Principles of Cultivar Development

🧭 Overview

🧠 One-sentence thesis

Effective cultivar development requires understanding and manipulating the components of genetic gain per year—especially heritability—to design breeding strategies that maximize selection reliability for quantitatively inherited traits.

📌 Key points (3–5)

• Core tool: A formula for genetic gain per year (from Chapter 17 of Principles of Cultivar Development) helps compare different breeding strategies by examining variance components and heritability.
• What heritability measures: the numerical reliability of selection for a quantitative trait; higher heritability means selection is more dependable.
• Realized heritability in practice: comparing selection among F₂ seeds vs. F₂ plants shows which stage gives more reliable prediction of progeny performance (F₂:₃ lines).
• Common confusion: heritability is not an absolute measure—it is used to compare strategies; breeders may still select even when heritability is low if it helps discard unpromising individuals efficiently.
• Practical threshold setting: instead of relying solely on heritability calculations, breeders often set minimum trait values (e.g., oleic acid %) to discard seeds or plants unlikely to meet breeding goals.

🧬 What is heritability and why it matters

🧬 Definition and purpose

Heritability: a numerical measure of the reliability of selection for a quantitative trait.

• It is not a measure of how much a trait is "genetic" in absolute terms; it quantifies how reliably selection at one stage predicts performance in the next generation.
• The excerpt emphasizes that heritability's primary value is to compare expected gain from different selection strategies.
• Example: Should a breeder select among individual seeds or among individual plants? Heritability calculations help answer this.

🔍 Heritability as a comparison tool

• The excerpt states that "no two breeding programs are designed the same" because resources (facilities, time, money) and traits of importance differ.
• Heritability helps breeders decide which method (e.g., seed vs. plant selection) will give more genetic improvement per year.
• Don't confuse: heritability is strategy-specific and context-dependent, not a fixed property of a trait.

🧪 Realized heritability: seed vs. plant selection

🌱 The experimental setup (Applied Learning Activity 1)

The excerpt describes an experiment to compare selection at two stages for oleic acid content (a quantitative trait):

1. F₂ seeds were analyzed non-destructively for oleic acid.
2. Each analyzed seed was planted; the resulting F₂ plants were harvested individually and analyzed (using a bulk sample of five F₃ seeds).
3. Progeny of each F₂ plant were grown as F₂:₃ lines in multiple locations, and oleic acid content was measured for each line.

The goal: determine whether selecting among F₂ seeds or F₂ plants is more reliable (i.e., which has higher realized heritability).

📐 How realized heritability is calculated

The excerpt provides a step-by-step method:

For F₂ seeds:

• Denominator = (mean of top 5 selected F₂ seeds) − (mean of all F₂ seeds)
  This is the selection differential at the seed stage.
• Numerator = (mean of F₂:₃ lines tracing to those 5 seeds) − (mean of all F₂:₃ lines)
  This is the response to selection in the progeny.
• Realized heritability for seeds = numerator ÷ denominator

For F₂ plants:

• Denominator = (mean of top 5 selected F₂ plants) − (mean of all F₂ plants)
• Numerator = (mean of F₂:₃ lines tracing to those 5 plants) − (mean of all F₂:₃ lines)
• Realized heritability for plants = numerator ÷ denominator

🔬 Why plant selection is expected to be more reliable

The excerpt asks: "Why would you expect the heritability to be greater for selection among individual plants than individual seeds?"

• Plants represent a later generation and have been grown in an environment; their phenotype reflects both genetic and environmental effects averaged over development.
• Seeds are measured before planting; their phenotype may be less representative of the genetic value that will be expressed in the field.
• The excerpt does not give the explicit answer, but the implication is that plant measurements integrate more information and reduce measurement error relative to seed measurements.

❓ Why heritabilities are less than 100%

The excerpt asks: "What are reasons to explain why the heritabilities were less than 100%?"

Possible reasons (implied by the context of quantitative genetics):

• Environmental variation: F₂:₃ lines were tested in multiple locations; environment affects trait expression.
• Sampling error: only five F₃ seeds were bulked to measure each F₂ plant; this introduces sampling variation.
• Genotype × environment interaction: the relative performance of lines may change across environments (this is a major theme in the next section, "Maximizing genetic gain II").

🎯 Practical selection without calculating heritability

🎯 Setting minimum thresholds

The excerpt emphasizes that breeders often decide on selection reliability without calculating a heritability value.

• Instead, they set a minimum acceptable trait value (e.g., 50% oleic acid).
• They then look at the seed and plant values for lines that met the threshold in the F₂:₃ generation.
• Example from the excerpt:
  • Lines with >50% oleic acid in F₂:₃ had a minimum seed value of 40.86% and a minimum plant value of 42.42%.
  • The breeder may decide to discard all future seeds below ~41% and plants below ~42%, reasoning that individuals below these thresholds have "little promise of being useful."

💡 Why select even when heritability is low

• The excerpt notes that a breeder may choose to practice selection "even if the heritability is low" in order to discard seeds or plants that have very little chance of being effective.
• This is a practical efficiency measure: it saves time and resources by eliminating the least promising individuals early, even if the selection is not highly reliable.

Don't confuse:

• High heritability = selection is reliable; chosen individuals are likely to produce superior progeny.
• Low heritability = selection is less reliable, but may still be worth doing to eliminate obvious poor performers.

📊 Variance components and the genetic gain formula

📊 The formula and its components

The excerpt refers to a formula for genetic gain per year in Chapter 17 of Principles of Cultivar Development, based on the variance component method of calculating heritability.

• The formula's components are derived from analyses of variance (ANOVA).
• The excerpt reproduces two ANOVA tables (Tables 17-6 and 17-7) for seed weight in soybeans, showing sources of variation:
  • Environments (E)
  • Replications within environments (R/E)
  • Lines (L) — this is the genetic variance among lines
  • Environment × Line interaction (E × L) — how line performance changes across environments
  • Replication × Line interaction within environments
  • Plants within plots — within-plot sampling error

🔢 What the tables show

Source	What it represents
Lines (L)	Genetic differences among lines; the target of selection
E × L	Genotype × environment interaction; lines perform differently in different environments
Plants/plots	Sampling variation within plots; measurement error

• Expected mean squares columns show how each variance component contributes to the observed mean square.
• The excerpt notes that the number of plants per plot (3), replications (2), and environments (2) are used in the formulas.

🧮 How breeders manipulate components

The excerpt states: "Each of the components in the formula can be influenced by the choices breeders make in carrying out their cultivar development programs."

Examples of breeder choices (implied by the ANOVA structure):

• Number of environments: testing in more environments reduces the relative size of E × L variance.
• Number of replications: more reps reduce error variance.
• Number of plants per plot: more plants per plot improve the reliability of line means.

Chapters 6, 7, 18, and 19 of Principles of Cultivar Development (mentioned in the excerpt) explain "how each of the components can be manipulated to maximize genetic improvement."

📋 Applied Learning Activity 1: step-by-step

📋 The task

The excerpt provides a 10-step assignment to calculate and compare realized heritabilities for seed vs. plant selection, using oleic acid data from 50 F₂ individuals.

Steps 1–3: Realized heritability for F₂ seeds

1. Highlight the 5 F₂ seeds with highest oleic acid; calculate their mean and the mean of all 50 seeds; subtract to get the selection differential (denominator).
2. Highlight the F₂:₃ lines tracing to those 5 seeds; calculate their mean and the mean of all 50 F₂:₃ lines; subtract to get the response (numerator).
3. Divide numerator by denominator to get realized heritability for seeds.

Steps 4–6: Realized heritability for F₂ plants 4. Highlight the 5 F₂ plants with highest oleic acid; calculate selection differential (denominator). 5. Highlight the F₂:₃ lines tracing to those 5 plants; calculate response (numerator). 6. Divide to get realized heritability for plants.

Steps 7–10: Interpretation 7. Explain why plant heritability is expected to be greater. 8. Explain why heritabilities are less than 100%. 9. Choose a minimum oleic acid % for discarding seeds and justify. 10. Choose a minimum oleic acid % for discarding plants and justify.

📊 The data

The excerpt includes a table (Table 1) with 50 entries, each showing:

• F₂ seed oleic acid %
• F₂ plant oleic acid %
• F₂:₃ line oleic acid %

Example rows:

• Entry 1: seed 43.43%, plant 37.51%, line 46.21%
• Entry 16: seed 65.28%, plant 62.70%, line 49.72%

The data show that seed and plant values do not always predict line values perfectly, illustrating the concept of heritability < 100%.

🔗 Context and next steps

🔗 Broader breeding program design

The excerpt opens by noting that "no two breeding programs are designed the same" because:

• Available resources (facilities, time, money) differ.
• Traits of importance differ.
• Breeding strategies differ (as seen in publications like Journal of Plant Registrations and Horticultural Science).

The goal of this section is to help breeders understand variables that need to be considered in designing an effective breeding program for selection of traits that are quantitatively inherited.

