Plant Tissue Culture Techniques

Plant Tissue Culture

• Refers to a set of techniques that maintain or grow plant cells, tissues, or organs
• This is done under sterile conditions using a nutrient culture medium of known composition
• Plant tissue culture is a widely used technique in plant biotechnology

Importance of Plant Tissue Culture

• Plants address major challenges like food, energy, and environment
• Research in plant biotechnology offers solutions
• Modern plant biotechnology has achievements in areas like agriculture, pharmaceuticals, germplasm conservation, and food security
• Plant cell culture is an important tool for the production of economically important plants

Applications of Plant Tissue Culture

• Includes In situ and Ex situ conservation (micropropagation, cryopreservation, synthetic seeds)
• Used for metabolite engineering (Agrobacterium-mediated gene transformation, COSTREL)
• Can enhance the production of active components like PGRs, bioreactors, additives, and elicitors
• Can be used for genome editing and synthetic biology using techniques like CRISPR/Cas9, TALLENS, and ZFNs
• Used to assess antimicrobial and antioxidant activities
• Involved in haploidy induction
• Can cultivate hairy roots
• Used for changing the chemical profile of plants
• Involved in artificial polyploidy induction

Advantages of Tissue Culture

• Allows for the production of a large number of plants from a single plant in a small space and short time
• Reduces growing space, labor, and plant maintenance requirements
• Eliminates viruses and systemic diseases by propagating the quickly dividing cells of the shoot tip
• Gives growers a means to produce plants with identical traits
• Horticultural cultivars can be improved by selecting plants with varying leaf shape, disease resistance, growth habit, and flower color
• Required for producing genetically engineered plants, as modified cells must divide and differentiate in vitro before maturity
• Results in excellent basal branching in some plants, giving excellent multi-stem characteristics

Plant Regeneration Capacity

• Plants have a great ability to develop in different ways and can regrow after damage
• They can close injuries and replace lost parts using dedicated developmental pathways
• This regeneration ability has been used for vegetative plant propagation

Differentiation, Dedifferentiation and Redifferentiation

• Plants can regenerate after injury or damage due to stem cells
• Stem cells allow plants to regrow cells of the roots or shoots
• Regenerative capacity is how much or how little a plant can regenerate
• Plants with high regenerative capacity can regenerate more by changing the injury site cells back to stem cells
• Differentiation is undifferentiated stem cells deciding their fate
• Meristems are stem cells in the plant body
• Differentiation is where a stem cell becomes a more specialized cell type, like a shoot or leaf cell
• After cell division, plant cells undergo maturation by differentiation and stop dividing
• When mature cells regain their ability to divide, that is dedifferentiation
• After dedifferentiation, mature cells become meristematic cells, leading to callus formation
• When secondary meristematic cells lose their ability to divide, that is redifferentiation

Trans-differentiation and Callus Formation

• Dedifferentiation can only occur within the same developmental lineage
• Trans-differentiation is cell fate changes independent of developmental potency
• Trans-differentiation leading to increased developmental potency is often called dedifferentiation, particularly during callus formation
• Callus formation is a result of over-proliferation/trans-differentiation of differentiated cells rather than a step back in the developmental lineage
• Callus tissue can exhibit increased developmental potency
• Callus is unorganized growth of a cell mass, wound responses, and callus formation have been observed
• Phytohormones auxin and cytokinin are required for in vitro cell proliferation
• The ratio of these hormones determines the morphogenetic pathway that the in vitro cultured tissue will follow

Plant Hormones (Plant Growth Regulators)

• Auxins promote cell elongation in stems and roots, stimulate adventitious root formation, regulate responses to light and gravity (phototropism and gravitropism), help in seedless fruit formation, and influence leaf and fruit drop
• Gibberellins promote internode elongation, stimulate seed germination, induce flowering in some plants, and enhance fruit size and growth
• Cytokinins stimulate cell division and differentiation, promote lateral bud and shoot growth, delay leaf aging, and aid in nutrient mobilization within the plant
• Abscisic Acid or ABA regulates stomata closure, induces seed dormancy, and mediates plant responses to environmental stresses such as drought, salinity, and cold
• Ethylene accelerates fruit ripening, promotes the shedding of leaves, flowers, and fruits, regulates plant aging, and plays a role in plant responses to mechanical stress, wounding, and pathogen attack

Auxins vs. Cytokinins

• Auxins promote cell elongation in stems and roots, stimulate adventitious root formation, regulate responses to light and gravity (phototropism and gravitropism), help in seedless fruit formation, and influence leaf and fruit drop
• Cytokinins stimulate cell division and differentiation, leading to increased shoot growth, promote the growth of lateral buds and shoots, delay leaf aging, and aid in nutrient mobilization within the plant

Protoplasts

• Plant cells that have their cell walls removed using cellulylitic enzymes

Totipotency

• Plants can regenerate from cultured plant cells via embryo formation
• Pathway termed "somatic embryogenesis"
• Initiation confined to single cells
• Considered experimental proof of "totipotency"
• Each somatic plant cell has the capability to regenerate into an entire plant
• Supported by isolation and culture of leaf protoplasts

History in Plant Tissue Culture

• 1904: Cultured embryos from several cruciferous species
• 1922: Cultured root and stem tips
• 1926: Discovered Indole acetic acid – first plant growth hormone
• 1934: First successful report of continuously growing cultures of tomato root tips
• 1939: Set up interminable proliferation of callus cultures
• 1941: Added coconut water to divide plant cells
• 1946: Raised plant by shoot tip culture
• 1952: Developed some virus-free plants using shoot meristem culture
• 1954: Breaked callus tissues into single cells
• 1957: Auxin: cytokinin ratio of plant organ formation
• 1959: First evidence of somatic embryogenesis with carrot cell
• 1960: Filtered cell suspension and isolated single cells by plating
• 1960: Developed test tube fertilization technique
• 1962: Developed Murashige and Skoog medium
• 1964: Produced first haploid plants from pollen grains
• 1966: Regenerated carrot plants from single cells of tomato
• 1971: Regenerated first plants from isolated mesophyll protoplasts
• 1972: Produced first interspecific hybrid by protoplast fusion
• 1974: Introduced biotransformation in plant tissue culture
• 1977: Integrated Ti plasmid DNA from Agrobacterium tumefaciens into plants
• 1978: Developed intergenic hybrid between potato and tomato
• 1983: Cytoplasmic hybridization between generations in Radish and Grapes
• 1984: Transfer of Agrobacterium mediated gene to create transgenic plants
• 1987: Plant biolistic gene transfer method
• 1994: First commercialization of transgenic crops (delayed-ripening tomato)
• 2000: First plant genome (A. thaliana) decoded
• 2002: Omics technologies adopted
• 2005: Rice genome sequenced
• 2012: Integrated breeding platform
• 2013: CRISPR first applied to plants

Explant

• Cell or tissue taken from a particular body, then placed in culture medium for growth to a whole plant

Meristem Cells

• All multicellular organism is characterized by a given number of genes
• None of their cells express all but only a portion of them
• The cells can be considered as genetically differentiated
• A genetically fully dedifferentiated cell expresses all genes coded in the genome does not exist
• Stem cells are also considered cells than somatic cells
• Stem cells are also differentiated