Plant Breeding and Genetics: Core Concepts and Applications

Domestication of Crops and Early Plant Breeding

Domestication of Crops

Domestication began with selecting for desirable traits, such as the non-shattering trait in crops, which prevents seeds from dispersing naturally and aids harvest. This process occurred at different times and locations for various crops:

• Barley (Hordeum vulgare): Fertile Crescent, before $9,000$ BC.

• Wheat (Triticum spp): Fertile Crescent, before $8,000$ BC.

• Beans (Phaseolus spp): Middle and South America, before $7,000$ BC.

• Rice (Oryza sativa): South of Himalaya Mountains, before $6,000$ BC.

• Maize (Zea mays): Mexico/Guatemala, before $3,000$ BC.

• Soybean (Glycine max): Central China, before $2,800$ BC.

• Peanut (Arachis hypogaea): South America, before $1,800$ BC.

• Grain Sorghum (Sorghum bicolor): Africa, before $1,000$ BC.

Historical Aspects of Plant Breeding

Plant breeding has a long history, progressing from early observations to modern scientific approaches:

• Hippocrates (460-377 BC): Proposed a theory similar to Darwin's pangenesis (approx. 2200 years earlier), suggesting the body produced "gemmules" as the basis of heredity, collected in semen.

• Egyptians/Mesopotamians (circa 700 BC): Practiced artificial pollination of date palms.

• 1676: Crew suggested the functions of ovules and pollen.

• 1716: Cotton Mather observed the effects of cross-pollination in maize.

• 1859: C. Darwin published Origin of Species.

• 1865: G. Mendel published his foundational work on segregation, independent assortment, dominance, and recessiveness.

• 1868: Beal proposed commercial F1 hybrid maize.

• 1875: ‘Pringle’s Progress’ became the first oats developed from artificial hybridization.

• 1879: Blount developed a maize cultivar using hand pollination.

• 1900: Mendel’s work was independently verified by de Vries, Correna, and von Tschermak.

• 1902: Garrod demonstrated the relationship between genes and enzymes.

• 1903: Johannsen developed the Pure Line theory of plant breeding.

• 1908: E.M. East reported on the effects of inbreeding in maize.

• 1908: Hardy and Weinberg independently described the expected relationship between gene frequencies, leading to the Hardy-Weinberg Equilibrium.

• 1913: Emerson and East published on the inheritance of quantitative traits.

• 1918: Jones proposed double crosses in maize.

• 1941: Beadle and Tatum’s work led to the 1-gene-1-enzyme theory.

• 1953: Watson and Crick elucidated the molecular structure of DNA.

• 1994: Genetically modified (GM) crops became commercially available. Examples include:

  • 1994: Soybean resistant to sulfonyl urea herbicides.

  • 1995: Bromoxynil-resistant cotton.

  • 1996: Bt cotton, maize, and soybean.

  • 1997: Roundup-resistant cotton, maize, and soybean.

Understanding Plant Breeding

What is Plant Breeding?

Plant breeding is defined as:

• The genetic adjustment of plants for the service of mankind.

• An applied science focused on improving plants for human use.

• Improvement in the quantity or quality of the economic product of plants.

Specific examples of improvement include:

• Increased fruit production (e.g., apples, beans, melons, nuts).

• Increased cotton fiber yield.

• Improvement in the entire plant, as seen in common Finger grass.

• Enhanced protein quality in maize or other grasses.

• Increased harvest efficiency (e.g., in peanuts by delaying deterioration of the peg—the elongated ovary).

Interdisciplinary Nature of Plant Breeding

Plant breeding is an applied science that integrates various basic sciences:

• Genetics

• Pathology

• Entomology

• Biochemistry

• Statistics

• Computer Science

• Agronomy

Modern plant breeders often work as part of a plant improvement team, which may include molecular geneticists, entomologists, pathologists, and plant physiologists.

Origins of Plant Breeding

Plant breeding is one of mankind's oldest accomplishments, dating back to when early humans transitioned from hunting to gathering. It originated in many places at different times with diverse crops:

• Fertile Crescent: Grains.

• China: Rice.

• Pakistan/India: Cotton.

• Central America: Beans, squash, cotton.

Domestication and Selection: Nature vs. Man

Nature's Selections

Natural selection favors traits that ensure a species' survival and propagation in the wild:

• Shattering: Generally a dominant trait that disperses seeds for the next generation. A domestic corn plant, for instance, would struggle to survive in the wild due to its non-shattering trait.

• Many small seeds: Increases the chance of survival and dispersal.

• Non-uniform maturity: Spreads risk, ensuring some seeds mature even under variable conditions.

• Dormancy: Allows seeds to survive unfavorable periods and germinate when conditions are ideal.

• Maintenance of variation: Ensures survival across varying years and environmental challenges.

• Nature typically discards most mutations or aberrant types that do not confer a survival advantage.

