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International Maize and Wheat Improvement Center - CIMMYT

Lisboa 27, Colonia Juárez, Apdo. Postal 6-641, 06600, México, D.F., México.

Perennial intergeneric F1 hybrids of durum wheat cultivars with alien Triticeae species: germ plasm status and use in breeding.

A. Mujeeb-Kazi, V. Rosas, and R. Delgado.

Intergeneric hybrids involving durum wheat cultivars and the annual/perennial Triticeae species have been produced to a great extent during the last two decades. Cross combination success was variable, but, in general, protocols involving bud-pollinations, pre and postpollination hormonal treatments, variations in embryo rescue media, and special handling of embryos after plating, together with seedling transplant care have provided adequate diversity to be assembled for durum breeders to address stress constraints encountered in the global cultivation of the crop. Some significant stresses for durums are F. graminearum, H. sativum, and BYDV, as well as the abiotic stress, salinity.

We list in Table 1 the combinations that were produced by us and are maintained as a living collection at CIMMYT, in El-Batan, Mexico. These F1s are of a perennial habit and are maintained in pots under screenhouse conditions. Each combination is physically cloned twice a year to maintain three potted plants. Of these, one is colchicine treated until a fertile amphiploid is obtained. The plants are cytologically analyzed after each cloning in order to ensure cytological stability during maintenance.

The self-sterile F1s in addition are cloned, vernalized, and transplanted for biotic stress screening in Mexico for H. sativum and S. tritici. Amphiploids, when obtained, are similarly vernalized, and the progeny screened for the above stresses as well as for head scab. Field, border-row, rust inoculations permit us to observe the performance of the germ plasm for leaf, stem, and stripe rusts in three locations in Mexico (El Batan, Toluca, and Poza Rica).

In those cases where we have not yet obtained amphiploids, the self-sterile F1 hybrids have been backcrossed to elite durum cultivars leading to BC1 seed. This seed can be tested directly for the stresses and serve for advancing the desired combination for applied purposes via addition/substitution lines and then introgressing the required trait by cytogenetic manipulation. The use of the ph genetic stock of Capelli is in its infancy in our program and is projected as a fast source to enforce alien transfers when it is the backcross parent for the F1 hybrid, yielding Phph heterozygote progeny. Selecting the ph recessive and achieving the alien transfer are anticipated and being studied.

Of the annual Triticeae species, those hybridized with durum cultivars are Ae. peregrina, Ae. ventricosa, Ae. geniculata, Ae. triuncialis, and Ae. speltoides. Amphiploids were produced from all the above hybrid combinations (see Table 1).

Intergeneric F1 hybrids of some bread wheat cultivars with annual and perennial Triticeae species: germ plasm status and utilization in wheat breeding.

A. Mujeeb-Kazi, V. Rosas, and R. Delgado.

In a bread wheat-based, intergeneric, hybridization program with a focus on applied agricultural objectives, outputs are necessary that address several biotic and abiotic stresses. This is indeed a tall order, because the initial hybrid production in itself is so complex but a necessary starting point. Next in order come the crucial steps of transfering the desirable stress genes and dealing with the genetic distance between wheat and the species involved. We have been involved in F1-hybrid production of wheat with annual and perennial Triticeae for over two decades. The annual species/wheat cultivar hybrids were relatively easy to produce, and their amphiploid products also obtained easily (Table 2). The complexity resided in the combinations of wheat with species that were not in the primary gene pool, hich is not the case for hybrid production alone, but is a major factor when alien genes have to be transferred from the distant species into wheat.

Our hybrid production protocols have been simplified enough that quite complex cross-combinations have been realized. The hybrids possessing a perennial habit are maintained with cytological documentation and physical cloning twice each year. Clones are further field evaluated for resistance/tolerance to stresses and production of amphiploids, wherever possible, is a routine biannual process. The complete list of these intergeneric combinations and some supporting details are provided in Table 2. The diversity is significant in that it allows researchers to produce desired BC1 progeny with virtually any bread wheat cultivar that may be site/country specific and to use our genetic-stock base more efficiently and avoid remaking the F1 hybrids; which is a difficult, if not cumbersome process. This information is shared to facilitate the broad usage of our germ plasm. To some extent, we have been producing special BC1 progeny for international colleagues who use their cultivars as the parents and our perennial F1 hybrids.

The most important stress traits linked to a species and combination are identified. The data that we have accumulated allow us to use a combination for multiple objectives with the same manipulation strategy. That strategy is pursued intensively and revolves around the alien species Th. bessarabicum. Combining this species with wheat has potential for salt tolerance and F. graminearum resistance, two major constraints on wheat production. Thinopyrum elongatum is another such preferred species. Tolerance/resistance in these species resides on more than one chromosome. A living collection allows us to swiftly go to the desired F1 hybrid that has the Ph gene and produce BC1 Phph heterozygote derivatives by crossing the Ph F1 with the Chinese Spring phph genetic stock. A PCR-based protocol then allows us to identify the progeny that possesses the ph recessive locus, and this becomes a source for multiple homoeologous transfers.

Agrotricum (2n = 8x = 56): potential for barley yellow dwarf virus resistance and its cytogenetics.

M. Henry, A. Cortes, V. Rosas, R. Delgado, and A. Mujeeb-Kazi.

Testing of various germ plasms for resistance to BYDV has been done over the past few years in CIMMYT both in the field and in controlled greenhouse conditions. The germ plasm was comprised of elite cultivars, Triticeae species of the three gene pools, amphiploids from some intergeneric hybrids, BC1 self-fertile derivatives of the intergeneric hybrids, and some partial amphiploids. One such partial amphiploid is Agrotricum (2n = 8x = 56), which was identified in Canada as being resistant to BYDV. In Mexico, we have obtained similar data to support the resistance and, after studying the cytogenetics of this genetic stock, have initiated a program to produce addition lines, identify addition lines that show resistance, and transfer the resistance genes to our spring wheat cultivars. We report here the cytogenetic progress and BYDV resistance data on the original 56-chromosome stock and its initial backcross derivatives. Appropriate controls were included in the study.

Cytology of Agrotricum. Somatic cytology of Agrotricum indicated some aneuploidy in the seedlings analyzed where 56-chromosome normal derivatives were present, but plants with 54, 55, and 57 chromosomes and some with telocentrics also were present. Plants with 56 chromosomes were Giemsa C-banded, and the stable lines were analyzed further by FISH. The 56-chromosome, partial amphiploid possessed 14 Thinopyrum chromosomes, 40 normal wheat chromosomes, and a pair of wheat chromosomes with a translocation between chromosome 3D of wheat and Thinopyrum. The exchange is at the terminal end of 3DL. Such plants were analyzed at meiosis, and all were normal with 29 bivalents at metaphase I and a normal anaphase separation. These plants were seed increased and tested for BYDV resistance. They were all resistant.

The euploid stock then was backcrossed to the susceptible wheat cultivars Prinia and Bagula, which resulted in BC1 progeny with 2n = 7x = 49 chromosomes. These progenies also tested positive for BYDV resistance. The BC1 progeny was advanced further by backcrossing to each of the two parental wheat cultivars and also by selfing to eventually identify plants with 43 chromosomes (21 bivalents +1 univalent). From these, 44 chromosome (22 bivalent) derivatives were obtained by selfing of the 43-chromosome plants or by producing 22-chromosome haploids after crosses with maize and then doubling these haploids.

The 44-chromosome progeny was C-banded, and five disomic additions were identified. One of these addition derivatives has a very low BYDV concentration when tested by ELISA, and further work is in progress to introgress the resistance into wheat. Backcross derivatives with 42 chromosomes (possessing the translocated pair) did not possess BYDV resistance.

BYDV screening-virus isolates and BYDV inoculation. The BYDV-PAV isolate used was collected in Mexico and maintained in CIMMYT's greenhouse through transmission by aphids. Inoculation was by infesting 7-day-old seedlings from the Agrotricum germ plasm, parental wheat cultivars, and a resistant check (TC14) with 10 viruliferous aphids (Rhopalosiphum padi) that had acquired BYDV by feeding on infected plants for 48 hours. The seedlings were isolated from each other by transparent plastic tubes. After a 2- to 5-day period, aphids were killed with Metasystox (Bayer). In each entry, two plants were kept free of aphids to serve as the uninoculated controls.

