Accelerated Plant Breeding

CAB Reviews 2014 9, No. 043

B.P. Forster1*, B.J. Till1, A.M.A. Ghanim1, H.O.A. Huynh1, H. Burstmayr2,3 and P.D.S. Caligari4,5

Address: 1 Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Division, IAEA Laboratories, A-2444 Seibersdorf, Austria. 2 University of Natural Resources and Life Sciences, Vienna, Austria. 3 Department for Agrobiotechnology (IFA-Tulln), Institute for Biotechnology in Plant Production, Konrad Lorenz Str. 20, A-3430 Tulln, Austria. 4 BioHybrids International Ltd, P.O. Box 2411, Earley, Reading RG6 5FY, UK. 5 Instituto de Ciencias Biolo´gicas, Universidad de Talca, 2 Norte 685, Talca, Chile.

*Correspondence: B.P. Forster. Email: [email protected]

Received: 22 April 2014 Accepted: 17 November 2014 doi: 10.1079/PAVSNNR20149043

The electronic version of this article is the deﬁnitive one. It is located here: http://www.cabi.org/cabreviews

The time to develop new cultivars and introduce them into cultivation is an issue of major importance in plant breeding. This is because plant breeders have an urgent need to help provide solutions to feed a growing world population, while in parallel, time savings are linked to profit- ability. Plant breeding processes may in general be broken down into the following five key elements: (1) germplasm variation; (2) crossing; (3) generation of new genetic combinations; (4) screening and selection (identification and subsequent fixation of desired allelic combinations); and (5) line/cultivar development. Each of these has implications in relation to the time taken to breed a new cultivar; a brief introduction is given for each to highlight the obstacles that may be targeted in accelerating the breeding process. Specific techniques that provide a time advantage for these elements are then discussed. Some targets for enhancing the efficiency of plant breeding, e.g., the manipulation of meiotic recombination, have proven to be recalcitrant. However, other methods that create new genetic variation along with improvements in selection efficiency com- pensate to a large extent for this limitation. Progress in accelerating the plant breeding process continues by exploiting new emerging ideas in science and technology.

Keywords: Accelerated breeding, Mutation breeding, GM breeding, Rapid generation cycling, Doubled haploidy, Marker-assisted selection, High-throughput screening.

Review Methodology: Searched various websites including the http://worldmeters.info/worldpopulation http://www.census.gov/ popclock; FAO/IAEA database of mutant varieties, http://mvgs.iaea.org. FAO Yearbooks and AGROSTAT, BBC News. Used the following key words in searches: world population, global forecasts, accelerated breeding. Discussed contents with colleagues.

Introduction – Issues and Drivers of Faster history, plant breeding has been a test bed for scientific Breeding Methods innovations, especially those in genetics, botany, physiology and biotechnology, thus plant breeding has capitalized The evolution of crop plants can be described as having on research in recombination, heritability, polyploidy, three phases: (1) gathering from the wild; (2) domestica- chromosome engineering, tissue culture, heterosis, gen- tion and agronomy and (3) plant breeding [1, 2]. The con- etic linkage mapping, molecular genetics, mutagenesis and version of wild plants to crop plants involves the selection transformation. Plant breeding has been a major factor in of suitable types, which developed in conjunction with increasing crop production [3–5]. Research and devel- suitable agronomic practices. Domestication and associ- opment achievements have added much to the toolbox, ated agronomy therefore played a significant and intimate but a major constraint and frustration that remains is the role in mankind’s early food security and thereby human time to generate de novo, a new cultivar for growers. evolution. However, this phase has been eclipsed by sub- Plants are grown for a wide range of uses, chiefly for sequent achievements in plant breeding. Throughout its food, feed, fibre and fuel, but also for specialized products

http://www.cabi.org/cabreviews 2 CAB Reviews (e.g., medicines), and social amenity such as recreation pedigree inbreeding method (PIM) as described by Briggs and ornamentation. The global human population is ex- and Knowles in 1967 in their book: ‘Introduction to plant pected to grow from current estimates of 7.1–7.2 billion breeding’ [9]. This method has, and continues, to serve (http://worldmeters.info/worldpopulation http://www. plant breeders well. Modern time-saving methods are census.gov/popclock) to 9.1 billion in 2040 [6]. Plant then described and may be compared with PIM. This breeders face a daunting [5] task as crop production is not means that we will tend to concentrate our focus and keeping pace with demands; crop yields are currently examples on inbreeding species, but in reality the princi- increasing at about 1.3% per annum, about half the rate that ples apply to species with other breeding systems and is required [7]. The area of land under cultivation is not the reader can easily apply the possibilities for the expected to increase; it has remained static over the past methodologies to those species 50 years at about 660 million hectares (data from FAO Homozygous lines, commonly referred to as genetically Yearbooks), and there is even concern that agricultural pure lines, or breeding pure lines, are important goals in land is degrading. Future increases in crop production are many plant breeding programmes and may be developed therefore dependent upon greater yields per unit area of through PIM. Pure lines not only form the end product for land area and therefore crops with high yield potentials self-pollinating species of the breeding programmes (lines must be developed and developed quickly. At the same that may be developed into cultivars in many crops, time, climate change is providing a major uncertainty as to notably cereals), but are exploited as parental lines in F1 how it will limit or change agricultural productivity. For hybrid cultivar production (notably in maize and many example, in temperate crops significant yield reductions vegetable crops). New homozygous lines are typically de- are expected as temperatures rise by more than 4C; while veloped from crosses between two distinct parental lines similarly, in tropical regions a rise of 2C will cause major that are complementary for desired traits. This provides yield losses [8]. Temperature is only one aspect of climate an opportunity for re-assortment and recombination of change. Other direct effects include salinity, drought, genes and alleles through meiosis and thereby the pro- waterlogging and storms, all of which bring with them new duction of segregating populations in subsequent gen- pest and disease problems. Some of the effects of climate erations, as first described in the experiments of Mendel change may be mitigated by the introduction of cultivars (1822–1864 [15]). In crosses between two pure lines, adapted to the new conditions, or growing new crops segregation is observed first in the F2 generation as the F1 species, both of which solutions require rapid breeding. individuals are presumed to be genetically identical but This review does not intend to go into details of plant heterozygous for all loci by which the parents differ. The breeding methods or comparisons between them. There plant breeder attempts to make improvements either are several excellent reference books that discuss theory negatively, by rogueing out inferior plants, or positively, by and methods in plant breeding in a wide range crops (e.g., selecting the best, starting with the segregating F2 popu- [9–14]). Here we provide a general overview of techni- lation. The next objective is to develop pure lines from ques that can accelerate the plant breeding process, from the selections and in self-pollinating species this is tradi- parental choice to cultivar release. tionally done by repeated rounds of selfing in the PIM (Figure 1). Seed from individual selected plants are sown out as F3 family rows; this is repeated in the F4 and F5. Components of Plant Breeding From F5 to F6 there is a shift from single plant selection to single family selection. Seed of an individual family are Plant breeding processes may be broken down into the grown out in small plots in the F6 bulk harvested, and then following elements: (1) access to germplasm or creation grown in larger replicated plots in F7––F10 generations. of genetic variation, (2) crossing; (3) generation of At about the F7 stage the term ‘line’ replaces the term new genetic combinations (normally through meiosis); ‘family’ as there should be no visible variation at this stage (4) generation of segregating populations, (5) selection (plants within a line look the same). Multi-locational trials (fixation of desired alleles); and (6) line development. Each often take place from F8 to F10. Larger strip tests take of these components introduces a time component place in F11 and F12 generations to bulk seed for potential and any, or all, of which can be a bottleneck in the plant commercial release, since at this stage the selected lines breeding process. The relevance and limitation of each must enter national, regulatory trials to achieve official component are described while specific techniques that cultivar status. This normally includes passing DUS tests, overcome these limitations are then discussed. i.e., the line must be distinct, uniform and stable, in The terms ‘traditional plant breeding’ and ‘accelerated addition it must normally give superior yields compared plant breeding’ are difficult to define as continual im- with standard checks or possess other important traits provements have taken place in deliberate plant breeding (such as disease resistance, superior quality, etc.). If the since Mendel first established the genetic principles line passes ‘is accepted’ it may be officially released and involved, principles that provided a scientific basis for grown by farmers. Thus, for an annual self-pollinating plant breeding. Therefore, to provide some perspective, crop, it can take between 10 and 15 years to produce a we take for our example of ‘traditional plant breeding’ the cultivar from an initial cross (Figure 1).