Mankind's Selections

Human selection, in contrast, targets traits beneficial for cultivation, harvest, and consumption:

• Non-shattering: Easier harvesting, prevents seed loss.

• Uniform maturity: Allows for efficient, single-time harvesting.

• Large seed: Increases yield and nutritional value per seed.

• Increased yield: Primary goal for food production.

• No dormancy: Ensures quick and predictable germination.

• Higher quality: Improved taste, nutritional content, or physical properties.

• Lodging resistance: Prevents plants from falling over, making harvest easier and reducing loss.

• Insect resistance: Reduces crop damage and pesticide use.

• Disease resistance: Protects crops from pathogens.

• Resistance to temperature extremes.

• Resistance to drought.

• Allelopathy: Ability of plants to suppress the growth of neighboring plants.

• Seedling vigor: Robust early growth for better establishment.

• Hard seededness (?).

• Anther culturability.

• Regeneration ability.

Plant breeding began as an art, involving simple selections by early humans for plants with desirable taste and high caloric return for the energy expended. Today, it is a complex art and science involving many disciplines.

The Power of Selection: Dog Breeding Example

Artificial selection demonstrates its immense power through the diversification of dog breeds. All dog breeds, from Terriers (selected for driving game out of burrows) to Retrievers (selected for retrieving game from water, often near freezing, requiring strong sense of smell and swimming ability), were produced by human intervention. This highlights that targeted selection can bring about dramatic changes.

When selecting for traits, the emphasis is on "screen, screen, screen and numbers, numbers, numbers." For example, improving the food quality of maize by increasing lysine and methionine content requires understanding existing variability, heritability, sources of variability, appropriate breeding techniques, and sampling methods (ear, kernel, individual plant, progeny row, and sample size).

The Value and Importance of Plant Breeding

Contribution to Yield Increases

Yield increases in agriculture are a result of multiple factors:

• Climate change

• Commercial fertilizer

• Herbicides

• Power equipment

• Plant Growth Regulators (PGR)

• GENETICS

The contribution of genetics is measured as Genetic Gain. While there are no hard universal numbers, observations show significant genetic contributions:

• Maize (1930-1980): $92 \text{ kg/ha/yr}$ or $89\%$ of total yield gain in Iowa attributed to improved genetics.

• Wheat: $50\%$ attributed to genetic gain.

• Sorghum (1956-1980): $39\%$ of yield increase attributed to genetics.

• Cotton: $7 \text{ kg/ha/yr}$ or $0.74\%/yr$ gain in yield potential from genetics.

Genetic gain is context-dependent, varying by location, time, and genotypes.

Plant Breeding and Food Security

Plant breeders developed crop varieties that enabled substantial increases in harvest yield (tons/hectare). This activity intensified in developed countries in the 1920s and in developing countries in the 1950s (the Green Revolution), led by figures like Norman Borlaug.

Approximately $50\%$ of these yield increases are due to better varieties (genetics), and the other $50\%$ to increased inputs (water, fertilizer, weed control, pest control).

Plant Cell Biology and Cell Division

Plant Cell Parts

Plant cells have specific organelles, many of which are distinct from animal cells:

• Cell Wall:

  • Stiff for protection and support.

  • Acts as a selective doorway.

• Cell Membrane (Plasma Membrane):

  • Located below the cell wall.

  • Provides additional protection and support.

• Nucleus:

  • Control center/brain of the cell.

  • Contains DNA (chromosomes - information for life).

• Cytoplasm:

  • Clear, thick, jelly-like fluid surrounding organelles.

• Mitochondria:

  • Cell powerhouse; makes energy (ATP).

• Vacuole:

  • Storage tank for food, waste, and water.

  • Plant cells typically have one large central vacuole.

• Chloroplasts:

  • Green colored organelles.

  • Capture sunlight for photosynthesis (makes glucose).

• Endoplasmic Reticulum (ER):

  • Transportation tubes within the cell.

• Ribosomes:

  • Protein factories, found on ER walls and in the cytoplasm.

Plant vs. Animal Cells

Key differences between plant and animal cells include:

Mitosis and Meiosis

These are the two main types of nuclear division.

Meiosis: Key Concept

Gametes (sex cells) have half the number of chromosomes that body (somatic) cells have.

Cell Types

• Body cells (Somatic cells): Make up the organism's body. In humans, they have $23$ pairs of chromosomes (diploid, $2n$).

  • Autosomes: Chromosome pairs $1-22$.

  • Sex chromosomes: X and Y, determine gender.

• Gametes (Sex cells): Egg and sperm. They are haploid ($n$), containing one copy of every chromosome. In humans, they have $22$ autosomes and $1$ sex chromosome. Gametes are formed from germ cells (located in ovaries/testes) and carry DNA passed to offspring.

Diploid and Haploid Cells

• Diploid ($2n$): Cells with two copies of every chromosome; one from each parent. Body cells are diploid.

• Haploid ($n$): Cells with one copy of every chromosome. Gametes are haploid.

