Transferring alien genes from related species and genera for wheat improvement
A. Mujeeb-Kazi, S. Rajaram

Wide hybrids in the Triticeae tribe have been attempted and studied for over a 100 years. The first such hybrid was between wheat and rye (Wilson, 1876). Rimpau (1891) described 12 plants recovered from seed of a wheat-rye hybrid that represented the first triticale. Farrer (1904) reported studies on wheat-barley hybridization; however, Shepherd and Islam (1981) considered it improbable that these were true intergeneric hybrids. Several perennial grasses were hybridized with wheat in the early 1930s with the objectives of transferring disease resistance and perenniality into annual crops. Many hybrids involving Triticum and several Aegilops species were also made during the 1920s and 1930s from which the genomic relationships of the two genera were derived. The large-scale practical use of the hybrids, however, was delayed until the advent of colchicine treatment in the late 1930s. The ability to double the chromosome number of hybrids using colchicine had both practical and theoretical consequences. The production of fertile amphiploids provided the way to develop X Triticosecale Wittmack as a new cereal crop. Also advanced were evolutionary studies when McFadden and Sears (1946) resynthesized T. aestivum, discovering that Ae. tauschii (syn. T. tauschii) was the D-genome donor to bread wheat.

With the advancement of hybridization techniques (Kruse, 1973) and embryo culture (Murashige, 1974), wide hybridization became a more common practice involving more perennial species. In reviews of the progress of wide hybridization, intense interest was expressed among breeding programmes in utilizing the genetic resources available in the perennial Triticeae for cereal improvement (Dewey, 1984; Sharma and Gill, 1983a; Mujeeb-Kazi and Kimber, 1985; Sharma, 1995; Wang, 1989).

Of the approximately 325 species in the tribe Triticeae, about 250 are perennials and 75 are annuals (Dewey, 1984). Relatively few perennials have been hybridized with wheat essentially because of the complexity of doing so and due to embryo rescue/regeneration constraints. The perennials, which include many important forage grasses, have the potential to serve as a vital genetic reservoir for the improvement of annual grasses. These include the major cereals: bread wheat, durum wheat, triticale, barley and rye. Perennials successfully utilized for improving wheat are predominantly in the Thinopyrum group.

This chapter will focus on achieving agricultural production targets with emphasis on bread and durum wheat. Such production targets are to be achieved by enforcing crop improvement protocols based upon the utilization of genetic diversity, crucial for durability of stress resistances and tolerances and for ensuring sustainability. Major emphasis is devoted to consideration of the exploitation of ‘alien’ genetic diversity, encompassing interspecific and intergeneric hybridization categories.

DISTRIBUTION OF GENETIC DIVERSITY: GENE POOLS

Though genetic diversity can be induced, for more controlled, well-directed incorporation, diversity naturally present in the annual and perennial Triticeae species has priority. This natural diversity resides in the conventional wheat germplasm and in closely or distantly related alien species sources. The species resources are distributed within gene pools, and genetic transfers can be realized for wheat improvement from these pools over short- or long-term time frames. The gene pools are structured upon the genomic constitution of the species and are comprised of three groups: primary, secondary and tertiary.

The primary gene pool species include the hexaploid landraces, cultivated tetraploids, wild T. dicoccoides and diploid donors of the A and D genomes to durum and bread wheats. Genetic transfers from these two genomes occur as a consequence of direct hybridization and homologous recombination with breeding protocols contributing different back-crossing and selection strategies. Some cross combinations require embryo rescue, but no cytogenetic manipulation procedures are necessary. The secondary gene pool is formed of the polyploid Triticum plus Aegilops species, which share one genome with the three genomes of wheat. The diploid species of the Sitopsis section are included in this pool, and hybrid products within this gene pool demonstrate reduced chromosome pairing. Gene transfers occur as a consequence of direct crosses, breeding protocols, homologous exchange between the related genome or through use of special manipulation strategies among the non-homologous genome. Embryo rescue is a complementary aid for obtaining hybrids. Diploid and polyploid species are members of the tertiary gene pool. Their genomes are non-homologous. Hence, genetic transfers require special techniques that assist homoeologous exchanges, facilitated by irradiation or callus culture mediated translocation induction.

