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International Maize and Wheat Improvement Center - CIMMYT
Lisboa 27, Colonia Juárez, Apdo. Postal 6-641, 06600, México, D.F., México.

 

Identification of highly transformable Bobwhite sister lines. [p. 96-97]

B. Skovmand, S. Rajaram, A. Pellegrineschi, and A. Mujeeb-Kazi.

Transformation protocols were used to screen 129 Bobwhite sister lines for their transformation ability. Some Bobwhite sister lines, generated at CIMMYT in the mid 1970s from the cross CM33203 (pedigree: Aurora//Kalyan/Bluebird/3/Woodpecker), have been reported to be highly transformable. Therefore, we screened all the Bobwhite accessions to identify the most transformable and responsive ones. Transformation efficiency was evaluated based on regeneration performance on selective medium. Healthy, fully differentiated embryogenic calli were scored (1 callus/embryo) as number of regenerating calli divided by the total number of immature embryos bombarded (one regenerating callus was scored as 1). Somatic embryo germination frequency was 0-89 %. Accessions responded in four different ways to bombardment: 1) no regeneration, 2) herbicide tolerance and bombardment susceptibility, 3) herbicide sensitivity, bombardment tolerance, and high regeneration, and 4) herbicide sensitivity and bombardment tolerance but low regeneration. Transformation efficiency was calculated as the effective number of transgenic plants obtained divided by the total number of immature embryos bombarded. The most efficient sister lines were SH-98 26 and SH-98 56 (Table 1). Other accessions (SH-98 15, SH-98 88, and SH-98 121) gave higher regeneration frequency but less overall efficiency due to escapes (plants surviving the selection process but not transgenic). The best Bobwhite sister lines for transformation were retested for their transformation ability and are listed in Table 1.

Table 1. Results of statistical analyses of the seven best Bobwhite lines for embryogenesis, regeneration in selective medium, and effective transformation rate. The data were pooled from three repetitions with over 2,000 embryos/transformation experiment.

Bobwhite line	T1BL·1RS	Somatic embryogenesis formation		Regeneration efficiency (%)		Transformation efficiency (%)
		Bombarded	Control	Bombarded	Control
SH 98 26	No	72.09 ± 13.34	75.35 ± 13.39	71.17 ± 14.68	0.00	70.86 ± 14.48
SH 98 29	Yes	59.23 ± 15.56	63.49 ± 18.22	61.90 ± 11.29	0.00	60.92 ± 11.58
SH 98 56	Yes	70.34 ± 9.82	70.14 ± 5.98	70.34 ± 9.83	0.00	69.02 ± 6.94
SH 98 96	Yes	69.17 ± 9.92	69.97 ± 4.73	69.17 ± 9.93	2.43 ± 1.55	66.96 ± 6.14
SH 98 97	Yes	90.96 ± 5.95	93.33 ± 4.92	76.63 ± 9.02	13.98 ± 4.54	66.96 ± 4.53
SH 98 110	No	81.07 ± 7.15	81.25 ± 5.23	70.95 ± 6.32	10.77 ± 4.87	60.80 ± 6.28
SH 98 128	Yes	91.15 ± 4.85	93.54 ± 4.22	78.13 ± 8.21	20.13 ± 5.61	58.27 ± 10.40
SH 98 129	Yes	80.55 ± 7.01	85.59 ± 6.11	71.82 ± 9.61	10.08 ± 3.58	60.04 ± 9.61
Materials and methods are described in Pellegrineschi A, Noguera LM, McLean S, Skovmand B, Brito RM, Velazquez L, Hernandez R, Warburton M, and Hoisington D. Identification of highly transformable wheat genotypes for mass production of fertile transgenic plants. Plant Cell Rep (submitted).

Acknowledgments. This research was funded in part by the Australian Cooperative Research Center for Molecular Plant Breeding, in which CIMMYT is a participant. The Bobwhite family, developed by Dr. S. Rajaram, is stored in CIMMYT's germ plasm bank, as 'in trust' material under a FAO/CIMMYT agreement and is consequently freely available to any bonafide user.

Synthetic bread wheat derivatives with Karnal bunt resistance. [p. 97]

R.L. Villareal, G. Fuentes-Davila, O. Bañuelos, and A. Mujeeb-Kazi.