Note: Chromosome number must be maintained in animals. Many plants can have more than two copies (polyploidy).

Mitosis

• Purpose: Makes more diploid cells (somatic cell division).

• Outcome: One parent cell divides into two daughter cells, each genetically identical and having both full sets of chromosomes.

• Phases:

  1. Interphase: Cell prepares to divide; genetic material doubles.

  2. Prophase: Chromosomes thicken, shorten, become visible (two chromatids joined by a centromere); centrioles move to opposite sides; nucleolus disappears; nuclear membrane disintegrates.

  3. Metaphase: Chromosomes arrange at the equator of the cell, attached to spindle fibers by centromeres; homologous chromosomes do not associate.

  4. Anaphase: Spindle fibers contract, pulling chromatids to opposite poles.

  5. Telophase: Chromosomes uncoil; spindle fibers disintegrate; centrioles replicate; nuclear membrane reforms; cell divides (cytokinesis).

• Result: Two daughter cells, identical to the parent cell.

Meiosis

• Purpose: Makes haploid cells from diploid cells (gamete cell division).

• Outcome: One parent cell undergoes two rounds of cell division to produce four daughter cells, each with half the chromosomes of the parent cell, leading to genetic diversity.

• Meiosis I: Divides homologous chromosomes.

  • Occurs after DNA has been replicated.

  • Prophase I: Homologous chromosomes pair up (synapsis) and crossing over occurs (exchange of segments), increasing genetic diversity.

  • Metaphase I: Homologous pairs align at the cell equator.

  • Anaphase I: Homologous chromosomes separate and move to opposite poles.

  • Telophase I: Cell divides, resulting in two haploid cells, but each chromosome still consists of two sister chromatids.

• Meiosis II: Divides sister chromatids.

  • DNA is not replicated between Meiosis I and Meiosis II.

  • Prophase II: Spindle fibers form.

  • Metaphase II: Sister chromatids align at the cell equator.

  • Anaphase II: Sister chromatids separate and move to opposite poles.

  • Telophase II: Cells divide, resulting in four haploid cells.

• Meiosis vs. Mitosis Differences:

  • Meiosis has two cell divisions; mitosis has one.

  • In mitosis, homologous chromosomes never pair up.

  • Meiosis results in haploid cells; mitosis results in diploid cells.

Mendelian Genetics and Inheritance Patterns

Mendel's Groundwork for Genetics

• Traits: Distinguishing characteristics that are inherited.

• Genetics: The study of biological inheritance patterns and variation.

• Gregor Mendel showed that traits are inherited as discrete units, contrasting with the then-prevailing belief that traits were blended.

Mendel's Experiments and Discoveries

Mendel's success with pea plants (Pisum sativum) stemmed from three key decisions:

• Use of purebred plants.

• Strict control over breeding (e.g., removing male parts, stamens, to prevent self-pollination).

• Observation of seven "either-or" traits (e.g., purple vs. white flowers, round vs. wrinkled peas).

Mendel's crosses:

• P generation (parental) crossed to produce F1 generation.

• He allowed F1 plants to self-pollinate to produce the F2 generation.

Key Observations and Conclusions

• In the F1 generation, all plants displayed the dominant phenotype (e.g., all purple flowers from purple x white parents).

• F1 plants are heterozygous.

• In the F2 generation, traits reappeared in specific ratios (e.g., $3/4$ purple, $1/4$ white).

From these patterns, Mendel drew three crucial conclusions:

1. Traits are inherited as discrete units.

2. Organisms inherit two copies of each gene, one from each parent.

3. The two copies segregate during gamete formation (Law of Segregation).

Mendel's Monohybrid Cross Results (Example Traits):

Genes, Alleles, Genotype, and Phenotype

• Gene: A segment of DNA that directs a cell to make a certain protein.

• Locus: A specific position on a pair of homologous chromosomes where a gene is located.

• Allele: Any alternative form of a gene at a specific locus. Each parent donates one allele for every gene.

  • Homozygous: Two identical alleles at a specific locus (e.g., AA or aa).

  • Heterozygous: Two different alleles at a specific locus (e.g., Aa).

• Genome: All of an organism’s genetic material.

• Genotype: The specific genetic makeup of an organism for a trait (e.g., AA, Aa, aa).

• Phenotype: The physical expression of a trait (e.g., red flowers, tall plant).

Dominant and Recessive Alleles

• Dominant allele: Expressed as a phenotype when at least one copy is present. Represented by uppercase letters (e.g., A).

• Recessive allele: Expressed as a phenotype only when two copies are present (homozygous recessive). Represented by lowercase letters (e.g., a).

• Both homozygous dominant (AA) and heterozygous (Aa) genotypes yield a dominant phenotype.

• Many traits show a range of expression and do not follow simple dominant-recessive patterns.

Laws of Probability and Genetic Crosses

Punnett Squares

The Punnett square is a grid system for predicting all possible genotypes resulting from a cross.