http://www.cabi.org/cabreviews B.P. Forster et al. 3

Initial cross Year P1 × P2 Progeny from cross: first generation. 1 F1

The second generation is normally the first opportunity to observe segregation and to select 2 F2 individual plants.

Single plant selections. 3 F3

Single plant selections, often includes check 4 F4 cultivars.

Single plant selections. Family rows should begin

F5 to appear uniform, if so rows may be selected as 5 lines.

Small plot evaluation and selection. First

F6 opportunity to estimate yield. Plots are bulked 6 harvested.

Large replicated plots; data on yield, quality and 7 F7 agronomic performance.

Three to 5 years of multi-locational trials. 8–10 F8 to F10

Seed multiplication and official testing and 11–15 F11 to F12 certification for cultivar release.

Figure 1 Scheme for PIM, redrawn and amended from Briggs and Knowles [9].

Genetic Variation to produced genetically modified cultivars (GM crops) may take a similar amount of time, the rate being depe- Plant breeding is based, ipso facto, on genetic variation. ndent upon the efficiency of selection and screening and Plant breeders aim to harness genetic variation to im- cleaning up the genetic background (see section on prove crop plants and the speed at which they can suc- Selection and Screening). ceed depends on whether the required variation exists (and if so in which gene pool) or needs to be created. The latter is not necessarily a disadvantage. The simplest Crossing scenario is where the desired genes are present in the primary gene pool, i.e., available in elite breeding lines. Crossing or hybridization is a basic tool in plant breeding. These can be used in crosses with other elite lines and This brings together genes of two or more parental the breeding can focus on the trait of interest as there is lines from which, it is hoped and expected, improved minimal disruption of the elite genetic background. If, plants will be produced. Crossing involves the application however, the desired variation is only available in sec- of pollen from the male parent to the stigmas of the ondary, tertiary or wild species germplasm then increasing female parent. It may involve emasculation to prevent efforts, such as repeated rounds of backcrossing, are re- self-pollination and isolation of the female flowers to quired to recover an elite genetic background which prevent uncontrolled pollination. Pollen is collected from expresses the trait of interest. Normally, the more distant the male parent (and in some species may be stored) and the genetic resource used the greater the delay in de- placed on receptive stigmas; in some case male sterile veloping a cultivar. Plant mutation induction and trans- lines can be used. Hybridization is not normally a time- formation can create novel variation directly in a favoured limiting step, but may be restricted by incompatibility genotype and selected lines can be developed into problems and the synchronous production of receptive cultivars relatively quickly. Plant mutation breeding is a stigmas and mature pollen, which in turn may be season well-established and rapid breeding strategy; generally dependent. In many countries, particularly developing taking 7–9 years to produce a cultivar in a self-pollinating countries, crossing can only be done during clement crop (see section on Mutation Breeding). Transformation conditions during seasonal crop production times and this

