Plant Tissue Culture Chapter Notes | Biotechnology for Class 12 - NEET PDF Download

Historical Perspective

• Plant tissue culture (PTC) involves cultivating undifferentiated plant cells, tissues, or organs on synthetic media under aseptic conditions with controlled physical environments.
• PTC is a key tool for basic research and commercial applications, relying on the totipotency of plant cells—the ability of a vegetative cell to divide, differentiate into specialized cells, or regenerate into a whole plant.
• In the 19th century, German scientists Theodor Schwann and Matthias Schleiden established that cells are the basic unit of life with the capacity to divide and grow.
• In the 1890s, Gottlieb Haberlandt, a German botanist, pioneered PTC by proposing that plant cells could achieve continuous divisions on nutrient media, earning him the title "Father of Plant Tissue Culture."
• In 1902, Haberlandt outlined key PTC principles, including the ability of plant cells to resume uninterrupted growth and regenerate embryos from vegetative cells, later experimentally confirmed.
• Between 1902 and the 1930s, efforts focused on culturing isolated plant tissues like root or shoot tips, leading to the establishment of continuously growing plant cell cultures.
• The discovery of vitamins and natural auxins as essential for plant tissue growth on synthetic media significantly advanced PTC.
• From the 1940s to 1970s, extensive studies improved techniques and optimized nutrient media components for plant tissue culture.
• Coconut water was found to stimulate young embryo development and was incorporated into nutrient media for in vitro cultures.
• Other natural supplements, such as corn milk and orange juice, were used to develop callus cultures for woody plants and herbaceous dicots.
• In the 1950s, adenine, kinetin, and high phosphate levels in nutrient media enabled successful culture initiation from non-meristematic tissues, leading to shoot or root production.
• Research established that the morphogenic fate of cultured cells depended on the balance of auxins and kinetin: high auxin-to-kinetin ratios promoted rooting, high kinetin-to-auxin ratios induced shooting, and intermediate levels led to callus proliferation.
• This understanding enabled plant regeneration through tissue culture using various plant tissues or organs, supporting commercial applications.
• The Murashige and Skoog (MS) medium, developed by Toshio Murashige and Folke Skoog, became the most widely used nutrient medium for culturing a large number of plant species.
• Major landmarks in PTC include:
  • 1902: Gottlieb Haberlandt proposed in vitro cell culture on artificial media.
  • 1904: Hanning cultured embryos from cruciferous species.
  • 1922: Kotte and Robbins suggested root and stem tips as explants for in vitro culture.
  • 1926: Went discovered Indole Acetic Acid (IAA), the first plant growth hormone.
  • 1934: White reported vitamin B as a growth supplement and established continuous tomato root tip cultures.
  • 1937: White formulated the first synthetic PTC medium (White’s Medium).
  • 1941: Johannes Van Overbeek introduced coconut water as a media component, demonstrating its benefits.
  • 1946: Ball raised whole plants from Lupinus shoot tips.
  • 1954: Muir induced cell division in mechanically isolated single cells.
  • 1955: Skoog and Miller discovered kinetin, a cytokinin promoting cell division.
  • 1957: Skoog and Miller described the chemical control of root and shoot differentiation via auxin-to-kinetin ratios.
  • 1962: Murashige and Skoog formulated the MS medium with high salt concentrations.
  • 1964: Guha and Maheshwari produced the first androgenic haploid Datura plant via anther culture.
  • 1971: Protoplasts were subcultured in vitro, and plants were regenerated.
  • 1972: Protoplasts from two Nicotiana species were fused to create somatic hybrids.
  • 1976: Gynogenic haploid plants were cultured from unfertilized barley ovaries by San Noeum.
  • 1978: Melchers and colleagues produced “Pomato,” a somatic hybrid of potato and tomato.
  • 1981: Larkin and Scowcroft introduced the term “somaclonal variations” for genetic variations in cultured plants.
  • 1981: Horsh and colleagues produced transgenic tobacco using Agrobacterium tumefaciens and leaf disc explants.
  • 1987: Klien and colleagues developed the biolistic gene transfer method for plant transformation.
  • 1987: Y. Fujita and Mamoru Tabata commercialized shikonin production using Lithospermum erythrorhizon cell cultures.
  • 1990: Monsanto produced transgenic Bt-cotton, approved for commercial production in India in 2000.
  • 1993: Kranz and Lorz produced fertile maize plants through in vitro fertilization.
  • 1995: The “Arabidopsis Floral-dip” method enabled plant transformation without tissue culture.
  • 1997: Golden Rice, engineered for provitamin A production, was developed.
  • 2012: An enzyme from tissue-cultured plants was approved for treating Gaucher’s disease.
  • 2015: Somatic embryogenesis was introduced in plant transformation via embryonic genes.

