Phenotyping for drought tolerance in grain crops: when is it useful to breeders?

Abstract

Breeding for drought tolerance isn't a one-size-fits-all solution due to variations in drought length, timing, and intensity.
Single-gene transformations for generic drought tolerance have been underwhelming, often focusing on plant survival under severe stress, which is rare in crops.
More effective strategies involve complex traits tailored to specific crop life stages: establishment, vegetative development, floral development, and grain growth.
The goal is to find affordable ways to identify promising phenotypes and align them with genomic data to pinpoint useful molecular markers for breeders.
Controlled environments provide stability for identifying phenotypes or genotypes suited to specific drought types.
Robots now enable measuring numerous plants, allowing for high-throughput phenotyping of genotypes.
However, controlled environments differ significantly from field conditions, making it difficult to replicate yield-determining processes, especially under water limitation.
Breeders are unlikely to value controlled environment research unless specific traits demonstrate worth in the field.
Translating lab research to the field requires developing novel genotypes expressing promising traits within breeding lines adapted to target field environments.
Breeders will only be interested if these novel genotypes perform well in the field.
High-throughput phenotyping is crucial in this process.
Additional keywords: deficit watering, floral resilience, germplasm, prebreeding, trait, water stress.

Introduction

In 2011, it was the centenary of Johannsen's introduction of 'genotype' and 'phenotype'.
Johannsen introduced these terms to counter the incorrect assumption by Galton and Pearson that variation in quantitative traits was solely genetic and hereditary.
Johannsen demonstrated significant 'phenotypical' variation in genetically-identical material, challenging their view.
The terms 'phenotyping' (noun), 'to phenotype' (verb), and 'phenome' (collective noun) emerged around the 1950s.
The use of these terms has increased exponentially in the last 10–15 years because genomic information requires allied phenotypic information to be effectively utilized.
Johannsen's insight highlights the challenge of using phenotypic information to identify desirable genotypes, as phenotypic variation within a genotype can obscure differences among genotypes.
Statistical analysis is essential to address this challenge.
Phenotypic information serves two overlapping purposes: understanding the functional significance of genes or gene clusters and developing selection tools for breeding, such as molecular markers or morphological characteristics.
Plant breeders use large segregating populations, creating interest in rapid phenotyping ('high throughput').
The key question is identifying traits of most interest to plant breeders.
Crop plant breeders are typically focused on producing cultivars attractive to farmers, prioritizing grain quality, disease resistance, and developmental alignment with target environments.
High yield is crucial but has low heritability in drought-prone environments, limiting selection to multisite, multiseason field trials of advanced breeding lines (Rebetzke et al. 2012).
Farm performance can differ from trials due to farmers' management constraints (Passioura 2010; Passioura and Angus 2010).
Bänziger et al. (2006) demonstrated how to increase heritability for water-limited yield in maize through carefully manipulated droughts.
Phenotyping for abiotic stress tolerance (drought, waterlogging, salinity, extreme temperatures) is vital in plant phenotyping facilities.
This involves rapid techniques to explore or discover traits that enhance stress resistance.
These traits are often initially examined in controlled lab environments but might not correlate with field behavior (Passioura 2010).
If there is not a strong correlation, breeders are unlikely to incorporate germplasm expressing such traits into breeding programs.
The practical rationale is compromised, although genetic and biochemical understanding improves, potentially informing future improvements.
Phenotyping for 'drought tolerance' (or 'resistance') has gained interest, with citations increasing by 40% annually in the past 5 years.
This paper analyzes breeding approaches for grain crops in water-limited environments, utilizing novel phenotyping to identify germplasm with potentially useful, selectable, and heritable traits.
The aim is to establish a continuous chain from research to cultivars that efficiently convert limited water into grain yield.
Success requires interactions across this chain from project inception, with breeders playing a pivotal role in developing and testing novel germplasm.