🔗 Preview of "Maximizing genetic gain II"

The excerpt ends with a brief introduction to the next section, which emphasizes genotype × environment interaction:

• The importance of this interaction is "highly dependent on the trait under selection."
• Example: days to maturity show much more consistent relative differences among genotypes across environments than yield does.
• As a result, the genotype × environment component in the genetic gain equation is smaller for maturity than for yield.
• Therefore, fewer environments are needed to obtain reliable values for maturity.

Don't confuse:

• Low G×E traits (e.g., maturity): genotype rankings are stable across environments; fewer test locations needed.
• High G×E traits (e.g., yield): genotype rankings change across environments; more test locations needed for reliable selection.

🧭 Overview

🧠 One-sentence thesis

Genotype × environment interaction determines how reliably a breeder can predict a genotype's performance across different conditions, with traits like yield showing much stronger interaction (and thus requiring more testing environments) than traits like maturity.

📌 Key points (3–5)

• What genotype × environment interaction measures: how consistently genotypes rank across different environments (locations, years).
• Trait-dependent importance: maturity shows low interaction (consistent ranking), while yield shows high interaction (rankings change across environments).
• Two types of interaction exist: one type is manageable, but the most difficult type occurs when the best genotypes in one environment perform poorly in another.
• Common confusion: a statistically significant interaction in analysis of variance does not automatically mean it matters to the breeder—the type of interaction determines practical importance.
• Testing strategy trade-off: breeders must balance number of environments vs. number of replications per environment to minimize interaction effects on genetic gain.

🌍 What genotype × environment interaction means

🌍 Core definition and importance

Genotype × environment interaction: the phenomenon where the relative performance (ranking) of genotypes changes across different environments.

• "Environment" includes different locations, years, and seasons.
• Low interaction = genotypes maintain similar relative differences across environments.
• High interaction = genotypes change their relative ranking across environments.
• The excerpt emphasizes this interaction is "highly dependent on the trait under selection."

🔍 How it affects prediction reliability

• From the farmer's perspective: performance data from one season is used to select cultivars for the next season.
• When interaction is low, relative differences in one season predict the next season well.
• When interaction is high, predictions become unreliable.

Example: A cultivar higher-yielding than another in one season may be lower-yielding in the next season (high interaction for yield).

📊 Trait-specific differences in interaction

📊 Maturity vs. yield comparison

Trait	Interaction level	Consistency across environments	Testing requirements
Days to maturity	Low (small component in genetic gain equation)	Relative differences "much more consistent"	Fewer environments needed
Seed or forage yield	High (large component)	Rankings change across environments	More environments needed

🌾 Maturity example

• A cultivar maturing 10 days earlier than another in one season "would be expected to mature earlier in the coming season."
• The exact difference may not be 10 days, but the direction (earlier vs. later) remains consistent.
• Don't confuse: the absolute number of days may vary, but the relative ranking stays similar.

🌾 Yield example

• One cultivar higher-yielding than a second in one season may be lower-yielding in the next season.
• This illustrates why yield requires more extensive testing across environments.

🔀 Types of genotype × environment interaction

🔀 Why statistical significance isn't enough

• A significant interaction may appear in analysis of variance.
• The excerpt states: "Whether or not the interaction is of importance to the breeder depends on which of the types...are involved."
• The breeder must identify the type of interaction, not just its statistical significance.

⚠️ The most difficult type

"The most difficult interaction to deal with is one that results when the best genotypes in one environment perform less well than others in another environment."

• This means genotypes that rank high in Environment A rank low in Environment B.
• This type creates the biggest challenge for selection decisions.
• When this occurs, "the breeder generally uses less stringent selection in one environment when deciding which genotypes to advance."

🛡️ Breeder's response strategy

• Less stringent selection in one environment = advancing more genotypes to the next testing season.
• This hedges against choosing genotypes that perform well in only one environment.
• The ultimate decision relies on "test results from multiple locations and years."

🧪 Applied Learning Activity 2 context

🧪 The research scenario

• Data from M.S. thesis evaluating total tocopherol (Vitamin E) content in soybean oil.
• 20 lines with mid-oleate content (~50%) tested at three Iowa locations.
• Randomized complete-block design with two replications per location.
• Breeding goal: develop cultivars with high Vitamin E content.

📈 Key finding

• Genotype × environment interaction was significant for population 2.
• Genotype × environment interaction was not significant for population 1.
• This sets up the learning activity questions about how to interpret and respond to these different patterns.

🔗 Phenotypic correlations (Table 1)

Population	Location pair	Correlation	Significance
1	Ames–Carlisle	0.20	Not significant
1	Ames–Rippey	0.41	Not significant
1	Carlisle–Rippey	0.25	Not significant
2	Ames–Carlisle	0.86	Significant (0.01 level)
2	Ames–Rippey	0.85	Significant (0.01 level)
2	Carlisle–Rippey	0.94	Significant (0.01 level)

• Low correlations in Population 1 suggest rankings change substantially across locations.
• High correlations in Population 2 suggest rankings remain more consistent.
• Don't confuse: Population 2 had significant interaction in analysis of variance but higher correlations—this illustrates why the type of interaction matters more than statistical significance alone.

🎯 Learning activity questions

🎯 Question themes

The activity asks students to:

1. Identify and explain the two primary types of genotype × environment interaction and their effects on selection.
2. Determine which type(s) caused the significant interaction in Population 2.
3. Compare selection difficulty between populations using phenotypic correlations, even when interaction is not statistically significant.
4. Make practical selection decisions: which lines to advance based on one environment's data, considering limited financial resources.
5. Use the genetic gain equation to decide whether to prioritize number of environments or number of replications per environment.
6. Relate trait heritability to testing requirements, using real examples of low- vs. high-heritability traits.

💰 Resource constraint emphasis

• "Any genotype you advance for additional testing will utilize your financial resources that always are limiting in a breeding program."
• This highlights the practical trade-off: advancing too many genotypes wastes resources; advancing too few risks missing the best genotypes.

📐 Genetic gain equation connection

📐 Role in decision-making

• The excerpt mentions the "genotype × environment component in the genetic gain equation discussed in the previous chapter."
• Question 5 explicitly asks students to "use the genetic gain equation to defend your answer" about environments vs. replications.
• The equation provides a quantitative framework for comparing breeding strategies.

🔗 Link to heritability

• Question 6 connects heritability to testing requirements.
• Lower heritability traits require more testing to determine genetic potential.
• Higher heritability traits require less testing because genetic differences are more easily distinguished from environmental noise.

🧭 Overview

🧠 One-sentence thesis

Maximizing genetic gain per year—not just per cycle—is the critical principle in plant breeding, and breeders can achieve this by choosing appropriate recurrent selection methods and minimizing the time required to complete each cycle.

📌 Key points (3–5)

• Core principle: genetic gain per year matters more than genetic gain per cycle; faster cycles yield more cumulative improvement over time.
• What the genetic gain equation does: it compares different methods of recurrent selection by quantifying how much improvement each method delivers annually.
• Classical vs. traditional breeding: classical recurrent selection uses only progeny from one closed population, but the genetic gain principles apply equally to traditional programs that cross superior progeny from multiple sources.
• Common confusion: don't assume that slow, careful cycles are acceptable as long as releases are consistent—a breeder completing cycles in 4 years will achieve substantially more gain over 24 years than one taking 8 years per cycle.
• Practical decisions: the best recurrent selection method depends on the crop species, the type of cultivar (e.g., hybrids vs. inbreds), and available resources (seasons, testing capacity, cost).

🔄 Recurrent selection and the genetic gain equation

🔄 What classical recurrent selection means

Classical recurrent selection: once a segregating population is formed, only superior progeny from that population are crossed together to begin the next cycle of selection.

• This is a closed-population approach: each cycle draws only from the previous cycle's best individuals within the same population.
• Many breeders do not follow strict classical recurrent selection; instead, they form new segregating populations each year by crossing superior progeny from different populations and sources.
• Key insight: the genetic gain equation's principles are valuable regardless of whether the breeder uses closed populations or mixes sources.

📐 Components of the genetic gain equation

• The equation includes multiple components (the excerpt does not list the full formula but emphasizes that breeders should be able to write it out and explain each part).
• Each component's value varies with:
  • The breeding population
  • The quantitative trait being improved
  • The plant species
• Breeders learn to derive these components in statistics and quantitative genetics courses.
• What matters for this lesson: understanding how the breeder can influence each component's magnitude to maximize gain per year.

🧪 Methods vary by cultivar type

• The practical usefulness of different recurrent selection methods depends on the type of cultivar being developed.
• Example: in maize (a crop where hybrids are used commercially), selfed families can be used for population improvement, but research shows that evaluating individuals as half-sib families for combining ability yields more genetic gain per year for hybrid yield.
• This aligns with how breeders develop improved inbreds: they rely on combining ability tests for yield, not the yield of an inbred line by itself.

⏱️ Time per cycle: the most important principle

⏱️ Why genetic gain per year trumps gain per cycle

• The excerpt emphasizes: "It is the genetic gain per year and not the genetic gain per cycle that is critical."
• Over a 24-year period:
  • A breeder completing a cycle in 4 years will make substantially more genetic gain than one taking 8 years per cycle.
  • The faster breeder completes 6 cycles; the slower breeder completes only 3 cycles.
• Common myth debunked: "It does not matter how long it takes to develop a cultivar, as long as you have new ones to release on a consistent basis."
  • This myth ignores the cumulative advantage of faster cycles.