Over the last 15 years, breeding for Karnal bunt-resistant germ plasm has received high priority at CIMMYT. Synthetic hexaploids derived from durum 'wheat/Ae. tauschii' crosses have shown highly resistant to immune (0 % infection) response to Karnal bunt and offer new genetic variability for resistance to the disease. The results summarized in this report focus on the advanced derivatives from this breeding effort. Four hundred thirty-eight, backcross-derived, synthetic hexaploid wheats from three crosses involving 'synthetic bread wheat/T. aestivum' were screened for resistance to Karnal bunt over two production cycles under field conditions at the Mexican Agricultural Research Center near Ciudad Obregon, Sonora. Each entry was planted in plots of two rows, 1-m long, and spaced 20 cm between rows. Two inoculation dates per cycle were employed. Ten random tillers of each entry at boot stage were injected with a suspension of sporidia in water (10,000 spores/ml of water). At maturity, the inoculated spikes were threshed individually and evaluated for percent Karnal bunt infection. Karnal bunt testing for 2 years showed 39 % of the derivatives with infection scores less than 3 % as compared to 74 % of the susceptible bread wheat check cultivar WL 711. The highly resistant backcross lines were mostly semidwarfs, intermediate in maturity, and of good test weight. The overall mean Karnal bunt score of the derivatives was 17.3 %, ranging from 0.35-70% infection as compared to 45.7 % of the three recurrent parents (Seri, Opata, and Bcn). A sample of the resistant lines from the three populations is presented (Table 2).

Table 2. Mean Karnal bunt infection score, grain yield and agronomic characteristics of BC1F1 lines derived from three populations.

Entry no.	BC1F1 sel. no.	KB infection (%)	Grain yield (kg/ha)	1,000-kernel weight (g)	Test weight (kg/hl)	Maturity (d)	Plant height (cm)
Altar 84/Ae. tauschii 219//2*Seri
137	-99Y-	0.35	5533	40.2	77.9	126	103
53	-51Y-	1.60	5885	40.1	80.1	132	101
52	-50Y-	1.65	5802	42.3	82.2	129	95
103	-41Y-	1.80	6231	44.2	79.7	129	91
Croc 1/Ae. tauschii 224//2*Opata
122	-125Y-	0.60	5694	37.9	80	128	106
11	-11Y-	0.70	6224	37.8	80	128	94
44	-45Y-	1.05	5834	37.6	78	126	89
22	-22Y-	1.15	5840	38.3	80	129	96
60	-62Y-	1.20	5611	38.9	77	129	94
15	-15Y-	1.65	5345	33.6	80	130	88
40	-41Y-	1.65	5720	37.0	81	135	101
32	-33Y-	1.75	6048	36.2	78	126	94
81	-83Y-	1.75	5273	34.6	80	125	93
Duergand/Ae. tauschii 214//2*Bcn
35	-36Y-	1.45	5365	40.2	80.2	123	97
121	-123Y-	1.95	4997	44.2	79.1	122	94
39	-40Y-	2.10	5201	38.9	80.0	136	98
16	-14Y-	2.35	5406	40.3	80.4	124	87

Heat tolerant synthetic bread wheat derivatives from CIMMYT wheat program. [p. 98]

R.L. Villareal, O. Bañuelos, J. Borja, S. Rajaram, and A. Mujeeb-Kazi.

Stability of performance under high-temperature environments is one of CIMMYTs most important breeding objectives. This report elucidates the results of screening backcross derivatives that have emanated from the use of the Ae. tauschii gene pool for tolerance to heat stress. One hundred fifty-six BC1F1s derived F5 advanced synthetic derivatives from the cross 'Duergand/Ae. tauschii 214//2*Bacanora' were evaluated for tolerance to heat stress over 2 years of field tests at the Mexican National Agricultural Research Station at Ciudad Obregon, Sonora. Materials were machine sown at 8-row plots, 20 cm apart and 5-m long in an alpha lattice design with three replications. Late planting (after 20 January) results in flowering in late March and physiological maturity in mid-May under relatively high temperatures and permits identification of high-temperature tolerant genotypes. This is similar to the Indian subcontinent conditions where late-planted wheats are continuously exposed to high temperatures, especially at the critical stages of flowering and grain filling. Seventy-eight percent of the lines showed similar yield to the recurrent parent Bacanora. Seven derivatives had superior biomass yield at maturity than the recurrent parent. The highest biomass yield was 14.6 t/ha, whereas the lowest was 6.5 t/ha. Mean 1,000-kernel weight for all derivatives ranged from 22.0-33.8 g, as compared to 23.2 g for Bacanora. Selected lines with their agronomic characteristics are presented in Table 3.

Table 3. Agronomic characteristics of 10 BC1F1 lines derived from the cross 'Duergand/Ae. tauschii 214//2*Becanora' selected under heat stress conditions.

Entry no.	BC1F1 sel. no.	Yield (kg/ha)	1,000-kernel Biomass (t/ha)	weight (g)	Test weight (kg/hl)	Maturity (d)	Height (cm)
105	-106Y-	3,933	11.6	31	72	92	67
119	-121Y-	3,834	13.3	24	70	89	64
96	-97Y-	3,812	10.7	31	73	87	64
112	-114Y-	3,798	11.3	33	69	87	74
92	-93Y-	3,796	14.2	30	71	89	75
5	-3Y-	3,718	12.6	31	70	88	64
93	-94Y-	3,666	10.6	31	70	85	73
142	-14Y-	3,655	10.2	31	75	88	60
87	-88Y-	3,654	12.5	26	71	89	63
63	-64Y-	3,651	11.2	28	67	89	66
1	Becanora	3,578	9.9	24	71	90	55

CIMMYTs basic and advanced wheat improvement training courses in 2000. [p. 98-99]

R.L. Villareal and O. Bañuelos.