http://www.cabi.org/cabreviews 4 CAB Reviews may be restricted to one opportunity per year, weather Screening methods are performed either by phenotyping permitting. Investment in controlled environment facil- or genotyping test materials. Selections are made based ities, such as a greenhouse with lighting and temperature on analysis of this data based on pre-determined criteria. controls allows for additional crossing opportunities per year. Phenotyping Plants exhibit doubled fertilization of the egg (which develops into an embryo) and of the central cell (which Screening by phenotyping has been the standard techni- develops into an endosperm). In wide crossing it is que in plant breeding to select improved crops since common for the endosperm to fail, necessitating the plants were first domesticated. In its basic form it entails rescue and in vitro culture of the hybrid embryo. Embryo growing up plants/populations and seeing how they per- culture can also speed up the time to growing the next form: are they high/low yielding, tall/short, early/late, generation (see section on Rapid Generation Cycling). In disease susceptible/resistant, drought sensitive/tolerance, extreme cases, the hybrid embryo loses chromosomes is the seed round/wrinkled, etc.? Tests may be performed of the male parent resulting in haploid embryos, these in simple growth chambers and greenhouse, or sophisti- too need to be rescued and cultured and are valuable cated phenomics facilities, or most commonly in the field. in the development of doubled haploids (DHs) which can The ability to phenotype effectively and efficiently is a shortcut the breeding process (see section on Doubled major concern in plant breeding and high-throughput Haploidy). methods need to be developed [18–20]. The best phenotypes are selected and advance to the next generation. In the PIM (Figure 1) lines are selected from segregating Generation of New Genetic Combinations families 5–6 years after the initial cross. In terms of time and cost savings, the sooner the screening the better, Meiosis provides a natural mechanism for re-assortment both between generations (early generations are pre- and recombination of genetic information [16]. Breeders ferred) and within generations (seedling screens are have a particular interest in recombinant events that better than adult plant screens). Phenotypic screening has improve the phenotype, but these are often rare. Large a disadvantage in that it may be influenced by environ- populations, or repeat crossing, may be necessary to mental factors, this is especially true when screening for achieve the desired recombinant. In wide crossing and disease resistance. In such cases genotypic screening may especially in inter-species crossing a large piece of an alien be more accurate. However, at some point, regardless of chromosome may be introduced. Although this may carry the screening method, selected lines must be tested and a desired gene for a target trait it is likely to be linked advanced in the field. to many other alien genes with negative effects. To complicate matters further the alien chromosomal seg- ment is often meiotically inert, i.e., it does not recombine Genotyping and is always linked to flanking genes leading to linkage drag. There is therefore interest in manipulating recom- The majority of the variation utilized by plant breeders bination rates, particularly in specific areas of the genome. comes in the form of heritable DNA changes that can be In polyploid species such as wheat there are genes monitored using molecular techniques. A wide range of that control chromosome pairing, notably Ph1, which techniques now exist. The use of molecular markers to ensure strict pairing of homologues at meiosis, when the assay for genetic variation in linkage disequilibrium with gene is absent, or recessive, pairing can take place desired traits, known as marker assisted selection (MAS) between non-homologous chromosomes and can break is now a common practice [21]. In developing markers for up alien segments [17]. The processes of meiosis can also traits the marker must be associated with the trait and be exploited in the development of chromosome trans- thus is dependent upon good phenotypic data [22]. The location lines, especially between crop and alien chro- advent of array hybridization methods and more recently mosomes, thus speeding up introgression (see section on next generation sequencing have allowed high-throughput Induced Recombination), but in general there are not approaches to measure inheritance of genetic variation many examples of stimulating meiosis to increase with tens of thousands of single nucleotide polymorph- the efficiency of plant breeding within a species, and this isms recoverable in a single assay. In addition to speeding remains a bottleneck in plant breeding, an area for future up traditional selection and removing potentially inter- developments. fering environmental factors, advanced methods speed up the process of genetic mapping, cloning and have provided new approaches to breeding in the form of genomic Screening and Selection selection, e.g., MutMap [23–25]. Advances in tools for the discovery of nucleotide variation have also enabled the Screening and selection are the mechanisms by which development of reverse genetic strategies. Reverse- breeders fix desired genes and gene combinations. genetics allows the identification of genetic lesions in

http://www.cabi.org/cabreviews B.P. Forster et al. 5 targeted gene regions hypothesized to be important for are distinct (carry new mutant trait), uniform and stable, is desired traits. One such example known as targeting reduced. A major bottleneck in plant mutation breeding induced local lesions in genomes utilizes induced point is the ability to screen for the desired mutant trait as mutations to alter gene function [26] for which protocols this is a rare event, occurring in 1 in 1000–1 in 100 000 have been developed [27–30]. Current issues in geno- individuals. Traditionally, mutant induction has deployed a typing include the cost and ease of DNA extraction and random approach (using physical or chemical mutagens) the large sample sizes that require analysis (typically 700 and screening has been done at the phenotypic level lines). by observations of plant traits, such as yield, height, flowering time, colour, disease resistance, tolerance to salt and drought etc. [32]. Recently, high-throughput Line Development phenotyping has been facilitated by the deployment of phenomics facilities [33]. Genotypic mutant screening A ‘line’ is defined as a group of genetically related siblings can also be deployed and this comes into play where which no longer exhibits trait segregation (which is a the target gene is known [34]; Szarejko review [35, 36]. characteristic of a family). Once selected, the line is Methods and trends in plant mutation breeding have been usually subject to field performance trials that increase reviewed recently by [37]; classic examples include the in scale as the line progresses (and as more inferior development of semi-dwarf barley in Europe, salt tolerant lines dropped). Line development in the PIM is given in rice in Vietnam, self-compatibility in fruit trees, seedless Figure 1, and may require 10 years of development before fruit in citrus. entry into national official DUS tests and cultivar release. A recent example is the development of mutant wheat Line development is costly in time, as well as in space and lines resistant to Ug99, a race of stem rust disease (Puc- official compliance. Breeders must focus their efforts on cinia graminis f. sp. tritici) first identified in Uganda in 1999. lines with a high potential of success and to advance these Historically this is the most feared disease of wheat as it as fast as possible. causes severe yield losses. It is a highly mobile disease As can be judged from above that while there is a need as its spores are carried by the wind over large distances. to develop new cultivars as quickly as possible into pro- The disease has now spread to neighbouring countries duction, there are numerous stages in the process that in Africa: Kenya (2002), Ethiopia (2003), Sudan (2006) need to work as efficiently as possible and be linked into and South Africa (2009). It has also reached Iran (2007), an integrated programme of breeding which is time effi- Yemen (2007) and Iran (2009) and is set to spread further. cient. Below we discuss some of the possibilities to Ug99 is of particular concern as there is little resistance in accelerate the steps in such programmes. wheat cultivars, in 2010 it was estimated that 80–90% of all global wheat cultivars were susceptible [38]. In 2009 Kenyan wheat cultivars were subject to gamma irradiation Techniques in Accelerated Breeding in an attempt to induce mutations for disease resistance. The first mutant generation (M1 population) was grown at Plant Mutation Breeding Eldoret, Kenya (a hot spot for the disease). Resistant mutant lines were selected in subsequent generations in Plant mutation breeding was heralded as providing a uni- both field and greenhouse conditions. Since two genera- versal solution to breeding problems but subsequently tions of wheat can be produced per year in Kenya with became more restricted to being used in rather specific the help of irrigation, it was possible to accelerate the circumstances, particularly easily obtained characteristics, development of resistant mutant lines. Four promising such as dwarf growth that could be readily selected mutant lines selected in the M4 population were advanced visually and thus in large numbers. However, it has gained and multiplied in 2010 and entered into National Perfor- more recent popularity and indeed provides a successful mance Trials in Kenya in 2012 and 2013. In 2014, one of and relatively fast breeding method. The Joint FAO/IAEA these, named ‘Eldo Ngano I’ was officially released as a Division of Nuclear Techniques in Food and Agriculture cultivar for farmers, this was achieved within 5 years of lists over 3000 officially released mutant cultivars in the initial mutation induction. over 200 crop species ([31]: http://mvgs.iaea.org). Over Plant mutation breeding using random mutation induc- 60% of these mutant cultivars have been derived from tion is considered to be a conventional breeding method gamma irradiation. Typically, mutation breeding takes and is non-regulated. 7–9 years as compared with 10–15 years in a standard PIM (Figure 1), for an annual crop. This is because the main objective in plant mutation breeding is to take Induced Recombination an already favoured cultivar and to improve it through induced mutation with minimal disruption of the elite Physical, chemical and biological mutagens can be used genetic background (Figure 2). Thus the breeding effort to to enhance the efficiency of homologous chromosome achieve cultivar status, i.e., the development of lines that recombination and induce chromosome translocations in

