Unit 5: Plant growth regulators - Subjective Questions

Plant Growth Regulators (PGRs) are organic compounds, either naturally occurring or synthetically produced, that in low concentrations influence and regulate physiological and developmental processes in plants.

Significance in in vitro organogenesis:

• Control of morphogenesis: PGRs direct explant cells to form shoots, roots, or callus depending on their type and concentration.
• Auxin:cytokinin ratio: The balance between these two classes determines the developmental fate of cultured tissue.
• Cell division and elongation: They stimulate cell division (cytokinins) and elongation (auxins/gibberellins).
• Dedifferentiation and redifferentiation: PGRs allow differentiated cells to revert to a meristematic state and then re-differentiate into organs.
• Commercial micropropagation: Enables rapid, large-scale clonal multiplication of elite plants.

Without appropriate PGR supplementation in the culture medium, organized development from explants generally does not occur.

Organogenesis is the process of de novo formation of organs such as shoots and roots from cultured plant cells, tissues, or explants under the influence of plant growth regulators.

Direct Organogenesis:

• Organs (shoots/roots) develop directly from the explant tissue without an intervening callus phase.
• Occurs from pre-existing meristematic or competent cells.
• Produces genetically stable (true-to-type) plants.
• Example: shoot bud formation directly from leaf or nodal explants.

Indirect Organogenesis:

• Organs form from an unorganized callus mass that first develops from the explant.
• Involves two stages: callus induction followed by organ differentiation.
• Higher chance of somaclonal variation (genetic instability).
• Useful for genetic transformation and mutation studies.

The auxin-to-cytokinin ratio is the single most important factor governing the direction of organogenesis in plant tissue culture. This principle was established by Skoog and Miller (1957) working with tobacco pith cultures.

Effect of the ratio:

• High cytokinin : low auxin → promotes shoot formation (caulogenesis).
• High auxin : low cytokinin → promotes root formation (rhizogenesis).
• Intermediate / balanced ratio → promotes callus proliferation (unorganized growth).

Skoog and Miller's findings:

• They demonstrated that morphogenesis in tobacco pith explants was not controlled by absolute concentrations but by the relative proportions of auxin (IAA) and cytokinin (kinetin).
• This established the classic paradigm that developmental fate is hormonally programmable.

Schematically:

The major classes of PGRs used in in vitro culture are:

1. Auxins

   • Promote cell elongation, root initiation, and callus formation.
   • Examples: IAA (Indole-3-acetic acid), IBA (Indole-3-butyric acid), NAA (Naphthalene acetic acid), 2,4-D (2,4-Dichlorophenoxyacetic acid).

2. Cytokinins

   • Promote cell division and shoot bud formation; delay senescence.
   • Examples: BAP/BA (6-Benzylaminopurine), Kinetin, Zeatin, 2-iP, TDZ (Thidiazuron).

3. Gibberellins

   • Promote shoot elongation and internode growth; break dormancy.
   • Example: GA₃ (Gibberellic acid).

4. Abscisic acid (ABA)

   • Involved in stress responses, dormancy, and somatic embryo maturation.

5. Ethylene

   • Gaseous regulator affecting senescence and, in some cases, morphogenesis.

In practice, auxins and cytokinins are the two most critical classes for regulating in vitro shooting and rooting.

In vitro Shoot Induction Procedure:

1. Selection of explant: Choose meristematic explants such as nodal segments, shoot tips, or leaf discs from a healthy mother plant.

2. Surface sterilization: Wash the explant and treat with disinfectants (e.g., 70% ethanol followed by 0.1% mercuric chloride or sodium hypochlorite) and rinse with sterile distilled water.

3. Inoculation on shoot induction medium:

   • Basal medium: MS (Murashige and Skoog) medium with sucrose and agar.
   • Supplement with a high cytokinin : low auxin ratio.
   • Common cytokinins: BAP (0.5–5.0 mg/L) or Kinetin; sometimes a small amount of auxin (NAA/IAA) is added.