Enzyme-linked immunosorbent assay (ELISA). The flag-1 leaf was collected at different dates after inoculation for the evaluation of the virus titer by ELISA. Double antibody sandwich ELISA (DAS ELISA) was used with a few modifications. Polystyrene microtiter plates (NUNC) were incubated at 37°C for 3 hours with coating polyclonal antibodies directed against the U.S. isolates provided by K. Perry (Purdue University, W. Lafayette, IN). Plant sap (1:10, in 0.1M phosphate buffer pH 7.0) was incubated for 3 hours at 37°C. Alkaline phosphatase-labeled, polyclonal, anti-PAV antibodies (1:1000) were incubated overnight at 4°C. P-nitrophenyl phosphate substrate (Sigma) was added at a concentration of 1 mg/ml, and the mixture was incubated for 1 to 2 hours at room temperature. Optical density (OD) was measured at 405 nm. A plant was considered infected when the OD was higher than twice that obtained for the uninfected control. The resistant line had low virus titers, which were equivalent to or slightly less than that obtained with TC14, the resistant check entry.

Summary of results. 1. The average OD was much lower in Agrotricum (OK 7211542) than in the susceptible and resistant wheat cultivars used in backcrosses. The values for Agrotricum were significantly lower than those of the resistant check (TC14 line) and those of the two susceptible cultivars, Prinia and Bagula (Table 3).

2. In the backcross derivatives, the low virus titers were conserved. Titers were not significantly different from each other in Agrotricum and its backcrosses to Prinia and Bagula but were different compared to those of Prinia and Bagula. The trend is elucidated in Fig. 1.

3. Analysis of advanced-backcross, selfed derivatives has identified one 44-chromosome line possessing low virus titers. All plants of this line are being characterizedcytologically, and seed is being increased. They will be tested further and subjected to cytogenetic manipulation to effect the resistance transfer in order to recover a euploid wheat with 2n = 6x = 42 chromosomes.

Thinopyrum bessarabicum (2n = 23x = 14, JJ) disomic chromosome addition lines in bread wheat: current germ plasm status.

A. Cortés, V. Rosas, S. Cano, R. Delgado, J. Zhang*, X. Li*, R.C. Wang*, and A. Mujeeb-Kazi.
*(USDA-ARS-FRRL, Logan, Utah, USA).

Thinopyrum bessarabicum is a self-fertile, maritime grass possessing salinity tolerance and resistance to wheat scab. These important abiotic and biotic characteristics make Th. bessarabicum an important Triticeae species to exploit for wheat improvement. We have been producing addition lines of the species in bread wheat. The addition lines were made in a mixed-wheat background (Chinese Spring/Th. bessarabicum//Genaro). For the homoeologous group 3, a homozygous 3JL, disomic addition line also was extracted.

The above disomic addition lines having Th. bessarabicum (J or Eb) chromosomes in a T. aestivum background (2n = 44; 21 II ABD + 1 II J) were analyzed using both AFLPs and RAPDs. Among the J-specific, AFLP fragments amplified from 32 selective amplification primer pairs, 195 fragments were single-chromosome specific. These included 44 AFLP markers for 1J, 46 for 2J, 39 for 4J, 37 for 5J, and 29 for 7J. Although no AFLP markers were specific for 3JL alone, we identified two RAPD markers specific to this chromosome arm. In addition, there were two RAPD markers for 1J, two for 2J, six for 4J, one for 5J, and two for 7J. Fifty-nine AFLP and two RAPD J-specific markers were present in the amphiploid but absent in all tested CIMMYT disomic addition lines making them potential putative markers for 6J or 3JS. The 50 AFLP markers and four RAPD markers were present in all (or at least five) J chromosomes. CIMMYT-derived 2J and 5J addition lines are distinguishable from those originating from the U.K. by 22 and 27 genotype-specific AFLP markers, respectively. All these molecular markers, whether genotype-, chromosome- or genome-specific, are useful in monitoring the introgression of J-chromosomal segments into wheat chromosomes.

Field testing of these addition lines for scab in particular posed a constraint associated with lateness of the germ plasm and also was a constraint for the salinity tests. Some instability of the lines also was encountered. Hence, an elite bread wheat cultivar Prinia was selected, and by the use of a backcross protocol, the addition chromosomes were transferred to Prinia. Four backcrosses to Prinia were made, and, after the final backcross, the 43-chromosome plants of each addition group were crossed with maize, yielding 21- and 22-chromosome haploids. Mitotic counts associated with C-banding identified 22-chromosome haploids of each group that then were treated with colchicine to yield 44-chromosome derivatives. Currently, homozygous disomic additions have been obtained for 1J, 2J, 3J, 4J, 5J, and 7J. These addition lines are now in a spring wheat, which has early maturity compared to Chinese Spring and Genaro, and will enable us to screen appropriately for the various stresses.

To complete this set, the 6J chromosome and the 3JL translocation chromosome still need to be added. Initial screening used the BC3-selfed, 44-chromosome progeny for evaluating scab infection in Toluca, Mexico. All lines except of 2J have adequate duration for days-to-flowering and physiological maturity. Type-II level of resistance to scab was present in additions 5J and 7J. Addition 2J was late and was not inoculated. With the one backcross advance now made, and with the doubled haploid base produced, the testing is anticipated to provide greater precision. A similar case would prevail for salinity testing of these lines under field conditions in some Asian locations where late lines are affected severely by heat during grain fill. The level of maturity of the cultivar Prinia is a suitable background to alleviate this constraint. So far, our observations support the trend of doubled haploid derivatives in wheat that is early maturing. This observation needs to be verified for the disomic additions. We do expect the allelic homozygosity to contribute to the stability of the addition lines during their maintenance.

A BC1 self-fertile intergeneric combination and the spontaneous production of alien multiple disomic chromosome additions.

A. Mujeeb-Kazi, A. Cortés, V. Rosas, and R. Delgado.

Fertile, BC1 intergeneric combinations by 'bread wheat/tetraploid alien Triticeae species//bread wheat' with 2n = 8x = 56 chromosomes are valuable alternatives for those F1 hybrids that generally do not yield fertile amphiploid progeny with ease. In contrast, producing BC1s on self-fertile hybrids is a very rapid procedure to exploit the hybrid for practical breeding advance until such time that the amphiploid is produced. In the course of this procedure of F1 advance to BC1 for a 'Chinese Spring/Th. bessarabicum' combination, the progeny possessed 2n = 7x = 49, AABBDDJ chromosomes that were meiotically associated as 21 bivalents and 7 univalents. Further advance of this BC1 material was by additional backcrossing in order to produce Th. bessarabicum addition lines. Simultaneously selfed seed also was harvested and kept in storage. Surprisingly, the BC1 49-chromosome plants were observed to be highly self-fertile, and more interesting, the BC1-selfed plants maintained a chromosome number that had a high frequency of derivatives with 49 chromosomes. The range in chromosome number was from 46 to 52. Where the alien species is a diploid, self-fertile BC1 progeny are rare, and retention of the alien haploid complement is more rare. This phenomenon was reported by us (Mujeeb-Kazi and Asiedu 1990) without extensive meiotic data.

More recently, Sharma (1996) published cytogenetic findings on a similar combination. In essence, a similar backcross, self-fertility trend was observed. The author concluded that the occurrence of plants with 49 chromosomes for several generations of selfing indicated that the seven chromosomes of Th. bessarabicum had a selective advantage and most likely were transmitted only through the female gametes.

With our recent priority being scab resistance, we identified a 'Chinese Spring/Th. bessarabicum' amphiploid to have type II resistance to scab. We also checked the BC1-selfed material that we had stored from our earlier addition line development stage in the early 1990s and found the derivatives to vary in resistance expression. A dosage effect dilution was anticipated, but a complete loss of resistance in individual plants is rather difficult to explain.

Because one spike of each plant had been collected for meiotic analysis, this check was initiated, and the data obtained revealed a totally different trend then that previously reported (Sharma 1996). Plants with 49 chromosomes rarely expressed the 21 II + 7 I associations; instead 22 II to 24 II + 5 I were frequent. In the occasional self-fertile, BC1 50-chromosome derivatives, we encountered some plants with perfect bivalent meiosis of 25-chromosome pairs.

Additional reserve seed was germinated, and precise cytological analyses involving a mitotic somatic count, FISH diagnostics, and meiotic analysis were made. Plants with 50 chromosomes with 25 II had four pairs of Th. bessarabicum chromosomes. These pairs were for chromosomes 2J, 3J, 5J, and 6J.So far, we have analyzed several hundred BC1-selfed plants and diversified the backcross production over different wheat cultivars. The trend of Th. bessarabicum bivalent associations has remained the same. Listed in Table 4 are some derivatives with their cytological detail and plant fertility seed number.

The differential response to scab of the BC1-selfed material is now explained by the absence/presence of the seven Th. bessarabicum chromosomes, although we initially determined in a separate test that the scab resistance is associated with up to three Th. bessarabicum chromosomes. The backcross, selfed derivatives with multiple disomics and superior type II scab resistance are good candidates for cytogenetic manipulation; a procedure currently underway.

References.