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Selected elite genotype, an example of a crop plant

with seven chromosomes (M0 generation)

After mutagenic treatment (e.g., gamma or X-ray irradiation) the population becomes the first

mutant generation, i.e., the M1 generation. This is made up of individuals with random mutated loci (an example of just four individuals from a population of normally several hundred individuals).

The M2 generation is produced by selfing the M1 population (four examples of individual M2 plants are given). Mutant events begin to become homozygous and thus recessive (most) mutant genes are unmasked and mutant traits observed. This is the first opportunity to select for desired mutants. Screening and selection can be carried out

in subsequent M3 family rows which provide information on segregation within the family.

Selection of (3) different mutant genotypes (individuals or lines) with a desired mutation (circled). The aim is to screen for plants or families with the desired mutation, but with the least distribution of the elite genetic background, i.e., low mutation load. Mutant lines may be developed

directly as cultivars (normally M7/8 generation) or used in cross-breeding.

Figure 2 Simplified scheme for plant mutation breeding using random mutagenesis in an inbreeding species. plants [16, 39]. The mode of action is primarily through in a closed irradiator, after treatment these need to double stranded breaks that are then subject to repair be maintained at least until mature pollen is available (for mechanisms. These can be exploited to speed up the intro- crossing) or, preferably, embryos are sufficiently devel- gression of same species of alien genes for crop improve- oped for rescue and culture. ment. Treatments have been applied to both somatic Mitotic intra-chromosomal recombination can be (mitotic) and gametic (meiotic) cells. The method has induced by stress treatments that cause double-strand been used most successfully in wheat, notably in the tran- breaks in DNA, such as physical and chemical mutagens sfer of disease resistance genes from alien species [40]. and heat stress [42, 43]. Lebel et al. found that low dose A classic example is the transfer of rust resistance into X-ray treatments could double the frequency of spon- bread wheat, Triticum aestivum from the wild, related taneous somatic recombination whereas treatment with species Triticum umbellulatum by ER Sears in 1956 [41]. In the chemical mutagen, mitomycin C increased the rate this work wheat aneuploid plants carrying an additional nine times in tobacco. Induced mitotic recombination T. umbellulatum chromosome were irradiated with events may therefore create new genetic variability that X-rays at the onset of meiosis. The irradiation stimulated can be harnessed by plant breeders. Induced recombina- double-strand breaks and the repair mechanism produced tion has obvious benefits as it allows recombination non-homologous chromosome recombination. Unfortu- of chromosomes that would rarely recombine in nature nately, the application of physical mutagenic treatments (normal meiosis). This can be exploited in transferring to pre-meiotic plants or plant parts is difficult due to desired genes into breeding materials and in breaking up sample space issues in conventional closed irradiators. linkage blocks where desired genes may be freed from It is possible to irradiate whole plants in specialized linked to deleterious genes (linkage drag). field or glasshouse facilities, where plants are arranged Despite its long history, induced recombination around a central mutagen such as a gamma or X-ray remains a largely academic practice as further develop- source, which is activated for a period of time to irradiate ments are needed for practical, large scale application in the sample, but such facilities, are limited. An alternative accelerated plant breeding. is to use detached floral parts (such as wheat spikes) In addition to enhancing recombination, supressing or short plants and placed these inside a sample chamber meiotic cross-overs is also of interest to plant breeders in