4. Incubation: Maintain cultures at 25 ± 2 °C under a 16 h light / 8 h dark photoperiod.

5. Shoot development: Multiple shoot buds emerge within 2–4 weeks.

6. Subculturing/multiplication: Transfer to fresh medium to increase shoot number.

7. Shoot elongation: GA₃ may be added to elongate short shoots before rooting.

The elevated cytokinin level is the key factor driving shoot bud differentiation.

In vitro Root Induction Procedure:

1. Selection of microshoots: Healthy, well-elongated shoots (2–4 cm) obtained from the multiplication stage are used.

2. Transfer to rooting medium:

   • Basal medium: usually half-strength MS medium (reduced salts favor rooting).
   • Supplement with an auxin at a low concentration.
   • Common auxins: IBA (0.5–2.0 mg/L), NAA, or IAA.
   • Cytokinins are usually omitted or kept minimal.

3. Incubation: Maintain at 25 ± 2 °C under suitable light.

4. Root emergence: Adventitious roots appear within 1–3 weeks.

5. Hardening/acclimatization: Rooted plantlets are gradually transferred to soil.

Role of auxins in rhizogenesis:

• Stimulate adventitious root initiation by promoting cell division in the pericycle/cambial region.
• Induce the formation of root primordia.
• IBA is often preferred as it is more stable and gives strong, healthy root systems.
• Excessive auxin, however, can cause callusing at the shoot base and inhibit root elongation.

Summary:

• Auxins and cytokinins act antagonistically and synergistically depending on their ratio.
• Their balance determines whether the explant forms shoots, roots, or callus, forming the basis of in vitro organogenesis.

Micropropagation is generally divided into the following stages (as described by Murashige):

Stage 0 – Selection and preparation of mother plant:

• Maintaining a healthy, disease-free stock plant. No PGR involvement in culture yet.

Stage I – Initiation/Establishment of aseptic culture:

• Explant is sterilized and placed on medium.
• Low levels of cytokinin (and sometimes auxin) help establish the culture.

Stage II – Multiplication (Shoot proliferation):

• High cytokinin (e.g., BAP) with low or no auxin promotes formation of multiple shoots.
• Repeated subculturing increases shoot number.

Stage III – Rooting (Shoot elongation and root induction):

• Shoots transferred to auxin-rich (IBA/NAA), low-salt medium.
• GA₃ may be used earlier to elongate shoots.
• Roots form on individual shoots.

Stage IV – Acclimatization/Hardening:

• Plantlets gradually transferred to ex vitro conditions (greenhouse/soil).
• PGRs generally not required; focus is on adaptation to environment.

Key point: The cytokinin-dominant phase drives multiplication while the auxin-dominant phase drives rooting.

Half-strength MS medium (where the concentration of macro- and micro-salts is reduced to half) is widely used during the rooting stage for the following reasons:

• Lower salt/nitrogen concentration favors root initiation, as high nitrogen (especially nitrate and ammonium) tends to inhibit root formation and promote shoot/callus growth.
• Reduced osmotic stress creates conditions closer to the natural rooting environment.
• Promotes better root elongation and quality rather than excessive vegetative growth.
• Reduces the risk of vitrification (hyperhydricity) of tissues.
• Works synergistically with auxins (IBA/NAA) to enhance adventitious root development.

Additional modifications at rooting stage:

• Reduced or omitted cytokinin.
• Sometimes reduced sucrose concentration.
• Addition of activated charcoal to adsorb inhibitory phenolics and darken the medium, favoring rooting.

Comparison of common rooting auxins:

Conclusion:

• IBA is generally the most effective and reliable rooting auxin because it is stable and gives good-quality roots with minimal callus.
• NAA is potent but may induce excessive callus at high concentrations.
• IAA is least favored due to rapid degradation.

Hyperhydricity (also called vitrification) is a physiological disorder of in vitro cultured shoots in which they become translucent, glassy, swollen, and water-soaked with poor development and reduced regeneration ability.

Causes:

• High cytokinin concentration in the medium.
• Excess ammonium ions and high salt levels.
• High relative humidity inside the culture vessel.
• Poor gas exchange (accumulation of ethylene).
• Low agar concentration (too soft/watery medium).

Control measures:

• Reduce cytokinin concentration.
• Increase agar/gelling agent concentration to lower water availability.
• Improve ventilation of culture vessels.
• Lower ammonium nitrate content in the medium.
• Add osmotic agents or reduce relative humidity.