• Axes represent possible gametes from each parent.

• Boxes show possible genotypes of offspring.

• Yields ratios of possible genotypes and phenotypes.

Monohybrid Crosses (one trait)

• Homozygous dominant x Homozygous recessive (e.g., AA x aa): All offspring are heterozygous (Aa), all express the dominant phenotype.

• Heterozygous x Heterozygous (e.g., Aa x Aa):

  • Genotypic ratio: $1 \text{ AA} : 2 \text{ Aa} : 1 \text{ aa}$.

  • Phenotypic ratio: $3 \text{ dominant} : 1 \text{ recessive}$.

  • Diagram:

    • Parent Ff x Ff

    • Gametes: F, f

    • Punnett Square:
      F f F FF Ff f Ff ff

• Heterozygous x Homozygous recessive (e.g., Aa x aa):

  • Genotypic ratio: $1 \text{ Aa} : 1 \text{ aa}$.

  • Phenotypic ratio: $1 \text{ dominant} : 1 \text{ recessive}$.

• Testcross: A cross between an organism with an unknown genotype and an organism with the recessive phenotype to determine the unknown genotype.

Dihybrid Crosses (two traits)

• Mendel’s dihybrid crosses with heterozygous plants yielded a $9:3:3:1$ phenotypic ratio.

• Led to Mendel’s Law of Independent Assortment: Allele pairs separate independently of each other during meiosis.

Beyond Simple Dominance and Recessiveness

Incomplete Dominance

• Where both genes contribute to the phenotype, resulting in an intermediate phenotype.

• Example (Flower color): Given AA = Red, aa = White, Aa = Pink.

  • AA x aa: All offspring Aa (Pink).

  • Aa x Aa: Genotypic ratio $1 \text{ AA} : 2 \text{ Aa} : 1 \text{ aa}$; Phenotypic ratio $1 \text{ Red} : 2 \text{ Pink} : 1 \text{ White}$.

  • Aa x aa: Genotypic ratio $1 \text{ Aa} : 1 \text{ aa}$; Phenotypic ratio $1 \text{ Pink} : 1 \text{ White}$.

Codominance and Multiple Alleles

• Codominance: Both alleles are expressed equally and distinctly in the phenotype (e.g., red x yellow flowers = orange, where both red and yellow pigments are visible).

  • If $P^R = \text{red}$ and $P^Y = \text{yellow}$:

    • $P^R P^R$ (red) x $P^Y P^Y$ (yellow) -> F1: $P^R P^Y$ (orange).

    • F1 x F1 ($P^R P^Y \text{ x } P^R P^Y$) -> F2: $1 \text{ red } (P^R P^R) : 2 \text{ orange } (P^R P^Y) : 1 \text{ yellow } (P^Y P^Y)$. This is a $1:2:1$ phenotypic ratio, not $3:1$. This example proves the rule of Mendelian inheritance while illustrating an important exception.

• Multiple Alleles: More than two alleles for a single gene (e.g., human hair color, where alleles like $H^B n$ (brown), $H^B d$ (blonde), $h^R$ (red), $h^{bk}$ (black) combine in various ways).

  • Important: Dominant does not mean frequent; recessive traits can be common.

  • Example: Many common pea traits (green seed, wrinkled seed, dwarf stature, white flower) are recessive ($gg, ww, dd, aa$).

Quantitative Inheritance

• Traits controlled by multiple genes (polygenic) and often influenced by environment.

• Examples: Crop yield, human skin color.

• Crop Yield (multiple genes): Highest yield (AABBCCDDEE), intermediate (AabbCCDdEe), lowest (aabbccddee).

• Human Skin Color: Darkest (AABBCCDDEE), intermediate (AaBbCcDdEe), lightest (aabbccddee).

  • A cross between two intermediate individuals (AaBbCcDdEe x AaBbCcDdEe) can produce a huge range of colors.

Phenotype = Genotype + Environment

Environmental factors significantly influence the expression of genetically determined traits.

• Crop Yield = Genotype + Minerals + Water + Light - Pests, etc. Optimizing these factors is key to agricultural productivity.

• Human Skin Color = Genotype + Sun (UV) Exposure - Aging Factors. The effect of sun exposure is most obvious in people with intermediate base skin color, but everyone can get "tan lines."

Inbreeding Depression vs. Hybrid Vigor

• Inbreeding Depression: Occurs when related parents contribute the same recessive deleterious alleles to offspring, leading to reduced vigor and increased incidence of genetic disorders.

  • Examples: Hemophilia in European royalty (Queen Victoria's mutation), Tay-Sachs in Jewish populations, Bipolar disorder in Irish populations, hip dysplasia in German Shepherds. This is the biological basis for the incest taboo.

• Hybrid Vigor (Heterosis): Occurs when offspring from genetically distinct parents exhibit superior qualities (e.g., growth, yield) compared to either parent. Recessive undesirable alleles from one family are "covered" by dominant desirable alleles from the other.