http://www.cabi.org/cabreviews B.P. Forster et al. 7 an approach called ‘reverse breeding’ (reviewed by [44]). classed as GM and whether they should be subject to In this approach high performing heterozygous genotypes genetically modified organism (GMO) legislation. These are selected and their meiosis is genetically engineered issues will have a significant effect on the uptake of NPBTs. to reduce or eliminate recombination events. The result Classic examples of first generation of GM crops of this is that male and female spores carry non- include: slow ripening tomato, herbicide resistant soya, recombinant parental haploid genomes, which may then insect resistant maize and pest resistant cotton. Future be manipulated to produce DHs (see section below targets are wide ranging and in addition to include stan- on Doubled Haploidy) and thus generate parental lines dard plant breeding goals of yield, quality, nutrition, pest for F1 hybrid breeding that reconstitute desired hetero- and disease resistance and abiotic stress tolerance, zygous genotypes. also include novel ventures such as the production of pharmaceuticals, biodegradable plastics, pigments and cosmetics. It also needs to be recognized that there are GM Breeding now huge hectarages of GM crops growing in a wide range of countries [47]. GM is normally defined as an alteration of the genotype by Issues in GM breeding, methods, benefits, safety, the insertion or alteration of a specific DNA sequence regulations and public concerns, have been reviewed by using ‘recombinant DNA technologies’ involving artificial Halford and Shewry [48]. delivery systems. Early GM technology focused on the insertion of DNA from a foreign species, but there has been a trend away from transgenics (foreign DNA inser- Rapid Generation Cycling tion) to cisgenics (same species DNA insertion) and most recently to targeted mutagenesis (genome editing) of a The production of new generations is a fundamental favoured genotype. In addition, methods in GM technol- component of plant breeding as this allows another round ogy are no longer confined to tissue culture methods (and of meiosis from which new recombinants can be pro- thereby tissue culture responsive genotypes), thus open- duced. The time taken to obtain new segregating materials ing the way for greater application [45]. Like mutation is often a major time constraint. Classic forms of speeding breeding, the starting point for GM breeding is typically a up generation times are single seed descent (SSD) and successful genotype, which can be improved for a specific shuttle breeding. SSD is an old technique first proposed trait (see Figure 2), but unlike random mutation induction by Goulden [49], later modified by [50]. In this method targets a specific gene. Farmers and end-users generally only one seed from a population of F2 plants is grown on favour cultivars/products they are accustom to as these to F3; this process is repeated in the next generations up have tailored agronomic, harvesting, shipping, processing to F5/6 when plants approach a high level of homozygosity. and marketing procedures. New, improved favoured Since only one seed is required per plant in early gen- cultivars have commercial advantages, especially if the erations these may be grown in small pots or densely in breeder has ownership of the target genotype. The more seed trays, which facilitate early flowering and can be sophisticated the GM technology used the less time it will grown in a small area. Greenhouses and off-season nur- take to clean up the genetic background, i.e., reduce the series may be used to produce more than one generation number of backcross generations; the ideal scenario being in a year. Disadvantages of SSD are that it does not allow targeted gene editing with no alteration of the genetic selection in early generations (although this means that background (no off-target effects). Thus GM breeding has more rounds of meiosis are undertaken before selection the potential to be very fast. A major disadvantage of GM is applied which can be an advantage) and it is important breeding is that the target gene must be known and that there is very low mortality/plant loss in each gen- sequenced. Currently this is a bottleneck, for example in eration or the risk of unconscious disadvantageous sel- 2012 only 702 functionally characterized genes were ection becomes real. Shuttle breeding was developed by annotated in rice (a well-researched crop), which repre- the famous plant breeder Norman Borlaug who won the sents < 2% of the predicted loci [46]. Until our knowl- 1970 Nobel Peace Prize for his involvement in the Green edge of genes improves, random mutagenesis remains a Revolution [51]. His idea was to speed up the breeding viable option. Other disadvantages of GM breeding are: it process by growing successive generation in the same requires specialized laboratories and is expensive, though year at different sites. His original work involved growing cheaper, easier options are being developed. Examples of wheat during the summer at high altitude sites in new plant breeding technologies (NPBTs) are zinc finger Chapingo and Toluca, Mexico and then almost 2000 km nucleases, TAL effector nucleases and clustered regularly north at Obregon, Mexico at sea level with irrigated interspaced short palindromic repeats technology [45]. conditions, and then back to the high-altitude sites. This NPBTs have yet to be proven, but have the potential to produced two generations a year and thus cut the produce GM cultivars that cannot be distinguished from breeding time by half. Shuttle breeding can cross greater those produced by conventional plant breeding, and this distances, such as UK$New Zealand, but then involves has sparked a debate about whether or not they should be certain regulations such as phytosanitary certificates and

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Repeated generation cycles (6–8/year)

Wheat Sorghum Barley Embryo rescue and transplant Planting in small pots, light watering and continuous of emerging plants into small lighting pots

Figure 3 Scheme for rapid generation cycling using wheat, sorghum and barley as examples, adapted from Zeng et al. [52]; Ghanim et al. [53]. In the above scheme plants (wheat, sorghum and barley) are grown in small pots in controlled environmental conditions (greenhouse). The aim is to limit vegetative growth and promote early flowering, but to provide sufficient seed for the next generation. Tiller production may be completely prevented in small pots, allowing growth of the main stem only. However, if preferred tiller production may be encouraged by growing in larger pots, this provides stronger plants and additional/spare spikes for crossing and more embryos, but increases the time to flowering. The time to flowering may also be promoted by growing under continuous lighting, limited watering and raised temperatures. The milk-ripe stage of endosperm development (15–20 days after fertilization) provides embryos that are easily detached from their endosperm in small grain cereals, and are sufficiently developed to germinate immediately (1–2 days) in culture on simple media containing basic nutrients (such as half-strength Murashige and Skoog (M&S) medium, [54]) and sugar [52, 53]. In vitro seedlings may be sampled for DNA analysis in MAS. In cases of winter genotypes, vernalization treatments may be applied to in vitro seedlings. material transfer agreements. Since shuttle breeding performed (see section on Marker Assisted Selection). exposes breeding lines to more than one environment, Some in vitro phenotypic screen may also be carried out which may include a range of biotic and abiotic stresses, such as salt tolerance and osmotic (drought) stress. Thus selection may be made for a wide range of traits, but this only preferred individuals are advanced to the next gen- may also be hazardous as the breeding lines may succumb eration cycle. to a new stress. Recently, a procedure was described that dramatically shortens the generation times of wheat and barley, High-throughput Phenotyping allowing the production of eight generations of wheat and nine generations of barley per year [52]. The procedure The ability to phenotype has lagged behind high- combines growing plants in greenhouse conditions, in throughput methods in genotyping, so much so that it small pots with limited watering and high light exposure. has led to the problem of bridging the ‘phenotypic gap’. These combine to speed up development. After flower- One response to this has been the emergence of large ing/crossing, embryos are excised and cultured, thus scale phenomics facilities which involve growing potted circumventing the need to grow seeds to maturity and plants in greenhouse with carefully regulated environ- avoiding any seed dormancy, thus saving several weeks. ments, These facilities are highly automated and equipped The methods are simple and have been extended to a with sophisticated data-capturing devices linked to large- wider range of cultivars and other crop species (Figure 3). scale computing and bioinformatics capacities, e.g., Table 1 shows examples of the time savings between Australian Plant Phenomics Facility and European Plant generations; these are for spring genotypes that do not Phenotyping Network. Phenomics facilities can measure require vernalization treatments. In the scheme presented and analyse phenotypic data at all stages in development. in Figure 3, the culture of embryos that germinate readily However, costs are high and since the growing environ- into seedlings can act as a convenient stage to apply ver- ment is artificial the trait data must be correlated with nalization (cold) treatments. The procedure has recently performance in the field, and this is notoriously difficult. been applied to sorghum with up to six generations being A more pragmatic approach has been the development of produced in one year using small pots, continuous lighting, low cost high-throughput field phenotyping systems [20]. limited watering and embryo rescue [53]. The in vitro stage Araus and Cairns [20] have reviewed recent advances is also ideal for taking tissue samples, e.g., leaf samples for in field-based high-throughput phenotyping which include: DNA extraction from which genotypic assays can be (1) field sensing and imaging; and (2) field sampling for