Plant Cell and Tissue Culture Techniques

• Virtually any plant part, such as leaves, apical meristems, embryos, cotyledons, or hypocotyls, can serve as an explant for in vitro culture to regenerate whole plants.
• Different plant organs and species respond variably to nutritional requirements and physical conditions in vitro, with immature embryos being more responsive than apical meristems, which are more responsive than leaf explants.
• Plant regeneration occurs via two morphogenetic pathways: organogenesis and somatic embryogenesis.
• Organogenesis involves inducing vegetative organ formation (e.g., shoots or roots) from cells or tissues.
• In organogenesis, specialized cells undergo dedifferentiation, forming a mass of undifferentiated cells (callus), followed by redifferentiation to form organ primordia like shoots or roots.
• The balance of growth hormones, particularly auxins and cytokinins, is critical for organogenesis, determining whether shoots, roots, or callus form.
• Somatic embryogenesis is the formation of embryos from somatic cells, producing somatic embryos that resemble zygotic embryos but originate from somatic tissues and are larger.
• Somatic embryogenesis follows pathways similar to zygotic embryogenesis.
• Basic requirements for PTC include:
  • A washing area for cleaning glassware, plasticware, and labware, stored in a clean, dry place.
  • Media components for preparing nutrient media.
  • Facilities to sterilize nutrient media and store them at low temperatures.
  • Controlled environments for maintaining cultures under aseptic conditions with regulated light, temperature, and humidity.
• Steps for in vitro plant tissue culture using tomato cotyledons as explants:
  • Select and sterilize a suitable nutrient medium by autoclaving or filtering through micropore filters to prevent microbial contamination.
  • Choose explants (e.g., root or shoot apical meristems, leaves, cotyledons, hypocotyls, or immature embryos) for tissue culture to regenerate dedifferentiated cells, tissues, organs, or whole plants.
  • Surface-sterilize explants using disinfectants like sodium hypochlorite, followed by washing with sterile distilled water; explants from aseptically grown seedlings may not require re-sterilization.
  • Inoculate explants onto nutrient media, as shown with tomato seedlings germinated on culture medium.
  • Grow cultures in a PTC room under controlled light, temperature, and humidity, allowing small calli to regenerate on cotyledon explants.
  • Transfer growing cultures to media for shoot regeneration and elongation.
  • Excise regenerated shoots and transfer them to rooting medium.
  • Transfer plantlets to sterilized soil in pots for hardening in a greenhouse or growth room, followed by field transfer.