Diploid and polyploid species with genomes that are non-homologous to wheat reside in the tertiary gene pool. Homologous exchanges cannot affect genetic transfers, but genomic homoeology of these species does permit the transfer of genes by somewhat complex protocols.

UTILIZATION OF GENE POOL DIVERSITY

For practical end products to be obtained, some transfer prerequisites that encompass all three gene pool species are hybrid production, embryo rescue, plant regeneration, cytological diagnostics, breeding methodology and stress screening, culminating in the stability of the advanced derivatives contributed by homozygosity. Based upon these prerequisites and genetic transfer ease, primary gene pool diversity is most important for wheat improvement. The species of the diploid A and D genomes contribute novel genes and allow direct recombinational exchanges with their respective genome partners to facilitate both durum and bread wheat improvement over a relatively short-term time frame than what is provided by the secondary or tertiary gene pool species. Setting the above as a basis for genetic introgression, the strategy and outcome of exploiting the diversity of the three gene pools and their accessions is illustrated.

PRIMARY GENE POOL

The A genome (Triticum boeoticum, T. monococcum, T. urartu; 2n=2x=14, AA)

One avenue of using the A-genome accessional diversity is via bridge-crossing of the AABBAA amphiploids (Figure 11.1a). In general, the durum parents x A-genome accession crosses are simplistic and of high frequency (Table 11.1). Meiosis of F1 hybrids (2n=3x=21, ABA) with up to six bivalents for metaphase I chromosome associations per meiocyte is indicative of genomic exchange among the A genomes (Table 11.2). The meiotic stability of the AABBAA, 2n=6x=42 amphiploids suggests an ease of maintenance of these genetic stocks (Table 11.3). The durum cultivars in these amphiploids are susceptible for the stresses being addressed in the authors’ studies. Hence, upon stress screening, a resistant amphiploid implies that the particular A-genome accession contributes the expressed resistance. So far, some diversity has been identified in the AAB-BAA amphiploids for Cochliobolus sativus, Fusarium graminearum (scab) and leaf rust resistance (Delgado et al., 2001). Diversity is more extensively observed for Septoria tritici resistance.

FIGURE 11.1
Schematics showing the production of A - and D - genome 2n = 6x = 42 chromosome stocks as a consequence of hybridizing durum cultivars with A-genome diploid (a) and D - genome diploid (b) accessions

The D genome (Aegilops tauschii = goat grass; 2n=2x=14, DD)

The A-genome strategy cover the D genome that demonstrates an genetic diversity for stresses as observed the AABBDD synthetic (MujeebKazi, 2001a). Crossing the resistant synthetic hexaploids with elite but susceptible bread wheat (BW) cultivars has yield resistant BW/SH derivatives (mujeeb-Kazi et al., 2001a, 2001b). Currently, 620 SH wheats (Plate 9, Table 11.4) have been produced, and a wide array of these wheats are being globally utilized for wheat improvement either at the SH or at the BW/SH advanced derivative level. The most advanced in the authors’ wheat breeding programmes are the D-genome resistances for Karnal bunt (Tilletia indica), S. tritici and C. sativus. Promise also exists for resistances and tolerances in this SH germplasm for leaf rust, stripe rust, mineral toxicities, drought, salinity (Pritchard et al., 2001), heat, cold, sprouting, waterlogging (Villareal et al., 2001), high molecular weight (HMW)/low molecular weight (LMW) quality subunits, powdery mildew, loose smut, cereal cyst nematode (CCN), yield and its components. The least accessional diversity observed so far in the D genome is for scab or F. graminearum (less than 1.0 percent), but under evaluation tests conducted at one location in Mexico, the observed scab resistance is promising and superior than that of the leading bread wheat cultivars Frontana and Sumai-3 with their assemblage of four genes (van Ginkel et al., 1996). No accessional diversity has been observed for aluminium tolerance so far after the screening of the authors’ elite synthetic hexaploid set of 95 entries.