http://www.cabi.org/cabreviews B.P. Forster et al. 9 Table 1 Normal and accelerated generation times with respective number of generations per year for wheat, barley and sorghum genotypes (data taken from [53])

Normal generation Normal number of Accelerated generation Generations Crop time (month) generations per year1 time (day)2 per year Wheat 4–5 1–2 45 7 Barley 4–5 1–2 40 8 Sorghum 5–6 1–2 60 6 1The number of generations is often limited to one per year (during the cropping season), in a few environments two generations may be grown per year in the ﬁeld. 2These are averages taken from 5 to 10 genotypes tested for each crop species with the aid of embryo culture.

Table 2 Methods in DH production in crop plants

Method Basics of methods Usage and crop examples References Androgenesis Culture of male haploid gametic Widely used: apple, asparagus, Maluszynski et al. [58]; cells using anthers or isolated aspen, brassicas, bread wheat, Touraev et al. [60] microspores barley, broccoli, citrus, durum wheat, flax, maize, oak, potato, rapeseed, rice, rye, ryegrass, swede, timophy, tobacco, triticale Gynogenesis Culture of female haploid gametic Specific use: sugar beet, onion Bohanec [61] cells using flowers, pistils, ovaries or ovules Specialized Production of haploid embryos after Wide crossing is commonly used in Maluszynski et al. [58]; crossing pollination with another species or many species: barley, bread wheat, Touraev et al. [60]; genus (wide crossing) or treated durum wheat, oat, triticale. Pollination Kosˇmrlj et al. [62] pollen. Involves embryo culture with treated pollen, e.g., irradiated pollen is more specialized: pumpkin Spontaneous The recovery of naturally occurring Specific use: oil palm Dunwell et al. [63]; haploids Nasution et al. [64] laboratory-based analyses. A method used in both is near- many crops (e.g., rice, bread and durum wheat and barley) infrared reflectance spectroscopy (NIRS). NIRS technology and satisfy two of the three DUS criteria for cultivar is rapidly advancing and aeronautical and portable status, i.e., uniformity and stability. Doubled haploidy devices are available for field screening. NIRS is also finding increased the efficiency of selection, especially for reces- application in grain analysis where it is normally applied to a sive traits from F1 or for mutant traits (which are gen- few grams of seed per samples [55]. However, single erally recessive) from M1 plants. DHs may also be used as seed NIRS methods are being developed and this would parental lines in the production of F1 cultivars, e.g., maize, allow for early generation screening and accelerate the pepper and rye. Doubled haploid techniques have now breeding process. The new generation of high-throughput been applied to over 200 plant species and have become a phenotyping screens for plant breeding are fast, cheap, standard tool in accelerating the breeding of a wide range non-destructive and accumulate large data sets; whereas of crops [58–60]. DHs are produced from haploid tissues, in high-throughput genotyping the data analysis requires organs and plants which may undergo spontaneous or good computing and bioinformatics support. A dis- artificial chromosome doubling. There are four general advantage of field screening is that it is season dependant. methods of haploid/DH production in crop plants; these For some traits simple high-throughput phenotypic are listed in Table 2. protocols are well established, such as tests for salt tol- Figure 4 shows a scheme for the production of DH erance and some disease resistance traits (e.g., [56, 57]). cultivars from an initial cross in an annual species; this These are generally performed in greenhouse conditions takes 5 years, about a third of the time for classic pedigree and on several hundred plants, usually seedlings and may inbreeding (Figure 1). Furthermore, the DH breeding or may not be season dependent. Tests are cheap, simple system does not suffer from biasing effects of dominance and quick and correlated to field performance. and competition between individual genotypes in early generations, thus selections can be made early, which has particular advantages for quantitatively controlled traits Doubled Haploidy such as yield. A feature and potential limitation of DH breeding is that recombination is confined to meiosis in Doubled haploidy is of interest to plant breeders as the F1 generation. This will help preserve linkage it is the fastest means of producing homozygous lines. blocks. However, if there is a need to break linkages it Homozygous lines are the final outcomes of breeding in will be necessary to provide greater opportunities for

http://www.cabi.org/cabreviews 10 CAB Reviews

P1 × P2 Initial cross Year The F is grown up and provides material for doubled haploid F 1 0.5 1 production, though this may be done at any generation.

The DH generation is grown up and seed recovered from 1 1 DH1 selected lines in glasshouse conditions.

Observation of rows or small plots in the field, selection for 2 DH2 agronomic performance.

Large replicated plots; data on yield, quality and agronomic 3 DH3 performance. Seed production.

Multi-locational trials, data on yield, quality and agronomic 4 DH4 performance. Identify best lines.

Seed multiplication and official testing and certification for 5 DH5 cultivar release.