Nutrient Media

• Optimal growth and development of explants require specific nutrients and environmental conditions, varying by plant species (monocot, dicot, domesticated, or wild) and tissue type.
• The success of in vitro culture depends on selecting the appropriate culture medium composition.
• Plant tissue culture media typically include:
  • Inorganic components: Macronutrients (required in millimolar concentrations) include carbon (C), calcium (Ca), hydrogen (H), potassium (K), magnesium (Mg), nitrogen (N), oxygen (O), phosphorus (P), and sulfur (S); micronutrients (required in micromolar concentrations) include boron (B), cobalt (Co), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), and zinc (Zn).
  • Organic supplements: Amino acids (e.g., arginine, asparagine, glycine, proline) serve as nitrogen sources; vitamins include thiamine (Vitamin B1), nicotinic acid (Vitamin B3), and pyridoxine.
  • Carbon source: Sucrose (2–5% concentration) is the preferred carbon source, with glucose, fructose, and mannose as alternatives.
  • Plant growth hormones: Auxins, cytokinins, gibberellins, abscisic acid, and ethylene are critical, with auxins and cytokinins most commonly used; their concentration ratios determine organ formation (e.g., high cytokinin promotes shoot regeneration).
  • Gelling agents: Agar is the most common gelling agent for solid media, ideal for routine applications.
  • Antibiotics: Used to suppress bacterial and fungal infections in cultures.
• The pH of nutrient media is adjusted to 5.8–6.0; higher pH increases medium hardness, while lower pH impairs solidification and affects nutrient uptake and salt solubility.
• Murashige and Skoog (MS) medium is the most commonly used media composition, with specific components compared to White’s medium:
  • Macronutrients (mg/L): 
    MS = MgSO₄·7H₂O (370) + KH₂PO₄ (170) + KNO₃ (1900) + NH₄NO₃ (1650) + CaCl₂·2H₂O (440)White’s = MgSO₄·7H₂O (750) + NaH₂PO₄·H₂O (19) + KNO₃ (80)
  • Micronutrients (mg/L):
    MS = H₃BO₃ (6.2) + MnSO₄·4H₂O (22.3) + ZnSO₄·7H₂O (8.6) + Na₂MoO₄·2H₂O (0.025) + CuSO₄·5H₂O (0.025) + CoCl₂·6H₂O (0.025) + KI (0.83) + FeSO₄·7H₂O (27.8) + Na₂EDTA·2H₂O (37.3)
    White’s = H₃BO₃ (1.5) + MnSO₄·4H₂O (5) + ZnSO₄·7H₂O (3) + CuSO₄·5H₂O (0.01) + KI (0.75)
  • Sucrose (g/L):
    MS = 30
    White’s = 20
  • Organic supplements (mg/L):
    MS = Thiamine HCl (0.5) + Pyridoxine HCl (0.5) + Nicotinic acid (0.5) + Myoinositol (100) + Glycine (2)
    White’s = Thiamine HCl (0.01) + Pyridoxine HCl (0.01) + Nicotinic acid (0.05) + Glycine (3)

Culture Types

• PTC is categorized into organ culture, callus culture, cell suspension culture, and protoplast culture.
• Organ culture involves cultivating plant organs (e.g., roots, anthers, ovaries, embryos, endosperms, seeds) under laboratory conditions, named according to the organ (e.g., root culture, anther culture).
• Callus culture involves inducing dedifferentiation in plant parts to form an unorganized mass of cells (callus), commonly used for plant regeneration and genetic transformation studies.
• Cell suspension culture involves culturing single cells, isolated mechanically or enzymatically from callus or other plant parts, in liquid medium, used for genetic transformation, secondary metabolite production, or somatic embryo induction.
• Protoplast culture involves culturing protoplasts (plant cells without cell walls), often used for somatic hybridization and genetic studies.

