Plant Biotechnology Notes

Introduction to Biotechnology

  • Biotechnology: Using biological principles to create products via biological agents (e.g., wine fermentation).

  • Coined by Karl Ereky in 1919.

  • Plant biotechnology: Modifying plants through engineering for human benefit.

  • Narrow definition: Genetic manipulation for specific purposes.

  • Branches:

    • Tissue culture: In vitro cultivation of plant cells, tissues, organs, embryos, seeds & protoplasts in a lab under sterile conditions.

    • Genetic engineering: Manipulating organisms at the molecular level, directly with DNA.

      • Modifies bacterial cells to produce new substances (hormones, vaccines).

      • Introduces novel traits into plants/animals.

History of Plant Tissue Culture

  • Gottlieb Haberlandt:

    • Father of plant tissue culture.

    • Predicted totipotency in 1902.

    • Attempted in vitro culture of mesophyll cells.

  • Totipotency: A plant cell's ability to multiply, differentiate, and grow into a complete plant.

  • Hanning (1904): First embryo culture attempted.

  • Laibach (1925): Recovered hybrid progeny in Linum using zygotic embryos.

  • F. Kogl et al. (1930s): Discovered indoleacetic acid (IAA), a plant hormone.

  • Philip White (1934): Cultured tomato roots on White’s medium.

  • Gautheret (1939): Cultured carrot tissues successfully.

  • Gautheret, White, Nobecourt (1939): Endorsed unlimited cultivation of plant tissues.

  • Miller and Skoog (1955): Discovered kinetin (a cytokinin).

  • Braun (1959): Regenerated the first plant from a mature plant cell.

  • Muir (1953-54): Demonstrated single-cell separation from callus tissues in liquid medium when shaken.

  • Bergmann (1960): Developed cloning method for single cells via suspension culture filtering (Plating technique).

  • Murashige and Skoog (1962): Published MS medium composition, widely used for tissue culture.

  • Guha, S and Maheshwari, S.C. (1964, 1966): First in vitro production of haploid plants from anthers of Datura.

Significance of Haploid Plant Production

  • Plants from doubled haploids are homozygous.

  • Express all recessive genes.

  • Ideal for pure breeding lines.

Further advancements

  • Steward (1966): Demonstrated totipotency of somatic cells through somatic embryos from carrot suspension cultures.

  • Morel: Rapid propagation of orchids/dahlias; developed virus-free orchid/dahlia plants from shoot meristems.

  • Cocking (1960): Produced protoplasts (cells without cell walls) using degrading enzymes.

  • Carlson et al. (1972): Produced first somatic hybrid plants via protoplast fusion of N. glauca x N. langsdorfli.

Development of Biotechnology in India

  • 1982: National Biotechnology Board (NBTB) established under the Department of Science and Technology.

  • 1986: Department of Biotechnology (DBT) was started.

  • International Center of Genetic Engineering and Biotechnology (ICGEB) established by the United Nations.

  • ICGEB centers: New Delhi and Trieste (Italy).

Scope and Importance of Biotechnology

  • Controlled use of biological agents for beneficial purposes.

  • Integrates biochemistry, molecular biology, and microbiology.

Biotechnology in Medicine

  • Monoclonal antibody production.

  • DNA/RNA probes for disease diagnosis.

  • Synthesis of insulin/interferon from bacteria.

  • DNA fingerprinting.

  • Recombinant vaccines (e.g., hepatitis B).

Industrial Biotechnology

  • Large-scale alcohol and antibiotic production by microorganisms.

  • Production of lactic acid and glycerine through genetic engineering.

  • Protein engineering (remodeling proteins/enzymes).

Biotechnology and Environment

  • Pollution control (detoxification of effluents, oil spill remediation).

  • Sewage treatment and biogas production.

  • Bio-pesticides.

Biotechnology and Agriculture

  • Plant cell, tissue, and organ culture for clonal multiplication (fruit/forest trees).

  • Production of virus-free genetic stocks.

  • Creation of genetic variations through somaclonal variation.

  • Transgenic plants with disease/herbicide resistance, increased fruit shelf life.

  • Molecular breeding (RFLP, SSR markers).

Landmark Discoveries in Molecular Biology

  • 1970: Smith & Nathans discovered HindIII restriction enzyme.

  • 1972: Berg et al. produced the first recombinant DNA molecule.