Searching for Drought-Tolerant Crops

'Drought' has multiple interpretations depending on the perspective of geographers, meteorologists, insurers, breeders, agronomists, plant physiologists, and molecular biologists (Passioura 2007).
Similarly, 'drought tolerance' varies, ranging from surviving severe water deficits (e.g., resurrection plants, cacti) to efficiently using limited water to maximize grain production.
This involves capturing available water (rainfall during the growing season and stored soil water), with many influencing morphological, physiological, and biochemical aspects, above- and belowground.
Breeders seek lines that yield well under water scarcity and maintain yield potential in favorable seasons (Fleury et al. 2010).
Agronomists and crop physiologists take a more analytical approach.
'Drought' in agronomy refers to low rainfall during the growing season (e.g., driest 10% of seasons) and its timing, including dryness episodes reducing yield.
Water limitations can occur at any stage: sowing, establishment, vegetative growth, floral fertility maintenance, and grain growth.
No universal traits address all possibilities, necessitating individual management by agronomists.
Flowering time is the most critical physiological trait in water-limited environments.
The optimal flowering time balances water use during canopy development with water use from flowering to grain harvest.
Winter and spring crops must flower late enough to avoid frosts, which can devastate grain production.
It is essential to:
- Develop enough biomass by flowering to achieve a good potential yield (potential number of grains m^{-2} is proportional to biomass).
- Ensure enough water remains available for photosynthesis and translocation during flowering and grain growth (Fischer 1979).
Each requirement can be further dissected to identify selection traits, breeding lines, and environmental tuning.
Traits should be easily selectable, heritable, and influential in large segregating populations.
Breeders typically ignore difficult-to-select traits whose worth hasn't been proven in realistic environments.
Popular approaches to improve droughted crop yields:
- Survival of severe water deficits (whole plant or floral organs).
- Productivity issues related to water capture and efficient use, focusing on available water rather than its absence, with observational and engineering approaches.
  - Observational: studies genotypic variation in plant responses to modest water supply changes.
  - Engineering: explores genotypic variation in specific 'designer' traits.

Survival of Severe Water Deficits

Ability of plants to survive severe water-deficits during vegetative growth

Studying genetics and plant performance under severe water deficits is a widely used approach, particularly in molecular genetics with Arabidopsis (~1000 journal papers, 40% on Arabidopsis).
This work involves transforming plants with genes that are though to protect from severe water stress, although increased survival of transformed has been seen when plants are smaller and use less water than wild types, Morran et al. 2011.
While surviving extreme water stress is crucial for turf grasses or perennial pastures in dry regions, it's less important for grain crops because severe drought will result in extremely low yields whether or not the crop survives, Sinclair 2011.
Farmers might harvest dying crops for forage.
However, this research has produced significant new insight into the cellular mechanisms of dehydration tolerance, such as the function of the DREB/CBF family of transcription factors, Shinozaki and Yamaguchi- Shinozaki 2000.
One potentially beneficial feature is young seedlings' capacity to resist substantial water shortages.
Climatic variability is predicated for an increase in the trend toward sowing at the best time, even if the soil is dry:
- Advantage: seeds are ready to grow as soon as it rains, and producers are under no time constraint.
- Disadvantage: if it rains and the seed germinates, seedlings may die if no additional rain falls in about two weeks, Finch-Savage 2004; Finch- Savage et al. 2010.

Maintenance of floral fertility despite water stress

Floral fertility is diminished by abiotic stresses such as decreased water potential while pollen mother cells meiosis and soon before and after anthesis, which impacts the integrity of both pollen and ovules (Saini and Westgate 1999; Boyer and McLaughlin 2007; Ji et al. 2010; Parish et al. 2012), also the timing of silking and anthesis in maize (Campos et al. 2006).
Poor seed set leads to low yields despite good vegetative growth, so this is an important topic.
Low yield may be caused by temporary water stress at a bad time, which would not affect the crop's vegetative phase.
In maize, stress-induced ovule abortion is complex, depending on stress duration and involving carbohydrate metabolism, Boyer and McLaughlin 2007.
Water stress-induced pollen failure also involves carbohydrate metabolism, Dolferus et al. 2011.
Wheat genotypes with good pollen survival have been found.

Physiological and morphological behaviour of plants in drought-prone environments

Genotypic variationin responsesto changing water supply

Robots that have been recent developed can measure the growth and water use of many plants at frequent intervals, which has aroused interest in the diversity of crop plant responses to changes in water supplies.
In general, genotypes that keep growing fast in the face of a decreasing water supply perform well in the field whenever there is adequate water in the subsoil and the prospects of rain or irrigation are good, so that there is productive advantage in the crop continuing to grow fast (Tardieu 2012); rice is a good example (Parent et al. 2010).
Conservative behavior during vegetative growth is often beneficial in semiarid environments, reserving subsoil water for flowering and grain growth.
This represents a distinct form of 'drought tolerance' where a limiting water supply is allocated to maximize productivity across the season.
Dual-purpose wheat crops, used for grazing and grain, sometimes yield more grain than ungrazed crops if grazed to ground level before stem elongation because water use is deferred (Harrison et al. 2011).