Controlling hyperhydricity is important because affected shoots root poorly and fail to acclimatize.

Effect of cytokinins on shoot multiplication:

• Cytokinins (e.g., BAP, Kinetin, TDZ) promote cell division and stimulate the outgrowth of axillary and adventitious shoot buds.
• They overcome apical dominance, allowing multiple shoots to develop from a single explant.
• Increasing cytokinin concentration generally increases the number of shoots produced per explant, up to an optimal level.
• They help maintain shoots in a juvenile, actively dividing state suitable for repeated subculture.

Effects at very high concentrations:

• Shoots become short and stunted with reduced elongation.
• Formation of abnormal, rosette-like clusters.
• Increased incidence of hyperhydricity (vitrification).
• Inhibition of rooting in later stages.
• Possible somaclonal variation and callus formation at the base.

Hence, an optimal cytokinin concentration must be determined for each species to balance shoot number against shoot quality.

Gibberellic acid (GA₃) is a gibberellin-class plant growth regulator with several roles in in vitro shoot culture:

• Shoot elongation: Its main use is to elongate short or stunted microshoots (especially those produced under high cytokinin), thereby making them suitable for rooting.
• Internode elongation: Stimulates cell elongation between nodes.
• Breaking dormancy: Helps break bud and seed dormancy in culture.
• Overcoming rosetting: Reverses the rosette (compact) growth habit caused by high cytokinin levels.
• Enhancing germination: Promotes germination of in vitro raised somatic embryos or seeds.

Precautions:

• Excessive GA₃ can cause abnormally elongated, weak shoots and may inhibit rooting.
• It is usually used at low concentrations (0.1–1.0 mg/L) and often only transiently before the rooting stage.

Callus is an unorganized, undifferentiated mass of proliferating parenchymatous cells that forms on an explant when cultured on a medium containing appropriate growth regulators (usually a balanced auxin and cytokinin combination, e.g., 2,4-D).

Inducing organogenesis from callus (indirect organogenesis):

1. Callus induction phase:

   • Explant is placed on medium with balanced auxin:cytokinin (often high 2,4-D) to promote dedifferentiation and callus proliferation.

2. Shoot regeneration:

   • Callus is transferred to medium with high cytokinin : low auxin to induce shoot bud differentiation (caulogenesis).

3. Root regeneration:

   • Regenerated shoots are transferred to high auxin : low cytokinin medium to induce rooting (rhizogenesis).

4. Plantlet formation:

   • Complete plantlets with shoots and roots are obtained and acclimatized.

Key point: By sequentially altering the auxin:cytokinin ratio, the totipotent callus cells can be redirected to form organized structures. However, prolonged callus culture increases the risk of somaclonal variation.

Totipotency:

• The inherent ability of a single plant cell to divide, grow, and regenerate into a complete, whole plant.
• It is the fundamental principle underlying all plant tissue culture and organogenesis.

Dedifferentiation:

• The process by which mature, differentiated (specialized) cells revert to a meristematic (actively dividing, unspecialized) state.
• Typically leads to callus formation.
• Induced by growth regulators such as auxins (2,4-D) and cytokinins.

Redifferentiation:

• The process by which dedifferentiated (callus) cells re-organize and differentiate to form new organs (shoots, roots) or embryos.
• Governed by manipulating the auxin:cytokinin ratio.

Sequence in indirect organogenesis:

Together these concepts explain how a cell reprograms its developmental fate under hormonal control.

Although PGRs are central, several other factors influence in vitro organogenesis:

1. Explant factors:

• Type (leaf, node, shoot tip, root) and age of explant.
• Physiological state and position on the mother plant.
• Juvenile tissues generally respond better.

2. Genotype:

• Different species and even cultivars vary greatly in regeneration ability.

3. Culture medium composition:

• Salt strength (full vs half MS), nitrogen source, sucrose (carbon source), and gelling agent.

4. Physical/environmental factors:

• Temperature (usually 25 ± 2 °C).
• Light intensity, quality, and photoperiod (commonly 16 h light).
• pH of medium (usually 5.6–5.8).

