Delving into the realm of advanced plant tissue culture techniques unveils new frontiers in plant biotechnology. This chapter explores sophisticated methodologies such as ​somatic embryogenesis, protoplast transfection, genetic transformation, and gene-editing. These advanced techniques significantly contribute to plant biotechnology, offering ​solutions for sustainable agriculture, enhanced crop traits, and new scientific discoveries in plant biology. By mastering these advanced procedures, researchers can push the ​boundaries of what is possible in plant science, addressing global challenges and fostering innovation. This chapter provides a comprehensive guide to these cutting-edge ​techniques, empowering you to elevate your expertise and impact in the field of plant tissue culture.

Genetic Transformation in Plant Tissue Culture

Genetic transformation involves introducing new genetic material into ​plant cells to alter their genetic makeup. This is a powerful tool in plant ​biotechnology for improving crop traits, studying gene functions, and ​producing genetically modified organisms (GMOs).

Methods of Genetic Transformation

Agrobacterium-mediated Transformation

Agrobacterium tumefaciens, is a soil-borne bacterium which naturally ​transfers a part of its DNA (T-DNA) into the plant genome via a tumor-​inducing (Ti) plasmid. During transformation, plant tissues (usually leaf ​discs, stem segments, calli or cells suspension) are infected with ​Agrobacterium containing the gene of interest. The bacterium transfers ​the T-DNA into plant cells, which integrate it into the plant genome.

Advantages: This method is highly efficient for both dicotyledonous ​and monocotyledonous plants and results in stable integration of the ​transgene, and generally produces low copy number of the gene.

Biolistics (Particle Bombardment)

DNA-coated microscopic particles (usually gold or tungsten) are ​physically delivered into plant cells using a gene gun. Plant tissues or ​callus cultures or ECS are bombarded with the DNA-coated particles ​under high pressure. The DNA penetrates the cell walls and ​membranes, integrating into the plant genome.

Advantages: Biolistics is versatile and can be used for a wide range ​of plant species, including monocotyledons which are less ​susceptible to Agrobacterium.

Applications and Examples

Plant tissue culture has diverse applications, including crop ​improvement with traits like pest resistance (Bt cotton), herbicide ​tolerance, and enhanced nutrition (Golden Rice). It aids functional ​genomics, biopharmaceuticals, phytoremediation, metabolic ​engineering, and abiotic stress tolerance. Additionally, it supports ​developing new flower colors and fragrances, improving crop yield, ​disease resistance, and producing nutraceuticals for health benefits.

Transgenic taro expressing GFP ​in roots.

Somatic Embryogenesis

Somatic embryogenesis involves the development of embryos from somatic or non-reproductive cells, leading to the formation of whole plants.

Induction and Development

• Somatic embryos are induced from explants (such as leaves, roots, or stem segments) cultured on a medium supplemented with plant growth ​regulators, typically auxins.

• The induced somatic embryos undergo stages similar to zygotic embryogenesis: globular, heart-shaped, torpedo, and cotyledonary stages. ​These embryos are then matured and germinated into complete plants.

Direct somatic embryogenesis: involves production of embryo from organized tissue without an intervening callus phase.

Indirect somatic embryogenesis: involves dedifferentiation of organized tissue into callus prior to embryo production. Callus can be used to ​establish embryogenic cell suspension (ECS) which produce somatic embryos.

Banana somatic embryos derived ​from embryogenic cell suspension ​(ECS) cultured in a petri dish, ​exemplifying somatic ​embryogenesis.

Applications

• Clonal Propagation: Somatic embryogenesis is used for the mass propagation of genetically uniform plants, which is especially valuable for ​high-value crops and forestry species.

• Genetic Engineering: The process facilitates the recovery of genetically transformed plants. Embryogenic cultures can be directly subjected to ​genetic transformation methods, ensuring efficient integration and expression of new genes.

• Synthetic Seed Production: Somatic embryos can be encapsulated in a protective coating to produce synthetic seeds, which can be stored ​and germinated later, providing a practical tool for the conservation and distribution of plant germplasm.

• Germplasm Conservation: Somatic embryogenesis allows for the cryopreservation of plant tissues, ensuring long-term storage and ​preservation of genetic diversity, especially for endangered or rare species.

• Mutation Breeding: Inducing mutations in somatic embryos to generate genetic variability, which can be selected for desirable traits, aiding in ​crop improvement programs.

• Pathogen-Free Plants: Producing plants free from viruses and other pathogens by starting cultures from meristematic tissues, which are ​typically virus-free, ensuring healthy propagation material.

Citruc somatic embryos derived ​from embryogenic callus ​cultured in a petri dish.

Protoplast

Protoplasts are plant cells with their cell walls removed, leaving the plasma membrane intact. They are used in various biotechnological applications ​such as genetic engineering, somatic hybridization, and studying cell biology.

Isolation, Transfection, and Culture

• In vitro plant-derived tissues such as leaves, petioles, calli, or cell suspension cultures are prepared for incubation in a sterile enzyme mixture ​consisting of cellulase, pectinase, and hemicellulase, solubilized in an appropriate buffer containing an osmotic stabilizer like mannitol or ​sorbitol, and calcium to stabilize the membrane.

Freshly isolated protoplasts, ​illustrating the initial stages of ​protoplast isolation before ​transfection and culture.

• The enzyme solution is prepared by combining cellulase, pectinase, and ​hemicellulase in the buffer containing the osmotic stabilizers and calcium, ​followed by filter sterilization to prevent contamination.

• Leaves and petioles are finely cut before immersion in the sterile enzyme solution, ​while calli and cell suspension cultures do not require cutting.

