Propagation and Nursery Management for Fruit Crop - Unit IV - Micro-propagation & Advanced Techniques

UNIT IV: Micro-propagation & Advanced Techniques

Micro-propagation - principles and concepts, commercial exploitation in horticultural crops. Techniques - in vitro clonal propagation, direct organogenesis, embryogenesis, micrografting, meristem culture. Hardening, packing and transport of micro-propagules.

Table of Contents

1. Principles of Micro-propagation 2. Concepts & Stages of Micro-propagation 3. Commercial Exploitation 4. Techniques: Organogenesis & Embryogenesis 5. Micrografting & Meristem Culture 6. Hardening, Packing and Transport 7. Important Exam Points & Sources

Micro-propagation – Principles, Concepts & Commercial Exploitation in Horticultural Crops

1. INTRODUCTION

1.1 Definition:
Micro-propagation = In vitro (lab-based) clonal propagation of plants using small plant parts (explants) under sterile and controlled conditions.
👉 It is a major application of plant tissue culture.

1.2 Key Concept

Based on Totipotency → Every plant cell has the ability to regenerate into a whole plant

1.3 Importance

• Rapid multiplication
• Disease-free planting material
• Year-round production
• Uniform plants

2. PRINCIPLES OF MICRO-PROPAGATION

2.1 Totipotency

• Single cell → whole plant
• Basis of all tissue culture techniques

2.2 Aseptic Conditions

• Sterile environment is essential
• Prevents contamination by: Bacteria, Fungi

2.3 Controlled Environment

• Temperature: 22–27°C
• Light: 1000–3000 lux
• Photoperiod: 16 hours light / 8 hours dark

2.4 Nutrient Medium

Most commonly used medium: 👉 Murashige and Skoog medium

• Components:
• Macronutrients (N, P, K, Ca, Mg)
• Micronutrients (Fe, Zn, Mn)
• Vitamins
• Carbon source (Sucrose: 2–3%)
• Growth regulators

2.5 Role of Plant Growth Regulators (PGRs)

Hormone	Function
Auxins (IAA, IBA, NAA)	Root formation
Cytokinins (BAP, Kinetin)	Shoot multiplication
Gibberellins	Shoot elongation

👉 Auxin : Cytokinin ratio controls organ formation

3. CONCEPTS OF MICRO-PROPAGATION

3.1 Explant Definition:
Small plant part used to initiate culture: Shoot tip, Meristem, Leaf, Node.

3.2 Stages of Micro-propagation

The standard stages involved in plant micro-propagation.

1. Stage I: Establishment
   • Selection of explant
   • Surface sterilization: HgCl₂ (0.1%) or NaOCl
   • Inoculation on culture medium
2. Stage II: Multiplication
   • Rapid shoot multiplication
   • Use of cytokinins (BAP)
   • Subculturing every 3–4 weeks
3. Stage III: Rooting
   • Transfer shoots to rooting medium
   • Auxins used (IBA, NAA)
4. Stage IV: Hardening (Acclimatization)
   • Transfer to soil/soilless media
   • Gradual exposure to external environment
   • High humidity initially (80–90%)

4. TYPES OF MICRO-PROPAGATION

1. Meristem Culture: Used for virus-free plants. Very small shoot tip (0.1–0.5 mm).
2. Callus Culture: Undifferentiated mass of cells. Can regenerate plants.
3. Somatic Embryogenesis: Embryo formation from somatic cells. Important for large-scale propagation.
4. Organogenesis: Direct formation of shoots/roots from explants.

5. COMMERCIAL EXPLOITATION IN HORTICULTURAL CROPS

5.1 Advantages for Commercial Use

• Rapid multiplication (1000s plants/year)
• Disease-free (virus-free) plants
• Uniform growth and yield
• Space-saving
• Export-quality planting material

5.2 Major Crops Propagated

• Fruit Crops: Banana, Strawberry, Pineapple, Apple
• Plantation Crops: Tea, Coffee, Oil palm
• Ornamentals: Orchid, Chrysanthemum, Gerbera
• Vegetable Crops: Potato (seed production)

5.3 Commercial Process Flow

Selection of elite plant → Establishment of culture → Mass multiplication → Rooting → Hardening → Nursery raising → Field planting

5.4 Industrial Scale Production

• Done in tissue culture labs
• Automated systems: Bioreactors, Controlled environment chambers