Targeting traits for optimising water balance to achieve water-limited potential yield

Traits have varying significance at different crop development phases, especially ensuring adequate water during floral development, flowering, and grain production.
These traits include morphological traits, such as root architecture (Hammer et al. 2009; Lopes and Reynolds 2010; Trachsel et al. 2011) and reduced unproductive tillers in cereals (Mitchell et al. 2012) and physiological, such as the retranslocation of pre-anthesis assimilate to the grain (Bidinger et al. 1977; Blum 1998).
A trade-off between root depth and amount of stored assimilate may be a concern (Lopes and Reynolds 2010), although Kirkegaard et al. (2007) showed that the uptake of only an extra 10 mm of water by wheat roots from the deep subsoil during grain growth led to an extra 600 kg ha^{-1} of grain yield, presumably largely because of more effective transfer of stored assimilate to the grain.
Reynolds and Tuberosa (2008) and Richards et al. (2010) and discussed a wide range of traits for improving water-limited yield in wheat and Salekdeh et al. (2009) have done so for a wide range of crops.
These traits include good seedling establishment (e.g., long coleoptiles), fast ground cover, leaf architecture, root vigor, transpiration efficiency (carbon-isotope discrimination), remobilization of stem carbohydrates to the grain, glaucousness to deflect heat, leaf rolling, and buffering against reproductive failure.
Maintaining green leaf area ('stay-green') and cool canopies during grain filling can also be promising, Jordan et al. 2012, Lopes and Reynolds 2012.
Vigorous growth of seedlings is strongly beneficial in unploughed soil, a characteristic of conservation agriculture (Watt et al. 2005).
Deep roots in rice are evidently beneficial (Henry et al. 2011) and in maize better buffering of floret fertility and early grain filling against water stress are likely to be the next areas of improvement after the earlier success in reducing the stress-induced gap between anthesis and silking (ASI) (Campos et al. 2006).
Further, promising new traits are sure to emerge as work in this area continues to blossom.
Some traits are constitutive and can be selected for in well-watered plants (Richards et al. 2010):
- Long coleoptiles:
  - Allow for better seedling establishment when seeds are sown deeply to access water below a dry surface soil.
  - Semi-dwarf wheats containing the dwarfing genes Rht1 and Rht2 are unable to elongate more than ~50 mm, so seeds sown more deeply than this produce seedlings that fail to emerge.
- Fast development of early leaf area:
  - Allows for use of water that might otherwise evaporate from a wet soil surface.
- Reduced tillering:
  - Avoids wasteful investment in unproductive tillers.
Given these diverse approaches for improving drought tolerance or water-limited yield in annual crops, how can we make good progress using controlled environments, which offer much better control for pursuing promising traits, but which are limited in their ability to reproduce the field conditions that most influence the final grain yield?

Phenotyping in Controlled Environments Aimed at Crop Improvement

Important yield-determining processes in the field occur gradually, such as the slow extraction of water from the subsoil over weeks.
Mimicking these processes in pot experiments in controlled environments is challenging.
Passioura (2006), Poorter et al. (2012a, 2012b) and Salekdeh et al. (2009) have discussed a wide range of issues in running pot experiments.
Experiments examining water stress face particular difficulties because:
- Pots are much smaller than the soil volume available to field plants (Poorter et al. 2012a).
- Soft growing media allow faster root exploration and leaf growth than typically occurs in the field (Stirzaker et al. 1996; Passioura 2002).
- The rate of water use can thus be many times faster than in the field, resulting in exhaustion over days rather than weeks or months.
Fast plant responses, like stomatal closure (effective over minutes to hours), overshadow slower but more influential processes, such as root growth into untapped wet areas and leaf area index modulation to adjust to slowly depleting soil water (Jordan 1983).
Integrative measurements are generally more useful than spot measurements in elucidating the main influences on water-limited yield (Masuka et al. 2012), although persistent patterns of stomatal behavior, for example, midday stomatal closure, may be beneficial in modulating water use (Rebetzke et al. 2003).
However, there are one-off fast events in the field that can strongly influence grain yield:
- Frost at flowering.
- Sudden hot, dry wind at flowering or during grain growth.
The risk of frost at flowering interacts with the risk of severe water deficits during grain growth of cereal crops growing during winter and spring (http://www.grdc.com. au/uploads/documents/GRDCFSTimeOfSowing_North.pdf? shortcut=1, accessed 17 August 2012) and is, therefore, pertinent to improving the water management by such crops.
These fast events may be easier to explore in controlled environments than in the field, although it has proven difficult to use frost chambers to replicate the relative behaviour of different genotypes experiencing damaging frost in the field (Chen et al. 2009; Frederiks et al. 2011).