TABLE 11.1
Mean production frequencies of F₁ hybrids (n=3x=21) from some Triticum turgidum (4x)/diploid A-genome species accessions

TABLE 11.2
Mean meiotic metaphase I chromosomal associations in some F₁ hybrids of Triticum turgidum (4x)/diploid A-genome species accessions

TABLE 11.3
Mean meiotic metaphase I chromosomal associations in some F₁ hybrids of Triticum A-genome (2n=2x=14) amphiploids (2n=6x=42, AABBAA)

TABLE 11.4
Crossability, plant regeneration and colchicine-induced doubled seed data for some synthetic hexaploids between Triticum turgidum and Aegilops tauschii accessions

^aAe. tauschii accession number in parenthesis.
^bCIGM = Cruza Intergenerica Mexicana (Intergeneric Mexican Cross).

SECONDARY GENE POOL

Included here are the Aegilops and Triticum species (mostly polyploid) that share one genome in common with wheat. Also included are the diploid Aegilops species of the Sitopsis section that are related to the B genome of wheat. Special emphasis is currently given to the use of Ae. speltoides accessions (2n=2x=14) via the hexaploid amphiploid bridge-crossing route (2n=6x=42, AABBBB) (Figure 11.2). These newly produced amphiploids have shown initial promise for resistances to C. sativus, F. graminearum, S. tritici, barley yellow dwarf virus (BYDV), leaf rust and stripe rust (Delgado et al., 2001). More testing for the above stresses together with others is required to exploit the Sitopsis species potential on a large scale; Ae. speltoides and its accessional genetic diversity is just one example.

TERTIARY GENE POOL

Of the approximately 325 species in the tribe Triticeae, about 250 are perennials (Dewey, 1984) with the rest being annuals. The bulk of these species have been difficult to exploit in cereal improvement primarily because their genomes are non-homologous to those of wheat, and genetic transfers cannot be made by homologous recombination. Genomic homoeology is prevalent, which enables gene transferring via an array of complex and tedious cytogenetic protocols. Hybridization per se is no longer a major obstacle within this pool and wheat (Sharma, 1995), though achieving swifter practical outputs from genera other than Secale and Thinopyrum is a definite constraint.

FIGURE 11.2
Schematic for AABBBB production involving Triticum turgidum and Aegilops speltoides (2n=2x=14, BB or SS)

TRANSFER PREREQUISITES ACROSS GENE POOLS

Some prerequisites for achieving gene transfers from the annual and perennial Triticeae across all three gene pools are related with varying degrees of complexity to hybrid production, embryo rescue, plant regeneration, hybrid validation through multiple diagnostic protocols, breeding methodology, biotic and abiotic stress screening and stability of the wheat/alien derivatives achieved by the use of sexual homozygosity inducing protocols.

HYBRID PRODUCTION

The production of the earliest interspecific and intergeneric hybrids was accomplished by the aid of the simplest techniques of emasculation and pollination that were in conventional use in wheat breeding programmes. With these techniques, many important hybrids were and still are made. A tabulation of hybrids involving wheat and its relatives is maintained by Kimber and Abu-Bakar (1979) and currently contains information on some 1 104 hybrids and 270 bibliographic references. In the of this large range of hybrids, considerable variation in the difficulty of making particular hybrids was observed.

In the Triticeae, hybridization of alien species with those of Triticum goes back to 1876 when Wilson consciously made the first wide hybrid involving wheat and rye. Rimpau in 1891 obtained seed on a presumably doubled sector in a wheat x rye hybrid and described 12 plants that must represent the first triticale. A more divergent hybrid (wheat x barley) was first reported by Farrer in 1904, which was considered rather improbable to be a true hybrid (Shepherd and Islam, 1981).

Though many hybrids involving Triticum and Aegilops species were produced during the 1920s and 1930s, it was the pioneering work of the late Anton Kruse reporting hybridization of T. aestivum x Avena sativa (Kruse, 1969), Hordeum vulgare x Secale cereale (Kruse, 1967) and H. vulgare x T. aestivum (Kruse, 1973) that led to an increase in research momentum in the area of intergeneric hybridization involving T. aestivum or T. turgidum with species of Agropyron, Aegilops, Elymus, Haynaldia, Heteranthelium or Hordeum, an intensity that has magnified over the last decade and a half (Mujeeb-Kazi and Hettel, 1995).