🌍 Off-season nurseries reduce cycle time

• Modern plant breeding programs extensively use off-season nurseries.
• Purpose: reduce the number of years from making a cross (forming a segregating population) to selecting superior individuals for use as parents in new populations.
• Example scenario (from the excerpt's Applied Learning Activity):
  • One season per year: test half-sib families in Ames in 2012, cross selected individuals in 2013, test new families in 2014 → 2 years per cycle.
  • Two similar seasons per year: test in Ames in 2012, cross in a winter season in Hawaii, test new families in 2013 → 1 year per cycle.
• Don't confuse: the number of years per cycle can be fractional (e.g., 1.5 years) depending on the number and type of seasons available.

🧬 Applied Learning Activity: comparing selection methods

🧬 Six methods evaluated

The Applied Learning Activity asks students to compute genetic gain per cycle and per year for six recurrent selection methods:

Method	Description
a. Recurrent phenotypic selection (before flowering, with gridding)	Select individuals based on phenotype before flowering; gridding organizes the field
b. Recurrent phenotypic selection (after flowering, no gridding)	Select individuals based on phenotype after flowering; no spatial organization
c. Half-sib selection (population as tester, recombine with half-sib seed)	Evaluate half-sib families; use the population itself as the tester; recombine using half-sib seed
d. Half-sib selection (inbred tester, recombine with selfed seed)	Evaluate half-sib families; use an inbred line as the tester; recombine using selfed seed
e. S₀:₁ line evaluation (1 intercrossing generation)	Evaluate lines derived from selfing once; one intercrossing generation between cycles
f. S₂:₄ line evaluation (2 intercrossing generations)	Evaluate lines derived from selfing to the S₂ generation, then bulked to S₄; two intercrossing generations between cycles

📊 Assumptions for the calculations

• Genetic variance: divided equally between additive (82) and dominance (82) variance.
• Genotype × environment interaction: additive × environment = 48; dominance × environment = 48.
• Evaluation setup: lines evaluated in 2 replications, 30 plants per plot, at 5 locations.
• Selection intensity: 10% for all methods.

🌦️ Four seasonal scenarios

Students must compute gain per year under four circumstances:

Scenario	Description	Impact on cycle time
a. One season per year	Testing, crossing, or selfing can only happen once per year	Longest cycle time
b. Two similar seasons per year	Testing, crossing, or selfing can be done in any season	Shorter cycle time; more flexibility
c. Two non-similar seasons per year	Testing possible in only one season; crossing and selfing in both	Intermediate cycle time; testing is the bottleneck
d. Three seasons per year	Testing, crossing, or selfing in one season; two consecutive seasons for crossing and selfing only	Shortest cycle time; rapid advancement

🎯 Decision criteria

• After computing gain per year for each method under each scenario, students must choose the best method considering:
  • Amount of gain per year
  • Cost of the gain
• Example: a method with high gain per cycle but long cycle time may be less effective than a method with moderate gain per cycle but short cycle time.

🔑 Key takeaways for breeders

🔑 Mastering the genetic gain formula

• For exams, students must be able to:
  • Write out the entire formula for genetic gain per year.
  • Explain how a breeder can influence the magnitude of each component.
• The formula is a tool for comparing methods and making strategic decisions, not just a theoretical exercise.

🔑 Practical experience matters

• The breeder's practical experience plays a major role in deciding which recurrent selection methods are more effective.
• Research and experience (e.g., the maize example with half-sib families vs. selfed families) guide these decisions.
• The genetic gain equation provides a framework, but real-world constraints (cost, labor, available seasons) shape the final choice.

🧭 Overview

🧠 One-sentence thesis

Clonal cultivar development relies on crossing heterozygous parents to create genetically unique hybrid plants, selecting superior individuals, and managing disease-free propagation through methods like virus-indexing to ensure commercial seedstock quality.

📌 Key points (3–5)

• Three basic steps: develop a segregating population by crossing heterozygous parents, select superior individual plants (clones), and prepare disease-free seedstock for commercial distribution.
• Every hybrid seed is unique: because parents are highly heterozygous, every gamete is genetically different, so every hybrid seed from a cross represents a potential new cultivar.
• Disease transmission challenge: vegetative propagules (tubers, rhizomes) and apomictic seed can carry viruses, requiring stringent virus-indexing and meristem culture methods.
• Common confusion—apomixis types: facultative apomicts produce both asexual and sexual seed; obligate apomicts rarely produce sexual seed, making crossing difficult.
• Why it matters: clonal cultivars allow propagation of genetically identical superior individuals, but require careful disease management to maintain seedstock quality.

🌱 Creating the segregating population

🌱 Parent characteristics and crossing

Heterozygous: parents have two different alleles at many loci.
Homogeneous: all plants of a parent cultivar are genetically identical.

• When two clonal parents are crossed, it does not matter how many plants of each parent are used because all plants of a parent are genetically identical.
• Every locus that is heterozygous will segregate, meaning every gamete produced is genetically different.
• When gametes from two parents unite, every hybrid seed is genetically different.
• The breeder attempts to obtain as many hybrid seeds as desired for sampling the genetic variation of the segregating population.
• Accidental self-pollination is generally not a concern because resulting plants will have inbreeding depression and inferior performance, so they will be discarded.

🌾 Apomictic species as a special case

Apomixis: reproduction where seed is produced asexually, genetically identical to the mother plant.

Two types of apomixis:

Type	Description	Example
Facultative	Seed produced both asexually and sexually; asexual seed is genetically identical to mother; sexual seed is heterogeneous	Kentucky bluegrass
Obligate	Rarely produces any seed sexually; almost all seed is asexual	Buffelgrass

• Sexual reproduction is necessary to form a segregating population in apomicts.
• Seed harvested from the female parent will include both asexual and hybrid seed.
• When planted, individuals phenotypically identical to the female parent are considered to have arisen through apomixis and are discarded.
• Those different from the female parent are assumed to have arisen through sexual reproduction and are used for selection.
• Challenge with obligate apomicts: limited sexual reproduction makes crossing difficult; special methods are needed to overcome this problem.

🔍 Selection of superior clones

🔍 Two-phase selection process

• Every hybrid plant obtained from the cross of two clonal cultivars is a potential new cultivar.
• Phase 1: Identify individual hybrid plants that have the characteristics desired in a new cultivar.
• Phase 2: Clonally propagate the selected individuals for more extensive evaluations over multiple locations and years.
• Example: potato evaluation process involves season-by-season testing with increasing numbers of locations and replications.

🦠 Disease management in seedstock production

🦠 The disease transmission challenge

• One of the main challenges in multiplication of clonally propagated cultivars is the potential for seedstock to carry diseases, particularly viruses.
• Diseases can be transmitted by:
  • Contact with an infected clone
  • Tools used for propagation
• Various methods of managing seedstock have been adopted to prevent disease transmission.

🧪 Virus-indexing and meristem culture

Virus-indexing: testing of seedstock to detect the presence or absence of transmissible viruses.

General process:

1. Collect small sections of tuber sprouts or shoot apical meristem
2. Regenerate plants in sterile medium supplemented with necessary nutrients
3. Take stringent measures to ensure meristem-cultured plants are disease-free
4. Transfer to greenhouses under controlled conditions

Why meristem tissue is virus-free:

• Meristem tissue lacks vascular bundle cells, eliminating the mode of virus transport and restricting spread
• Cells in meristem tissues divide more rapidly than the rate of virus replication, so new meristem tissue escapes virus infection

Additional quality control:

• Rare mutations can occur during tissue culture
• Molecular markers (such as SSR) can be used for cultivar identification to ensure plants are of high quality and uniformity as their parents

🥔 Example: Potato virus-indexing

• Virus-indexing is a requirement in North America for potato seedstock (tubers)
• Process involves meristem culture to produce disease-free plantlets
• Plantlets are transferred to greenhouse and monitored for disease symptoms
• Tubers produced in greenhouse are grown in field for tuber increase
• Other methods include heat treatment or combination of meristem culture and heat treatment

🍠 Example: Sweet potato

• Uses meristem culture to produce plants
• Plants are tested by grafting onto a virus-sensitive indicator relative (Ipomoea setosa)
• After grafting, the indicator plant (rootstock) is monitored for disease symptoms
• If no symptoms are observed on indicator plant, the meristem-cultured plants (scions) are considered virus-free

🍌 Example: Banana

• All banana diseases are thought to spread through exchange of vegetative stock (suckers) among farmers
• Process:
  1. Collect suckers from field
  2. Grow them in disease monitoring facilities
  3. Screen plants for diseases
  4. If negative, use for tissue culture production of disease-free stocks
• Genetic tests using DNA markers may be applied to establish that tissue culture plants are genetically the same as their parent
• Virus- and disease-free stocks are distributed to farmers

🌿 Example: Kentucky bluegrass (Awesome cultivar)

• Derived from cross between 'Limousine' (female) and 'Midnight' (male)
• Limousine seed was sown in flats and germinated in greenhouse
• Seedlings transferred to field nursery
• Plants that looked different from Limousine were flagged and their seed harvested individually
• Awesome is different from Limousine in size, shape, and color of seedheads
• Average level of apomixis in Awesome is about 95% (meaning 95% of seed is produced asexually)
• Protected under United States Plant Variety Protection (PVP) Act

📦 Commercial seedstock preparation

📦 What "seed" means for clonal cultivars

• The term "seed" refers to:
  • Vegetative propagules (tubers, rhizomes)
  • True seed of species that reproduce by apomixis (Kentucky bluegrass, buffelgrass)

📦 Key information for commercial distribution

For each clonal cultivar, breeders must document:

1. What part of the plant is used for commercial propagation
2. Method used to multiply and prepare seedstock for distribution
3. Who distributed the seedstock commercially
4. What legal protection was sought (e.g., Plant Variety Protection)

🧭 Overview

🧠 One-sentence thesis

Pure-line cultivar development follows a four-step process—creating a segregating population, deriving pure lines, testing them as potential cultivars, and purifying/multiplying seed—with key decisions at each stage affecting the speed, cost, and genetic uniformity of the final cultivar.