5.5 Role of Institutions

• ICAR → Research & protocols
• National Horticulture Board → Promotion & subsidy
• Private tissue culture labs → commercial supply

6. LIMITATIONS

• High initial cost
• Requires skilled labour
• Risk of contamination
• Somaclonal variation (genetic variation)

Techniques: In vitro Clonal Propagation, Direct Organogenesis, Somatic Embryogenesis

1. IN VITRO CLONAL PROPAGATION (MICROPROPAGATION)

1.1 Definition: Production of genetically identical plants (clones) using tissue culture under sterile conditions.

1.2 Principle: Based on Totipotency. Controlled use of growth regulators → shoot & root formation.

1.3 Steps

• Explant selection (shoot tip/node)
• Surface sterilization
• Inoculation on medium
• Shoot multiplication
• Rooting
• Hardening

1.4 Key Features

• Rapid multiplication (10–1000 plants/explant/year)
• Uniform plants
• Used in banana, potato, ornamentals

2. DIRECT ORGANOGENESIS

2.1 Definition: Formation of shoots or roots directly from explant without callus stage.

Direct organogenesis bypassing the intermediate callus stage.

2.2 Principle: Cells differentiate directly into organs. Avoids genetic variation.

2.3 Process

• Explant placed on medium
• Cytokinin induces shoot formation
• Shoots elongated
• Rooting induced

2.4 Characteristics

• Faster than indirect method
• Genetically stable
• High success in nodal cultures

2.5 Applications

• Micropropagation of fruit crops
• Rapid multiplication of elite genotypes

3. SOMATIC EMBRYOGENESIS

3.1 Definition: Formation of embryos from somatic (non-reproductive) cells.

Developmental stages in Somatic Embryogenesis.

3.2 Principle: Somatic cells behave like zygotic embryos. Develop into complete plants.

3.3 Types

• Direct embryogenesis (without callus)
• Indirect embryogenesis (via callus)

3.4 Stages

• Induction → callus formation
• Embryo formation: Globular → Heart → Torpedo
• Maturation
• Germination → plantlet

3.5 Advantages

• Large-scale propagation
• Synthetic seed production
• Automation possible

3.6 Applications

Banana, Oil palm, Coffee

4. MICROGRAFTING & MERISTEM CULTURE

4. MICROGRAFTING

4.1 Definition: Grafting of very small scion (meristem/shoot tip) onto a seedling rootstock under in vitro conditions.

In vitro Micrografting method.

4.2 Principle: Combines Tissue culture + Conventional grafting. Used for disease elimination.

4.3 Process

• Rootstock grown in vitro
• Tiny shoot tip excised
• Grafted onto rootstock
• Cultured in sterile conditions

4.4 Applications

Citrus (very important), Apple, Grapes.

4.5 Advantages

• Produces virus-free plants
• Rapid multiplication
• Overcomes incompatibility

5. MERISTEM CULTURE

5.1 Definition: Culture of apical meristem (0.1–0.5 mm) to produce disease-free plants.

Excision and culture of the apical meristem.

5.2 Principle: Meristem is free from viruses. Viruses do not easily invade actively dividing cells.

5.3 Process

• Excise meristem under microscope
• Culture on nutrient medium
• Shoot development
• Rooting
• Hardening

5.4 Applications

Potato (virus-free seed), Banana, Strawberry.

5.5 Advantages

• Virus-free plants
• Maintains genetic purity

6. COMPARISON OF TECHNIQUES

Technique	Key Feature	Advantage	Limitation
Clonal propagation	Mass multiplication	Uniform plants	Costly
Direct organogenesis	No callus	Stable	Limited explants
Somatic embryogenesis	Embryo formation	Large scale	Variation possible
Micrografting	Tiny graft	Virus-free	Skill required
Meristem culture	Shoot tip culture	Disease-free	Slow initial growth

Hardening, Packing and Transport of Micro-propagules

1. INTRODUCTION

Micro-propagules = plantlets produced through tissue culture (in vitro), which are:
• Delicate
• Non-functional cuticle
• Poor stomatal control
• Weak root system
👉 Therefore, hardening (acclimatization) is essential before field transfer.