What measurements to make

There is a wide range of physiological and morphological measurements that can be made on plants growing in pots: water potential, stomatal conductance, leaf temperature, various characteristics of photosynthesis, multispectral reflectance, thermal imaging, biomass, rate and amount of water use, relative growth rate and leaf area development, root growth and distribution and many others.
Some of these can be done effectively and non-destructively with remote sensing (Furbank and Tester 2011; Masuka et al. 2012; Nagel et al. 2012).
Choosing measurements that enable rapid selection of plants in populations segregating for such traits is likely to result in more rapid progress if the eventual goal is to incorporate promising traits into commercial breeding lines.
Often, promising traits are first identified in the field, for example, the need for long coleoptiles in wheat, to enable better establishment of seedlings when there is a dry start to a season (Rebetzke et al. 2007).
Others have arisen from laboratory or theoretical studies, such as the use of carbon isotope discrimination to select for intrinsic water-use efficiency (Condon et al. 2004), that is, the ratio of biomass produced to the amount of water transpired.
The appropriate measurements to make are those that either directly concern the given trait or that are a good surrogate for it.
Cool canopies during grain filling, easily measurable by thermal imagery, may be a useful surrogate for root depth (Lopes and Reynolds 2010).
Similarly, the diurnal pattern of stomatal conductance, also rapidly measurable by thermal imagery, may be an indicator of parsimonious and efficient water use during the vegetative phase (Rebetzke et al. 2003).
Phenomics, although in its infancy, is developing new measurement techniques, sometimes with no specific traits in mind.
Some of these techniques are sure to prove valuable, but they are likely to be most rapidly effective if, during their development, they are related to traits of known or potential importance.
How best to simulate in controlled environments a drought experience or modulation of water use that gets close to what happens in the field?
There are many aspects of pot experiments that involve the water (and oxygen) relations of crop plants and can strongly influence behaviour.
At best, these experiments are based on hypotheses about what traits are likely to be influential in improving yield in a range of circumstances in the field, or on what new procedures can speed up the rate of selection of traits that may be important, but are currently too cumbersome to be useful in breeding programs.
There are many traits, outlined earlier, that are currently of interest, many of which are amenable to selection in controlled environments and some of which are best selected for in well watered conditions.
Various watering regimes have been tried for reducing the water supply to plants growing in pots with the aim of looking for useful genetic variation.
Each has its advantages and disadvantages in relation to the specific aims of the research.
In some studies, good technique requires that the surface of the medium be covered so that there is little loss of water by direct evaporation from the soil.
Doing so enables the amount of water transpired by the plants to be measured, which in turn, can be related tothe performance ofthe plant in relation toits water use.
If this is not done, a slowly growing genotype will lose a larger proportion of its water supply to direct evaporation, yet might have a larger ratio of biomass accumulation to water transpired.
In these circumstances it would be difficult to interpret any variation of the plants’ behaviour in relation to its water economy.
Tables 1 and 2 summarise some features of pot experiments and of the water relations of the growth medium that can influence interpretations of how the plants behave.
They compare common techniques for establishing well-watered controls and for applying deficit watering.