📌 Key points (3–5)

• Four-step process: develop a segregating population → develop pure lines → test lines as potential cultivars → purify and multiply seed for the new cultivar.
• Population types: two-parent crosses are most common; three-parent or backcross populations are used when a two-parent cross is unlikely to produce enough desirable segregates.
• Timing trade-offs: deriving lines in early generations (F₂ or F₃) is faster but yields less homogeneity; later generations take more time but produce more uniform lines.
• Common confusion—seed production methods: mass selection is faster and cheaper but yields less uniformity; progeny testing takes longer and costs more but produces greater genetic and phenotypic uniformity.
• Concurrent vs sequential seed increase: starting seed multiplication during testing speeds release but wastes resources on lines that may not be released; waiting until release is cheaper but delays availability to producers.

🌱 Creating the segregating population

🌱 Two-parent crosses

Segregating population: the initial population obtained by crossing parents, from which pure lines will be derived.

• Most common approach: cross two elite parents.
• Key assumption: parents are homozygous and homogeneous for quantitative traits, so only enough plants are used to obtain the desired number of hybrid seed.
• All F₁ seeds are considered genetically identical in a two-parent cross; the breeder obtains enough to generate the desired F₂ population size.

🔀 Three-parent and backcross populations

• When to use: when a two-parent cross is unlikely to have adequate frequency of segregates with one or more desired traits.
• How it works: after the initial two-parent cross, the F₁ hybrid plants are crossed to a third parent or to the recurrent parent (in backcrossing).
• Sampling strategy: the breeder tries to obtain as many hybrid seeds as possible to sample the segregating gametes from the two-parent F₁ individuals.
• Example: In soybean, a cycle 0 population for iron chlorosis resistance was obtained by intermating 10 elite lines and 10 plant introductions with the highest resistance; recurrent selection using S₀:₁ lines developed genotypes with exceptionally high resistance.

🔄 Complex populations

• Generally limited to situations where the breeder chooses recurrent selection for multiple cycles to improve a particular trait.
• Not the standard approach for routine cultivar development.

🧬 Deriving pure lines

🧬 Methods and resources

• The excerpt directs readers to review alternative methods discussed in the "Inbreeding chapter" in Plant Breeding Methods.
• Method choice depends on: resources available and the breeder's personal experience.

⏱️ Generation of line derivation

Aspect	Early generation (F₂ or F₃)	Advanced generation
Advantage	Fewer years to develop a cultivar	Higher frequency of lines with desired homogeneity
Disadvantage	Lower frequency of homogeneous lines	Takes more years

• Don't confuse: speed vs uniformity—early derivation is faster but less uniform; later derivation is slower but more uniform.
• Breeding schedule influence: often influenced by the use of local environment and off-season nurseries.

🌾 Soybean example

• Crosses made at Ames during summer.
• F₁ seeds planted near Santa Isabel, Puerto Rico, in mid-October.
• F₁ plants harvested in January.
• F₂ seeds planted in early February.
• F₃ seeds harvested in May by the multiple-seed procedure of single-seed descent.
• F₃ seeds planted at Ames in May.
• Individual F₃ plants harvested individually in fall to form F₃-derived lines for subsequent evaluation.
• This schedule uses off-season nurseries to accelerate the breeding cycle.

🧪 Testing pure lines

🧪 Initial evaluation strategy

• Objective: discard lines that are too inferior to warrant further evaluation.
• Testing conditions: limited number of replications and environments, relatively small plot size.
• Selection factors:
  • Quality of parents used to form the population
  • Number of traits a line must have to be selected
  • Frequency of lines with adequate homogeneity
  • Stringency of the breeder in making selections
  • Number of lines that can be tested the next season
• Practical experience with a crop is needed to manage these variables effectively.

🌾 Seed handling for testing

• For most self-pollinated species, the seed used for planting a field test is the self-pollinated seed harvested from the previous season of testing.
• Assumption: harvest equipment is adequately self-cleaning to minimize seed mixtures from one plot to the next.
• Specialized harvest equipment is available for this purpose.

🌾 Seed purification and multiplication

🌾 Timing decision

Strategy	Advantage	Disadvantage
Delay until release	Least expensive; no wasted effort on non-released lines	Delays seed availability to producers
Concurrent with testing	Seed available to producers as soon as possible	Funds spent on lines that ultimately are not released

• Most breeders prefer to have seed available to producers as soon as possible, so they carry out seed purification and multiplication concurrently with field testing.

🔍 Mass selection method

Mass selection: seed of a line inspected for uniformity is planted in an increase; plants are inspected, off-types removed, and remaining plants harvested in bulk to obtain breeder seed.

• Seed source: commonly obtained from the previous year of yield testing; may or may not be inspected for uniformity before planting.
• Process: plants in the increase are inspected for uniformity → off-types removed → remaining plants harvested in bulk.
• Advantages: requires only one season; minimum labor and expense.
• Disadvantage: genetic and phenotypic uniformity of breeder seed generally less than with progeny testing.
• Example: mass selection was used for breeder seed production of the spring barley cultivar 'Transit' (JPR 5:270-272).

🧬 Progeny testing method

Progeny testing: individual plants are selected from a line, a progeny row is grown of each, and rows are evaluated for uniformity and similarity to each other for phenotypic traits.

• Process:
  • Individual plants selected from a line
  • Progeny row grown of each
  • Rows evaluated for uniformity and similarity
  • Selected progeny rows may be harvested in bulk or separately
  • If harvested separately, each may be inspected for seed traits before bulking
• Optional second season: seed from progeny rows may be grown separately a second season to further examine uniformity before bulking.
• Advantage: greater genetic and phenotypic uniformity of breeder seed than mass selection alone.
• When important: when a cultivar must meet standards for genetic purity for seed certification.
• Disadvantages: takes at least two seasons; requires more labor and expense than mass selection.
• Example: progeny testing was used for breeder seed production of the lentil cultivar 'Essex' (JPR 5:19-21).

⚖️ Choosing between methods

• Don't confuse: the choice is not about which method is "better" in absolute terms—it depends on the balance between speed/cost (mass selection) and uniformity/certification requirements (progeny testing).
• Breeders may choose based on the crop's certification standards and the resources available.

🧭 Overview

🧠 One-sentence thesis

Hybrid cultivars are commercially viable when a species exhibits sufficient high-parent heterosis and when breeders can reliably eliminate pollen from the female parent, transfer pollen from the male parent, and produce hybrid seed economically.

📌 Key points (3–5)

• High-parent heterosis is the commercial standard: the hybrid must outperform both parents, not just their average, to justify the cost of hybrid seed production.
• Four requirements for commercial hybrids: sufficient heterosis, elimination of fertile pollen from the female parent, effective pollen transfer from the male parent, and economical seed production.
• Multiple methods to eliminate female pollen: manual emasculation, genetic male sterility, gynoecious inbreds, self-incompatibility, cytoplasmic-genetic male sterility (CMS), and transgenic systems.
• Heterotic groups maximize performance: crossing inbreds from different heterotic groups (e.g., Stiff Stalk Synthetic vs. non-Stiff Stalk in maize) generally produces superior hybrids compared to crosses within the same group.
• Common confusion—mid-parent vs. high-parent heterosis: mid-parent heterosis (hybrid exceeds the parental average) is scientifically interesting but not commercially useful, because one parent may still outperform the hybrid; only high-parent heterosis (hybrid exceeds both parents) matters for commercial viability.

🌱 What is heterosis and why it matters

🌱 Definition and types of heterosis

Heterosis (hybrid vigor): the phenomenon where a hybrid's performance exceeds that of its parents.

• Mid-parent heterosis: hybrid performance exceeds the mean of the two parents.
  • Commonly measured in research but not commercially relevant.
  • Problem: one parent may still perform better than the hybrid.
• High-parent heterosis: hybrid performance exceeds both parents.
  • This is the commercially important type.
  • A species must exhibit sufficient high-parent heterosis to justify the cost of hybrid seed production.

Don't confuse: Mid-parent heterosis with commercial viability—only high-parent heterosis ensures the hybrid is better than either parent alone.