Applications of Plant Cell and Tissue Culture

• PTC is routinely used for various plant science applications, including micropropagation, artificial seed production, haploid/triploid production, somatic hybridization, virus-free plant production, somaclonal variations, and secondary metabolite production.
• Micropropagation:
  • A tissue culture technique for multiplying plants asexually, producing genetically identical clones without seeds.
  • Traditional methods (cuttings, budding, grafting, corms, tubers) are laborious, environmentally dependent, and less successful, while micropropagation enables rapid multiplication in a small space, independent of seasons.
  • Useful for multiplying non-fertile, rare, endangered, or elite plants where sexual reproduction cannot maintain desired traits.
  • Successfully applied in agriculture, horticulture, and forestry for crops like potato, banana, carnation, and chrysanthemum.
  • Banana tissue culture is a major industry in India, producing over 400 million disease-free plantlets annually, benefiting farmers.
• Artificial (Synthetic) Seed Production:
  • Synthetic or somatic seeds are produced by encapsulating somatic embryos in a protective matrix (e.g., calcium alginate) with nutrients and growth regulators, mimicking conventional seeds.
  • Somatic embryos are generated on callus tissue using nutrient media with appropriate hormones.
  • Artificial seeds enable long-term storage and rapid, mass propagation of elite and hybrid plant varieties, supporting asexual propagation.
  • Successfully used for crops like carrot, grapes, and sandalwood.
• Haploid or Triploid Production:
  • Haploid plants have one set of chromosomes (n), unlike diploid plants (2n), and are used to produce homozygous diploid plants (double haploids) by doubling chromosomes with colchicine, allowing recessive traits to express.
  • Produced through anther, pollen, or ovary culture, used in cross-breeding for crops like broccoli, brassica, sorghum, rice, and tobacco.
• Somatic Hybrids:
  • Somatic hybridization overcomes species barriers in sexual reproduction by fusing protoplasts from distantly related species, enabling the transfer of desirable traits.
  • Protoplasts are isolated by digesting cell walls with enzymes (cellulases, pectinases) and fused using polyethylene glycol (PEG), regenerating into somatic or parasexual hybrid plants.
  • First achieved in 1972 by Carlson and associates, applied to crops like potato, rice, and brassica.
  • Cybridization combines the nuclear genome of one parent with the cytoplasmic genome (mitochondrial or plastid) of another, creating cybrids (e.g., for cytoplasmic male sterility or photosynthetic proteins).
• Production of Virus-Free Plants:
  • Viruses in vegetatively propagated crops reduce yield and quality, as infections persist in clonal populations.
  • Apical or axillary meristems (less than 1 mm) are typically virus-free due to uneven virus distribution and lack of vascular tissue for viral replication.
  • Using small meristem explants for culture produces virus-free plants, successfully applied to sugarcane, banana, and potato.
• Somaclonal Variations (Genetic Variability):
  • Genetic variations in terminally differentiated somatic cells are not passed to offspring in sexual reproduction but can be expressed in plants regenerated from cultured somatic tissues, termed somaclonal variations.
  • Long-term callus or cell suspension cultures may introduce additional genetic variations, expressed in regenerated plants (somaclones).
  • Somaclonal variations can be beneficial for engineering novel traits or detrimental if clonal uniformity is desired, affecting commercial value.
  • Screening somaclones has led to agronomically valuable cultivars, such as disease-resistant sugarcane, banana, and tomato, and improved wheat yields.
• Production of Secondary Metabolites:
  • Secondary metabolites (e.g., alkaloids, flavonoids, tannins, steroids, latex, resins) are specialized compounds for plant defense and environmental interactions, with industrial applications (drugs, flavors, dyes, insecticides, fragrances).
  • Plant cell or tissue cultures produce specific secondary metabolites under controlled conditions, independent of environmental, seasonal, or disease variations.
  • Hairy root systems are used industrially for high-quality secondary metabolite production.
  • Cultured cells or tissues can accumulate higher metabolite concentrations than parent plants, addressing overharvesting and cost issues.
  • Examples include artemisinin (antimalarial, Artemisia sp.), azadirachtin (insecticidal, Azadirachta indica), berberine (antibacterial, Coptis japonica), capsaicin (pain treatment, Capsicum annum), codeine (analgesic, Papaver sp.), digoxin (cardiac tonic, Digitalis lanata), diosgenin (antifertility, Dioscorea deltoidei), scopolamine (antihypertensive, Datura stramonium), quinine (antimalarial, Cinchona officinalis), shikonin (antimicrobial, Lithospermum erythrorhizon), taxol (anticarcinogenic, Taxus sp.), and vincristine (anticarcinogenic, Catharanthus roseus).