• Mujeeb-Kazi A and Asiedu R. 1990. Wide hybridization - Potential of alien genetic transfers for Triticum aestivum improvement. In: Biotechnology in Agriculture and Forestry, Vol. 13, Wheat (Bajaj YPS ed). pp. 111-127.
• Sharma HC. 1996. Maintenance of haploid genome of Agropyron junceum in wheat. Wheat Inf Serv 83:15-18.

Current status of D-genome based, synthetic, hexaploid wheats and the characterization of an elite subset.

A. Mujeeb-Kazi, G. Fuentes-Davila, R. Delgado, V. Rosas, S. Cano, A. Cortés, L. Juarez, and J. Sanchez.

Bridge crosses utilizing synthetic hexaploids (T. turgidum/Ae. tauschii) provide a potent means of improving bread wheats. The procedure enables incorporation of the genetic diversity of T. turgidum cultivars together with that contributed by the Ae. tauschii accessions. From the 521 SH wheats produced since 1995, an elite set of 95 synthetics was prepared and has been characterized for some morphological, growth, biotic, and abiotic attributes. All SH wheats are cytogenetically stable. The elite set possesses an agronomically more desirable grown habit under two Mexican locations; Obregon (27°20'N,105°55'W, 39 masl) and El Batan (19°31'N, 98°50'W, 2,249 masl). Growing the synthetics in these locations enabled selections to be made with the assistance of our breeding colleagues (S. Rajaram and R.L. Villareal) for the elite set of 95 SH entries. These SH entries were studied for several growth parameters and screened for diversity towards some stresses. Observations based on an Obregon planting were recorded for days-to-flowering, presence of pubescence on spikes, tiller anthocyanin pigmentation, plant height, awn color, days-to-physiological maturity, leaf and stem rust response, and 1,000-kernel weight. Reactions to stress evaluations associated with leaf/stem rust, H. sativum, F. graminearum, S. tritici, and N. indica conducted at various Mexico locations were tabulated. Quality parameters (HMW- and LMW-glutenin subunits) also were analyzed. These elite set attributes are elucidated here.

Establishing some descriptors. From the wide array of SH wheats produced, field plantings were utilized for the evaluation of agronomic parameters including the assessment of yield potential and its components. Based upon these characteristics, Villareal et al. (1994) demonstrated extensive genetic diversity for plant height, flowering date, grain-fill duration, days-to-physiological maturity, aboveground biomass at maturity, 1,000-kernel weight, spikes/m2, and higher grain yield. The grain yield ranged from 0.89 up to 8.01 t/ha. Utilization of this select germ plasm for wheat improvement presumably will be an advantage, if the more agronomically desirable SH wheats are exploited that further express high levels of resistance to biotic/abiotic stresses as opposed to using resistant but poor agronomic types. Days-to-flowering ranged from 85 to 119, and physiological maturity between 115 to 152. Plant height was between 85 to 140 cm, and 1,000-kernel weight was from 30.2 to 67.6 g. Lodging was fairly common and was associated with the taller SH entries with higher 1,000-kernel weight.

None of the SH wheats were free-threshing. There was great diversity for pubescence on the spikes. Awn color ranged from white to a light and dark brown to black. Variation for anthocyanin pigmentation was well distributed. Similar diversity was observed for leaf and stem rusts in the SH elite set. From a screening of 95 SH wheats in Poza Rica for H. sativum, we observed diversity of resistance in the elite SH wheats. The durum cultivars involved in these SH combinations were susceptible both for the leaf infection and seed blemish parameters. Hence, resistance in an SH wheat was interpreted as being due to the involvement of the respective Ae. tauschii accession.

Those SH wheats with a leaf score of 95 or less and a seed damage of 3 or less (data not shown) are the preferred resistance-gene donors for wheat improvement. Scores for the respective durum parents involved in the SH wheats were 97 to 99 for leaf damage and 3 to 5 for grain blemish. This SH bridge is advantageous for crop improvement, because it not only allows the Ae. tauschii resistance to be exploited but also incorporates the genetic diversity of the A and B genomes of the respective durum wheat cultivars. Desirable levels for scab are 15 % or less (Type II), S. tritici 5-4 and less, and N. indica less than 3 %. The scoring scales are elaborated in the footnote of Table 5.

Conclusions.

• Crosses between T. turgidum cultivars and several accessions of Ae. tauschii have so far led to the production of 790 synthetic hexaploid wheats.
• An elite set of 95 SH wheats based upon growth habit under two locations in Mexico has been prepared, and seed has been increased and transferred to our germ plasm bank for global distribution.
• Several stress descriptors are being established that should facilitate utilization of SH wheats in crop improvement. Some of these descriptors are elucidated along with a few morphological features.

Reference.

• Villareal RL, Mujeeb-Kazi A, Rajaram S, and Del Toro E. 1994. Morphological variability in some synthetic hexaploid wheats derived from Triticum turgidum x T. tauschii. J Genet Breed 48:7-16.

New synthetic hexaploids (Triticum dicoccum/Aegilops tauschii): their production, cytology, and utilization as a source for Russian Wheat Aphid resistance.

A. Mujeeb-Kazi, B. Skovmand, M. Henry, R. Delgado, and S. Cano.

Use of the dicoccom group in wheat improvement has been limited but recently received attention in our program, particularly because potent Russian wheat aphid resistance was identified in several accessions. Triticum dicoccum accessions were hybridized with some Ae. tauschii diploids, and fertile synthetics derived and screened for RWA resistance. This screening led to a candidate set of SHs for utilization in transferring the resistance to bread wheat cultivars (Table 6). These aspects of the germ plasm characterization and utilization are described.The standard vernalization procedure resulted in very vigorous growth of the Ae. tauschii accessions with a flowering range of 90 to 135 days. Crossing with the two transplanted batches of the vernalized T. dicoccum accessions for a majority of the Ae. tauschii accessions was successful. Embryos were rescued at 18-20 days postpollination from all crosses. The small, translucent embryos had a definitive shape and were floating in a watery endosperm cavity. The embryos were plated on MS medium and given a 21 day cold shock (dark) at 4°C. The cold treatment allowed better seedling regeneration. The embryo-culture tubes were kept further in the dark at 22°C after the cold treatment. The embryos usually germinated within 30 days, after which the plantlets were transplanted into a soil medium and maintained in the greenhouse for examining cytology, and inducing amphiploidy. Crossability data for some combinations indicate the general trends for seed set, embryo recovery, plant regeneration, and the seed number of doubled plants (Table 6). C0 seed from SHs that arose by spontaneous doubling had a greater cytological normalcy than their colchicine-doubled counterparts. All F1 hybrids were stable with 2n = 3x = 21 (ABD) chromosomes. After colchicine doubling, the C0 synthetic seed generally possessed 42 chromosomes, though some hypo- or hyperploidy was observed and was subsequently purified by additional cytology and seed increase.

That Ae. tauschii is the source of the D genome was discovered by McFadden and Sears (1944, 1946), who also described the origin of T. spelta. Kihara (1944) also ascertained this D-genome source independently. A spelt-type hexaploid results when the wild tetraploid T. turgidum subsp. dicoccoides or its cultivated derivative (dicoccom group) is crossed with Ae. tauschii, and amphiploidy is induced. Cultivated emmers were in existence by the time the hexaploid forms appeared (2,000 years later than emmer cultivation). Several independent events combined different tetraploids, and Ae. tauschii led to the hexaploid gene pool. Recognizing the nature of occurrence of these events, it is not surprising that the production of the presently reported synthesis was relatively simple and the spontaneous doubling events for each hybrid combination were of a high frequency.

Although our agricultural focus is on stress resistance transfers, the basic information that has surfaced warrants reporting and demonstrates: 1) ease of crossability, 2) excised embryos with a well-defined shape, 3) rapidly growing vigorous hybrid seedlings, 4) high capacity of all hybrids to double spontaneously, and 5) presence of nominal aneuploidy in the spontaneously doubled synthetics. These categories of basic information drastically contrast the earlier observations related to synthetics that involve elite T. turgidum cultivars instead of T. dicoccum (Mujeeb-Kazi et al. 1996).

Russian wheat aphid screening of synthetic hexaploids. The T. dicoccum parents were rated highly resistant or resistant to the aphid, with a majority scoring 1, which indicates high resistance. Comparing the disease reactions of the 56 T. dicoccum-based synthetics and their T. dicoccum parents showed that five of the synthetics were rated highly resistant, 44 were rated resistant/moderately resistant, and seven moderately susceptible. None were rated susceptible or highly susceptible. All the emmer parents were rated highly resistant or resistant. This demonstrates that the emmer resistance was expressed in the synthetics. However, in certain combinations, it may not be expressed to a high degree as shown by the parental emmer wheat. Table 7 shows the pedigrees of the synthetics rated as highly resistant, of which two SHs have the same Ae. tauschii parent.