A wide hybrid production procedure in which wheat is the maternal parent is routinely adopted with significant success (Mujeeb-Kazi et al., 1987). The procedure involves early or bud pollinations, post-pollination gibberellic acid treatment (75 ppm aqueous) of the maternal floret tissue, embryo excision from 14 to 18 days post-pollination, embryo culture on Murashige and Skoog’s (1962) or Taira and Larter’s (1978) media and cold treatment to break dormancy that eventually culminates in plantlet differentiation. Despite this protocol, hybridization success is limited, and an array of manipulative techniques become essential in order to obtain viable hybrids. These range from pre-pollination to post-pollination hormonal treatments. Genotypes, polyploidy level, crossing procedure and cross direction all seem to contribute to hybrid production success (Mujeeb-Kazi and Kimber, 1985; Sharma and Gill, 1983a). Most hybrids (particularly in the tertiary pool) in the Triticeae have been predominantly produced with the T. aestivum cultivar Chinese Spring. The highly successful role of Chinese Spring is attributed to the kr₁kr₁kr₂kr₂kr₃kr₃ crossability genes that the cultivar possesses (Falk and Kasha, 1981; Fedak and Jui, 1982) for its crossability with rye (Riley and Chapman, 1967). The loci are located on chromosomes of homoeologous group 5 in kr₁ (5B), kr₂ (5A) and kr₃ (5D) respectively, acting in a complementary manner, with the kr₁ kr₁ loci contributing most to the crossability frequencies. The kr₄ locus is now added as are some other crossable cultivars (Asakazikomugi and Fukuhokomugi) with both possessing a superior agronomic type than Chinese Spring (Fedak, 1998; Jauhar, 1995).

HYBRID VALIDATION: DIAGNOSTICS

Initial hybrid identification is based upon mitotic counts in root tips collected at various hybrid development stages (Mujeeb-Kazi and Miranda, 1985). A normal intergeneric F1 hybrid possesses half the chromosome number of each parent involved in the combination. For hybrids of different polyploidy levels, a mere number count is adequate initial verification. There are, however, cases where the alien species are hexaploids, such as wheat, and hybrids would then have 42 chromosomes. These may be difficult to classify categorically as hybrids, but with superb primary and secondary constriction resolution of wheat 1B, 6B and 5D chromosomes (Mujeeb-Kazi and Miranda, 1985), identification of hexaploid hybrids is simplistic. Additional identification can be made by employing chromosome-banding techniques. Karyotypic differences play a part, but positive claim to hybridity must be accompanied by clear meiotic analyses.

In some situations, the alien genome may be totally or partially eliminated, resulting in the production of polyhaploid/haploid or aneuploid F₁ hybrids. The two aspects are classified under: (i) genome elimination; and (ii) aneuploid F₁ hybrids.

A modified hybrid phenotype is also good proof of hybridity, but often a hybrid may not express the features of one parent as in the case of wheat x barley or its reciprocal combination (Islam et al., 1978). In general, however, a majority of the F₁ wheat x alien (reciprocal also) hybrids involving all the A-B- and D-ge-nome diploids, annual and perennial, Triticeae genera and species exhibit a co-dominant phenotype (Plate 10) (Mujeeb-Kazi et al., 1987, 1989). Similar is the phenotype modification of their amphiploids. More recent diagnostic procedures utilized for the detection of hybrids and/or their advanced derivatives are in situ hybridization (FISH, GISH, etc.), electrophoretic analyses (biochemical) and molecular detection of the presence of alien chromatin.