🧬 Genetic basis remains unresolved

• Scientists have not yet determined the exact genetic and molecular mechanisms underlying heterosis.
• Current hypothesis: heterosis may result from a combination of dominance, overdominance, and epistatic interactions at different loci.
• Some loci may contribute through dominance effects, others through overdominance, and still others through interactions between genes.

🔗 Combining ability

• Combining ability: the ability of a parent to produce superior hybrids when mated with other parents.
• Specific combining ability: how well one parent combines with a specific other parent.
• General combining ability: the average ability of one parent to combine with a group of other parents.
• These concepts are central to breeding hybrid and synthetic cultivars.

🧩 Heterotic groups and their role

🧩 What are heterotic groups?

Heterotic groups: distinct germplasm pools that, when crossed to each other, produce hybrids with greater performance than crosses within the same pool.

• Breeders divide parent germplasm into heterotic groups for some species.
• Crossing inbreds from different heterotic groups generally yields better hybrids than crossing within the same group.

🌽 Example: Maize heterotic groups

• Stiff Stalk Synthetic: developed by USDA-ARS and Iowa State University breeders.
• Non-Stiff Stalk groups: include Lancaster and Reid Yellow Dent populations.
• Best hybrid performance is typically obtained by crossing Stiff Stalk inbreds with non-Stiff Stalk inbreds.

🔄 How heterotic groups arise

• From breeding systems: Use of cytoplasmic-genetic male sterility (CMS) leads to separate female (A-line) and male (R-line) germplasm pools, which evolve into distinct heterotic groups.
• From self-incompatibility: Separate germplasm pools with different self-incompatibility genes are maintained, leading to unique heterotic groups.

🚫 Requirement 2: Eliminating pollen from the female parent

🚫 Why pollen elimination is necessary

• To produce hybrid seed, the female parent must not self-pollinate.
• Fertile pollen must be eliminated from the female parent so that only the male parent's pollen fertilizes it.

✋ Manual emasculation

For species with perfect flowers (e.g., tomato):

• Hand removal of anthers before pollen is released.
• Requirements for practicality:
  1. Low labor costs (often done in countries with cheaper labor).
  2. High seed yield per pollination (tomato produces ~200 seeds per fruit).
  3. Hybrid value must justify the cost.

For monoecious species (e.g., maize):

• Manual removal of staminate flowers (detasseling).
• Commonly practiced for commercial maize hybrid seed production.

Don't confuse: Manual emasculation in perfect flowers (removing anthers from the same flower) with detasseling in monoecious plants (removing entire male flower structures).

🧬 Genetic (nuclear) male sterility

Genetic male sterility: major nuclear genes (often recessive) that render pollen inviable.

• Used in vegetable crops (tomato, pepper, okra) as an alternative to hand emasculation.
• Problem: the female parent segregates for male-fertile and male-sterile plants.
  • Male-fertile plants must be individually identified and removed before pollination—labor-intensive and expensive.
  • Only economical for high-value crops.

Example: Pepper:

• Controlled by recessive allele ms.
• Female parent genotype is Msms (segregates 50% male-sterile msms and 50% male-fertile Msms).
• Male-fertile plants are rogued by hand before pollination.
• Male parent is MsMs (fully fertile).
• Hybrid seed from male-sterile plants pollinated by male parent is male-fertile (Msms).

Ideal but unrealized: A tightly linked gene producing a visible phenotype before flowering would allow easy identification and removal of male-fertile plants, but no such linkage has been used commercially.

🌸 Gynoecious inbreds

Asparagus (dioecious species):

• Sex determination: XX = female, XY = male, YY = supermale.
• Commercial hybrids: gynoecious female (XX) × supermale (YY) → androecious male hybrid (XY).
• Male hybrids yield more and live longer than females.
• Inbred development methods:
  1. Self-pollination of andromonoecious (XY) plants that produce some perfect flowers.
  2. Anther culture to generate homozygous XX females and YY supermales faster.

Cucumber (monoecious species):

• Three major genes control flower type: M, F, and A.
• Key flowering types:
  • Gynoecious (MMFF): only pistillate flowers.
  • Monoecious (MMff): pistillate and staminate flowers on the same plant.
  • Hermaphroditic (mmFF): perfect flowers.
  • Andromonoecious (mmff): mostly staminate flowers with some perfect flowers.
  • Androecious (ffaa): only staminate flowers.

Common hybrid types in cucumber:

Hybrid cross	Female parent	Male parent	Hybrid genotype	Use
Gynoecious × Gynoecious	MMFF	MMFF (treated to produce staminate flowers)	MMFF	Commercial field production; ~15% monoecious pollinators blended in
Gynoecious × Monoecious	MMFF	MMff	MMFf	Commercial field production; ~15% monoecious pollinators blended in
Gynoecious × Hermaphroditic	MMFF	mmFF	MmFF	Not used in USA (unacceptable fruit shape)
Monoecious × Monoecious	MMff	MMff (female treated with ethephon to suppress staminate flowers)	MMff	Home gardens (extended harvest period)

Selfing gynoecious plants: Treat with silver nitrate or silver thiosulfate to stimulate staminate flower production for inbreeding.

🔒 Self-incompatibility (SI)

Self-incompatibility: a genetic system that prevents self-pollination; each parent has a different set of SI genes, allowing cross-pollination but not self-pollination.

• Used in cabbage, broccoli, kohlrabi, cauliflower.
• One parent has one set of SI genes, the other parent has a different set.
• Inbreeding method: bud pollination—apply pollen to the stigma before the SI mechanism activates.
• Limitations:
  1. Producing enough self-pollinated seed of each parent for hybrid seed production fields.
  2. Maintaining stable SI in inbred lines to avoid self-pollination during hybrid seed production.
• Restricted to vegetable species where hybrid seed value justifies the cost.

🧪 Cytoplasmic-genetic male sterility (CMS)

CMS system: uses cytoplasm that renders pollen male-sterile unless the nucleus contains restorer genes (Rf) that overcome the cytoplasmic sterility.

Three line types:

• A-line: male-sterile female parent (sterile cytoplasm + non-restorer rf genes).
• B-line: maintainer line (normal cytoplasm + non-restorer rf genes); used to produce seed of the A-line.
• R-line: restorer line (normal or sterile cytoplasm + restorer Rf genes); male parent in hybrid seed production.

Key principle: Cytoplasm is transmitted only through the egg cell, not through pollen.

Developing an elite A-line:

1. Develop a superior B-line by crossing B-line parents and selecting from the segregating population.
2. Backcross the B-line (recurrent parent) to an existing A-line to transfer the sterile cytoplasm while maintaining the B-line's nuclear genes.
3. The resulting A-line is genetically identical to the B-line except for the cytoplasm.

Seed production:

• A-line seed: A-line × B-line in strips; destroy B-line rows after pollination; harvest A-line seed.
• B-line and R-line seed: plant in isolation and allow natural pollination.
• Hybrid seed: A-line × R-line in strips; harvest A-line seed, which is the hybrid seed sold to farmers.

Hybrid plants: Male-fertile because the R-line's Rf genes restore fertility in the presence of sterile cytoplasm.

🧬 Transgenic methods for male sterility

🌾 SeedLink Invigor system (canola)

Genes used:

• barnase (BS): from Bacillus amyloliquefaciens; produces a protein that destroys RNA, causing male sterility.
• barstar (BR): produces a protein that neutralizes barnase, restoring male fertility.
• bar (L): confers glufosinate (Liberty) herbicide tolerance.

Line development:

• A-line: heterozygous for BS-LL transgene; male-sterile; sprayed with Liberty to kill 50% of plants lacking the transgene.
• B-line: lacks transgene; used to produce A-line seed by crossing in strips.
• R-line: homozygous for BR-LL transgene; male-fertile; restores fertility in the hybrid.

Hybrid seed production:

• A-line × R-line in strips.
• A-line sprayed with Liberty at least three times to kill male-fertile plants.
• Hybrid seed harvested from A-line is male-fertile (BR dominant to BS) and glufosinate-tolerant.

🌽 Seed Production Technology (SPT) system (maize)

Genes used:

• DsRed2 (Alt1, "F"): produces fluorescent pinkish-red color in seed.
• Ms45 ("Ms"): wild-type nuclear male-fertility gene.
• zm-aa1 ("A"): produces α-amylase enzyme that destroys pollen by breaking down starch; does not affect egg viability.

Line development:

• B-line maintainer: genotype F-Ms-A/f-ms-a ms/ms (hemizygous for transgene, homozygous for recessive male-sterility ms gene).
• A-line: genotype f-ms-a/f-ms-a ms/ms (lacks transgene, homozygous for ms); male-sterile.
• R-line: wild-type Ms/Ms (lacks transgene); male-fertile.

Seed production:

• A-line seed: A-line × B-line in strips; B-line pollen segregates 50% viable (f-ms-a ms) and 50% non-viable (F-Ms-A ms); all A-line seed lacks transgene and is male-sterile.
• B-line seed: plant in isolation; harvest fluorescent red seeds (hemizygous for transgene) for future B-line planting; discard yellow seeds.
• R-line seed: plant in isolation; all seed lacks transgene and is male-fertile.
• Hybrid seed: A-line × R-line in strips; hybrid seed lacks transgene and is male-fertile.