  • 1972: Termin discovered reverse transcriptase.

  • 1974: Reinhard: Biotransformation in plant tissue culture.

  • 1974: Zaenen et al., Larebeke et al.: Discovered Ti plasmid as tumor-inducing agent.

  • 1977: Chilton et al.: integrated Ti plasmid DNA into plants using Agrobacterium tumefaciens.

  • 1977: Maxam, Gilbert: DNA sequencing methods (degradation-based).

  • 1978: Melchers et al.: Pomato production using somatic hybridization (potato + tomato).

  • 1980: Eli Lilly and Co.: Commercial insulin production in bacteria (genetic engineering).

  • 1980: Restriction Fragment Length Polymorphism (RFLP) technique developed.

  • 1981: Larkin & Scowcroft coined the term "somaclonal variation".

  • 1983: Kary Mullis conceived Polymerase Chain Reaction (PCR).

  • 1984: De Block et al., Horsch et al.: Transformation of tobacco with Agrobacterium, transgenic plants.

  • 1984: Jeffreys: Genetic fingerprinting via DNA polymorphism analysis.

  • 1987: Barton et al.: Isolated Bt gene (Bacillus thuringiensis).

  • 1990: Human Genome Project launched.

  • 1990: Williams et al., Welsh & McClelland: RAPD (Random Amplified Polymorphic DNA) technique.

  • 1991: Fodor: DNA microarray system.

  • 1995: Vos et al.: AFLP (Amplified Fragment Length Polymorphism) technique.

  • 1997: Blattner et al.: Completed E.coli genome DNA sequencing.

  • 2001: Craig Venter et al.: Completed Human Genome Project.

Nutritional Requirements of Tissue Culture

  • Explant: Isolated plant tissue grown on nutrient medium.

  • Nutrient medium:

    • Support system.

    • Macronutrients.

    • Micronutrients.

    • Carbon source.

    • Organic supplements.

    • Growth regulators.

Support System

  • Liquid Medium:

    • Suspension Culture.

    • Cultured in water with nutrients; frequent agitation for aeration.

  • Solid Medium:

    • Gelling agents.

    • Agar (0.5-1.0%): Most widely used, resistant to enzymes, doesn't react with media components.

    • Agarose, gellan gums.

  • Alternative supports: Perforated cellophane, filter paper bridges/wicks, polyurethane foam, polyester fleece.

Macronutrients

  • Required concentration: >0.5 ml/liter

  • Elements: N, P, K, Ca, Mg, S

  • Typical Media Concentrations:

    • N, K: 20-30 mM.

    • P, Mg, S, Ca: 1-3 mM.

Micronutrients

  • Required concentrations: <0.5 ml/liter.

  • Elements: Fe, Mn, Zn, B, Cu, Mo.

  • Iron: Most critical micronutrient.

  • Iron and zinc: Commonly used in chelated form.

Carbon Source

  • Sucrose (2-3%): Most preferred.

  • Glucose and fructose.

  • Fructose less effective.

  • Other carbohydrates: Myo-inositol, maltose, lactose, galactose, raffinose, starch.

Organic Supplements

  • Vitamins: thiamin (B1), nicotinic acid, pyridoxine (B6), myo-inositol

    • Thiamin: Required by all cells for growth.

  • Amino Acids: Stimulate cell growth (especially in protoplast cultures).

    • Glycine (most common).

    • Glutamine, asparagine, arginine, cystine.

  • Other Organic Supplements:

    • Protein (casein) hydrolysate, coconut milk, yeast & malt extract, ground banana, orange/tomato juice, activated charcoal.

    • Activated charcoal: Removes toxic compounds.

  • Antibiotics: Prevent microbial infections (e.g., Streptomycin, Kanamycin).

Growth Regulators

  • Four main classes:

    • Auxins.

    • Cytokinins.

    • Gibberillins.

    • Abscisic Acid (ABA).

Auxins

  • Naturally occurring: IAA (Indole 3-Acetic Acid).

  • Functions:

    • Cell division/elongation.

    • Stem/internode elongation.

    • Tropism, apical dominance, abscission, rooting.

  • Commonly used auxins:

    • IAA, IBA (Indole 3-Butyric Acid), 2,4-D (Dichloro Phenoxy Acetic Acid), NAA (Naphthylene Acetic Acid), NOA (Naphthoxy Acetic Acid).