Figure 4 Scheme for plant selection and rapid advancement using doubled haploidy. recombination. This may be done by increasing the size of where single plants, rows and plots are subject to the the DH population or reverting to the SSD method which ‘breeder’s eye’. Individual plants, families and lines are has the potential to generate new recombinants at selected that appear to perform better than control each generation until homozygosity is reached. Compar- checks. Target traits have typically focused on yield, isons of the DH, SSD and PIM breeding methods have quality and agronomic fitness, but also pest and disease been made by several workers with respect to speed, resistance and tolerance to drought and salinity where recombination frequency, population size, practicalities, fields are subject to these environmental influences, inputs and costs (list of references has been compiled or where glasshouse screens are available. This is a slow [59]). Some negative features of DH breeding that need processes often confined to seasonal growing periods to be considered are: (1) variation is generated at the and, for many traits with a strong environmental com- beginning of the process and the breeder must therefore ponent, often unreliable. MAS offers solutions. Biochem- choose suitable parental lines that will generate the ical markers (proteins and isozymes) were among the first desired variation (these may be generated from a PIM markers used in genetic studies and plant breeding, these scheme); (2) not all genotypes are responsive to DH were both practical and cheap, e.g., endopeptidase marker production methods. Low-cost molecular methods can be for eyespot disease in wheat [66] and in some cases moni- applied to diversity allele selection in newly created DH tored the trait directly, e.g., storage proteins involved in lines [65]. bread-making quality [67]. However, biochemical markers DH can also be used in accelerating backcross con- were limited and have been eclipsed by DNA markers. version (BCC). The aim in BCC is to introgress a desired Over the past decades the most widely used markers gene into a favoured cultivar/genotype (see also Mutation in plant breeding have been simple sequence repeats Breeding). The most successful BCC schemes involve (SSRs, also known as microsatellites [68]). SSRs are simple genotyping for both the introduced gene of interest and and cheap to use, reliable, co-dominant and have a high the elite genetic background integrated MAS, thus well- level of polymorphism. As a consequence high-density defined genetic marker maps and marker–trait associa- SSR maps have been produced and SSR markers linked tions must be known or readily obtained. A standard BCC to genes of interest have been found. Other commonly scheme is described in Figure 5. used markers include sequence-tagged sites, sequence Alternatives to BCC are mutation and GM breeding characterized amplified regions and single nucleotide (see Plant Mutation Breeding and GM Breeding sections polymorphisms. An historical account of the development above). of DNA markers in plant breeding is given by Moose and Mumm [69]. Perfect markers are those that define the alleles of the target gene, however, linked markers are Marker Assisted Selection good enough for plant breeding purposes especially if a pair of tightly linked flanking markers ( < 1–5 cM on either Plant breeding has been and continues to be based on side) are deployed. Advantages of MAS are: they can phenotypic selection. This is generally done in the field replace unreliable phenotypic screens, can be used at any

http://www.cabi.org/cabreviews B.P. Forster et al. 11

Initial cross between recipient and donor parents (2n=14 P1 X P2 chromosomes).

X P F1 1 Progeny from cross: first generation (50% DNA from P1).

Example of one genotype from the BC2 population (population BC2 X P1 has 75% DNA from P1), with selection for a marker/trait.

Example of one genotype from the BC3 population (population X P BC3 1 has 75% DNA from P1), with selection for a marker/trait.

Example of one genotype from the BC4 population (population BC4 X P1 has 75% DNA from P1), with selection for a marker/trait.

Example of one genotype from and advanced BC population, with selection for a marker/trait. This is then selfed to produce a BCn-1 Selfing near isogenic line for P1.

BCn Near isogenic line of P1 carrying the marker/trait introgressed inbred line from P2.

Figure 5 Scheme for backcrossing conversion using marker-assisted or trait selection. time (not season dependent), reduce the number of lines breeding lines are not contaminated with GMOs. A major for promotion to the next generation and can be used in limitation to MAS is the cost of DNA extraction. Costs rapid generation cycling (more generations per year, see, have been coming down with time and recently low-cost e.g., Figure 1). Thus selected lines can be fast-tracked with methods have been developed [74]. High-throughput savings in time and costs. genotyping methods have recently been used to develop In addition to MAS for target genes, genetic finger- a new approach for breeding natural quantitative alleles. printing can be deployed to monitor and select for the Called genomic selection, the method employs thorough desired genetic background. This is particularly important phenotyping and high density genotyping of a ‘training in crossing programmes where an elite line is crossed to population’. Through an iterative process, a set of mar- one or more donor lines that are genetically diverse. In kers associated with the trait(s) of interest in the popu- such cases gene pyramiding and backcrossing may be lation are defined (‘model training’). These prediction deployed to introgress the desired genes (using markers, models are then applied in selecting the most promising ‘offensive breeding’) and genetic fingerprinting used novel breeding lines from the ‘breeding population’ based to select for the desired genetic background (‘defensive on their marker genotypes only [25]. A major advantage breeding’). Fingerprinting markers should have wide gen- of genomic selection is that it allows increased numbers of ome coverage. Amplified fragment length polymorphisms breeding cycles per unit time. As outlined above, conven- and restriction fragment length polymorphisms were tional breeding requires 10 or more years from crossing commonly used fingerprinting methods, but have given before the best progeny, those which carry an improved way to hybridization based and more recently sequence- allelic combination, are identified and can be recombined based procedures [70] Rapid developments in genotyping for the next breeding cycle. Genomic selection, once a have promoted the concept of ‘molecular breeding’ predictive model is well established and validated, allows and more recently ‘genomics assisted breeding’ whereby the identification of superior individuals or lines much selection is done in a marker laboratory rather than in the earlier, even superior F2 plants can be selected based on field [71–73]. their genetic fingerprint and immediately recombined. Such Despite the advantages of MAS there are relatively a procedure is expected to increase the frequency of few examples of practical use in plant breeding. Examples desired alleles in a breeding population much faster, are typically confined to high value traits, such as disease increasing the selection gain per unit time, but experi- resistance or publicly sensitive issues, such as ensuring mental results supporting this hypothesis are still pending.

http://www.cabi.org/cabreviews 12 CAB Reviews Table 3 Historical perspective of innovations providing efﬁciency gains in plant breeding