Conclusion. T. dicoccum/Ae. tauschii F1 hybrids were produced with high frequency, gave vigorous seedlings, and all spontaneously doubled to yield fertile synthetic hexaploids. The synthetics involving several T. dicoccum/Ae. tauschii accessions were produced to serve as a source for RWA-resistance transfers via bridge crossing to bread wheat cultivars. The RWA resistance of T. dicoccum accessions was expressed over different categories of disease scoring in all synthetics tested. Four SHs exhibited high RWA resistance and are superior candidates for a bread wheat improvement program.

References.

• Kihara H. 1944. Die Entdeckung der DD-Analysatoren beim Weizen. Agr and Hort (Tokyo) 19:889-890.
• McFadden ES and Sears ER. 1944. The artificial synthesis of Triticum spelta. (Abstr) Rec Genet Soc Amer 13:26-27.
• McFadden ES and Sears ER. 1946. The origin of Triticum spelta and its free-threshing relatives. J Hered 37:81-89, 107-116.
• Mujeeb-Kazi A, Rosas V, and Roldan S. 1996. Conservation of the genetic variation of Triticum tauschii (Coss.) Schmalh. (Aegilops squarrosa auct. non L.) in synthetic hexaploid wheats (T. turgidum L. s.lat. x T. tauschii; 2n=6x=42, AABBDD) and its potential utilization for wheat improvement. Genet Res Crop Evol 43:129-134.

New synthetic hexaploids and a set of bread wheat/synthetic hexaploid derivatives as sources for scab resistance.

A. Mujeeb-Kazi, L.I. Gilchrist, and R. Delgado.

Fusarium head blight is one of the most devastating diseases of cereal crops that affects wheat, barley, and maize worldwide. Also known as scab, the disease reduces both grain yield and quality and also increases toxins in the grain that pose serious health risks to human and animal consumers. Head blight infections have caused several billion dollars worth of losses to the U.S. wheat sector alone over the last 5 years, not to mention its impact elsewhere in the world.

In bread wheat, limited resistance has been identified, and the diversity is not excessive in the conventional sources available. Predominant resistant sources are the cultivars Frontana, Sumai 3, and Ning. The potential for identifying resistance in diverse alien sources hence ranks high and has been an aspect that we have been exploring in Toluca, Mexico for the past several years. Among the Triticeae gene-pool species, one avenue is to exploit the primary D-genome donor grass Ae. tauschii, which has several hundred accessions. Several of these accessions have been combined with elite durum wheat cultivars to result in a synthetic hexaploid germ plasm resource. So far, a total of 790 synthetics have been produced. These synthetics have been screened in Mexico using the Type II-evaluation protocol. A new batch of synthetics with Type II (spread) scab resistance was identified in the summer of 1999 and are reported in Table 8. Bread wheat cultivars Frontana and Sumai-3 were the resistant checks, and Flycatcher was the susceptible check.

Also provided are the data for advanced derivatives from resistant synthetic/bread wheat combinations that show resistance for other types, i.e., Types I (penetration), III (toxin), and IV (test weight) (Table 8) over two summer cycles in Mexico.

The line 'Mayoor//TK SN 1081/Ae. tauschii (222)' in addition to being a good source for all four types of scab resistances, also possesses resistance to leaf, stem, and stripe rusts; Karnal bunt; S. tritici; and H. sativum. The line is free-threshing type and has a spring habit. F1s of this line have been made with a bread wheat Flycatcher, which is susceptible for all the above attributes. The F1 currently is being used to produce a doubled haploid-based mapping population in order to obtain molecular information.

Practical applications of a gene pyramiding strategy with D genome-based synthetic hexaploids for two major biotic stress resistances: wheat head scab and spot blotch.

A. Mujeeb-Kazi, R. Delgado, and S. Cano.

Among the primary gene pool species we have focused on initially to exploit the genetic diversity of the D-genome for wheat improvement are the diploid species accessions of Ae. tauschii. The procedure followed has been to cross durum wheats with Ae. tauschii accessions and the then double the F1 hybrids (2n = 3x = 21, ABD) to obtain hexaploid wheats called synthetic hexaploids or SHs. Several such combinations have been produced over the last decade, and have been seed increased, and evaluated for biotic stresses. Two of these stresses are of major global importance for wheat production; head scab and spot blotch (H. sativum or C. sativus). In field evaluations done in Mexico over the past 10 years, we have observed that the best lines selected for spot blotch in Poza Rica also performed adequately for scab in Toluca. Consequently the best performers of the synthetic hexaploids for spot blotch were intercrossed in order to combine the diversity of different Ae. tauschii accessions, select superior F2 segregates, and make these stable by the maize doubled protocol using the detached tiller approach. Results of these DH synthetic/synthetic F2 resistant selections for spot blotch were reported in the Annual Wheat Newsletter, Vol. 45, 1999.

The above DH spot blotch germ plasm was tested in the summer of 1999 in Toluca for head scab, and a few lines were identified that possessed superior Type 11 (spread) resistance (Table 9). Because stripe rust also is prevalent at this site, the selected lines also were screened for the stress, and all lines reported (Table 9) possess good stripe rust resistance. The lines also possess leaf and stem rust resistance based upon screening in Obregon, another location in Mexico. The DH derivatives are considerably early to flower, reach physiological maturity earlier, and are significantly shorter than their tall synthetic parents.

Such lines with pyramided Ae. tauschii accessional diversity are advantageous for incorporation in wheat breeding, because diverse genes may be incorporated simultaneously, thereby enhancing breeding efficiency. In addition, our approach also is fostering the use of those synthetics that possess multiple disease resistances. Such germ plasm is being identified readily, and we anticipate that it also will make a significant contribution in wheat improvement by providing rapid outputs either by individual usage of the synthetics or by the above-described pyramiding route option.

The contribution of the D genome to efficiency of doubled haploid production in wheat.

J. Ahmad, R. Delgado, S. Cano, and A. Mujeeb-Kazi.

Bread wheat haploids are being produced by us routinely via the wheat/maize crossing protocol for use in wheat cytogenetics, wide crosses, wheat breeding, and genetic analyses, with extension of the application into genetic engineering and molecular mapping. The mean frequency percentage data at this stage for embryo recovery, plantlet differentiation, and colchicine doubling range between 20-25 %, 80-90 %, and 80-95 %, respectively, over long-term experiments involving various bread wheat cultivars crossed with various maize sources. We can infer that haploid production for spring bread wheats is 100 % effective.

However, similar success has not been realized for the durum wheats where genotypic specificity is prevalent. The absence of the D genome in durum wheats is reportedly a factor apart from the durum genomic variation. These inferences also have been noticed by other researchers. We report here rather extensive data obtained on the durum genotypic diversity for haploid production across several cultivars. Furthermore, we elucidate the contribution of Ae. tauschii to these same durums as measured by the haploid production frequencies in their synthetic hexaploids T. turgidum/Ae. tauschii (Table 10). Data categories reported are florets pollinated, seed set, embryos excised, and embryo formation (percentage). Embryo differentiation is not reported but ranged from 50-70 % (much less than 80-90 % obtained for bread wheats ).

Durum cultivars are generally poor in haploid production and the significant D-genome contribution to haploidy is observed readily. Table 10 elucidates this trend; each group is separated to compare the durum parent (top line, bold) with its synthetic (second line) hexaploid derivative e.g., Croc 1 (line 1) and 'Croc 1/Ae. tauschii (210)' (line two).

A doubled haploid, bread wheat-transformed cultivar: its use in conventional transfer to elite spring wheats and development of germ plasm for detecting the location of the transgene.

A. Mujeeb-Kazi, S. McLean, A. Pellegrineschi, R. Delgado, and S. Cano.

Wheat transformation has been limited by the inability to transform and regenerate the proper cells in the target tissue and also by problems related to gene expression, plus stability of expression after several cycles of selfing the transformant. We were provided a Bar-Gus-transformed bread wheat winter/facultative cultivar (K-39) by Dr. Richard Brettell after his sabbatical stay at CIMMYT, El-Batan, Mexico. Our objectives were as follows:

1. To produce doubled haploids on the K-39 transformed stock and test the DHs for expression. If the DHs were positive for expression, a seed increase would be made for further studies. Selfed progeny could be analyzed for gene expression and compared with the selfed derivatives of the original stock, which might indicate if gene silencing occurred was the DH material more stable. Theoretically, this must be the case, because all alleles will be homozygous and if the original C0 DH expressed resistance, then this should be expressed permanently.

 

2. To conventionally transfer the transgene to three elite, spring-habit, bread wheat cultivars (Attila, Kauz, and Luan) using the backcross protocol coupled with diagnostic assays to detect the positive plants at each BC generation.