  • 2,4-D: Callus induction.

  • Other auxins: Root induction.

Cytokinins

  • Stimulate cell division.

  • Induce shoot formation/axillary shoot proliferation.

  • Inhibit root formation.

  • Activate RNA/protein synthesis, enzymatic activity.

  • Commonly used cytokinins: BAP (6-Benzyl Amino Purine), BA (Benzyl adenine), 2ip (Isopentyl adenine), Kinetin, Zeatin.

Effect of Growth Regulators

  • More cytokinin/low auxin: Shoot regeneration.

  • Low cytokinin/more auxin: Root regeneration.

  • Medium cytokinin/medium auxin: Shoot and root regeneration.

  • Medium cytokinin/low auxin: Callus regeneration.

  • Concentrations and ratios of auxins/cytokinins are important.

Gibberillins and Abscisic Acid

  • GA3 (Gibberellic Acid): Most commonly used.

  • Enhances callus growth, stimulates elongation of stunted plantlets.

  • ABA: Can stimulate or inhibit culture growth based on species; inhibits late embryo development.

pH

  • Suitable range: 5.0-6.0.

  • Autoclaving reduces pH by 0.3-0.5 units.

  • pH > 6.0: Hard medium.

  • pH < 5.0: Prevents agar gelling.

  • Adjust pH with 0.1N NaOH or HCl.

Nutrients and Physiological Role

  • Nutrient requirements vary among species.

  • Basal media: MS medium, B5 (Gamborg et al), White media.

  • No single media suits all cultures.

Key elements

  • Nitrogen (N): Proteins, nucleic acids.

  • Phosphorus (P): Nucleic acids, energy transfer.

  • Potassium (K): Regulates osmotic potential.

  • Calcium (Ca): Cell wall synthesis, membrane function.

  • Magnesium (Mg): Enzyme co-factor, chlorophyll.

  • Sulfur (S): Amino acids (methionine, cysteine).

  • Chlorine (Cl): Photosynthesis.

  • Iron (Fe): Electron transfer.

  • Manganese (Mn): Enzyme co-factor.

  • Cobalt (Co): Some vitamins.

  • Copper (Cu): Enzyme co-factor, electron transfer.

  • Zinc (Zn): Enzyme co-factor, chlorophyll synthesis.

  • Molybdenum (Mo): Enzyme co-factor, nitrate reductase.

MS medium Composition

  • Macronutrients (g/L):

    • NaNO_3: 1.65

    • KNO_3: 1.90

    • CaCl2 ewline 2H2O: 0.44

    • MgSO4 ewline 7H2O: 0.37

    • KH2PO4: 0.17

  • Micronutrients (mg/L):

    • FeSO4 ewline 7H2O: 27.80

    • Na2EDTA ewline 2H2O: 33.60

    • Kl: 0.83

    • H3BO4: 6.20

    • MnSO4 ewline 4H2O: 22.30

    • ZnSO4 ewline 7H2O : 8.60

    • Na2MoO4
      ewline 2H_2O: 0.25

    • CuSO4 ewline 5H2O: 0.025

    • CoCl2 ewline 6H2O: 0.025

  • Organic supplements (mg/L):

    • Myo-inositol: 100.00

    • Nicotinic acid: 0.05

    • Pyridoxine HCl: 0.05

    • Thiamine HCl: 0.05

    • Glycine: 0.20

  • Sucrose: 20 g/L

  • Growth regulators: As needed

  • Gelling Agent:

    • Agar: 6-8 g/liter (0.5-1%)

  • pH: 5.8

Methods of Media Preparation

  • Using commercially dry powdered media is preferable.

    • Dissolve in distilled water (10% less than final volume), add sugar, agar, and supplements. Adjust to final volume & pH, then autoclave.

  • Prepare Concentrated Stock Solutions:

    • Reduces repetitive steps and errors.

    • Essential for micronutrients and hormones (mg or µg quantities).

    • Components: Dissolve in distilled water and dilute into final media.

  • Storage of Stock Solutions:

    • Proper containers in refrigerators at 2°-4°C.

    • Macronutrients: 10X concentration.

    • Calcium: Separate stock to prevent precipitation.

    • Micronutrients: 100X concentration (up to 1 year in refrigerator).

    • Vitamins: 100X or 1000X concentration (freezer at -20°C for 2-3 months).