Era Innovation Landmark papers and recent reviews 1822–1865 Progeny prediction: Mendel studies publishes laws on Mendel [15]; Weiss [80]; Ellis et al. [81] inheritance which provide a scientific basis for plant breeding 1901–1934 Mutation breeding: dawn of plant mutation breeding, from De Vries et al. [82]; Kharkwal [83]; de Vries’ ideas to the first mutant variety. Mutagenesis allowed Shu et al. [35, 36] the production of new biodiversity directly in elite germplasm 1902–1960 Plant tissue culture: the dawn of plant tissue culture; from the Haberlandt [84]; Vasil [85] concept of cell totipotency, to a wide range of cell, tissue and organ culture methods 1921–1952 Doubled haploidy: dawn of haploid/DH technologies; from Blakeslee et al. [86]; Touraev et al. [60] first observations of haploids to applications in plant breeding. Provided a quick means of producing homozygous lines 1923–present Screening: Phenotypic and genotypic screening; from the dawn Sax [87]; Varshney et al. [88]; Araus of selection by phenotypic associations (seed weight in beans) and Cairns [20] to high-throughput phenomic and genomic screening 1941–present Rapid generation cycling: speeding up generation times; from Goulden [49]; Brim [50]; Zeng et al. SSD to the incorporation of a range of methods in shortening [52]. This review; [89]; De La Feunte the time between successive generations et al. [90] 1980s–present GM plant breeding: genetic modification: use of recombinant Bevan et al. [91]; Fraley et al. [92]; DNA technologies to provide novel genetic variation. Herrera et al. [93]; Halford and Shewry Techniques are becoming increasingly sophisticated and [48]; James [47]; Varshney et al. [94] include targeted mutagenesis and other NPBTs 1980s–present Molecular plant breeding: based on developments in Paterson et al. [95]; Tanksley [96]; high density genetic maps, genetic fingerprinting, robust Collard and Mackill [72]; Moose and marker–trait associations and bioinformatics Mumm [69]; Varshney et al. [94]

Other Issues potential for food security, particularly for less developed regions where, for many crops, only landraces with low Preferred cultivars productivity are available. Many crops have ‘market preferred’ cultivars. This is true for high-value crops such as coffee, banana and citrus Patents, IP and legislation and local subsistence cultivars or landraces. Such crops Intellectual property rights and innovations in plant are grown extensively and often become susceptible to breeding are often protected by patents [75], for example, disease or small changes in the environment. Breeders Dunwell [76] lists over 30 granted patents related to plant therefore have a dilemma in attempting to breed a new haploids [76]. This is not normally a problem for breeding improved cultivar. On one hand they need to produce a companies who live in a commercial world and can pay for new improved cultivar, on the other they do not want to their use. GM breeding however is particularly restricted disrupt a winning genotype, which conventional crossing by legislation and is currently a hot topic for action and will entail. Subtle changes are therefore desirable and this re-action; some GM breeding companies have indicated will promote the use of gene modification methods such they may withdraw from crop production in some as mutation and GM breeding, which are also faster than countries and conversely some countries have called for a conventional breeding. reduction in legislation to promote GM crops (https:// www.gov.uk/government/publications/genetic-modification- Participatory plant breeding gm-technologies). Several countries promote ‘farmer participatory plant breeding’ and in such cases the farmers have early access Accessing foreign germplasm to (pre-release) seed. Participatory breeding has huge Germplasm needed for breeding may not be available in- potential particularly in less developed regions of the house and may need to be accessed from other breeders, world. Breeders working in close cooperation with gene banks or collections. The transfer of materials across farmers can thus include regionally or culturally desired boundaries often requires the use of phytosanitary certi- cultivar traits much better in their selection programme, ficates, Standard Material Transfer Agreements and quar- such as taste, aroma, shape or other important characters antine. A further problem may arise from restrictions in in addition to typical breeder’s traits such as yield per- germplasm exchange among breeders. In the past ex- formance and stress tolerance. Participatory breeding change of novel breeding lines was common practice and does not necessarily speed up the breeding scheme itself, included the reciprocal agreement to use exchanged lines but facilitates the adoption of cultivars by growers, in crosses. This has altered recently: breeding lines are especially those in rural areas. This approach had high often exchanged with a ‘test only’ restriction. There is

http://www.cabi.org/cabreviews B.P. Forster et al. 13 also an International Treaty on Plant Genetic Resources Table 3 provides a list of some signiﬁcant innovations in in Food and Agriculture, (http://www.planttreaty.org/). plant breeding from 1822 to the present day. These compliances come with costs and time delays.

Pragmatism Summary Breeders will always use useful material, and have been effective in developing cultivars with novel traits using Food, feed and fuel security, climate change and profit- conventional methods. For example, the 1B/1R trans- ability are major issues which demand rapid plant breeding location was bred into many successful wheat cultivars responses. Although some issues have remained recalci- because it carried desirable disease resistance. The cause trant to improvement (such as the ability to manipulate of the resistance, the wheat/rye translocation, was not meiotic recombination effectively), thus far these have discovered until later [77]. Similarly, graphical genotyping been compensated for by developments in selection for [78] applied to foundation barley cultivars over time has desired phenotypes/genotypes, including: rapid generation revealed the introduction of novel markers in successful cycling, doubled haploidy and MAS. Since the time cultivars, and once introduced are retained in subsequent of Mendel plant breeders have applied innovations in successful cultivars. This will also be true for genes intro- science and biotechnology to promote plant breeding. duced by GM techniques – once they are released, they Plant breeding continues to be a dynamic process and can be used in conventional crossing programmes. Today based more and more firmly on inter-disciplinary science, we know that these are associated with useful traits, such yet more techniques are likely to arise and old ones find as disease resistance [79], but for the breeder perfor- new application within these. Mutation breeding perhaps mance in the field was the main selection criterion. is a good example of this, of where it was heralded ori- For the future, genotypic data, and pedigree information ginally as an all-encompassing solution to plant breeding of successful cultivars could provide useful information in problems, failed to fulfil its initial promise (in large part genotypic breeding, especially if it can be linked to target because of the inability to target any changes and traits. National list trials provide an ideal opportunity to the generation of a plethora of unwanted undesirable combine data on performance, pedigree and genotype affects), to rise again with the possibility of controlled and of elite lines. However, since plant breeding is also a site-directed changes and methodologies for massive commercial enterprise, these data are now seen as pro- screening. prietary and not openly available.

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