 

3. To use the original K-39 DH germ plasm to develop stocks that might enable us to locate the transgene. This attempt would use the conventional monosomic procedure.

Our progress for these three steps is provided. All experiments were conducted in biosafety greenhouses and were monitored stringently by the CIMMYT Biosafety Committee.

Results.

1. Our doubled-haploid protocol with spring bread wheat cultivars generally allows us to obtain at least five embryos per spike, of which four generally differentiate, and all double. Hence, obtaining four DHs per spike is considered normal for bread wheat. The spikes of the K-39 plants were weak in growth and small. This cultivar has a winter habit, which may explain why we obtained only 11 embryos, from which seven differentiated into plants, and six were doubled successfully. All DH plant seed were germinated, given limited vernalization, tested for the transgenes expression, and found to be positive. The DH germ plasm now can continue to be tested alongside its parental K-39 cultivar for studying the stability of gene expression after continuous selfing.

2. During DH production with the K-39 winter-habit cultivar, we also pollinated six spikes by three elite spring bread wheat cultivars (two spikes/cultivar of Attila, Kauz, and Luan). Three F1 progenies were formed, and the F1s were grown, tested positive for transgene expression, advanced to the F2, and also used for producing BC1 seed from each combination by pollinating each F1 by Attila, Kauz, and Luan. The F2 generation was planted and tested for the transgene presence. The population tested for each of the F2s studied showed a perfect 3:1 (resistant : susceptible) ratio. The BC1 similarly tested gave the expected (resistant : susceptible) 1:1 ratio. The BC1 plants have been advanced to the BC3, and one more BC is planned for making the derivative phenotypes akin to Attila, Kauz, and Luan. From the transgene-positive, BC4 plants, DHs will be produced and after testing for the transgene, these DHs will represent the culmination of this study. This result will demonstrate one option for transferring an initial stable event to elite cultivars, in this case, from K-39 to Attila, Kauz, and Luan.

3. Transgene location. Using FISH is a swift procedure to locate transgenes on wheat chromosomes. The procedure requires chromosome banding as an indicator to identify the wheat chromosomes. So far, we have not been successful with this approach. Simultaneously, the conventional monosomic analysis route utilized the Chinese Spring monosomic stocks has been pursued. The 41-chromosome monosomes of each of the 21 wheat chromosomes have been hybridized with the stable DH K-39 transformed cultivar. F1 seed from each monosome have been obtained, and hence the germ plasm necessary to locate the transgene has been produced. Our approach will follow a protocol that identifies at least five F1 plants with 41 chromosomes for each of the 21 combinations. From these 41-chromosome, F1 plants, haploids will be produced and will be tested for gene expression. Segregating and nonsegregating haploids will indicate the transgene locations.

Our contention is that once the desired transgene or the futuristic 'clean' transgene events have been obtained, the practical utilization of the material can be integrated with conventional breeding procedures mediated by the homozygosity DH protocol. Inheritance studies considered crucial for basic information can be pursued independently.

Performance of advanced bread wheat x synthetic hexaploid derivatives under reduced irrigation.

R. Trethowan, M. Van Ginkel, and A. Mujeeb-Kazi.

The annual increase in genetic potential in drought environments is only about half (0.3-0.5 %) of that obtained in irrigated, optimum conditions. Attempts by many researchers to produce wheat adapted to semiarid environments have had limited success. At CIMMYT, we follow a system for drought tolerance in which yield responsiveness is combined with adaptation to drought conditions.

The T1BL·1RS translocation wheats have a demonstrated advantage in dryland wheat areas, and the search for other diverse sources to exploit continues. One such unique gene pool resides in the primary Triticeae diploid Ae. tauschii. We have combined this diploid grass with elite durum cultivars to produce synthetic hexaploids. Field testing under reduced irrigation over the past several years has led to the identification of some synthetics classified as drought tolerant. The best five of these SHs have been crossed with a drought susceptible cultivar Opata, and the resulting F1s are being used to develop doubled haploid mapping populations.

Utilizing a few drought-tolerant synthetics, some crosses with Opata were advanced beyond the F1, and the performance of these advanced derivatives was studied in Obregon, Mexico. Very little rainfall was recorded during the 1998-99 crop cycle, resulting in good evaluation for drought tolerance (Table 11). A number of synthetic derivatives yielded more than Baviacora, the long-term check used in drought trials sown in Obregon. These synthetic derivatives are free threshing with large, bold white grain. From the five SH-based advanced derivatives, 25 doubled haploid derivatives per entry have been produced in anticipation that complete homozygosity may have a beneficial contribution in future evaluations of this germ plasm in Mexico and globally.

In order to combine drought tolerance with late heat tolerance, replicated trials of the candidates for HTWYT and WAWSN were sown under drought with the purpose of identifying potential parental material for the crossing program. Surprisingly, a number of lines, primarily synthetic derivatives, performed well under moisture stress (Table 12). A possible relationship between drought and late heat tolerance selected under optimally irrigated conditions is indicated. Table 12 shows the performance of these Bacanora derivatives in relation to Bacanora itself. The derivatives yield up to 23 % higher than Bacanora. This relationship needs further examination and will be handled by our physiology program.

A sea-water based salinity testing protocol and the performance of a tester set of accumulated wheat germ plasms.

J. L. Diaz De Leon*, R. Escoppinichi*, R. Zavala*, and A. Mujeeb-Kazi.
(* Universidad Autónoma de Baja California Sur, Department of Agronomy, Apdo. Postal 19-B, 23054 La Paz, Baja California Sur, México).

Abiotic stresses are static mechanisms that tend to be more durable due to the absence of pathogen influence. Three stresses of significance are heat, drought, and salinity, and all still pose a major challenge. Focusing on salinity with wheat as the main crop, we have accumulated a number of land races and cultivars from global collaborators to form a tester set. We have developed a field-screening protocol using a dilution of sea water as the irrigation source. This setup was initiated in 1996 and initially reported by us in 1997. We are now providing an update after several investigations, particularly after we implemented the use of a well-designed field layout and were able to make projections for discriminating saline-tolerant germ plasm under our conditions at this stage.

The tester set is comprised of 12 bread wheat cultivars and one durum wheat (PBW 34) cultivar. The bread wheat cultivars include land races (Kharchia 65 and Shorawaki); conventional cultivars (KRL 1-4, Lu 26 S, Sakha 8, SNH-9, and WH-157); a wheat cytogenetic-stock parental line (Chinese Spring); an intergeneric hybrid-derivative cultivar (Pasban 90); and the elite bread wheat lines Oasis, Galvez, and Yecora as checks. The test saline regimes were 0, 8.0, 12.0, 16.0, and 20.0 dS/m with observations recorded for leaf area, plant height, days-to-anthesis and physiological maturity, and 1,000-kernel weight.

Germ plasm details. Details of the 13 entries included in the tester set are given in Table 13. The durum wheat PBW 34 is a susceptible line, whereas Oasis, Galvez, and Yecora are the three wheat check cultivars. Oasis and Yecora are separated by Oasis in having the Lr19 gene. Both are dwarf and high-stress levels readily influence this trait. Kharchia and Shorawaki are tolerant but rust susceptible, tall land races from India and Pakistan, respectively. The cultivar Chinese Spring is a line used in intergeneric hybridization primarily because of its superior crossability with alien Triticeae species and is notable for its superior salt tolerance. Chinese Spring is a tall, awnless, facultative winter wheat and susceptible/highly susceptible to leaf/stem rust. Pasban 90, a variety released in Pakistan for irrigated agricultural areas plus saline sodic soils, is an intergeneric derivative with the pedigree Inia66/Th. distichum//Inia 66/3/Genaro81. The cultivars KRL 1-4, SNH-9, WH-157, Lu 26S, and Sakah 8 originate from India, Pakistan, and Egypt.

Seeds of the above lines are maintained by the wheat wide crosses program in CIMMYT, and 10-g samples can be provided to researchers upon request to the second author of this article.

Field screening using sea-water dilutions. The germ plasm screening of the tester lines for salinity was conducted under field conditions in La Paz, Baja California Sur, Mexico. Sea water in close proximity to this field site was trucked in and stored in 1,200-liter dark Nalgene containers. Mixing sea water with normal field-site irrigation water provided the desired EC levels of 8, 12, 16, and 20 dS/m, representing treatments T2 to T5. The control nonsaline treatment T1 (1.5 dS/m) utilized well water as the irrigation source.

