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I am currently working on a project focused on protoplast culture and somatic fusion for crop improvement, with a particular interest in identifying practical protocols and published case studies where new varieties have been successfully developed using this technique.

As I had to work in protoplast culture for crop improvement, in that case I am looking for literature on , with a particular interest in identifying practical protocols and published case studies where new varieties have been successfully developed using this technique.

Hello,

Here are key references and case studies on protoplast culture for crop improvement, including practical protocols and hybrid development:

1. Protocols & Practical Guides

• A comprehensive overview of isolation and culture techniques (enzymatic digestion, viability assays, culture media), useful for setting up protocols in your lab slideshare.net+15plantcelltechnology.com+15tandfonline.com+15researchgate.netplantcelltechnology.com.
• A ScienceDirect chapter details protoplast use in somatic hybridization and genetic manipulation, including examples of viable regeneration systems plantcelltechnology.com+5sciencedirect.com+5researchgate.net+5.

2. Case Studies of Crop Improvements

• Swapan K. Datta’s work on Indica rice: Developed transgenic pollen- and homozygous lines regenerated from protoplasts, including Golden Rice and iron-enriched rice youtube.com+15en.wikipedia.org+15sciencedirect.com+15.
• Duboisia species: First mesophyll protoplast-to-plant regeneration protocol; optimized enzyme treatment and plating density achieved 33% division and 17.9% microcalli induction vtechworks.lib.vt.edu+2mdpi.com+2bmcplantbiol.biomedcentral.com+2.
• Buckwheat (Fagopyrum spp.): Used antioxidants/activated charcoal to enhance protoplast regeneration, laying groundwork for future genetic enhancement slideshare.net+15bmcplantbiol.biomedcentral.com+15vtechworks.lib.vt.edu+15.

3. Applications in Crop Improvement & Biotechnology

• Somatic hybridization and cybridization: Fusion of protoplasts across species enables creation of novel hybrids—used in tobacco, wheat × soybean hybrids researchgate.net+4plantcelltechnology.com+4researchgate.net+4.
• Gene function studies & editing: Protoplasts allow transient transformation (e.g., CRISPR, RNAi) and pathogen defense analysis in tomato, Arabidopsis, wheat, and rice .

4. Practical Training & Lab Tips

• SlideShare tutorials and video demos detail protocols for protoplast isolation/culture, including enzyme digestion setups, media formulations, viability assays, and regeneration techniques youtube.com+4slideshare.net+4slideshare.net+4.
• Lab-based tips include enzyme concentrations, osmotic stabilisers, plating density, and culture additives (activated charcoal, antioxidants, auxin/kinetin ratios) bmcplantbiol.biomedcentral.com+2plantcelltechnology.com+2plantcelltechnology.com+2.

Suggestions for Your Work

• Choose species-specific enzyme mixes and optimize plating density (e.g., 0.5×10⁵ cells/mL for Duboisia).
• Use additives (activated charcoal, ascorbic/citric acid) to reduce browning and improve division/callus formation.
• Tailor media (MS/B5) with correct auxin-to-cytokinin ratio for callus and shoot regeneration.
• Explore protoplast fusion or transient gene editing (e.g., CRISPR RNP delivery) to develop improved or hybrid varieties.

Hello,

Here is a curated collection of literature and case studies on protoplast culture for crop improvement, including somatic hybridization and modern gene-editing applications:

1. Reviews & Protocol Overviews

• Reed & Bargmann (2021) review the use of protoplast regeneration in next-generation plant breeding (e.g., CRISPR/Cas delivery via protoplasts) as a DNA-free approach to produce edited plants in a single generation pmc.ncbi.nlm.nih.gov+15frontiersin.org+15arcjournals.org+15.
• A thorough technique guide outlines isolation, culture, and fusion methods of protoplasts, covering enzymatic digestion, PEG fusion, and regeneration steps .
• Springer mini-review (2025) highlights modern isolation workflows, high-throughput approaches, and integration with CRISPR for tailored protoplast applications arcjournals.org+8link.springer.com+8pmc.ncbi.nlm.nih.gov+8.

2. Somatic Hybridization & Case Studies

• Begna (2021) provides a review of somatic hybridization, especially across sexually incompatible species, for trait introgression (e.g., disease resistance, abiotic stress tolerance) pmc.ncbi.nlm.nih.gov+4researchgate.net+4mdpi.com+4.
• Mackowska et al. (2023) report successful interspecific carrot hybrids using dual fluorescent labeling and micro‑selection—yielding novel hybrid plants sciencedirect.com+2plantmethods.biomedcentral.com+2pmc.ncbi.nlm.nih.gov+2.
• MDPI study on Brassica oleracea explores the development of somatic hybrids and cytoplasmic male sterility (CMS) lines for hybrid seed production mdpi.com+1mdpi.com+1.

3. Regeneration in Crop Species

• BMC Plant Biology (2024) optimized protocols for protoplast-to-plant regeneration in Brassica oleracea, including collards, with high viability and shoot formation efficiency bmcplantbiol.biomedcentral.com.
• Ongoing improvements in legumes and grapes highlight protoplast culture's use in genome editing and trait improvement sciencedirect.com+4tandfonline.com+4frontiersin.org+4.

4. Key Practical Insights

• Efficient protoplast culture requires species-specific enzyme blends, optimized plating density or immobilization (e.g., alginate layers), and careful addition of antioxidants and phytohormones pmc.ncbi.nlm.nih.gov+15plantcelltechnology.com+15bmcplantbiol.biomedcentral.com+15.
• Selectable markers like dual-fluorescence in carrot, or genetic tools (CRISPR/Cas9 RNPs), enhance selection and regeneration efficiency plantmethods.biomedcentral.com+1pmc.ncbi.nlm.nih.gov+1.

Tohid Nooralvandi Thank you so much for sharing valuable information