WIDE-CROSS BREEDING METHODOLOGY

In wide-crosses (intergeneric and interspecific), classically the self-sterile F1 hybrids, on colchicine treatment, result in fertile amphiploids that may then have practical utility. The fertile amphiploids are sources of back-cross I (BCI) derivatives (amphiploid/Triticum sources) with eventual production of alien disomic addition lines leading to subtle alien genetic transfers by subsequent cytogenetic manipulation. The method of F₁ hybrid advance under those circumstances where amphiploids are not produced is by pollinating the F₁ hybrid by T. aestivum and attaining the crucial BCI advanced derivative. This BCI derivative may be of the classical type, where the BC parent is the same as that involved in the F₁ hybrid pedigree. There are modifications to this conventional process in that different wheat cultivars than those present in the F₁ hybrid are pollen parents in the F1 hybrid’s advanced cross progeny. This process could also be applied when back-cross derivatives are to be produced from the amphiploid. A uniform wheat background is reportedly advantageous for morphological or biochemical marker applications, but since Chinese Spring is the wheat cultivar most commonly involved in intergeneric crosses, the disomic additions in this background are of little immediate practical value and complicate the field evaluation of the lines under field conditions. Top-crossing with a different wheat several times, followed by a final selfing, finishes the addition lines in a commercial wheat background and results in a plant type that is readily manageable under the additional necessary field screening conditions. When two different, but not too divergent, wheat cultivars are involved in top-crosses, the resulting progeny simulates the F1 top-cross process of the conventional breeding programme. The material at this stage is amenable for field testing, has superior agronomic appearance, an excellent segregation differential and retains an adequate number of alien chromosomes (generally random) for subsequent controlled manipulation, as is the case for most intergeneric combinations.

SCREENING AND STABILITY

Wheat x alien species advanced derivatives improved for agronomic plant type according to the choice of breeding methodology are subjected to stress screening. The interspecific combinations lend themselves favourably to rapid crop improvement and yield-superior plant types that can be selected in shorter time durations. Intergeneric combinations, if aided by an appropriate breeding input (caution in using susceptible wheat cultivars in crosses), yield genetic stocks that blend adequately with field screening protocols requiring normal maturity and quality plant type. Once resistant stocks are selected, genetic manipulation strategies form an essential aspect of integrating the alien chromatin into a euploidlike (2n=6x=42 or 2n=4x=28) wheat product. Genetic recombination between wheat and alien chromosomes determines the simplicity or complexity of the programme.

Filial generation advances coupled with selection give stable derivatives, but the recent sexual protocols involving haploid production and inducing colchicine mediated doubled derivatives offer excellent homozygosity outputs (Mujeeb-Kazi and Riera-Lizarazu, 1996). The protocols are being put to extensive use in molecular mapping (Mujeeb-Kazi and Delgado, 1998), cytogenetic stock development (Riera-Lizararu and Mujeeb-Kazi, 1993), breeding (Inagaki et al., 1998a; Mujeeb-Kazi, 1998), genetic studies (Mujeeb-Kazi, 1998) and gene localization (Mujeeb-Kazi et al., 1998) and is being extended to durum wheats (Almouslem et al., 1998; Inagaki et al., 1998b), as well as X Triticosecale Wittmack (Inagaki et al., 1997).

GENETIC MANIPULATION AND ALIEN TRANSFERS

Chromosome 5B mechanism

There seems to be no parallel to the chromosome 5B-like manipulative approach that encompasses mono-5B, phph or nulli-tetrasomic stocks as the maternal wheat sources in wide-crosses. These stocks enhance wheat/alien recombinations in the F₁ hybrids and all involve the ph system (Sharma and Gill, 1983a, 1983b, 1983c; Darvey, 1984; Mujeeb-Kazi et al., 1984; Forster and Miller, 1985; Sharma and Bäenziger, 1986). The resultant F₁ hybrids exhibit a high meiotic chromosome pairing frequency, but obtaining back-cross derivatives was considered to be a major problem. Sharma and Gill (1986) encountered similar constraints when T. aestivum x Aegilops species hybrids were produced. Subsequently, Ter-Kuile et al. (1988) reported success with the ph maternal system using T. aestivum x Ae. variabilis as the test cross. Since then, numerous ph manipulative high pairing F₁ hybrids have been routinely produced and advanced to BCI or BCII (Rosas et al., 1988). However, as an alternative, since a general constraint prevails, it may be appropriate to produce the F₁ hybrid with a highly crossable wheat (PhPh) and either back- or top-cross it with the phph stock (Sharma and Gill, 1986). Additional options for influencing the PhPh locus are associated with this locus being suppressed by Ae. mutica or Ae. speltoides; a procedure that could be incorporated at the F₁ stage with a low recombination hybrid or on desired alien disomic addition lines. Achieving high recombination is emphasized primarily because the T. aestivum crop species with its phenomenal cytogenetic flexibility via Ph manipulation offers remarkable opportunities for alien gene transfers and incorporation of homoeologous segments introduced in the best location in the recipient wheat chromosomes.