Field plots measured 3 m2 and were separated from each other by 1 m on all sides by black plastic line dividers. Each plot was flood-irrigated individually according to its treatment category with 200 liters twice a week. The electrical conductivity (EC) of each irrigated plot was measured and, if necessary, precisely adjusted. Soil samples were taken randomly from two places in each plot after 24 hours for EC analysis. The established extraction procedures included measuring the fresh soil weight, drying samples at room temperature, taking 100-g samples/plot, extracting salts from the 100-g plot sample with 30 ml distilled water, and reading the EC level of the filtrate an Orion® conductivity meter. Plots were fertilized with urea once a week up to 8 weeks after germination in each plot. Each entry was planted in four rows, 4-m long and 15 cm apart, and replicated in triplicate in a lattice design.

Results and establishing criteria to discriminate the tolerance of the entries. Salinity levels from 10 dS/m and above are considered by researchers as being unsuitable for wheat, and if cultivars grow well then these are assumed to possess tolerance to such elevated EC levels. Hence, a good cutoff point in screening may be at about 12.0 to 12.5 dS/m. At this level, some reductions in measured traits should appear. These reductions will become more pronounced as the EC levels are increased further.

In our test, significant reductions were initiated in the cultivars at 12.5 dS/m, became pronounced at 16.5, and reached a maximum at 20.5 dS/m, where 50 % reductions across all traits were widespread. Shorawaki and Kharchia exhibited reductions between 20 to 35 % at 20.5 dS/m and were the least affected, indicating their superior tolerance. They were unaffected at 12.5 dS/m.

Oasis, Yecora, and PBW 34 had maximum reductions in leaf area (50 %), which confirmed their salinity sensitivity. A height reduction of 15 % was observed at 12.5 dS/m for each cultivar, which translated to between 20 to 30 % at 20.5 dS/m. The reductions for the two land races, even though around 25 %, was acceptable because the reduced height adequately supports its plant habit, and a beneficial biomass/harvest index could be realized. The height of Chinese Spring was unaffected at 12.5 dS/m, was reduced 12 % at 16.5 dS/m, and reduced nearly 20 % at T5. This trend was the same as observed for the two other land races, Kharchia and Shorawaki. In general, high saline concentrations induced earliness and physiological maturity for all cultivars except for Chinese Spring, Shorawaki, and Kharchia. This characteristic may influence grain-filling quality and yield. Reductions in grain yield and 1,000-kernel weight across treatments and cultivars followed a similar trend as observed for the traits above they were between 15 and 20 % were at 12.5 dS/m for the check cultivars Yecora, Galvez, and PBW 34; whereas the three superior entries Kharchia, Shorawaki, and Chinese Spring exhibited this reduction at the highest salinity test level of 20.5 dS/m.

In general, all the tester entries reported to be salt tolerant were indeed so at the 12.5 dS/m EC level. The performance of the checks showed highly pronounced reductions at this level. The best lines performed well and demonstrated reduction trait levels observed for the checks at 12.5 only at the highest EC level of 20.5 dS/m. We suggest that a level of 12.5 dS/m may be ideal for future field screening of germ plasm and breeding populations.

The above germ plasms also are being tested under controlled conditions in hydroponics (50 mM NaCl) in order to determine their K:Na discrimination trends.

Conclusions.

1. The evaluation of the tester-set that we accumulated from different locations may provide information that will facilitate the development of a common ground, from which inferences relative to salinity tolerance can be made by its global testing.

2. Promising lines identified by colleagues could be added to this tester set, with new sets possessing reasonable number of entries made available for evaluations.

3. Breeding populations could be screened initially at 12.5 dS/m and then tested stringently under in vitro conditions and at higher EC levels for comparisons with the best three wheats identified at this stage (Chinese Spring, Kharchia, and Shorawaki).

4. The most superior entries in the current set in addition to Kharchia 65, Shorawaki, and Chinese Spring are WH-157 and KRL 1-4 based upon their performance at 12.5 and 20.5 dS/m

Identification of four bread cultivars tolerant to salinity following sea-water field evaluations as varietal candidates for Baja California, Mexico.

J. L. Diaz De Leon*, R. Zavala*, R. Escoppinichi*, and A. Mujeeb-Kazi.
(* Universidad Autónoma de Baja California Sur, Department of Agronomy, Apdo. Postal 19-B, 23054 La Paz, Baja California Sur, México).

Several elite bread wheat lines from CIMMYT's bread wheat program were screened under in vitro conditions in an MS medium supplemented with NaCl levels of 50, 100, 150, and 200 mM. Seed of the lines were soaked in distilled water for 24 hours and the embryos excised and plated in the above medium. The control had no NaCl. After 50 % germination in the controls, further growth was allowed for 10 days, and then a final germination count and seedling height were recorded. The seedling performance was estimated similarly to the mutation-breeding radiosensitivity determinations for estimating LD50 levels. Using this as a basis, and also observing other phenology parameters, we identified a few lines that showed a minimum degree of growth reduction at a NaCl level equal to 12.5 dS/m EC, a level that we have proposed allows selection of tolerant lines.

With the in vitro selections made and some promising lines identified, we advanced this germ plasm for field tests using sea-water dilutions of 12.5 dS/m, following the experimental design, observation, and inference protocols identical to those described in the preceding article. The germ plasms evaluated included four promising lines from the in vitro tests, the tolerant land races Kharchia and Shorawaki, and the susceptible durum wheat PBW 34 (Table 14).

Leaf area, plant height at maturity, 1,000-kernel weight, and yield showed a general trend of reduction between 10 and 15 % at 12.5 dS/m EC. Reductions in these parameters for the susceptible PBW 34 were around 25 % and less than 15 % for the two tolerant land races. In our valley, the salinity levels approach a maximum of 6.0 dS/m where wheat is grown. Thus, the simple approach of acquiring a bread wheat screening nursery from CIMMYT, testing lines in the valleys where wheat is cultivated, and identifying the best performers may lead to new varieties for this area. However, growers often erroneously associate the best wheat performers in valley cultivation locations with salt tolerance. However, salinity tests, if not made over stringent EC levels, cannot classify the best lines as salt tolerant. We are aware of this discrepancy in advanced line performance and have gathered data that do not correlate the best high yielding lines in our location with salinity tolerance; i.e., in tests where stringent EC levels are maintained and are within the desired tolerant discrimination range over 12.5 dS/m.

The four lines that we are reporting here do possess salinity tolerance and are adapted to the local growing conditions. We have named them Cochimi, Mepuchi, Pericu, and Calafia. They are high yielding and capable of release as varieties in the near future. The germ plasm has been distributed for agronomic testing, and we will include at least two (Mepuchi and Calafia) in the tester set mentioned in the previous article. Currently, we are testing these four lines at higher EC levels in order to ascertain if their tolerance is as good as some of the best from the tester set.

Current progress in assessing the long-term implications of conservation-tillage cropping systems on wheat and maize root diseases and yield in six trials at CIMMYT, Mexico.

Monica Mezzalama, Julie M. Nicol, Ken Sayre, and Peter Grace.

Six management trials have been assessed for root rots caused by soilborne fungi and nematodes. These trials represent two separate megaenvironements (ME) as defined by CIMMYT. The ME1 is at the CIMMYT field station in the NW of Mexico, Obregon, which is the favorable, irrigated, low-rainfall environment. The ME2 is found at two CIMMYT field stations in the high valley of central Mexico, Toluca, and El Batan and is a high summer rainfall environment (> 500 mm rainfall during the crop cycle). The soils at all sites have medium to high clay content.

Work has been conducted on the survey of various root pathogens at these sites, and the results are summarized in Table 15. The presence of the different pathogens seems to be correlated with environmental conditions. A higher incidence of G. graminis tritici was found in Toluca than at the other locations, most likely associated with higher rainfall. Other soilborne pathogens commonly found in our survey were Fusarium spp. and the root lesion nematode P. thornei.

A 1-3 year (1997-99) program has been undertaken to monitor the dynamics of such pathogens under different crop management systems (including rotation, tillage, nutrition, and straw management). A summary of the results is presented in Table 16.

To date some generalized conclusions are:

Tillage and straw management. In wheat, zero and conventional tillage with straw removal increases root disease symptoms, while straw retention decreases. The effect of straw management under continuous maize rotation was not clear; it sometimes increases and other times decreases root disease. The number of nonparasitic nematodes (including fungal and bacterial feeders) was generally higher with straw retention than straw removal. For continuous wheat, there was little difference in yield with zero or conventional tillage. The yield of maize, averaged over the different rotation, tillage, and residue management treatments, was better under conventional tillage than zero tillage, but much larger, significant interactions between the management treatments occurred for maize as compared to wheat. A lower incidence of weeds also was found in zero tillage compared to conventional tillage.

Crop rotation. The crop rotations investigated so far did not clearly affect the incidence of disease on root systems of both maize and wheat. The numbers of the root lesion nematode were generally found to increase under continuous wheat than a maize/wheat rotation. The wheat-maize rotation gave better yields than continuous wheat or continuous maize. The use of vetch in winter in rotation with wheat also gave better yields compared to the use of rape.

