PRACTICAL MANNUAL OF PLANT BREEDING FOR B.Sc.VTH SEM

  EXPERIMENT -1 

Professional Plant Breeder’s Kit

Used in research stations, universities, and seed companies.

• Field Tools

  • Hand lens (magnifying glass) for examining floral structures

  • Forceps, scissors, scalpel for emasculation and pollination

  • Brushes or applicators for controlled pollination

  • Labels, waterproof markers, and notebooks for record-keeping

  • 
    
    
    

  • Bags (butter paper, muslin, or plastic) for covering flowers after crossing

  • 
    
    
    

• Lab Tools

  • Micropipettes and tips

  • DNA extraction kits

  • PCR reagents and primers

  • Gel electrophoresis apparatus

  • Seed germination trays and growth chambers

• Consumables

  • Ethanol, buffers, and staining solutions (e.g., tetrazolium for seed viability)

  • Fertilizers and soil media

  • Desiccants for seed storage

🌱 Plant Breeding Kits

These are often marketed online as DIY kits for students or gardeners.

• Seed starter kits (trays, soil pellets, seeds)

• Pollination brushes

• Labels and markers

• Instruction manuals for simple crossing experiments

• Sometimes, hydroponic or sprouting systems are used for controlled growth

 Moisture content is one of the most critical parameters in seed quality testing because it directly affects storability, germination, and longevity. Here’s a clear breakdown of how it is calculated:

 EXPERIMENT -2

🌱 Methods of Determining Seed Moisture Content

1. Hot Air Oven Method (Standard ISTA Method)

• Principle: Moisture content is the weight loss when seeds are dried under controlled conditions.

• Procedure:

  • Weigh a sample of seeds (initial weight).

  • Dry in a hot air oven at a specified temperature (usually 103 ± 2 °C for 17 hours, or 130 ± 2 °C for 2 hours, depending on seed type).

  • Cool in a desiccator and reweigh (final weight).

• Formula:

$$\text{Moisture Content (\%)}=\frac{\text{Initial Weight - Final Weight}}{\text{Initial Weight}}\times 100$$

2. Moisture Meter Method

• Uses electronic devices that measure electrical conductivity or resistance, which correlates with moisture.

• Quick and non-destructive, but less precise than oven drying.

• Often used for routine checks.

3. Distillation Method (Karl Fischer Titration)

• Chemical method using Karl Fischer reagent.

• Highly accurate, especially for oily seeds where oven drying may not remove all moisture.

📊 Expression of Moisture Content

• Wet basis (common in seed testing): Percentage of water relative to the original sample weight.

• Dry basis: Percentage of water relative to the dry matter weight (used in some scientific contexts).

⚠️ Key Considerations

• Different seeds require different drying temperatures to avoid decomposition or loss of volatile compounds.

• Replicates are often tested to ensure accuracy.

• Proper storage conditions (low humidity, cool temperatures) are essential to maintain seed viability after testing

•  EXPERIMENT -3

• To test seed vigour.

• Seed vigour is a critical quality trait in crop plants because it determines how well seeds germinate, establish seedlings, and perform under diverse field conditions. It goes beyond simple viability (whether a seed is alive) and focuses on the strength and resilience of the seed lot. Here are the main methods used to determine seed vigour:

  🌱 Common Seed Vigour Tests

  Method	Principle	What It Reveals
  Standard Germination Test	Seeds are germinated under optimal conditions.	Basic viability and uniformity of germination.
  Accelerated Aging Test	Seeds are exposed to high temperature and humidity, then tested for germination.	Predicts storability and field performance under stress.
  Cold Test	Seeds are germinated under low temperatures.	Ability to perform in early spring or cool soils.
  Electrical Conductivity Test	Measures electrolyte leakage from soaked seeds.	Membrane integrity; higher leakage = lower vigour.
  Tetrazolium Test	Seeds are stained with tetrazolium chloride to assess living tissues.	Rapid viability and vigour assessment.
  Seedling Growth Rate Test	Measures speed and uniformity of seedling growth.	Energy reserves and metabolic efficiency.
  Stress Tests (Salt, Drought, Pathogen exposure)	Seeds are germinated under stress conditions.	Tolerance to adverse environments.