    • Auxins: 100-1000X concentration.

      • NAA and 2,4-D stable, store at 4°C for months.

    • Iron: Amber bottles.

  • Substances unstable in frozen state: Add fresh at medium preparation.

  • Avoid: Contaminated/precipitated stock solutions.

Plant Tissue Culture Techniques

  • Depending on the explant, techniques are: meristem, embryo, anther, ovule culture. Main stages are:

    • STAGE 0: Selection of Explant.

    • STAGE I: Initiation of Aseptic Culture.

    • STAGE II: Proliferation of Axillary Shoots

    • STAGE III: Rooting.

    • STAGE IV: Hardening.

Stage 0: Selection of Explant

  • Plant part used to initiate tissue culture i.e explant.

  • Success depends on location, age, developmental phase.

  • Shoot primordia-containing explants (meristems, node buds, shoot apices) preferred.

  • Younger plants are more successful.

Stage I: Initiation of Aseptic Culture

  • Surface sterilize explant to remove microbial contaminants

  • Use disinfectants: sodium hypochlorite, calcium hypochlorite, ethanol, mercuric chloride (HgCl2).

  • Incubate in growth chamber (light or dark conditions).

Stage II: Proliferation of Axillary Shoots

  • Induce axillary shoot proliferation by adding cytokinin to the shoot culture medium.

  • Cytokinin to auxin ratio of 50:1 produces shoots with minimal callus formation.

  • Subculture new shoots at ~4-week intervals.

Stage III: Rooting

  • Addition of auxin to the medium induces root formation.

  • Roots induced on shoot to produce plantlets for transfer into soil.

  • Rooting can occur in same media for multiplication or requires a separate media.

Stage IV: Hardening

  • Plant transferred to substrate (sand, peat, compost etc.).

  • Acclimatize to field conditions (low humidity, high light intensity) in greenhouse.

Applications of Plant Tissue Culture

  • Generating large numbers of identical individuals from a mother plant.

  • Rescuing hybrid embryos in wide hybridization.

  • Conserving rare or endangered plant species.

  • Creating transgenic plants.

  • Propagating orchids by immature embryo culture.

  • Screening cells for advantageous characters (herbicide resistance).

  • Producing haploid plants by anther/pollen culture.

  • Large-scale growth in bioreactors for secondary metabolites & recombinant proteins.

  • Crossing distantly related species by protoplast fusion.

  • Biosynthesis of secondary products and biotransformation.

  • Evaluating physiological, biochemical, and reproductive mechanisms (e.g., stress tolerance).

  • Chromosome doubling for polyploidy induction (colchicine or oryzalin).

  • Meristem tip culture for virus-free plants.

  • Producing identical, sterile hybrid species.

Maintenance of Asepsis

  • Absence of contamination as definition.

  • Types and sources contamination knowledge is essential for detection and prevention.

Sources of Contamination

  • Laboratory ware and Media:
    *If not properly sterilized, can cause contamination.
    *The medium contains nutrients for microbial growth.

  • Explant:

    • Primary source of contamination.
      *Microorganisms on the surface, between cells within cells (endogenous/systemic contamination e.g., Corynebacterium, Xanthomonas).

  • Environment of Transfer Area:

    • Air quality is important.

    • Atmospheric dust particles, fungal spores & bacteria are contaminants

  • Worker:

    • Dirty hands & attire contamination sources.

    • Talking/sneezing spreads microbes.

Types of Contamination

  • Bacteria: Killed at high temperatures otherwise heat resistant endospores are produced (e.g., Clostridium). *Common genera: * Agrobacterium, Bacillus, Beijerinckia, Pseudomonas, Staphylococcus, Acinetobacter.

    • Identification involves morphology, biochemistry and physiology.

  • Fungi:
    *Reproduce by spores (sexual/asexual).
    *Common contaminants:
    *Neurospora, Pensillium, Fusarium, Cladosporium.
    *Identification based on colonies, mycelia, fruiting body morphology.
    *Yeast - Common genera in plant tissue culture are Candida and Rhodotorula.

  • Actinomycetes - Are also prokaryotic that looks like fungi produces spore causing contaminations.

  • Viruses:
    *Presence cannot be visually seen.
    *Infected plants may carry virus, but look healthy will express later.

  • Insects

    • Mites and thrips are not serious unless microbes carried by them contaminate.