Nutrition. Effects of nutrition on root disease incidence were variable. Obregon tended to have a higher number of root lesion nematodes and greater root disease incidence with increasing nitrogen fertilizer application.

Crop management practices in some cases obviously have a marked effect on the incidence of root diseases. To date, none of the changes in the incidence or presence of the root diseases monitored in these experiments appeared to be correlated with yield. However, trends indicated that both factors have increased with certain management practices. More time is needed for these trials to show evidence of yield losses associated with root pathogens that are correlated with environmental conditions and soil chemistry. Work will continue to determine the most appropriate crop management practices for a range of MEs.

Exploiting synthetic hexaploids for improving wheat resistance to crown rot (Fusarium graminearum - Group 1) and common root rot (Bipolaris sorokinana).

J.M Nicol and A. Mujeeb-Kazi.

Background. Root rots caused by F. graminearum and B. sorokinana are common and are implicated in causing significant yield losses of wheat (Tinline et al. 1988; Wildermuth et al. 1992; Diehl et al. 1983). The fungi are particularly important in marginal environments of low rainfall and poor soil nutrition, such as large regions in west Asia and North Africa. CIMMYT has begun a new program for screening and breeding resistance for these soilborne pathogens. These pathogens are difficult to work with, because they are soilborne and cannot be screened easily in the field; hence, a laboratory/field breeding strategy has been established. Select groups of germ plasm have been screened. In particular, the synthetics (T. turgidum/Ae. tauschii) have been emphasized, because they provide a wide array of resistances to a range of other biotic stresses including F. graminearum group 2, S. tritici, and H. sativum.

Methodology. In controlled greenhouse conditions, 46 synthetic derivatives (synthetic wheats crossed with improved bread wheats) were screened against both soilborne pathogens. A randomized, complete block design with eight replicates per genotype was used. Plants were grown in open-ended, electrical conduit tubes (12.5 cm x 2.5 cm) in a large tray of sterile soil. Plants were inoculated 1 week after planting with a prepared, cultured, oat-seed inoculum (initially derived from monosporic cultures of these pathogens), which was applied above the ungerminated sterile seeds (one per tube) and covered with soil. After 1 month, the plants were scored visually for lesion development (on roots (RS), shoots (SS) and coleoptiles (CS)) using a qualitative scale adopted from the methods developed by Wildermuth (1994) and (Wallwork, pers. comm.). The scale is based on a 0-5 rating of lesions on either the roots, shoots, or coleoptiles; where 0 = no lesions, 1 = 0-25 %, 2 = 25-50 %, 3 = 50-75 %, 4 = 75-100 %, and 5 = dead. Data was analyzed with and ANOVA. Known resistant and susceptible checks were included to identify promising new lines.

Results and conclusions. The data are illustrated in Table 17. Certain synthetic hexaploid derivatives appear to be potential sources of resistance to the root pathogens studied. Three derivatives indicated resistance as good as currently available; 'Sabuf/3/BCN//Ceta/Ae. tauschii (895)' against both crown rot and common root rot, 'Altar 84/Ae. tauschii (224)//YACO/6/CROC1/Ae. tauschii (205)/5/BR12*3/4/ 224)' for crown rot, and 'MAYOOR/TKSN1081/Ae. tauschii (222)' for common root rot. Interestingly, these three lines also offer good resistance to F. graminearum Group 2 (Mujeeb-Kazi et al. 1999), perhaps inferring some association between the different Fusarium pathogens (foliar and root). We now plan to confirm these results in the field, to verify their resistance under those conditions and in the adult plant stage.

Table 17. Mean resistance scores for roots (RS), shoots (SS), coleoptile (CS), and an overall total score (TS (RS+SS+CS/3)) for synthetic derivatives and the resistant and susceptible check lines when inoculated with crown rot and common root rot fungi. Data based on a 0-5 ranking of lesioning; 0 = no lesions, 1 = 0-25 %, 2 = 25-50 %, 3 = 50-75 %, 4 = 75-100 % and 5 = dead.

Material	Crown rot				Common root rot
	RS	SS	CS	TS	RS	SS	CS	TS
Pedigree of synthetic derivative
Sabuf/3/BCN//Ceta/Ae. tauschii (895)	0.38	0.31	0.63	0.44	0.81	0.44	3.0	1.42
Altar 84/Ae. tauschii (224)//YACO/6/CROC 1/Ae. tauschii (205)/5/BR12*3/4/ 224)	0.31	0.94	1.95	1.09	1.19	1.06	3.12	1.79
MAYOOR/TKSN1081/Ae. tauschii (222)	1.25	0.81	1.38	1.15	0.69	0.56	3.5	1.58
Check lines for resistance
2-49 for crown rot	0.63	0.81	1.13	0.85	2.00	0.31	3.88	2.10
302-5 for common root rot	1.06	0.69	1.22	0.68	1.50	0.44	3.50	1.90
Check lines for susceptibility
Batavia for crown rot	1.06	1.19	1.75	1.33	2.30	0.88	3.88	2.19
Timgalen for common root rot	1.44	1.81	2.11	1.77	3.31	1.19	4.00	2.71
Durati durum wheat for crown rot and common root rot	2.88	2.69	3.25	2.77	2.38	1.19	3.75	2.44
SED (standard error for difference of the means)	0.47	0.48	0.62	0.44	0.47	0.41	0.32	0.29

References.

• Diehl JA, Tinline RD, and Kochhann RA. 1983. Perdas em trigo causadas pela podridao comum de raizes no Rio Grande do Sul, 1979-1981. Fitopatol Bras 8:507-511.
• Mujeeb-Kazi A, Gilchrist LI, Fuentes-Davila G, Velazquez C, and Delgado R. 1999. Fusarium graminearum resistance in alien germplasm and in a bread wheat/alien species derivative with multiple biotic stress resistances. Ann Wheat Newslet 45:107-108.
• Tinline RD, Wildermuth GB, and Spurr DT. 1988. Inoculum density of Cochliobolus sativus in soil and common root rot of wheat cultivars in Queensland. Aust J Agric Res 39:569-577.
• Wildermuth GB, Tinline RD, and McNamara RB. 1992. Assessment of yield loss caused by common root rot in wheat cultivars in Queensland. Aust J Agric Res 43:43-58.
• Wildermuth GB. 1994. Testing wheat seedlings for resistance to crown rot caused by Fusarium graminearum group 1. Plant Dis 78:949-953.

Cultivar susceptibility to the root-lesion nematode Pratylenchus thornei and wheat yield loss in the state of Sonora, Mexico, and wider implications.

Julie M Nicol and Ivan Ortiz-Monasterio.

Background. The root-lesion nematode P. thornei is a polyphagous, migratory, endoparasitic nematode causing necrotic lesions on the root system. A known pathogen of wheat in many parts of the world, P. thornei was reviewed by Nicol et al. (2000) and has been shown to cause major yield reductions on susceptible, intolerant, wheat cultivars of up to 32 % in Sonora, Mexico (Van Gundy et al. 1974) and 44-85 % in various states of Australia (Doyle et al. 1987; Thompson and Clewett 1986; Thompson et al. 1993; Eastwood et al. 1994; Nicol et al. 1999).

One of the roles of CIMMYT, to improve wheat germ plasm for developing countries, is achieved through a shuttle-breeding program between central and northern Mexico. Although this process has been occurring for about 30 years in some areas infested with P. thornei, there is little knowledge of the resistance (ability of the plant to limit the multiplication of the nematode) or tolerance (ability of the plant to yield despite attack by the nematode) of CIMMYT cultivars to P. thornei. Tolerance, although effective, does not necessarily reduce nematode numbers, and sources of resistance coupled with tolerance are recommended. Reports in the literature also suggest that water limitation is an important factor in determining yield loss with P. thornei and closely related species (Grandison 1972; Orion et al. 1984).

Attempts are made here to:

• establish whether CIMMYT has been involuntarily selecting for resistant and tolerant germ plasm against the nematode,
• determine susceptibility of various CIMMYT cultivars to P. thornei and yield loss, and
• establish the importance of water availability, i.e., drought, or sufficient irrigation on yield loss.

Methodology. Two experiments were established at CIMMYTs experiment station in Cd. Obregon, Sonora, during the 1998-99 wheat crop cycle. The experiments were designed as two split-plots, one under drought (one irrigation) and the other with full irrigation (five irrigations). Each trial consisted of three replicates with the main plots being with and without chemical fumigation of the soil, and the subplots included a selection of seven CIMMYT wheat cultivars released over the past 30 years plus one known, susceptible variety from Australia.