  • Examples: "Mutt" dogs often have better health, "half-breeds" can be superior, Wild maize A x Wild maize B can produce high-yield hybrid maize. This concept suggests that human "melting pots" cultures might be superior.

Finding Genes in Wild Relatives & Beyond Species Boundaries

• Wild relatives (Lycopersicum peruvianum) can be crossed with cultivated species (Lycopersicum esculentum) to introduce desirable traits, leading to offspring with superior characteristics.

• Introgression (backcrossing) can transfer specific desirable genes from wild relatives into cultivated crops.

Bread Wheat (Polyploidy Example)

• Bread wheat (Triticum aestivum) is a fertile hexaploid (meaning it has six sets of chromosomes), resulting from a complex evolutionary history involving multiple hybridization events and chromosome doubling.

• Ancestral Process (estimated ~7500 BC, spontaneous DNA doubling):

  1. AA (Diploid wheat, e.g., Triticum urartu) x DD (Diploid grass, e.g., Aegilops tauschii) -> AD (sterile diploid, similar to a mule).

  2. Spontaneous or colchicine-induced doubling -> AADD (fertile tetraploid).

  3. BB (Diploid grass, e.g., Aegilops speltoides) x AADD -> BADD (sterile triploid).

  4. Spontaneous or colchicine-induced doubling -> AABBDD (Fertile hexaploid, Bread Wheat Triticum aestivum).

Plant Breeding Methods and Challenges

Plant Breeding Definitions

Reproduction

• Definition: Sequence of events involved in the multiplication and perpetuation of cells and organisms.

• Types:

  • Sexual: Increase of plants through seeds formed from the union of male and female gametes.

  • Asexual (Vegetative): Increase of plants through ordinary cell division and differentiation (e.g., cuttings, stolons).

Genetics Terminology

• Chromosome: Structural units in the nucleus that carry genes in linear order. Made of DNA (deoxyribonucleic acid).

• Gene: Unit of inheritance determining the sequence of a given protein. Proteins are made by reading RNA (ribonucleic acid): messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA).

• Allele: Alternate form of a gene.

• Homozygous: Alleles for a trait are identical (e.g., TT, tt).

• Heterozygous: Alleles for a trait are not identical (e.g., Tt).

• Genotype: Organism’s genetic constitution.

• Phenotype: Outward expression of the genotype.

• Dominant gene: When genes are heterozygous, one is expressed.

• Recessive gene: When genes are heterozygous, one is not expressed.

• Complete dominance: The dominant gene is the only one expressed.

• Incomplete dominance: Both genes influence the phenotype, leading to an intermediate phenotype.

Other Key Definitions

• Gamete: Sex cell or nucleus (egg cell or sperm nuclei) produced during meiosis.

• Self-pollination: Transfer of pollen from the male structure to the female structure of a flower on the same plant.

• Cross-pollination: Transfer of pollen between flowers on different plants that differ genetically.

• Fertilization: The actual union of male and female gametes.

• Hybrid: The first generation offspring (F1) of a cross between two individuals differing in one or more genes.

Challenges in Breeding Grasses

Breeding grasses is often more difficult than breeding cash crops due to several factors:

1. Cross-pollination: Many grasses are obligate cross-pollinators, making controlled crosses complex.

2. Self-sterile: Inability to self-pollinate or self-fertilize.

3. Apomictic: Formation of seeds without the combination of male and female gametes (asexual seed production), which can complicate genetic improvement but also fix desirable genotypes.

4. Small flower organs: Manual manipulation for pollination is difficult.

5. Low seed production or poor germination: Can limit breeding efforts.

6. Weak seedlings: Requires careful management.

7. Row planting: Does not resemble natural veld conditions, which can skew evaluations.

8. Clean available soil: Specific soil conditions needed for trials.

9. Mixtures difficult: Evaluating mixed grass stands is challenging.

10. Long lifespan of most grasses: Requires long evaluation periods.

11. Lack of basic information: Fundamental genetic and biological knowledge is often missing for many grass species.

12. Breeders work with many species: Requires broad expertise.

Kinds of Reproduction in Grasses

1. Sexual

   • Cross-pollinators (most South African grasses).

   • Self-pollinators.

2. Apomictic: Formation of seed without the combination of male and female gametes. Many important available grasses are apomictic.

3. Vegetative reproduction.

Grass Breeding Methods

Methods depend on the species and breeding goals:

A) Self-Pollinating Grasses

• Most plants are homozygous.

• Relatively pure breeding lines can be derived from outstanding parents.

• Usually annual plants.

• Of less economic importance than cross-pollinators.

B) Cross-Pollinating Grasses

• General procedure:

  • Start with a large variety of material.

  • Mass selection.

  • Hybridization.

  • Develop synthetic varieties.

  • Multiply seed.

C) Apomictic Grasses

• Mother plant and progeny are genotypically and phenotypically identical.