Prebreeding for Effective Use of Phenomic Information – the Link to Breeding Programs

Prebreeding, previously genetic enhancement (Duvick 1990), is described by FAO’s Global Partnership Initiative for Plant Breeding Capacity Building (GIPB) as:
- '… all activities designed to identify desirable characteristics and/or genes from unadapted materials that cannot be used directly in breeding populations and to transfer these traits to an intermediate set of materials that breeders can use further in producing new varieties for farmers. It is a necessary first step in the use of diversity arising from wild relatives and other unimproved materials.'
Implicit in this description is that the worth of the modified breeding populations needs to be well demonstrated in the field if they are to attract the attention of commercial breeders.
Richards et al. (2010) describe how near-isogenic or recombinant inbred lines (RILs) can be developed in which given traits are either present or absent in a common background.
These lines can then be grown in the field, preferably with managed water deficits, so that the worth of a given trait can be assessed in different types of drought (Campos et al. 2004; Rebetzke et al. 2012).
Commercial breeders of annual crops are unlikely to take much interest in evidence from controlled environments that a given trait may be beneficial.
Given that different traits assume greater or lesser importance in different types and timing of drought, they are much more likely to take interest in a trait that has passed these additional tests:
- Climatic analysis has shownthatitis likelyto beimportant in a substantial proportion of growing seasons over a large target area (e.g. Chenu et al. 2011).
- The relevant genes have been incorporated into cultivars or advanced breeding lines that are adapted to the target areas (Rebetzke et al. 2012).
- Lines contrasting in expression of the trait (high and low) have been grown in plots in the field and the presence of the trait has evidently improved the water-limited yield of the plants.
- The trait is not deleterious in seasons without drought or is not excessively prone to trade-offs in different types of drought (Tardieu 2012).
- The trait is genetically stable, persists across generations and is expressed in different genetic backgrounds.
- The trait is easy to select for in breeding populations, either with robust molecular markers, rapid visual appraisal, or automated remote sensing.
Traits that concern the performance of plants at different developmental stages are likely to be effective in only a minority of growing seasons, e.g. one in four.
For example, if conditions at the time of sowing are good, then traits that help establishment in poor conditions are not relevant.
Similarly deep roots may not be of much importance during droughts in which only the top 50 cm of the soil profile gets water and similarly in good seasons, with ample well distributed rainfall, when water deep in the subsoil, though present, is not needed (Lilley and Kirkegaard 2007).
There is an important corollary to this.
In commercial breeding programs, selection is strongly driven by the essentials of disease resistance, grain quality, right flowering time and, finally, in the generation or two before the release of a new cultivar, yield (although obviously underperforming lines are culled in all generations).
This means that specific traits that have been deliberately incorporated into breeding lines are often not explicitly selected for in later generations.
Thus, if there is a majority of growing seasons in which a given trait is of little value, it may slowly disappear by attrition during the selection process.
A historical example isthat of osmotic adjustment, which can be of substantial value in water-limited wheat (Morgan and Condon 1986), yet there is no evidence that its frequency in breeding populations has increased.
Hammer et al. (2005) and Messina et al. (2011) have provided frameworks for guiding breeding goals where there are known interactions of this sort between genotype and target environment.