Some other novel systems for genetic manipulation in intergeneric hybridization have experimental priority and are targetted to yield a tremendous agricultural impact via germplasm developed by this procedure. The following are strategies that the authors feel must be pursued vigorously in the quest to keep product development at a pace ahead of the population surge anticipated during the next two decades. Exploiting these strategies is anticipated to render available diverse genes from alien sources that have been insufficiently utilized and that the authors feel shall provide a durable and sustainable practical focus through gene pyramiding. The need to match or exceed the impact of the T1BL.1RS spontaneous translocation (Rajaram et al., 1983) and the induced T1AL.1RS germplasm (Islam-Faridi and Mujeeb-Kazi, 1995; Villareal et al., 1996) ranks high. With the relative ease of hybridization (Sharma, 1995) and the range of hybrids and alien chromatin exchange stocks available (Jiang et al., 1994; Friebe et al., 1996), some diversification seems appropriate as the next century begins. Can intergeneric hybridization protocols be modified to yield faster returns of quality end products?

Though closely related genomes hold a priority for wheat improvement, additional genes from ‘diverse’ gene pools also offer unique resistance durability and are anticipated to contribute to sustainable cropping systems. The various gene sources contributing to C. sativus resistance elucidates this concept. These genes, when pyramided, have the potential to ensure resistance durability across several locations where C. sativus is a wheat production constraint. From the earlier BH1146 cultivar resistance, the improvement present in the Th. curvifolium plus Chinese wheat cultivars has been significantly dramatic (cultivars Chirya and Mayoor) and has remained durable across several countries for approximately 12 years. However, complacency in not introgressing more diverse genes must not prevail for C. sativus or for any other biotic stress. In essence, the progress from BH1146 to Th. curvifolium and/or usage of Chinese wheat cultivar derivatives (Chirya and Mayoor), coupled with the extensive variation identified in the A-, B- and D-genome accessions, is seen as a guarantee for stability over years to come. It is a path being followed for C. sativus and can be extended to address other stress objectives. The above approach, as the next century begins, is totally removed from some wide hybridization views. Fedak et al. (1994) expressed that wide-crossing for purposes of gene transfer be done as a last resort when the variability for a particular trait is exhausted or is non-existent in the primary gene pool. The authors, however, view wide-crossing as a complementary approach, conducted simultaneously and integrated within conventional breeding programmes. This approach contributes multiple diverse novel genes from all gene pools by pyramiding them with the conventional genetic resource present in the primary pool commonly used by breeders. Even if these are ‘major’ alien genes, their multiplicity and diversity shall provide an advantage when pyramided with the conventionally available ‘minor’ genes that recognizably contribute to durable resistance.

Distantly related species (e.g. tertiary gene pool) are complex to exploit, but their potential use in crop improvement is very high. To exemplify, the contributions of S. cereale in wheat improvement cannot be overlooked (Islam-Faridi and Mujeeb-Kazi, 1995; Mujeeb-Kazi et al., 1996), and a major role of Thinopyrum species for BYDV resistance further attests to the use of this distant diversity (Henry et al., 1996). Involvement of other tertiary gene pool species in wheat germplasm has been the subject of a few recent reviews (Jiang et al., 1994; Sharma, 1995; Friebe et al., 1996). A modified tertiary gene pool transfer strategy, which should receive greater emphasis in the future, is aimed at providing maximum recombination between wheat and alien species chromosomes in the early hybrid generation stages. The enhanced recombination will be a consequence of cytogenetic Ph locus manipulation, irradiation, callus induction, etc. Ph manipulations will involve the use of chromosome 5B genetic stocks and use of the relatively newer PhI germplasm option (Chen et al., 1994). The latter warrants more exploitation. The sooner the wheat/alien chromosomal exchanges occur in an intergeneric hybridization programme involving tertiary gene pool species, the sooner appropriate breeding protocols will be incorporated, stress screening coupled with homozygosity will find its place and, with current molecular diagnostic strength, alien introgression(s) identification will occur and be exploited. The authors’ vision is to shorten the tertiary gene pool conventional genetic transfer protocols to a short-term product-oriented programme akin to the interspecific approach that capitalizes upon the primary and secondary gene pool species. The vision projected above will better address the incorporation of polygenically controlled traits, hopefully enbloc, into wheat of which the alien Th. elongatum chromosomal control (3E, 4E and 7E) for salinity tolerance is one example (Dvorak et al., 1988).