• General procedure:

  • Introduce a large number of accessions for desirable characteristics.

  • Select the most desirable characteristics.

  • Multiply seed.

Interspecific Breeding

• Crossing of closely related species to transfer desired characteristics from one to another (e.g., Ryegrass and Sorghums).

Tetraploidisation (Example: Ryegrass)

• Chromosome numbers:

  • Haploid: $n=7$ chromosomes.

  • Diploid: $2n=14$ chromosomes.

  • Tetraploid: $4n=28$ chromosomes.

  • Common annual (diploid) ryegrass has seven pairs of chromosomes.

• Process: Treatment of chromosomes with Colchicine leads to the doubling of the chromosome number.

• Effects of Tetraploidisation:

  • Broader leaves.

  • Longer leaves.

  • Higher water content.

  • Thicker but less stolons.

  • Larger seed.

• Caveat: Doubling of chromosomes leads to the doubling of both good and bad characteristics.

Crop Improvement Objective

The ultimate goal of crop improvement is to enhance a specific trait within a species, or to introduce/remove a particular trait.

• Increased productivity.

• Increased quality.

This ability relies on two assumptions:

1. Genetic variation exists: Available from adapted cultivars, breeder stock, wild plants, mutations, previously crossed plants, and genetic introductions from other parts of the world.

2. Techniques exist: For achieving a favorable combination of genes.

Breeding Methods:

Methods for Self-Pollinating Crops

(e.g., Cotton, Barley, Sorghum, Soybean, Peanut)

• Crossing followed by selection:

  • Mass Selection:

    • Year 1: Cross plants with desired traits.

    • Year 2 (F1): Grow heterozygous seed from cross in bulk.

    • Years 3-7 (F2-F6): Grow several plants from previous generation's seed. Harvest and bulk seed from desirable plants.

    • Year 8 (F7): Grow seeds from individual plants in separate rows. Harvest and check for desired trait.

    • Years 9-13 (F8-F12): Continue yield testing. Retain seed from best rows.

    • Years 14-15 (F13-F14): Increase seed from best rows for commercial release.

  • Pedigree Selection:

    • Year 1: Cross plants with desired traits.

    • Year 2 (F1): Grow heterozygous seed from cross in bulk.

    • Year 3 (F2): Grow several plants from F1 seed. Harvest seed from desirable individual plants.

    • Year 4 (F3): Grow rows of plants from selected F2 seed. Harvest seed from desirable rows.

    • Years 5-7 (F4-F6): Grow rows of plants from selected F3 rows. Harvest seed from desirable rows.

    • Year 8 (F7): Grow seed from selected rows and measure yield on each row.

    • Years 9-13 (F8-F12): Continue testing and increase seed from the best one for commercial release.

  • Backcross Method (Introgression):

    • Goal: To transfer a specific desirable gene from a donor parent (wild relative) to a recurrent parent (elite cultivar) while maintaining most of the recurrent parent's genome.

    • Process: Repeatedly cross the F1 generation and subsequent backcross generations with the recurrent parent (A), coupled with selection for the desired trait from the donor parent (B).

    • Year 1: Cross desirable plants (e.g., A x B).

    • Year 2 (F1): Plant F1 seed and backcross with A (approx. $50\%: 50\%$ A:B genes).

    • Year 3 (BC1): Plant BC1 seed and backcross with A (approx. $75\%: 25\%$ A:B genes).

    • Year 4 (BC2): Plant BC2 seed and backcross with A (approx. $87.5\%: 12.5\%$ A:B genes).

    • Year 5 (BC3): Plant BC3 seed and backcross with A (approx. $93.75\%: 6.25\%$ A:B genes).

    • Year 6 (BC4): Plant BC4 seed and backcross with A (approx. $96.88\%: 3.12\%$ A:B genes).

    • Year 7 (BC5): Plant BC5 seed and backcross with A (approx. $98.44\%: 1.56\%$ A:B genes).

    • Year 8 (BC6): Plant BC6 seed and backcross with A (approx. $99.22\%: 0.78\%$ A:B genes).

    • Year 9 (BC7): Plant seed from BC7 plants in rows. Select plants with desired characteristics, harvest and bulk seed.

    • Year 10: Increase seed for commercial release.

    • Introgression details: With each backcross, the percentage of genes from the donor parent decreases. Field selection is crucial at each step. While percentages theoretically halve, crossing over means the actual percentage of donor genome can vary considerably among individuals. Molecular mapping and marker-assisted selection are crucial to track desired loci and determine gene percentages accurately.

Methods for Cross-Pollinating Crops

(e.g., Lucerne, Maize, Fescue)

• Pedigree selection (as in self-pollinated crops).

• Mass selection (as in self-pollinated crops).

• Backcross method (as in self-pollinated crops).

• Hybrid breeding.

Hybrid Production

• Definition: Production of heterozygous populations from crossing homozygous (inbred) lines.