  🔑 Why Seed Vigour Matters

  • Ensures uniform crop establishment.

  • Improves yield stability under variable environments.

  • Helps farmers decide seeding rates and storage strategies.

  • Supports seed industry quality control.

  🌾 Practical Note

  In crop production, vigour testing is often combined with germination and viability tests to give a complete picture of seed quality. For example, a seed lot may show high viability but poor vigour, meaning it will struggle in real field conditions despite looking fine in the lab

 EXPERIMENT -4 To test seed viability

Materials Needed

• Seeds of the crop to be tested

• 2,3,5-triphenyl tetrazolium chloride (TTC) solution (usually 1%)

• Distilled water

• Petri dishes or small containers

• Scalpel or blade

• Filter paper or tissue

• Incubator (25–30 °C)

Step-by-Step Procedure

1. Pre-conditioning

   • Soak seeds in distilled water for 12–24 hours at room temperature.

   • This softens the seed coat and activates metabolic processes.

2. Preparation

   • Carefully cut or pierce the seed to expose the embryo (radicle and cotyledons).

   • For small seeds, puncturing may be enough.

3. Staining

   • Immerse the seeds in 1% TTC solution.

   • Keep them in the dark at 25–30 °C for 2–4 hours (sometimes longer depending on species).

4. Observation

   • Remove seeds and rinse with water.

   • Examine embryos under a microscope or magnifying lens.

   • Living tissues will stain red/pink due to enzyme activity (dehydrogenases reducing TTC to formazan).

   • Dead tissues remain unstained (white).

5. Interpretation

   • Fully stained embryos → viable seeds.

   • Partially stained embryos → weak or damaged seeds (low vigour).

   • Unstained embryos → non-viable seeds.

🌾 Other Common Viability Tests

• Germination Test: Place seeds under optimal conditions and record % germination.

• Float Test: Seeds placed in water; viable ones usually sink, but this is a rough method.

• X-ray Test: Used in labs to check embryo integrity without cutting seeds.

• 
  

  • EXPERIMENT-5 To study genetic diversity using molecular markers

    Assessing genetic diversity using molecular markers is a cornerstone of modern plant breeding and conservation biology. It allows researchers to quantify variation at the DNA level, which is more precise than relying only on morphological traits. Here’s a structured overview:

    🔬 Types of Molecular Markers Used

    Marker Type	Principle	Advantages	Limitations
    RAPD (Random Amplified Polymorphic DNA)	Uses random primers to amplify DNA segments.	Quick, inexpensive, no prior sequence info needed.	Low reproducibility.
    AFLP (Amplified Fragment Length Polymorphism)	Restriction digestion + selective PCR amplification.	High resolution, detects many polymorphisms.	Technically demanding.
    SSR (Simple Sequence Repeats / Microsatellites)	PCR amplification of short tandem repeats.	Highly polymorphic, co-dominant, reproducible.	Requires sequence info.
    SNP (Single Nucleotide Polymorphism)	Detects single-base changes in DNA.	Abundant, amenable to high-throughput genotyping.	Costly, requires advanced platforms.
    ISSR (Inter Simple Sequence Repeats)	Amplifies regions between microsatellites.	Simple, reproducible, no prior sequence needed.	Lower resolution than SSR.

    🧪 General Procedure for Genetic Diversity Assessment

    1. Sample Collection

       • Collect representative plant material (leaves, seeds, or tissues) from different populations or varieties.

    2. DNA Extraction

       • Isolate high-quality genomic DNA using standard protocols (CTAB method or commercial kits).

    3. Marker Selection & PCR Amplification

       • Choose appropriate markers (SSR, RAPD, AFLP, etc.).

       • Amplify DNA fragments using PCR.

    4. Electrophoresis & Visualization

       • Separate amplified fragments on agarose or polyacrylamide gels.

       • Visualize bands using staining (ethidium bromide, SYBR Green).

    5. Data Scoring

       • Record presence/absence of bands (dominant markers) or allele sizes (co-dominant markers).

    6. Statistical Analysis

       • Use software (e.g., NTSYS, STRUCTURE, GenAlEx) to calculate genetic similarity, polymorphism information content (PIC), and cluster analysis.