Immediate priority has been assigned to a doubled haploid wheat/maize-based manipulation protocol that utilizes the Ph F1 wheat/alien hybrids maintained at the International Maize and Wheat Improvement Center (CIMMYT) as a living herbarium. The protocol is applicable to amphiploids and fertile BCI combinations.

The double haploidy role in salvaging Ph-based F₁ hybrids has become an option to enable ph-mediated alien introgression(s) without having to remake complex F₁ hybrids using the ph genetic stock (Sears, 1977) as the maternal parent. Because of CIMMYT’s living F₁ herbarium involving wheat and several alien species (Ph locus present), BCI derivatives can be produced by pollinating these Ph F₁ wheat/alien hybrids with the Chinese Spring ph ph wheat genetic stock. The BCI progenies (Ph ph) are crossed with maize to yield poly-haploids that possess the Ph or ph locus. The entire wheat and alien chromosomal complement is represented. The haploids derived from the Ph ph BCI derivatives possessing the ph recessive gene get identified at the seedling stage by a polymerase chain reaction (PCR) based diagnostic analysis (Gill and Gill, 1996; Qu et al., 1998), which enhances programme efficiency (Figure 11.3) and sets a crop improvement programme in place using an integration of the breeding methodologies. The ph locus facilitates wheat/alien chromosomal exchanges and generates translocation stocks that are between homoeologous and non-homoeologous chromosomes. They are Robertsonian translocations generally, but smaller alien exchanges are also produced. The diagnostic protocols are FISH followed by Giemsa C-banding. Derivatives of interest possess the critical translocation chromosome(s), but also have entire alien chromosomes that are eliminated via back-crossing (Mujeeb-Kazi, 2001b).

FIGURE 11.3
Molecular marker - assisted genetic manipulation involving the ph locus on chromosome 5B mediated by maize induced doubled haploidy.

BCI=back-croos I

Current emphasis has shifted for delivering farm crop products at a fast pace. Such impacts shall be a consequence of gene transfers from the closely related diploid species and their accessional diversity. In some cases, even swifter results can be obtained as exemplified by the D-genome direct crossing procedures (Figure 11.4) (Alonso and Kimber, 1984; Gill and Raupp, 1987). Greater efficiency emerges by mediating direct transfers with sexually induced double haploidy (DH) for achieving rapid homozygosity (Mujeeb-Kazi and Riera-Lizarazu, 1996).

FIGURE 11.4
Schematic showing the crossing scheme of Triticum aestivum /Aegilops tauschii direct hybridization and back-cross advance

BCII=back-cross II

FIGURE 11.5
Schematic ahowing steps involved in pyramiding accessional diversity of Aegilops tauschii

SH=synthetic hexaploid

Identifying genes in different resistant accessions and pyramiding these prior to crossing with wheat also appears challenging (Figure 11.5, Figure 11.6). One approach is to intercross resistant synthetic hexaploids for a stress, advance the F₁, select resistant F₂/F₃ derivatives that combine the effect of the two divergent Ae. tauschii accessions, incorporate double haploidy and end up with a homozygous stock. Use of this stock in wheat improvement will simultaneously permit incorporating two different genes from the Ae. tauschii accessions (Figure 11.5). Another approach is intercrossing two genomically divergent resistant diploid accessions and developing tetraploid stocks. Use of resistant A- and D-genome accessions is an example (Figure 11.6). In order to hasten such gene identifications, the DH approach has provided further usage and is now being advantageously utilized for modified complete or partial monosomic analyses. The partial analysis is conducted when resistance is associated with the D genome of SH wheats (Figure 11.7). The F₁ monosomics of 1D to 7D chromosomes (2n=6x=40 + 1D to 40 + 7D) when crossed with maize yield 21 chromosome polyhaploids with the 1D to 7D contributions coming from resistant SH wheats. Doubling these n=3x=21 polyhaploid plants with colchicine results in stable 42 chromosome double haploids. Each DH now possesses the homozygous 1D to 7D chromosomes of the resistant SH parent being analysed for the chromosomal location(s) of the resistant gene(s). Upon screening, the non-segregating resistant DHs are attributed with having the gene(s) in them. The stable monosomic derived DH germplasm, apart from simplifying the conventional monosomic analyses, also facilitates global distribution of the developed germplasm. The germplasm enables experimental repetition without havnecessary when the conventional monosomic analytical procedure is followed.