Five weeks prior to planting, the fumigant Basamid® (60 g/m2) was applied to moist soil on the specified areas and incorporated with a disc plough to 30 cm. The trial was planted in plots of eight rows (20 cm apart) by 5 m. To establish the initial nematode density, six individual soil and root samples were taken from each plot at two depths (0-20 cm and 20-40 cm) 1 month after planting between plant rows 2 and 3, 4, and 5, and 6 and 7 at 1.5 and 3.5 m of the plot length. The P. thornei were extracted from one composite, homogenous, 200-g sample using the Whitehead tray extraction method for 3 days at room temperature. The numbers of P. thornei were counted from a water suspension using a 1-ml dilution with a Doncaster dish. All numbers were converted to numbers of P. thornei per 200 g oven-dried soil. A similar sampling was conducted 4 months later (after harvest) to determine the final density of nematodes per plot. Throughout the growing season, a number of plant variables were measured. Three meters of the six central rows were harvested for yield determination. The trial was analyzed as a split plot in conjunction with the use of orthogonal comparisons to compare CIMMYT cultivars against the Australian check variety Warigal.

Results and discussion. The application of the fumigant effectively controlled the nematode by significantly lowering numbers under drought (82 %) and irrigation (91 %). Under irrigation, yield and most other plant parameters for all varieties were not affected by the density of P. thornei, suggesting that the importance of P. thornei is limited under full irrigation. However, the yield of some cultivars was affected significantly under drought conditions with and without Basamid® application (Fig. 2). Yield losses varied from 2-40 % with no indication of a relationship to time of release of the various CIMMYT cultivars, which suggests that CIMMYT has not been selecting for or against tolerance over time.

Orthogonal comparisons comparing the intolerant check variety Warigal against CIMMYT cultivars (Fig. 2) indicated a range of tolerance within the CIMMYT germ plasm, with Baviacora 92 showing the highest tolerance (no yield loss), and Seri 82 being highly intolerant (40 % yield loss). Although the CIMMYT germ plasm tested offers genetic variation for tolerance, P. thornei multiplied on all varieties over the growing season, indicating that they are susceptible.

Conclusions and continuing work. Work is currently underway to repeat this trial. In addition, a range of advanced lines and synthetic derivatives with good performance under drought are being investigated for their resistance and tolerance to P. thornei.

The data collected indicate that the nematode is not a major problem when materials are optimally irrigated, at least in Obregon. However, the implications for yield loss in the presence of the nematode under limited moisture (as a result of reduced irrigation or rain-fed environments) should not be understated. Furthermore, it is documented that in many regions where the nematode is present, root-rotting fungi may play a role in further reducing yield. In particular, evidence exists in western Asia and northern Africa that implicates both nematodes and root rots in some cases to cause severe yield losses (H. Braun, CIMMYT).

CIMMYT germ plasm appears to offer high-yielding sources of tolerance against P. thornei; however, the current results have not indicated any sources of resistance. Ongoing work continues at CIMMYT to screen in the greenhouse and confirm in the field possible sources of resistance from both landraces and advanced CIMMYT germ plasm. Lines that have been identified with resistance and tolerance have been introgressed actively within the wheat breeding program.

As a result of the wide host range (including cereal, legumes, and weeds) of this nematode, we highly recommend that cereal germ plasm be screened to identify sources of resistance. This screening is essential to ensure the long-term control of this pathogen, particularly where rotational regimes do not involve certain legumes that can be used as a source of resistance to lower nematode numbers.

References.

• Doyle AD, McLeod RW, Wong PTW, Hetherington SE, and Southwell RJ. 1987. Evidence for the involvement of the root lesion nematode Pratylenchus thornei in wheat yield decline in northern New South Wales. Aust J Exp Agric 27:563-570.
• Eastwood DR, Smith A, and Wilson J. 1994. Pratylenchus thornei is causing yield losses in Victorian wheat crops. Aust Nemat Newslet 5:2.
• Grandison GS. 1972. The population ecology of the lesion nematode Pratylenchus thornei and root-knot nematode Meloidogyne javanica. Ph.D. Thesis, Waite Agricultural Research Institute, University of Adelaide.
• Nicol JM. 2000. Important nematode pests of cereals in wheat production and improvement. In: FAO Plant Production and Protection series (Curtis BC ed). In press.
• Nicol JM, Davies KA, Hancock TW, and Fisher JM. 1999. Yield loss caused by Pratylenchus thornei on wheat in South Australia. J Nemat 31(4):367-376.
• Orion D, Amir J, and Krikun J. 1984. Field observations on Pratylenchus thornei and its effects on wheat under arid conditions. Revue de Nématologie 7(4):341-345.
• Thompson JP and Clewett TG. 1986. Research on root-lesion nematode. In: Queensland Wheat Research Institute Biennial Report 1982-1984, Qld Dept Primary Industries, Qld Govt, Qld. Wheat Research Institute, Toowoomba, Qld. p. 32-35.
• Thompson JP, Clewett TG, and O'Reilly MM. 1993. Tolerance and resistance in wheat to Pratylenchus thornei. In: Proc Pratylenchus Workshop, 9th Biennial Conf Australasian Plant Pathology Soc, 8-9 July, 1993, Hobart, Tasmania. Australia.
• Van Gundy SD, Gustavo J, Perez B, Stolzy LH, and Thomason IJ. 1974. A pest management approach to the control of Pratylenchus thornei on wheat in Mexico. J Nemat 6:107-116.

Publications.

• Ayala L, Khairallah M, González-de-León D, Van Ginkel M, Mujeeb-Kazi A, Keller B, and Henry M. 2000. Identification and use of molecular markers to detect barley yellow dwarf virus resistance derived from Th. intermedium in bread wheat. Theor Appl Genet (submitted).
• Mujeeb-Kazi A, Cortes A. Rosas V, William MDHM, and Delgado R. 1999. Some Triticum turgidum L. cultivars possessing the chromosome T1BL·1RS substitution. Cereal Res Commun 27:373-376.
• Mujeeb-Kazi A, Cortes A, Rosas V, William MDHM, and Delgado R. 1999. Development of near-isogenic sets of derivatives with T1BL·1RS or 1B chromosome substitutions in bread wheat. Wheat Inf Serv 89:13-16.
• Mujeeb-Kazi A, Gilchrist LI, Villareal RL, and Delgado R. 2000. Registration of ten wheat germplasm lines resistant to Septoria tritici leaf blotch. Crop Sci (in press).
• Mujeeb-Kazi A, William MDHM, Villareal RL, Cortés A, Rosas V, and Delgado R. 2000. Registration of 11 new isogenic T1BL·1RS chromosome translocation and 11 extracted chromosome 1B lines in Triticum turgidum L. cv. 'Altar 84'. Crop Sci (in press)
• Mujeeb-Kazi A, William MDHM, Villareal RL, Cortes A, Rosas V, and Delgado, R. 2000. Registration of 10 isogenic chromosome 1B and 10 T1BL·1RS chromosome translocation bread wheat germplasms. Crop Sci (in press).
• Mujeeb-Kazi A, Cortes A, Rosas V, Cano S, and Delgado R. 2000. Registration of 17 isogenic chromosome 1B and 17 T1BL·1RS chromosome translocation bread wheat germplasms. Crop Sci (in press).
• Mujeeb-Kazi A, Cortes A, Rosas V, Cano S, and Delgado R. 2000. Registration of six isogenic T1BL·1RS chromosome translocation and six chromosome 1B durum germplasms. Crop Sci (in press).
• Mujeeb-Kazi A, Cano S, Rosas V, Cortes A, and Delgado R. 2000. Registration of five synthetic hexaploid wheat and seven bread wheat germplasm lines resistant to wheat spot blotch. Crop Sci (in press).
• Mujeeb-Kazi A, Fuentes-Davila G, Villareal RL, Cortes A, Rosas V, and Delgado R. 2000. Registration of 10 synthetic hexaploid wheat and six bread wheat germplasms resistant to Karnal bunt. Crop Sci (in press).
• Mujeeb-Kazi A and Rajaram S. 2000. Transferring alien genes from related species and genera for wheat improvement. FAO. Book chapter (in press).
• Mujeeb-Kazi A and Villareal R. 2000. Wheat: an adaptive, descriptive and futuristic treatment (Chopra VL ed). Book chapter (in press).
• Mujeeb-Kazi A, Cortes A, Rosas V, Delgado R, Shafiq FA, and Vahidy AA. 2000. Three new monosomic chromosome 5B genetic stocks of Triticum aestivum L. cultivars. Wheat Inf Serv (in press).
• Villareal RL, Sayre K, Banuelos O, and Mujeeb-Kazi A. 2000. Registration of four synthetic hexaploid wheat (Triticum turgidum/Aegilops tauschii) germplasm lines tolerant to waterlogging. Crop Sci (in press).