Advantage over CMS: Avoids cytoplasmic factors that could make hybrids susceptible to disease (e.g., Southern corn leaf blight in the 1970s).

🧪 Chemical hybridizing agents (CHA)

CHA: chemicals that induce male sterility without reducing female fertility.

• Potential advantage: no need to incorporate specific genetic factors for male sterility into inbred lines.
• Extensively studied in wheat as an alternative to CMS.
• Key attributes:
  1. Cause male sterility without reducing female fertility.
  2. Complete male sterility.
  3. Effective across a range of genotypes.
• Used commercially in crops with unreliable CMS systems (e.g., wheat).

🌬️ Requirement 3: Transferring pollen from male to female parent

🌬️ Natural pollination methods

• Wind pollination: most cost-effective for some species (e.g., maize).
  • In wheat, seed set ranges from 10–70%, limiting hybrid cultivar use.
• Insect pollination: bees or other insects brought to fields to ensure adequate pollination (e.g., alfalfa, sunflower, canola).
  • Example: Leafcutter bees used in alfalfa hybrid seed production.

✋ Manual pollination

• Used for some species with perfect flowers (e.g., tomato).
• Labor-intensive; only economical for high-value crops.

🚧 Barriers to hybrid cultivar use

• Soybean: insufficient and unreliable insect pollination prevents large-scale hybrid seed production.
• Wheat: variable wind pollination (10–70% seed set) limits commercial hybrid use.

💰 Requirement 4: Reliable and economical hybrid seed production

💰 Profitability for all stakeholders

• Hybrid cultivars must be profitable for:
  1. The breeder.
  2. The seed production company.
  3. The end user (farmer).

💰 Cost considerations

• Greater seed cost due to:
  1. More expensive breeding of hybrid parents.
  2. Higher cost of producing hybrid seed.
• Hybrid must outperform other cultivar types to justify the higher seed cost.

💰 Why farmers buy hybrid seed every season

• Harvested seed from a hybrid segregates like an F₂ population (loses uniformity and performance).
• Farmers must purchase new hybrid seed each planting season.
• This ensures seed companies have a reliable market, unlike pure-line or synthetic cultivars, where farmers can save and replant seed.

💰 Example: Wheat hybrids

• Breeding research on hybrid wheat has continued for over 50 years.
• Area planted to hybrid wheat is far less than pure-line cultivars.
• Factors limiting widespread use:
  1. Insufficient high-parent heterosis in some environments.
  2. Challenges with pollen elimination methods (CMS, CHA).
  3. Unreliable pollination (wind pollination variability).
  4. Higher seed production costs not offset by hybrid performance gains.

🧭 Overview

🧠 One-sentence thesis

Hybrid cultivar development requires four distinct steps—population development, inbred line creation, combining ability testing, and commercial seed production—with careful management of heterotic groups to maximize hybrid performance.

📌 Key points (3–5)

• Four-step process: population development → inbred line development → combining ability testing → commercial seed production.
• Heterotic groups are critical: breeders maintain independent programs for each heterotic group (e.g., Stiff Stalk vs. non-Stiff Stalk in maize) and avoid crossing between groups during population development.
• Two testing philosophies: early-generation testing (test combining ability during inbreeding) vs. finishing lines first (test only completed inbreds); each has trade-offs in resource use and success rate.
• Common confusion: inbred line performance per se (the line grown alone) vs. combining ability (the line's performance when crossed to another line)—yield per se is generally not a good predictor of combining ability.
• CMS systems require parallel programs: one breeding program develops A-lines and B-lines (one heterotic group), another develops R-lines (the other heterotic group), and crosses between B-lines and R-lines are avoided to preserve heterotic patterns.

🌱 Population development for hybrid breeding

🌱 Cross types and heterotic group management

• Crosses for hybrid cultivar populations are similar to those for pure-line cultivars: single crosses between elite inbreds are most common, though three-way or backcrosses are used when appropriate.
• Major difference from pure-line breeding: heterotic groups are central to hybrid cultivar development.
• The breeder runs independent programs for each heterotic group.
  • Example: in maize, one program develops inbreds related to Stiff Stalk Synthetic; another develops inbreds related to non-Stiff Stalk germplasm.
• Crosses between heterotic groups are avoided during population development; the goal is elite inbreds from each group that produce superior hybrids when crossed together.

🧬 CMS-specific population strategies

Cytoplasmic-genetic male sterility (CMS): a system requiring one breeding program for A-lines and B-lines (male-sterile and maintainer lines) and another for R-lines (restorer lines).

• A-lines and B-lines constitute one heterotic group; R-lines constitute another.
• A-lines cannot be crossed to each other (they are male sterile), so B-lines are crossed to form breeding populations for improved A-lines.
• R-lines are crossed independently to develop populations for improved R-lines.
• B-lines and R-lines are not crossed because it disrupts heterotic groups and causes undesirable segregation of restorer and non-restorer genes.

🔬 Inbred line development

🔬 Methods of inbreeding

• Methods for developing inbred lines for hybrids are the same as for pure-line cultivars (pedigree, single-seed descent, doubled haploids, etc.).
• The only difference may be in early-generation testing.

🧪 Early-generation testing: two types

Type 1: Testing inbred lines per se

• Evaluate the performance of lines grown alone during the inbreeding process.
• Used for traits the inbred must have to be a useful parent (e.g., disease resistance).
• Example: select for resistance among F₂:₃ or F₃:₄ lines, then continue inbreeding the desirable ones.

Type 2: Testing for combining ability (yield)

• Inbred line yield per se is generally not a good indicator of combining ability for yield.
• Instead, cross individuals (plants or lines) to a tester from the other heterotic group and evaluate the testcross seed.
• Individuals with superior testcross performance are saved; inferior ones are discarded.

⏱️ Simultaneous inbreeding and testing

• Breeders often conduct inbreeding and testing in parallel to save time.
• Example workflow:
  • Summer in Iowa: grow F₂:₃ lines, select for desirable traits, cross lines to a tester from another heterotic group.
  • Winter in Hawaii: grow F₃:₄ lines (no selection possible), harvest random F₄ plants.
  • Next summer in Iowa: evaluate testcross seed at multiple locations for yield; grow F₄:₅ lines and evaluate them per se; harvest F₅ plants from selected lines.
  • After yield data arrive: advance F₅ plants tracing to superior F₂:₃ lines to Hawaii; discard those from inferior F₂:₃ lines.

⚖️ Balancing inbred traits per se vs. combining ability

• Breeders must balance expected traits of the inbred per se with its combining ability.
• Don't confuse: a line with poor traits per se may still have outstanding combining ability.
• Example: Dr. Wilbert Russell discarded the line that became B73 (a maize inbred related to Stiff Stalk Synthetic) because its traits per se seemed inadequate. When yield test data showed outstanding combining ability in testcrosses with non-Stiff Stalk testers, he retrieved seed from storage and continued developing the line. B73 became extensively used in commercial hybrids and as a parent for subsequent populations.

🌾 Obtaining testcross seed for B-lines and R-lines

For R-line development:

• Cross experimental R-lines to an elite male-sterile A-line tester.
• The A-line's lack of fertile pollen facilitates crossing, even by hand.

For B-line development (more difficult):

• Both B-line and R-line tester are male fertile, making hand emasculation impractical for obtaining enough testcross seed.
• Options:
  • Treat B-lines with a chemical hybridizing agent (e.g., as in wheat).
  • Convert B-lines to their A-line counterparts during inbreeding (described for sorghum on pages 436–437 of the text).

🧪 Testing inbred lines for combining ability

🧪 Two breeding philosophies

Strategy	Description	Advantage	Disadvantage
Early-generation testing	Test unfinished lines during inbreeding for combining ability	Greater percentage of finished lines have good combining ability	Resources spent on testing unfinished lines instead of finished lines
Finish lines first	Develop finished lines (via single-seed descent, pedigree, doubled haploids, etc.) without early testing	Resources focused on testing finished lines	Lower percentage of finished lines may have good combining ability

🔍 Multi-stage testing of finished inbreds

Regardless of the strategy used, finished inbred lines undergo extensive testing across multiple locations and years.

Stage 1: Initial screening

• Mate each line to one or a few inbred testers from another heterotic group.
• Testers are generally elite inbreds currently used in commercial hybrids.
• This test evaluates both general combining ability (GCA) and specific combining ability (SCA):
  • GCA: lines that perform well with one tester are likely to perform well with other testers.
  • SCA: if the experimental line is ultimately used with the same tester to produce a commercial hybrid.
• Example: maize inbred B73 was commonly used as a tester for evaluating combining ability of lines from the non-Stiff Stalk group.
• Testing is done at a limited number of locations in one year.
• One replication per location is common because it provides more information about performance across environments than replicated plots in fewer environments.

Stage 2: Expanded testing

• Reduce the number of lines.
• Increase the number of testers and locations.
• Emphasis remains on as many locations as possible rather than replication.

Subsequent stages:

• Continue reducing the number of lines.
• Continue increasing the number of testers and locations.
• At final stages, use strip tests planted and harvested by farmers to obtain data from more environments than the breeder could conduct alone.