5. Gaseous environment:

• Accumulation of ethylene and CO₂ in closed vessels affects morphogenesis.

6. Additives:

• Activated charcoal, coconut water, amino acids, vitamins, and antioxidants can influence response.

Optimizing these factors together with PGRs is essential for successful organogenesis.

Acclimatization (Hardening) is the process of gradually adapting in vitro raised plantlets from the controlled, aseptic, high-humidity culture environment to the natural ex vitro (greenhouse/field) conditions.

Why in vitro plantlets need hardening:

• They grow under high humidity, low light, and heterotrophic (sucrose-fed) conditions.
• They possess poorly developed cuticles, non-functional stomata, and weak root systems.
• They are not autotrophic and are prone to desiccation and transplant shock.

Steps in acclimatization:

1. Remove plantlets from culture and wash off agar from roots.
2. Transplant into a sterile potting mix (peat, vermiculite, sand).
3. Maintain high humidity initially (mist chamber/polytunnel) and gradually reduce it.
4. Gradually increase light intensity and expose to normal atmospheric conditions.
5. Transfer to greenhouse and finally to the field.

Critical importance: Without proper hardening, plantlets wilt and die due to water loss and inability to photosynthesize independently, causing failure of the entire micropropagation effort.

Somaclonal variation refers to the genetic and phenotypic variation observed among plants regenerated from cultured somatic cells, tissues, or callus.

Relation to growth regulators and organogenesis:

• Variation is more frequent in indirect organogenesis (callus-mediated) than in direct organogenesis.
• Auxins such as 2,4-D, commonly used for callus induction, are known to be strongly mutagenic and promote chromosomal abnormalities.
• Prolonged culture and repeated subculturing in the presence of high PGR levels increase the frequency of variation.

Causes:

• Chromosomal changes (aneuploidy, polyploidy, breaks).
• Point mutations, gene amplification/deletion.
• Epigenetic (DNA methylation) changes.

Significance:

• Disadvantage: Undesirable when true-to-type clones are required (commercial propagation).
• Advantage: Useful as a source of genetic variability for crop improvement and selection of novel traits.

To minimize it, use direct organogenesis, avoid strong auxins like 2,4-D, and limit the number of subcultures.

Problem: Good shoot multiplication but poor/failed rooting.

Possible reasons (PGR-related):

• Carry-over of high cytokinin from the multiplication medium inhibits root initiation.
• Insufficient or inappropriate auxin in the rooting medium.
• Too high auxin causing basal callusing instead of roots.
• Full-strength MS medium with high salts/nitrogen suppressing rooting.
• Accumulated phenolics inhibiting rhizogenesis.

Remedies:

• Transfer shoots to a cytokinin-free medium before rooting.
• Use an appropriate auxin such as IBA (0.5–2.0 mg/L) or NAA at optimized concentration.
• Switch to half-strength MS medium to lower salt/nitrogen levels.
• Add activated charcoal (0.1–0.3%) to adsorb inhibitory phenolics and darken the medium.
• Give a brief auxin pulse treatment (dipping shoot bases in concentrated auxin) rather than continuous exposure.
• Optimize sucrose concentration and provide suitable light conditions.

Adjusting the auxin:cytokinin balance in favour of auxin is the key corrective step.

Activated Charcoal (AC):

• Added to the medium (usually 0.1–0.5% w/v).
• Functions:
  • Adsorbs toxic phenolic compounds and browning exudates released by explants.
  • Adsorbs excess or inhibitory growth regulators, balancing their effective concentration.
  • Darkens the medium, mimicking soil conditions and favoring root induction.
  • Reduces vitrification and promotes healthy growth.
• Caution: Being non-selective, it may also adsorb essential nutrients and vitamins.

Coconut Water (Coconut Milk):

• A natural liquid endosperm used as a complex organic supplement (typically 10–15% v/v).
• Functions:
  • Rich source of natural cytokinins (e.g., zeatin), amino acids, sugars, and vitamins.
  • Stimulates cell division and growth, historically used to induce proliferation in cultures.
  • Enhances callus growth and morphogenesis in many species.
• Limitation: Its composition is variable and undefined, so results can be inconsistent.

Both are used to improve regeneration efficiency where fully defined media are inadequate.