• Mostly used for cross-pollinated crops.

• Hybrid seed must be reproduced each year as the favorable heterozygosity breaks down in subsequent generations.

• Steps:

  1. Produce inbred lines.

  2. Cross the inbred lines:

     • Single cross hybrid: Cross two inbred lines (e.g., A x B). Harvest seed from the female rows.

     • Double cross hybrid: Cross two single cross hybrids (e.g., (A x B) x (C x D)).

     • Three-way cross hybrid: Cross an inbred line with a single cross hybrid (e.g., (A x B) x C).

Weaknesses of Traditional Plant Breeding

• Time-consuming: Many years required to produce a commercially marketable seed.

• Limited crosses: Crossing generally occurs only within the same species.

• Inefficient selection: Often depends primarily on phenotypes, which can be influenced by environment.

• Space limitations: Finding desirable traits requires large numbers of crosses, demanding significant field space.

Single-Gene vs. Quantitative Traits

• Single-gene traits (Monogenic):

  • Often controlled by a single gene.

  • Example: Disease resistance. Transferring the resistance locus from a wild relative or landrace to a cultivar often makes the cultivar resistant. Selection can be based on a clear phenotype (presence/absence of disease) in the field.

• Quantitative traits (Polygenic):

  • Controlled by multiple genes located at different positions on chromosomes, often influenced by environment.

  • Examples: Yield, flowering time, drought resistance.

  • Transferring these Quantitative Trait Loci (QTLs) by introgression (backcrossing) is more difficult because differences are often smaller and not simply "present/absent." Molecular markers and chromosome maps facilitate the transfer of QTLs.

Screening Existing Genotypes (Landraces)

• Screening diverse existing genotypes (landraces) and wild relatives is essential for identifying useful traits for breeding programs.

• Some traits can be directly identified, and seeds distributed to farmers.

• For other traits, introgression into elite cultivars may be necessary.

• Examples: Screening wheat genotypes on low P calcareous soils (high pH, iron unavailable) or soybean genotypes on low P acidic soils (low pH, aluminum toxicity) to find adapted lines.

Biotechnological Modified Crops (GM Crops)

Context for GM Crops

• Food Security Challenge: More crops are needed to feed a rapidly growing global population (projected 9 billion by 2050).

• Environmental Change: Agricultural production faces increasing challenges from climate change.

• Fuel Security: Demand for biofuels puts pressure on food crops.

• FAO (2009) prediction: Need $70\%$ more food by 2050, with limited opportunities to increase cultivated land or theoretical yield potential.

• Need for Innovation: Plant science, modern plant breeding (quantitative genetics), and genetic modification are crucial to increase "yield stability" (e.g., drought, salinity tolerance) and annual food production (from **$32$ to *$44$ MT*, a *$38\%$* increase sustained for $40$ years).

Currently Commercialized GM Traits

• Primary GM traits in cotton, corn, and soybean are herbicide resistance and pest resistance.

• Industry Soybean Portfolio (Example): Includes traits like Roundup Ready (RR2Y), Liberty Link, Bt, omega-3, low saturated fat, high oleic, dicamba tolerant, herbicide tolerance to 2,4-D, rust resistance, high beta-carotene, low phytate, and disease/nematode resistance.

Biotech Crop Countries (2008 Data Examples)

• Mega-countries (>$50,000$ hectares):

  • USA: $62.5$ M ha (soybean, maize, cotton, canola, squash, papaya, alfalfa, sugarbeet)

  • Argentina: $21.0$ M ha (soybean, maize, cotton)

  • Brazil: $15.8$ M ha (soybean, maize, cotton)

  • India: $7.6$ M ha (cotton)

  • Canada: $7.6$ M ha (canola, maize, soybean, sugarbeet)

  • China: $3.8$ M ha (cotton, tomato, poplar, petunia, papaya, sweet pepper)

  • South Africa: $1.8$ M ha (maize, soybean, cotton)

• Many other countries growing smaller areas of specific GM crops (e.g., Spain, Germany, Portugal, Czech Republic, Poland, Slovakia, Romania, Mexico, Honduras, Colombia, Bolivia, Chile, Uruguay, Paraguay, Philippines, Egypt, Australia, Burkina Faso).

Why GM Matters

• World food production must increase.

• Conventional approaches are making decreasing relative impacts.

• GM is one additional tool to contribute to food security and is not "the answer" alone.

• GM has the potential to increase food quality and environmental sustainability.

Marker Assisted Selection (MAS)

Definition and Assumption

• Definition: Marker-assisted selection (MAS) refers to the use of DNA markers that are tightly linked to target loci as a substitute for or to assist phenotypic screening.

• Assumption: DNA markers can reliably predict phenotype.

MAS vs. Conventional Plant Breeding

• Conventional Plant Breeding (Phenotypic Selection):

  • Involves large populations (thousands of plants).

  • Requires extensive field trials and glasshouse trials.