🎯 Final hybrid selection

• After initial testing with one or a few testers, selected lines are crossed to other elite inbreds from the opposite heterotic group to find the best inbred combinations for a new commercial hybrid.
• In some cases, selected lines are crossed to the original tester itself (e.g., to B73) to produce a commercial hybrid.

🌾 Commercial hybrid seed production

🌾 Seed purification and increase

• Seed of an inbred line selected for use in a hybrid must be purified and increased.
• Timing considerations are the same as for other cultivar types:
  • Delaying seed increase until the final decision is made is less expensive but delays availability of hybrid seed for end users.
• Options for purifying and increasing seed are the same as for pure-line cultivars.
• High uniformity is required for inbred lines so off-type plants can be readily identified and removed in hybrid seed production fields.
• Progeny testing of individual plants is commonly done to obtain pure seed.

🔄 Converting B-lines to A-lines (for CMS systems)

• B-lines must be converted to their A-line counterpart, generally by backcrossing.
• The A-line is the female donor parent; the B-line is the recurrent parent.
• All offspring in each backcross should be male sterile.
• Key evaluation: whether backcross progeny are completely male sterile.
  • If not completely sterile, the B-line and backcross progeny must be discarded (incomplete sterility in a seed production field is unacceptable).

🌽 Example: Maize hybrid seed production

Parent selection:

• Female inbreds: selected for yield of high-quality seed, good silk production, good plant health.
• Male inbreds: selected for good pollen production, good plant health.

Eliminating female self-pollination: Three methods to eliminate fertile pollen from the female parent:

1. Detasseling: physical removal of the tassel (male flowers) manually, mechanically, or both.
2. CMS system: female parent is the male-sterile A-line; male parent is the male-fertile R-line.
3. SPT system (Pioneer Hi-Bred): female parent is homozygous for a nuclear ms gene (male sterile); male parent is homozygous for the wild-type MS allele.

Field design:

• Most common planting pattern: four rows of female parent, one row of male parent.
• After pollination is complete, the male parent row is cut down to prevent accidental mixing of its seed with the female parent's seed at harvest.

Isolation:

• Effective isolation requires sufficient distance between the seed production field and other maize fields.
• Example: in Washington state, the isolation distance for hybrid maize is 415 feet.

Harvest and processing:

• The entire ear (including husks) is picked and elevated into a wagon; husks protect the seed during harvest.
• Loaded wagons are transported to a processing facility.
• At the facility: ears are conveyed to a building where husks are removed and ears are inspected for mold or disease.
• Seed is dried while still on the cob (ears are harvested at relatively high moisture to minimize damage).
• After drying, seed is removed from the cob, conditioned, sized, treated, bagged, and stored until delivery to farmers.

🌼 Example: Canola hybrid seed production

Male sterility systems:

• Production relies on male sterility systems.
• Field design: alternating strips of male and female rows (about 8 male : 24 female).
• Each acre has about two beehives housing alfalfa leaf-cutter bees for pollination of the female parent.
• Isolation distance: 800 m.

Field management:

• After pollination, rows of the male parent are removed to prevent contamination of hybrid seed (produced in the female parent) with seed from self-pollination (produced in the male parent).

A-line seed production:

• Same field arrangement is used.
• The female line (A-line) is pollinated by the male line (R-line).

🧭 Overview

🧠 One-sentence thesis

Synthetic cultivars are developed by selecting and intermating superior clones from heterogeneous populations of cross-pollinating perennial species, primarily used for forage and turfgrass in the United States.

📌 Key points (3–5)

• Primary use: Synthetic cultivars are mainly used for forage and turfgrass species in the U.S., though other regions use them for crops like maize.
• Key biological traits: These species have perfect flowers but minimize self-pollination through self-incompatibility; they are highly heterozygous, heterogeneous, perennial, and suffer severe inbreeding depression.
• Four-step development process: (1) develop a segregating population, (2) select superior clones, (3) evaluate experimental synthetics, (4) produce commercial seed.
• Common confusion—broad vs. narrow based: Broad-based synthetics use 20–100 parent clones selected phenotypically; narrow-based use fewer than 20 clones selected both phenotypically and genotypically.
• Syn. generation management: Commercial seed is typically Syn. 3 or Syn. 4; earlier generations (Syn. 1) may not reliably predict later performance due to linkage disequilibrium.

🌱 Biological characteristics of forage and turfgrass species

🌸 Reproductive system

Self-incompatibility: a mechanism that minimizes self-pollination even though flowers are perfect (contain both male and female parts).

• Cross-pollination is the norm; self-pollinated seed produces plants lacking vigor.
• Each plant in a population is genetically unique.
• Don't confuse: "genotype" and "clone" are used interchangeably here because individual plants can be propagated vegetatively.

🧬 Genetic structure

• Highly heterozygous: individual plants carry diverse alleles at many loci.
• Highly heterogeneous: the population as a whole contains many different genetic combinations.
• Severe inbreeding depression: selfing drastically reduces vigor, so inbreeding is avoided.
• Perennial life cycle: plants live multiple years, enabling repeated seed harvests from the same field.

🔄 Four-step development process

🔄 Step 1: Develop a segregating population

Two common approaches:

1. Use existing synthetic cultivars as the starting population (already heterozygous and heterogeneous).
   • Example: Freedom red clover was used to select Freedom MR!
2. Intermate clones from different sources by planting them in a polycross and allowing natural cross-pollination by wind or insects.
   • Parent clones are chosen for complementary traits.
   • Example: Recovery wheatgrass was derived from three parent clones (Rosana, D2945, WW117FC) differing in vigor, seedling establishment, sod-forming ability, and forage/seed production.

🔍 Step 2: Select superior clones

Selection begins as soon as seed is available; no inbreeding is done.

🔍 Phenotypic selection

• Thousands of plants are evaluated visually for traits of importance.
• Stepwise selection improves efficiency: e.g., select for disease 1, then disease 2, then field traits like winter hardiness and vigor.
• Artificial infection in controlled environments (greenhouse, growth chamber) is common for disease/insect resistance.

🔁 Recurrent phenotypic selection

• One or more cycles of selection improve the population for target traits.
• The improved population itself may become a new cultivar.
• Example: TifQuik bahiagrass resulted from four cycles of selection for fast germination from cultivar Tifton.

🧪 Genotypic selection

• Used after phenotypic selection to assess general combining ability for hard-to-measure traits like yield.
• Clones are grown in a polycross; half-sib seed is harvested from each clone.
• Half-sib families are tested in replicated trials across multiple environments and years.
• Example: Nelson annual ryegrass was tested at Overton and Beaumont, Texas, across 3 years.
• Selected clones are maintained in a maintenance nursery for later seed production.

🧪 Step 3: Evaluate experimental synthetics

🧪 Broad-based vs. narrow-based synthetics

Type	Number of parent clones	Selection method
Broad-based	20–100	Phenotypic only
Narrow-based	<20	Phenotypic + genotypic

🌾 Syn. generations

Syn. 0: the selected parent clones themselves.

Syn. 1: seed produced by planting parent clones in a polycross and harvesting half-sib seed.

Syn. 2: seed produced by planting Syn. 1 seed in isolation, allowing natural intermating, and bulk harvesting.

• Key decision: test with Syn. 1 or Syn. 2?
  • Syn. 1 is faster to produce but may not reliably predict performance of commercial Syn. 3 or Syn. 4 seed due to linkage disequilibrium effects.
  • Syn. 2 is a better predictor but takes more time.

🏭 Step 4: Produce commercial seed

• Vegetative propagules from the maintenance nursery are planted in a polycross to produce Syn. 1 seed.
• Syn. 1 seed is planted in the field with mechanical equipment, usually in rows for weed control and irrigation.
• Plants cross-pollinate naturally; Syn. 2 seed is bulk-harvested with a combine.
• Process repeats to produce Syn. 3 (most common commercial class) or Syn. 4.
• Harvest limits: fields are harvested for a limited number of years to avoid genetic drift (some plants may be lost due to competition, or volunteer seedlings may alter the population).

🌽 Synthetics of maize (contrasting case)

🌽 Key biological differences

Maize differs from forage/turfgrass species in three important ways:

Trait	Forage/turfgrass	Maize
Inbreeding depression	Severe	Much less severe
Self-incompatibility	Present	Absent (some selfing occurs)
Life cycle	Perennial	Annual

🌽 Development and seed production

• Maize synthetics can be developed using any recurrent selection method.
• Commercial seed is produced by growing the cultivar in isolation each year (not from perennial fields).
• Example: maize population HIS1.

🔬 Practical breeding procedures (alfalfa example)

🔬 Initial evaluation

• Thousands of seedlings (each genetically different) are germinated in greenhouse or growth room.
• Seedlings may be screened for traits like disease resistance before field transplanting.

🔬 Field evaluation

• Seedlings are space-planted in the field and evaluated for multiple years.
• Traits assessed: winter hardiness, spring regrowth, forage yield, insect/disease resistance.
• Some clones do not survive; superior ones are selected.