       • Construct dendrograms or principal coordinate analysis (PCA) plots to visualize diversity.

    🌱 Applications in Crop Science

    • Breeding Programs: Identify diverse parents for hybridization.

    • Conservation: Detect genetic erosion and guide germplasm preservation.

    • Variety Identification: DNA fingerprinting for cultivar protection.

    • Trait Mapping: Link markers to desirable traits (disease resistance, yield).

    ⚖️ Key Insight

    Molecular markers provide a neutral, reliable, and reproducible way to measure diversity compared to morphological traits, which can be influenced by the environment. SSRs and SNPs are currently the most widely used in crop breeding due to their high resolution and reproducibility

  • Facilitates hybrid seed production without manual emasculation.

  • Maintains genetic diversity in populations.

EXPERIMENT-6🧪 Tetrazolium (TTC) Seed Viability Test

Materials Needed

Seeds of the crop to be tested

2,3,5-triphenyl tetrazolium chloride (TTC) solution (usually 1%)

Distilled water

Petri dishes or small containers

Scalpel or blade

Filter paper or tissue

Incubator (25–30 °C)

Step-by-Step Procedure

Pre-conditioning 

Soak seeds in distilled water for 12–24 hours at room temperature.

This softens the seed coat and activates metabolic processes.

Preparation

Carefully cut or pierce the seed to expose the embryo (radicle and cotyledons).

For small seeds, puncturing may be enough.

Staining

Immerse the seeds in 1% TTC solution.

Keep them in the dark at 25–30 °C for 2–4 hours (sometimes longer depending on species).

Observation

Remove seeds and rinse with water.

Examine embryos under a microscope or magnifying lens.

Living tissues will stain red/pink due to enzyme activity (dehydrogenases reducing TTC to formazan).

Dead tissues remain unstained (white).

Interpretation

Fully stained embryos → viable seeds.

Partially stained embryos → weak or damaged seeds (low vigour).

Unstained embryos → non-viable seeds.

🌾 Other Common Viability Tests

Germination Test: Place seeds under optimal conditions and record % germination.

Float Test: Seeds placed in water; viable ones usually sink, but this is a rough method.

X-ray Test: Used in labs to check embryo integrity without cutting seeds.

✅ Why the TTC Test is Preferred

Rapid (results in hours vs. days/weeks for germination tests).

Reliable for many crop species.

Detects viability even in dormant seeds that may not germinate immediately.

EXPERIMENT -7 To study self-incompatibility

 

Studying self-incompatibility (SI) in crop plants is important because it prevents self-fertilization, promotes outcrossing, and is widely used in hybrid seed production. About 40–60% of flowering plants exhibit SI, and it is particularly significant in families such as Brassicaceae (cabbage, cauliflower, broccoli) and Solanaceae (tomato, eggplant).

• 🔬 Methods to Study Self-Incompatibility

  Method	Procedure	What It Shows
  Controlled Pollination	Perform self-pollination and cross-pollination under controlled conditions, then observe fruit/seed set.	Direct evidence of SI (selfed flowers fail to set seed).
  Microscopic Pollen Tube Growth Test	Stain pistils after pollination (e.g., with aniline blue) and observe pollen tube growth under fluorescence microscopy.	In SI plants, pollen tubes stop growing in the style after self-pollination.
  Fruit/Seed Set Analysis	Compare the seed set after selfing vs. crossing.	Quantifies SI strength.
  Molecular Marker Studies	Use DNA markers to identify S-alleles (genes controlling SI).	The genetic basis of SI is useful for breeding programs.
  Biochemical Assays	Detect proteins or RNases involved in SI response (e.g., S-RNase in Solanaceae).	Mechanistic understanding of SI.

  🌱 Types of Self-Incompatibility

  • Gametophytic SI (GSI): Pollen’s own genotype determines compatibility (common in Solanaceae, Rosaceae).

  • Sporophytic SI (SSI): Pollen compatibility determined by the parent plant’s genotype (common in Brassicaceae).

  • 
    
    
    

  ⚖️ Importance in Crop Breeding

  • Prevents inbreeding depression.

  • Facilitates hybrid seed production without manual emasculation.

  • Maintains genetic diversity in populations.

•