  • Selection is based on observable traits (phenotype) directly (e.g., salinity screening in phytotron, bacterial blight screening, phosphorus deficiency plot).

• Marker-Assisted Selection (MAS):

  • Phenotypic selection is based on DNA markers.

  • Can quickly identify resistant/susceptible individuals even in large populations.

Advantages of MAS

• Simpler method compared to laborious phenotypic screening.

• Can save time and resources.

• Selection at the seedling stage: Important for traits like grain quality, or for selection before transplanting in rice.

• Increased reliability: Eliminates environmental effects on trait expression.

• Can discriminate between homozygous and heterozygous individuals and select single plants accurately.

Potential Benefits from MAS

• More accurate and efficient selection of specific genotypes.

• May lead to accelerated variety development.

• More efficient use of resources, especially field trials (e.g., reducing the need for extensive backcross nurseries).

Overview of Marker Genotyping Process

1. Leaf tissue sampling.

2. DNA extraction.

3. PCR (Polymerase Chain Reaction) to amplify marker DNA.

4. Gel electrophoresis to separate DNA fragments by size.

5. Marker analysis.

Marker-Assisted Selection in Pedigree Breeding (SLS-MAS)

• Traditional Pedigree Method (Example):

  • F1 -> F2 -> F3 (individual plant selection) -> F4-F6 (progeny row selection, preliminary yield trials) -> F7 (further yield trials) -> F8-F12 (multi-location testing, licensing, seed increase, cultivar release).

  • Phenotypic screening is constant throughout this process.

• Single-Large Scale Marker-Assisted Selection (SLS-MAS):

  • F1 -> F2 -> MAS at F3.

  • Only desirable F3 lines (identified by markers) are planted in the field.

  • F4 (progeny row selection) -> F5-F7 (pedigree selection based on needs) -> F8-F12 (multi-location testing, licensing, seed increase, cultivar release).

  • Benefit: The breeding program can be efficiently scaled down to focus on fewer promising lines early on.

Requirements for Sustainable Use of Biotechnological Modified Crops

Refuge Area Requirements for Bt Maize

Refuge areas are cultivated with non-modified maize to manage pest resistance, particularly against the stork-borer, to preserve the effectiveness of Bt technology.

1. Two Options for Refuge Size:

   • Option A: $80\%$ modified maize with a $20\%$ refuge area, where chemical control (using non-Bt related products) may occur in the refuge.

   • Option B: $95\%$ modified maize with a $5\%$ refuge area, where no chemical stork-borer control may occur in the refuge.

2. Distance: No modified maize crop may be further than $400$m from the refuge area.

3. Planting Time: Refuge area and modified maize crop may not be planted more than seven days apart.

4. Growth Season: Must be derived from hybrids with the same growth season duration.

5. No Seed Mixing: Seed of modified maize and refuge maize cannot be mixed in seeding trays, as this would allow stork-borers to mature on non-modified plants and then cross to modified plants, circumventing control.

6. Agronomic Conditions: Both areas must be cultivated under the same agronomic conditions (e.g., irrigation, pest control practices).

7. Refuge Width: Refuge area must be at least six rows wide (planted at the edge or as strips).

8. Farm Responsibility: Every farm must have a refuge area meeting these requirements. A neighbor’s maize field does not serve as a refuge.

   • Note: "Yield Guard" or "Genuity Yield Guard II" refers to stack-gene hybrids; "refuge area" includes Roundup Ready hybrids.

Inspection of Refuge Area After Cultivation

Inspections span multiple seasons:

• Selection: A random number of clients (e.g., $200$) are selected; an independent inspector obtains purchasing records and schedules appointments.

• Visit: Inspectors visit selected clients from December to March. Clients found non-compliant in previous seasons are automatically included.

• Evaluation: The inspector randomly selects fields to evaluate adherence to regulations.

• Follow-up: Seed suppliers contact clients if regulations are not adhered to.

• Repetition: Steps 1-4 are repeated annually. Non-compliant clients are automatically included in subsequent evaluations.

• Planning Document: Non-compliant clients must submit a comprehensive planning document before the next planting season to ensure adherence.

Seed Legislation and Certification

Plant Improvement Act 53 of 1976

• Governs seed quality and certification.

• SANSOR: South African National Seed Organisation.

• Basic seed: Seed certified in terms of the scheme as basic seed.

• Certified seed: Seed certified in terms of the scheme as certified seed.

Conditions for Certification (Highlights)

1. Land of production must be registered under the scheme.

2. Produced by the seed grower to whom the certificate of registration for the unit was issued.

3. Parent seed used must have been planted according to established requirements.

4. Production unit must be isolated according to isolation requirements.

5. Plants must comply with required inspections.

6. Seed must comply with physical requirements (Section 14).

7. Containers must comply with requirements and be labeled as required.

8. Presented for certification according to the provisions of the scheme.

9. All other provisions of the scheme regarding the seed and its production processes must be complied with.