🔬 Crossing methods

• Manual crossing: stem cuttings are taken, rooted, and grown in pots; pollen is manually transferred among clones by tripping flowers with folded paper.
• Cage crossing: parent clones are planted in a cage where bees perform pollination.
• Pollinated flowers produce pods; each seed in a pod is genetically different because pollen is heterogeneous.
• Seed from each parent clone is harvested separately (for half-sib evaluation) or mixed (for population formation).

🔬 Field testing

• Experimental synthetics are evaluated with Syn. 1 or Syn. 2 seed in multiple locations over several years.
• Trials are planted with self-propelled equipment.
• Visual evaluation for disease, insect resistance, winter hardiness, regrowth.
• Forage yield measured with self-propelled harvester; samples analyzed for digestibility.

🔬 Seed production

• Alfalfa seed production often occurs in Idaho.
• Leafcutter bees are used for pollination in production fields.

🧭 Overview

🧠 One-sentence thesis

Seed blends combine the best existing cultivars of one or more species for a specific purpose, and breeders typically develop cultivars for individual use rather than specifically for blending.

📌 Key points (3–5)

• What seed blends are: mixtures of multiple plant species or multiple genotypes of a species, using components best suited for the intended purpose.
• How cultivars are chosen for blends: breeders develop cultivars to be used individually; the same cultivars can then be used in a blend without special breeding.
• Refuge-in-a-bag example: a recent seed blend application in maize that mixes transgenic and non-transgenic hybrids for insect resistance management.
• Common confusion: the transgenic and non-transgenic hybrids in refuge-in-a-bag are phenotypically the same (near-isogenic lines), so farmers cannot see a difference unless insect damage appears.
• Commercial rationale: blends are used for pastures, lawns, and insect resistance refuges to meet specific agronomic or regulatory needs.

🌾 What seed blends are and how they work

🌾 Definition and component selection

Seed blends: mixtures of multiple plant species or multiple genotypes of a species, where the components used are those best suited for the intended purpose.

• The excerpt emphasizes that breeders generally do not breed cultivars specifically for use in a blend.
• Instead, cultivars are developed to be used individually, and the same cultivars can be used in a seed blend.
• Example: A company may sell a single synthetic cultivar of alfalfa to some farmers, or blend the cultivar with a synthetic cultivar of bromegrass to sell to other farmers.

🔍 How components are chosen

• For a seed blend of Kentucky bluegrass and ryegrass for planting a lawn, the mix involves:
  • The best clonal cultivars of Kentucky bluegrass
  • The best synthetic cultivars of ryegrass for the geographical region of interest
• The key is selecting cultivars that are already the best for individual use in that region.

🌽 Refuge-in-a-bag: a recent seed blend application

🌽 What refuge-in-a-bag is

• The most recent use of seed blends is in maize for the concept referred to as refuge-in-a-bag.
• It involves blending transgenic and non-transgenic hybrids together.
• The rationale (from the Applied Learning Activity) is to meet insect resistance management requirements without planting a separate refuge in a field of corn.

🧬 How the hybrids are developed

According to industry sources, the process is:

1. Seed companies first develop inbreds for use in non-transgenic hybrids.
2. The transgenes are backcrossed into the inbreds of a hybrid.
3. Since multiple transgenes are used for insect resistance, some can be incorporated in one inbred and others in the other inbred.
4. The inbreds with the transgene derived from the backcross will be near-isogenic lines of the original inbred.

🔬 Phenotypic similarity (key point)

• Inbred plants with the transgene should be the same phenotypically as the plants without the transgene.
• Similarly, the hybrids with and without the transgenes should be phenotypically the same.
• Result: A farmer will not be able to see a difference between hybrid plants with or without the transgene, unless the plants without the transgene show symptoms of insect damage.
• Don't confuse: "phenotypically the same" means the plants look identical under normal conditions; the difference only becomes visible if insect damage occurs on the non-transgenic plants.

🏭 How the seed blend is produced

• Hybrid seed is produced independently with and without the transgenes.
• After the seed of each is conditioned, it is mixed in the proportion desired by the company.
• This is a straightforward physical mixing step, not a breeding step.

🌿 Commercial uses of seed blends

🌿 Pastures and lawns

• Seed blends of forage species are used for planting pastures and lawns.
• The Applied Learning Activity directs readers to the Bailey Seed Company and Warner Brothers Seed Company websites to investigate the rationale for commercial use of blends for pastures and different species.
• The excerpt does not provide specific rationales, but implies that blends are tailored to meet specific agronomic needs (e.g., combining species with complementary traits).

🐛 Insect resistance management

• The refuge-in-a-bag concept is used in corn hybrids that contain transgenes for insect resistance.
• The Applied Learning Activity references a Farm Industry News article about single-bag refuge seed.
• The rationale (not detailed in the excerpt) is to simplify compliance with regulatory requirements by mixing refuge seed directly into the bag, instead of requiring farmers to plant a separate refuge area.

🧭 Overview

🧠 One-sentence thesis

The release and distribution of new cultivars involves institutional decision-making processes, legal protection mechanisms, branding strategies, and voluntary seed certification systems that together govern how plant germplasm moves from breeder to commercial market.

📌 Key points (3–5)

• Decision processes differ: public institutions (e.g., universities) and private companies use different structures to decide whether to release a new cultivar.
• Two legal protection methods: patents provide full control over commercial use and breeding; plant variety certificates offer less protection due to research and farmer exemptions.
• Branding vs. cultivar names: in the U.S., one cultivar can have multiple brand names from different sellers, which can confuse buyers who may unknowingly purchase the same genetic material under different labels.
• Common confusion: certified seed classes (Foundation, Registered, Certified) are voluntary third-party verification, not a legal requirement—companies may choose internal quality control instead.
• Why it matters: these systems determine who can grow, sell, and breed new plant varieties, affecting both commercial seed markets and farmer access.

🏛️ Decision-making processes

🎓 Public institutions (university example)

• At Iowa State University, germplasm developed by breeders is considered intellectual property of the university.
• The university discloses the invention to the Iowa State University Research Foundation (ISURF), which becomes the owner and handles marketing.
• ISURF distributes information to companies that may want to license the germplasm for commercial use.
• A royalty may or may not be charged depending on the germplasm.
• Some public institutions use an internal committee to review germplasm merits and recommend release; others contract with companies for marketing services.

🏢 Private companies

• The decision process involves more people than in public institutions.
• An "advancement meeting" typically includes:
  • Breeders
  • Individuals responsible for seed production
  • Marketing personnel
• This broader group evaluates whether to release a cultivar commercially.

🔒 Legal protection mechanisms

⚖️ Patents vs. plant variety certificates

Protection type	Rights granted	Limitations
Patent	Developer controls who can grow commercially or use for breeding	None mentioned; strongest protection
Plant variety certificate	Developer has some control	Research exemption (unclear scope) + farmer exemption (limited seed sales)

📜 Plant variety certificate exemptions

• Research exemption: not clearly defined; whether it prevents breeding use has never been tested in courts. The excerpt strongly advises getting written permission before using any germplasm for breeding.
• Farmer exemption: defined by U.S. Supreme Court—farmers can produce and sell only the amount of seed needed to plant their own farm.
  • Example: if a farmer needs 200 units to plant their farm, they can only sell the unused portion of those 200 units to another farmer.

🤝 Alternative: license agreements

• Applying for patents or certificates is time-consuming and expensive.
• Enforcement is the developer's responsibility.
• Some developers skip legal protection and instead use license agreements with seed producers/marketers that restrict who can produce, sell, or breed with the germplasm.

🏷️ Branding and naming

🏷️ How branding works in the U.S.

• A cultivar can be licensed to multiple companies, each giving it their own brand name.
• Legally, a cultivar can have only one cultivar name, but unlimited brand names.
• Labeling requirement: must notify buyers it's a brand, e.g., "Brand XYZ – Variety not stated."
• This system does not exist in some other countries (e.g., Canada allows only a single name per cultivar).

⚠️ Disadvantage for buyers

• Buyers cannot tell if seed from different companies is genetically identical.
• A buyer wanting to plant several different cultivars could accidentally purchase the same one under different brand names from different companies.
• Don't confuse: a brand name is not the same as a cultivar name; multiple brands may represent the same genetic material.

🌾 Seed certification system

🌾 What certification provides

Seed certification: a voluntary system where a third party verifies the genetic identity and purity of seed for sellers.

• The international organization is the Association of Official Certifying Agencies (AOSCA).
• In the U.S., state-level units are commonly called Crop Improvement Associations.
• Services vary by state; each state's association can be found by searching "[State name] Crop Improvement Association."

📋 Certification classes and tags

• Breeder seed → produces Foundation class (white tag)
• Foundation → produces Registered class (purple tag) or Certified class (blue tag)
• Registered → can also produce Certified class in some states
• Standards and inspected traits vary by crop; search "[Crop name] Seed Certification Standards" for details.

✅ Voluntary nature

• Use of certification is optional for seed developers and marketers.
• Advantage for buyers: third-party assurance of cultivar identity.
• Companies not using certification rely on internal systems and consumer trust to verify cultivar names or brands.
• Don't confuse: certification is not a legal requirement; it's a market service that companies choose to use or not use.