies, and developing somatic embryos to regenerate whole plants, using CRISPR-Cas9 for precise genomic alterations.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If protoplast culture is used to produce non-chimeric gene edited plants in clonally propagated species, then genetic modification precision is improved, but plant regeneration capability deteriorates

Engineering Contradiction:

Improvegenetic modification precisionVSAvoidplant regeneration capability

Core Design Contradiction:

Manufacturing precisionVSReliability

Solution Approach 1:

The patent uses encapsulation matrices as an intermediary environment to support protoplast survival and regeneration. The matrix provides structural support and controlled microenvironment that facilitates cell wall reformation and division, enabling plant regeneration from protoplasts that would otherwise fail to regenerate

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent optimizes multiple parameters including osmotic potential (using mannitol and sorbitol), pH levels, nutrient composition, and hormone concentrations in the culture medium. These parameter changes create optimal conditions for protoplast stability, cell wall reformation, and subsequent plant regeneration

Inventive Principle:

Principle #35Parameter changes

2Productivity

If CRISPR-Cas9 DNA is introduced into protoplasts using PEG or electroporation, then gene editing efficiency is improved, but protoplast stability deteriorates due to osmotic stress

Engineering Contradiction:

Improvegene editing efficiencyVSAvoidprotoplast stability

Core Design Contradiction:

ProductivityVSStability of the object's composition

Solution Approach 1:

The patent pre-treats protoplasts with osmotic protectants (mannitol, sorbitol) and antioxidants before CRISPR-Cas9 delivery. This cushioning protects protoplasts from osmotic shock during PEG or electroporation treatment, maintaining membrane integrity and cellular stability throughout the gene editing process

Inventive Principle:

Principle #11Beforehand cushioning (Prior cushioning)

Solution Approach 2:

The patent converts the potentially harmful osmotic stress of PEG/electroporation into a beneficial effect by using controlled osmotic conditions. The same osmotic agents that could cause damage are used in controlled amounts to facilitate DNA delivery while simultaneously protecting cellular structures through osmotic balance

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

3Reliability

If cell wall reformation is allowed to occur in 48-72 hours, then protoplast recovery is improved, but time for subsequent plant regeneration deteriorates

Engineering Contradiction:

Improveprotoplast recoveryVSAvoidtime for plant regeneration

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The patent performs preliminary actions during the 48-72 hour cell wall reformation period by pre-supplementing the culture medium with all necessary nutrients, hormones, and growth factors. This preliminary preparation ensures that once cell walls are reformed, protoplasts are immediately ready for rapid division and plant regeneration without requiring additional adjustment periods

Inventive Principle:

Principle #10Preliminary action

4Reliability

If encapsulation in gel matrix is used to support protoplast culture, then protoplast survival is improved, but diffusion of nutrients and gases deteriorates

Engineering Contradiction:

Improveprotoplast survivalVSAvoidnutrient diffusion efficiency

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The patent uses porous gel matrices that provide a three-dimensional network structure with controlled pore sizes. This porous structure physically supports protoplasts while allowing efficient diffusion of nutrients, gases, and waste products through the matrix, resolving the contradiction between structural support and mass transfer

Inventive Principle:

Principle #31Porous materials

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 the regeneration of non-chimeric gene-edited grape plants by ensuring single-cell descent and efficient genomic modifications, overcoming previous limitations in grapevine regeneration from protoplasts.

Implementation Method 1

encapsulating an isolated grape protoplast in a gel matrix

Methodology Applied

Scientific EffectGel matrix encapsulation: Gel

Implementation Method 2

culturing the encapsulated protoplast in the presence of an osmotically conditioned grape cell suspension culture or osmotically adjusted conditioned medium

Methodology Applied

Scientific EffectOsmotic pressure: Osmotic Pressure

Data Source

PatentUS12593767B2Protoplast isolation and regeneration of plants

Publication Date: 2026.04.07 RGT UNIV OF CALIFORNIA

AI summary

The present disclosure provides methods for inducing callus formation and plant regeneration from isolated protoplasts of grape. This technology allows for the production of non-chimeric gene edits in grape plants by allowing the delivery of DNA through the plant cell membrane with the recovery of whole plants from a single edited cell.