FIGURE11.6
Schematic indicating a novel gene pyramiding strategy associating stress-resistant diverse genomes (AA of Triticum monococcum and DD of Aegilops tauschii)

FIGURE 11.7 Steps associated in conducting a partial monosomic analysis, where resistance is located within the synthetic hexaploid (2n=6x=42) D-genome chromosomes

SH=synthetic hexaploid
*Seven such double haploids result

ALIEN TRANSFERS FOR DURUM WHEAT IMPROVEMENT

Progress in durum wheat/alien species hybridization has not been as exhaustive as bread wheat. However, the need to diversify the durum genetic base is crucial and can be achieved by incorporating the diversity of the primary, secondary and tertiary gene pools. The intergeneric recombination constraints can presumably be overcome by using the ph1c Capelli genetic stock that requires greater investigating to fit agricultural goals.

For interspecific durum wheat improvement, the A and the B genome diversity through their AAAABB/AABBBB amphiploid routes allows for cross combinations to be made between the resistant amphiploids and elite durum cultivars. This facilitates introgression and exploitation of resistant traits in breeding programmes by utilizing appropriate breeding protocols. This alien diversity based durum improvement programme is currently in its infancy, but the authors do anticipate contributions for resistant transfers to be achieved for durums.

Challenging is the exploitation of D-genome resistances for durum wheat improvement, and at a high priority would be the transfer of scab (F. graminearum) resistance genes. Additionally, it must be mentioned the potential been assigned to these diseases.of D-genome resistance transfers to durum wheats of genes associated with salinity tolerance, drought tolerance, S. tritici, C. sativus and BYDV resistance, with quality being an integral part in all A-, B- and D-genome accessional transfers. These genomic transfers would be a consequence of recombinational events due to the preferential A- and D-genomic chromosome pairing represented as seven bivalents in the presence of the ph locus (Plate 11). The bivalents are generally of the A- and D-genome chromosomes and univalents of the B genome, as inferred separately from meiotic C-banding data (unpublished). The authors’ current tester system to demonstrate the D- to A-genome genetic exchange efficacy is for C. sativus and S. tritici from some D-genome resistant accessions. Durum wheat cultivars are highly susceptible for both of these biotic stresses, and since CIMMYT has ideal screening protocols with reliable screening locations in Mexico, priority has

From the secondary gene pool, the potential of using Ae. speltoides resistance diversity for several stresses does also exist for durum wheat improvement.

CONCLUDING REMARKS

The tribe Triticeae includes 25 genera, some 400 species with a wealth of accessional variation distributed over three gene pools that offer ample genetic diversity for wheat improvement. This genetic resource blends itself with research strategies to yield quality short-term products. The complex protocols associated with genetic transfers from the more distant alien species are also amenable for simplification through genetic manipulation that is anticipated to permit minimal introgression of desired alien chromatin into wheat.

To meet target crop development goals, integrated research that encompasses multilocational testing and multidisciplinary teams is crucial. Novel protocols aided by molecular inputs are demonstrating promise to deliver agricultural products in a time frame that will address the population food needs projected for the next two decades. A 2.7 percent increase in grain production is considered essential each year through the year 2020; a challenging goal set to provide for the approximately 8.5 billion people by 2025 (N.E. Borlaug, personal communication, 1988). The authors feel that the present approach where, contrary to most views, alien genetic diversity strongly complements conventional germplasm, shall provide durable and sustainable products through multifaceted integration of activities.

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