Prospects for Good Progress

High-throughput phenotyping for drought tolerance is still developing.
There are now many tools that can be used for both for understanding how particular genes work and for selecting desirable quantitative traits in prebreeding and breeding programs (Furbank and Tester 2011; Masuka et al. 2012; Nagel et al. 2012; White et al. 2012) both in the laboratory and in the field.
How does it compare to other breeding methods for water-limited environments?
These methods include genetic modification and the 'black-box' statistical method of genomic selection (Goddard 2009; Cabrera-Bosquet et al. 2012).
Single-gene transformation has worked well with simple constitutive qualities like pest or herbicide resistance, which address exogenous compounds not involved in plant processes that contribute to yield (Sinclair 2011).
In that it can remove mistakes that might prevent higher yield possibilities from being selected, pest or disease resistance is priceless.
Progress in boosting aluminum tolerance has been made with aluminum because smart molecular and physiological research discovered and replicated significant genes, so genetic transformation is most effective at figuring out membrane and cellular functions (Pereira et al. 2010).
Due to the current cloning of rust-resistance genes in wheat (Risk et al. 2012), there are possibilities for greatly enhancing long-lasting fungal pathogen resistance.
The advancements from genetic transformation have been very low with complex traits like drought resilience.
Araus et al. (2008), Passioura (2010), and Sinclair (2011) outline the reasons for the lack of effectiveness.
Of the ~1000 papers that have been published that mention ‘drought tolerance (or resistance) and transformation and gene’ in their abstracts very few report evaluation in field plots.
Two recent and notable examples are Castiglioni et al. (2008) and Saint Pierre et al. (2012).
The first of these claimed some improvement of yield when maize transformed with a bacterial RNA chaperone experienced drought at flowering; however, little agronomic information was supplied with which to gauge the nature of the improvement and its likelihood of being usefully robust across a range of environments – for example, was flowering time precisely the same across the transformants and wild type?
The second showed that wheat plants transformed with DREB did not yield more when grown with limiting water in the field, although their ratio of shoot biomass to water used was greater in pot experiments.
The main reasons for this general failure of genetic transformation in relation to drought tolerance in the field is that, first, most of the initial selection and identification of candidate genes has relied on survival of extreme water deficits and, as noted earlier in this paper, survival of severe water deficits is not agronomically important, except possibly for very young seedlings; and, second, it is rare for single genes to contribute much to improvements in quantitative traits: the recent success of genomic selection highlights that, for its power has been in selecting for many quantitative trait loci (QTL) of small but additive effect (Goddard 2009).
Genomic selection is proving to have great abilityin predicting breeding value within existing breeding populations.
It requires densely-mapped genotypes accompanied by rich phenotypic information of a reference population.
If the markers are dense enough to be closely associated with useful QTL, they can be used to select for favourable alleles at each QTL without needing to identify the QTL or its functional significance (Goddard 2009).
It has the potential, despite the low heritability of yield in drought-prone environments, of identifying the best genotypes and perhaps of aligning them with different target environments (Burgueño et al. 2012).
Its efficacy may be further improved by using overt knowledge of the roles of known QTL in dealing with genotype-environment interactions (Messina et al. 2011).
Nevertheless, making progress in strongly drought-prone environments in which yields can vary several- fold across years will be difficult (Richards et al. 2010), especially given the agronomic flexibility – particularly in sowing time and fertiliser application – that farmers show in dealing with drought (Passioura and Angus 2010).
High-throughput phenotypinginthe field using remote sensing of traits that are associated with water- limited yield (White et al. 2012) may help structure a reference population for genomic selection.
Genomic selection is limited in its power to the reference population on which it is trained.
Thus, the pursuit of new traits may require deliberate broadening of a breeding population.
A classic example of such broadening is the use of Norin 10 to develop semi-dwarf wheats in the 1950s and ’60s (Hall and Richards 2012) and a more recent example is the search for alternative dwarfing genes that do not overly restrict the length of wheat coleoptiles as the Norin 10 genes do (Ellis et al. 2005).
It is in the exploration of novel traits that phenotyping in controlled environments offers fastest initial progress in identifying genotypes from which proof of concept in water- limited field conditions can eventually by explored.
Successful examples, mentioned earlier, include transpiration efficiency, coleoptile length and resilience (or not, depending on the target environment) of leaf growth to water deficits.
Where will new ideas for traits effective in water-limited environments come from?
Observations of field scientists – breeders, agronomist, crop physiologists – have been a fertile source and will surely remain so.
These can be thought of as top down (in relation to the rest of the biological spectrum) and come with the advantage that they are demonstrably relevant in the field; long coleoptiles and the association between yield and cool canopies during grain filling come into this class.
Such observations stimulate more detailed work in laboratory and controlled environments, which in turn often results in greater insights into limiting processes in the field.
At the same time, tuning suites of traits to target environments will be needed (Chenu et al. 2011).
Meanwhile, phenomics is going through a phase of rapid development in which new techniques are being developed to explore morphological and physiological traits in a range of genotypes, by direct robotic measurement, remote sensing or both.
In the context of breeding better cultivars of grain crops for drought-prone environments, the challenge ahead is to make best use of these techniques to explore, perhaps uncover, novel traits and associated genotypes that can contribute to faster genetic improvement.
Cross fertilisation between laboratory and field is needed (Salekdeh et al. 2009).
Given that the rates of genetic improvement required in the coming decades are greater than those currently being achieved (Fischer and Edmeades 2010) and that the lead times for trait- based breeding have typically been ~20 years (Hall and Richards 2012), well-focussed high-throughput phenotyping will be crucially important in developing novel effective traits.
The latter will require developing enhanced germplasm in adapted genetic backgrounds that can be used in the field to obtain proof- of-concept for such traits.
Further development will then depend on commercial breeders being sufficiently impressed with the novel germplasm to use it in their breeding programs.