• The prepared tissue is incubated at 23-25°C with gentle shaking (50-100 rpm) for ​3-18 hours, depending on the tissue type and enzyme efficiency.

• During incubation, the tissue is periodically checked under a microscope for ​protoplast release.

Protoplast Transfection

Polyethylene Glycol (PEG)-mediated Transfection

• Mix protoplasts with plasmid DNA containing the gene of interest and add PEG ​solution to facilitate DNA uptake.

• Incubate for a specific time, then wash to remove PEG. Dilute the PEG mixture ​with washing solution gradually to avoid osmotic shock.

• Centrifuge and resuspend the protoplasts in culture medium.

• Optimise the amount of DNA, PEG molecular weight, concentration of PEG, and ​incubation time for maximum efficiency.

Protoplast Collection

• Once released, filter the enzyme-tissue mixture through a ​nylon mesh to remove undigested tissue.

• Centrifuge the filtrate at low speed (e.g., 100 g for 5-10 ​minutes) to pellet the protoplasts.

• Wash the protoplast pellet with a washing solution (buffer ​with osmotic stabilizers) and centrifuge again.

• Resuspend the protoplasts in a suitable volume of washing ​solution or culture medium and determine the yield using a ​Haemocytometer.

Electroporation

• Mix protoplasts with DNA and subject them to an electric pulse to create ​temporary pores in the plasma membrane.

• Optimize voltage and pulse duration for maximum efficiency and viability.

Protoplasts pelleted by ​centrifugation at low ​speed.

Protoplast Culture

Culture Media, Plating and Regeneration

• Use media containing osmotic stabilizers (e.g., mannitol or sorbitol and Calcium) to maintain protoplast integrity.

• Media components: macro and micronutrients, vitamins, amino acids, plant hormones (auxins and cytokinins), and a carbon source (sucrose ​or glucose).

• Plate the transfected protoplasts in a thin layer of liquid or agar-solidified culture medium.

• Incubate the cultures in the dark or low light at 25-28°C.

• Maintain high humidity to prevent desiccation.

• Monitor for cell wall regeneration and cell division.

• Transfer dividing protoplasts to fresh medium to promote colony formation.

• Gradually change the culture conditions to encourage shoot and root formation (adjust hormone ​levels).

Applications

• Genetic Engineering: Introduction of new genes to create genetically modified plants.

• Somatic Hybridization: Fusion of protoplasts from different species to create hybrid plants.

• Functional Genomics: Studying gene function and regulation in plant cells.

• Metabolic Engineering: Manipulating metabolic pathways to produce desired compounds.

Challenges and Considerations

• Protoplast Viability: Ensuring high viability during isolation and transfection.

• Efficiency: Optimizing conditions for high transfection efficiency.

• Regeneration: Successful regeneration of whole plants from protoplasts.

Protoplast culture: Aggregates ​of protoplasts cultured in a ​small petri dish with a thin ​layer of medium, illustrating ​their clustering during culture.

Protoplast in agarose bead at ​first stage of cell division.

Challenges

• Off-Target Effects: CRISPR/Cas9 may cause unintended mutations.

• Efficiency and Specificity: Ensuring precise edits without off-target effects.

• Delivery: Challenges in delivering CRISPR components to target cells efficiently.

• Immune Response: Cas9 protein can trigger immune responses.

• Large DNA Insertions: Inserting large DNA fragments is less efficient.

• Ethical and Regulatory Concerns: Concerns regarding potential misuse and regulatory ​frameworks.

• Epigenetic Changes: CRISPR/Cas9 can induce unintended epigenetic effects.

CRISPR/Cas9 and Plant Tissue Culture

• CRISPR/Cas9 is a revolutionary gene-editing technology that allows precise ​modifications to the plant genome.

• The CRISPR/Cas9 system uses a guide RNA (gRNA) to direct the Cas9 ​nuclease to a specific DNA sequence, where it induces a double-strand ​break.

• The break is repaired by the plant's natural DNA repair mechanisms, which ​can be harnessed to introduce or delete specific genetic sequences.

Protocols for Gene Editing in Plants

• Designing gRNAs: gRNAs are designed to target ​specific genes of interest.

• Construct Assembly: The gRNA and Cas9 gene ​are assembled into a plasmid vector suitable for ​plant transformation.

• Delivery: The construct is delivered into plant ​cells via Agrobacterium-mediated transformation ​or biolistics.

• Selection and Regeneration: Transformed cells ​are selected on a medium containing antibiotics ​or herbicides and regenerated into whole plants.

• Screening and Validation: Edited plants are ​screened using PCR and sequencing to confirm ​the presence of the desired genetic changes.

The Future of Plant Biotechnology: Integrating CRISPR/Cas9 with Tissue Culture

• The integration of CRISPR/Cas9 technology with plant tissue culture marks a ​significant leap in plant biotechnology. CRISPR/Cas9 enables precise genome ​editing, enhancing crop traits, disease resistance, and environmental adaptability. ​Combined with tissue culture, it allows for the efficient regeneration of genetically ​uniform plants from edited cells or tissues.

• This powerful combination accelerates genetic research and crop development, ​crucial for addressing global food security challenges. While promising, it is vital to ​manage off-target effects and navigate regulatory frameworks to ensure safe use. ​Ongoing research will further refine these technologies, driving innovation in ​sustainable agriculture.

• In summary, CRISPR/Cas9 and plant tissue culture together offer transformative ​potential for crop improvement, poised to make a lasting impact on plant science ​and agriculture.

Visualizing gRNA and Cas9 ​complex: Illustration showing how ​the gRNA and Cas9 complex ​precisely target DNA for gene ​editing with CRISPR/Cas9.

https://www.thermofisher.com/au)