Detection of Contamination

  • Contamination occurs in tissue culture process.

  • Testing of contamination happens out whenever necessary.

  • Autoclave Contaminated cultures before discarding.

  • Bacterial contamination:

    • Recognized by turbidity in liquid media with unusual odor.

  • Yeast growth appears as heavy milky turbidity with a distictive odour.

  • Fungi appear forms mycelia which looks like ‘balls’ in liquid media.

Effect of Contaminants on Tissue Culture Plants

  • Microbes overrun explants.

  • Carbohydrate fermentation produce toxic metabolites like acetic acid and ethanol.

  • pH of the medium decreased below 3.0.

  • pH of the medium causes not availability of certin nutrients.

Micropropagation

  • Vegetative propagation involves mitotic cell divisions.

  • Progeny are a clone.

  • Micropropagation is in vitro clonal propagation.

  • Select plant material from healthy mother plant as explant.

  • Produce genetically identical plants.

Pathways of Regeneration

  • Proliferation from pre-existing meristems (Axillary bud proliferation).

  • Organogenesis.

  • Somatic embryogenesis.

PROLIFERATION OF PRE-EXISTING MERISTEM/AXILLARY BUD PROLIFERATION

  • Use already existing meristem to initiate in vitro culture (e.g. shoot-tip / nodal explant).

    • Shoot has already differentiated.

    • Elongation and root differentiation required.

  • Shoot tip ranges between 1 and 10 mm in length.

  • Cytokinin in the media stimulates meristem (apical meristem in shoot tips and axillary buds in nodal explants) to develop into shoots.

  • Shoots sub-cultured nodes after 4-6 weeks onto a fresh medium

  • One explant produces 5-6 shoots in 4-5 weeks which would result in 510 to 612 plants in one year from a single explant.

Single node culture example blueberry

  • Shoot bud is excised and cut into small pieces then subcultured to initiate a new cycle of micropropagation.

  • Shoot tips are genetically stable and have high success rates. Contain preformed shoot and are phenotypically homogeneous.

  • Axillary and terminal buds are the advantages of shoot tips but they are more difficult to disinfect.

Organogenesis

  • Formation of plant organs (roots/shoots) directly on the explant.

  • Lacks preformed meristem or de novo origin from callus and cell suspension culture.

  • Plant production achieved by two modes:

    • Emergence of adventitious organs directly from the explant (Direct organogenesis).

    • Emergence of adventitious organs through callus formation (Indirect organogenesis).

  • Direct organogenesis:

    • Also known as Adventitious regeneration. Development of organs directly bypassing callus stage, less common.

  • Indirect organogenesis:

    • Explant gives rise to callus, an unorganized mass of undifferentiated cells, from which organs are formed.
      *Formation of callus from mature explant (by dedifferentiation).
      *Formation of various organs from the callus/meristems (by redifferentiation).

  • Ratio of hormones is critical.

  • High ratio of cytokinin to auxin stimulated the formation of shoots in tobacco callus.
    *High auxin to cytokinin ratio induced root regeneration.

  • Negative side:
    *Callus introduces mutations in vitro (somaclonal variations making it technically challenging).

Somatic Embryogenesis

  • Embryos are regenerated from somatic cells, tissues or organs from non-zygotic embryos.

  • Somatic embryogenesis is the opposite of zygotic or sexual embryogenesis.

  • Somatic embryos formed from a single cell with hormonal signal to induce a bipolar structure.

  • Bipolar structure of somatic embryo contains shoot and root meristems.

  • Embryos develop through structural steps of globular, heart, torpedo, cotyledonary and mature stages.

  • It requires two different hormonal signals to induce first a shoot organ and then a root organ.

  • No endosperm or seed coat is formed around a somatic embryo.

  • Two Types
    *Direct - initiated directly from explants through pre- embryogenic determined cells found in embryonic tissues of scutellum, hypocotyls and nucellus.
    *Indirect - done through the establishment of callus from which embryos are developed with induced determining cells
    *Somatic embryogenesis encompasses various stages from callus initiation, development of somatic embryos, maturation, plantlet formation and transfer to soil.

  • Soimatic embryogenesis has been reported in cactus, grapes, rose etc

Advantages of Somatic Embryogenesis

  • Clonal propagation of genetically uniform plant material.

  • Elimination of viruses.