2. HARDENING (ACCLIMATIZATION)

2.1 Definition: Gradual adaptation of in vitro plantlets to external (ex vitro) conditions.

2.2 Need for Hardening

In vitro plantlets have:

• Thin cuticle → high water loss
• Non-functional stomata
• Low photosynthetic capacity
• High humidity adaptation

👉 Sudden transfer → high mortality

2.3 Types of Hardening

1. Primary Hardening: Done in Growth chamber, Mist chamber, or Polyhouse. 👉 Controlled conditions.
2. Secondary Hardening: Done in Nursery (shade net house). 👉 Gradual exposure to natural conditions.

2.4 Steps in Hardening Process

• Step 1: Removal from Culture: Remove plantlets from culture vessels. Wash roots to remove agar medium.
• Step 2: Fungicide Treatment: Dip in fungicide (Carbendazim 0.1% or similar). 👉 Prevent fungal infection.
• Step 3: Transplanting to Medium: Plant in sterilized medium (Cocopeat, Vermiculite, Perlite). 👉 Ideal mixture: Cocopeat : Vermiculite = 1:1.
• Step 4: High Humidity Maintenance: Maintain 80–90% RH initially. Use Mist chamber or Fogging system.
• Step 5: Gradual Exposure: Reduce humidity gradually. Increase light intensity stepwise. Open vents slowly.
• Step 6: Nutrient Supply: Foliar spray of dilute nutrient solution (e.g., ½ MS). Helps growth establishment.

2.5 Environmental Conditions

Factor	Ideal Range
Temperature	25–30°C
Humidity	80–90% (initial)
Light	Low → moderate
Shade	50–75% shade net

2.6 Duration & Importance

• Primary hardening: 2–4 weeks | Secondary hardening: 4–6 weeks
• Improves survival rate (up to 80–95%)
• Develops functional: Roots, Stomata, Photosynthetic system

3. PACKING OF MICRO-PROPAGULES

3.1 Objectives

• Prevent mechanical damage, moisture loss, and contamination.
• Maintain plant health during transport.

3.2 Packing Methods

1. Polybag Packing: Plantlets packed in polybags with growing medium. Most common method.
2. Tray Packing: Plug trays used for bulk transport. Suitable for short distances.
3. Moist Packing: Roots wrapped in moist sphagnum moss or tissue paper.
4. Box Packaging: Corrugated boxes with ventilation holes and cushioning material.

3.3 Precautions

Avoid overpacking. Maintain moisture (not waterlogged). Labeling of varieties. Use ventilated packaging.

4. TRANSPORT OF MICRO-PROPAGULES

4.1 Objectives & 4.2 Key Requirements

• Deliver plants safely, maintaining physiological condition.
• Temperature Control: Maintain 15–25°C. Avoid heat stress.
• Humidity Maintenance: Prevent desiccation. Use moist packing.
• Ventilation: Prevent CO₂ accumulation. Avoid fungal growth.
• Handling: Gentle handling. Avoid shaking and damage.

4.3 Modes of Transport & 4.4 Post-Transport Care

• Road (short distance) or Air transport (export material).
• Immediate unpacking, light irrigation, and keep in shade initially.

5. PROBLEMS DURING HARDENING & TRANSPORT

1. High Mortality: Due to sudden environmental change
2. Desiccation: Poor humidity control
3. Fungal Infection: Poor sanitation
4. Mechanical Damage: Improper packing

7. IMPORTANT EXAM POINTS (REVISION)

• Micro-propagation based on totipotency
• MS medium is most widely used
• 4 stages: Establishment, Multiplication, Rooting, Hardening
• Cytokinin → shoot
• Auxin → root
• Widely used in banana, potato, ornamentals
• Micropropagation → cloning via tissue culture
• Direct organogenesis → no callus stage
• Somatic embryogenesis → embryo from somatic cells
• Micrografting → in vitro grafting
• Meristem culture → virus-free plants
• Hardening = adaptation from in vitro → ex vitro
• Two stages: Primary (controlled), Secondary (nursery)
• RH initially 80–90%
• Cocopeat + vermiculite commonly used
• Packing → prevent moisture loss & damage
• Transport → maintain temperature & humidity

8. SOURCES

• Hartmann & Kester – Plant Propagation: Principles and Practices
• Bhojwani & Razdan – Plant Tissue Culture: Theory and Practice
• ICAR – Handbook of Horticulture
• NHB Guidelines – National Horticulture Board
• ICAR Tissue Culture Manuals – ICAR
• ICAR Protocols – ICAR
• FAO Tissue Culture Manuals

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