  • Source tissue for genetic transformation.

  • Generation of whole plants from single cells (protoplasts).

  • Development of synthetic seed technology.

  • Develop plants resistant to various environmental stresses.

  • Introduce genes by genetic transformation (e.g., cotton resistant to Fusarium, Verticillium wilt).

Problems Associated with Micropropagation

  • Microbial contamination:

    • Prevented by growing the donor plant in growth chamber, by effective sterilization of explants, by performing inoculation in the laminar air flow cabinets and by using sterilize surgical instruments and by addition of Fumigation with dilute formaldehyde solution.

  • Callusing:

    • Effects the normal development of shoots and roots and variability. Addition of tri-iodo-benzoic acid, flurogauicinol and flurorizin into the culture medium/ reduction of inorganic salt concentration

  • Tissue culture-induced variation:

    • Exhibit genetic (or) epigenetic control by careful selection of initial explant, and controlling the cultural environment favoring slow multiplication rates).

  • Browning of medium:

    • Substance leach from the cut surfaces of explain lead to brown color and necrosis/death of cultures.
      prevented by frequent subculture, growth of culture in liquid medium, use of antioxidants like citric acid, use of adsorbents like activated charcoal (or) PVP (poly vinyl pyrrolidon) and incubation of cultures in dark.

  • Vitrification:
    *Appear brittle, glassy and water soaked. Reduce by Humidity, levels of cytokinins or NH4, or salt concentration, by addition of flurorizin, fluroroglucinol or CaCl2.

  • Transplantation shock:

    • High mortality rate by creating moisture by creating good humidity, and the by transplanting ahs given good result.

Solution to Some Common Problems in Tissue Culture

  • Insoluble precipitates can be avoided by dissolving each compound completely before adding the next compound otherwise each can be dissolved separately and added as a solution rather than as the salt.

  • colour of the final medium is a Golden (Media with agar, usually are usually of golden colour whereas Liquid media are very pale yellow colour). Any abnormal change in callouts indicates that the autoclave or media composition problem

  • If agar cannot solidify, The reason could be improper mix or the pH is too acidic in nature.

  • Un clean cultures are the result of low sterilization and can be fixed by Increased prewashing time, use o strong detergent, increased time in ethanol, concentration of bleach solution and time in bleach solution.

  • Black or brown explants with growth come from the wrong use of sterilization procedures and reduced by reducing the strength of the detergent or decrease the amount of handling during pre-washing, eliminate exposure to ethanol and decrease the time in bleach or concentration of bleach solution.

  • endophyte microorganism: Bleach and antibiotic treatment will work.
    *lack of shoot multiplication due to lack of a true shoot meristem use large pieces.
    *Large plantlets survive better in the culture to soil vessel step as they are better developed while for Herbaceous plants, watering should be regulated.

Advantages of Micropropagation

  • Production of large number of genetically uniform plants

  • A small explant is enough to produce millions of true to type plants rapidly

  • The technique is possible alternative in plants species which do not respond to conventional bulk propagation.

  • Useful to obtain virus free stocks

  • selective multiplication of plants
    *carried throughout the year independent of seasons.

Classification of Tissue Culture Techniques

  • Seed culture: Growing seeds in vitro
    *Embryo Culture: culture of embryos
    *Cell culture, culture of cells

  • Protoplast, protoplast culture

  • Organ culture (named after organ ex. Meristem = Meristem culture)

MERISTEM CULTURE & SHOOT TIP CULTURE

  • Meristem culture is regeneration of whole plant from tissues of actively dividing plant parts (stem tip, root tip or axillary bud). The apical meristem tip of 0.25 to 0.30 mm in length and 0.1 mm in diameter produces virus free plants
    *In contrast, For shoot tip culture requires large explants measuring up to 2 cm in length.

  • used in sugarcane, potato, banana, and several timber species.

  • Morel and Martin (1952) produced virus-free shoots in dahlia.

  • Virus free clones of potato, sugarcane have been produced from valuable virus infected stocks through meristem culture.

Procedure for production of virus free plants by Meristem culture

Dissect out the shoot apical meristem (100-500 µm in length) with one or two leaf primordia.
Viruses can be decreased by thermotherapy of whole plants, cryotherapy
OR chemotherapy.

Applications of meristem culture in crop improvement

Micro propagation of banana, strawberries, citrus