CIMMYT training programs are characterized by close working relations between the Center's senior scientists and a number of participants from NARSs. In the basic wheat improvement course, young researchers, usually with none to little experience in the national agricultural programs, spend one wheat cycle in Mexico fully participating in research or the development of production practices. The advanced course focused on person-to-person exchange of ideas and more formal information-sharing of activities between and among active senior wheat visiting scientists from the developing countries and CIMMYT staff. Most important, participants learned the skills on how to manage efficiently a germ plasm improvement program and the opportunity to select new wheat germ plasm. This course was offered for the first time at CIMMYT. In 2000, the CIMMYT wheat improvement courses provided practical training to 22 wheat researchers from 11 countries. The in-service trainees in 2000 were Jahangir Alam Chowdhury and Ali Babar, Bangladesh; Zhou Kuanji and Wu Xiaohua, China; Dario Novoselovic, Croatia; Alshoraz Abzhappar and Shpigun Sergey, Kazakhstan; U Aye Po, Mayanmar; Pashkov Sergey, Russian Federation; Olaf Müller and Khayalethu Ntushelo, South Africa; Kosimov Farkhod, Tadjikistan; Fevzi Partigoc, Sait Ceri, and Mustafa Yildirim, Turkey; and Radivoje Jevtic, Yugoslavia. The participants in the 2000 advanced wheat improvement coure were Dr. Md. Abdus Samad, Wheat Principal Scientific Officer, Bangladesh; Mr. YaoJinbao, wheat breeder, China; Prof. Zou Yuchun, Senior Plant Breeder, China; Dr. Minura Yessimbekova, Head of Cereal Crops Department, Kazakstan; Mr. Dhana Bahadur Gharti, Senior Scientist (Plant Pathology), Nepal; and Dr. Kenan Yalvac, Head of Breeding and Genetics Department, Turkey. Fellowships to these activities were provided by Mathile Family Foundation.Officer, Bangladesh; Mr. YaoJinbao, wheat breeder, China; Prof. Zou Yuchun, Senior Plant Breeder, China; Dr. Minura Yessimbekova, Head of Cereal Crops Department, Kazakstan; Mr. Dhana Bahadur Gharti, Senior Scientist (Plant Pathology), Nepal; and Dr. Kenan Yalvac, Head of Breeding and Genetics Department, Turkey. Fellowships to these activities were provided by Mathile Family Foundation.

Irrigated, wheat-production systems: excess tillage, excess nitrogen, and inadequate water. [p. 99-102]

K.D. Sayre, J. Cruz, S. Sanchez, and M. Cano.

Irrigated, wheat-production systems (spring, facultative, and winter wheat) comprise nearly 55 % of the wheat area and roughly 65 % of wheat production in the developing countries. Between 35 and 45 % of these production systems involve wheat in rotation with flooded paddy rice and the rest with wheat in rotation with a large number of potential upland crops including maize, soybean, and cotton. The vast majority of this area is characterized by: 1) use of intensive tillage systems, often with crop residue removal or burning; 2) largely inefficient irrigation water delivery by gravity systems (mainly by flooding), and 3) use of comparatively high levels of N fertilizers.

Excessive tillage, especially when residues are removed or burned, is clearly contributing to a 'wearing down' of the foundation for sustainable production through degradation of soil productivity and/or through creation of conditions leading to diminishing input use efficiencies. However, the problems associated with marked reductions in tillage combined with high levels of surface retained crop residues for surface/gravity irrigation water delivery systems (especially flood irrigation systems) have discouraged most irrigation researchers and farmers from trying to reduce tillage and retain residues. Nonetheless, CIMMYT agronomists, in collaboration with NARS scientists and farmers, have developed new technologies and machinery to allow zero/reduced till planting with crop residue retention, which are being extended to South Asia, including to small-scale farmers. Furrow irrigated, bed-planting systems have greatly facilitated the scope to manage crop residues as well as dramatically reduce tillage.

The efficiency of irrigation water use in wheat production needs continuing improvement, because water presents a major production cost to most farmers. Yet, more importantly, a worldwide accelerating competition for scarce water resources and agriculture will undoubtedly lose the battle to maintain even its current share, especially because most irrigation systems and farmer irrigation practices are notoriously inefficient, wasting excessive amounts of water. It is a foregone conclusion that marked increases in the efficacy of irrigation water use must be achieved if production levels are to be maintained or increased, since we will need to produce more from less.

Similarly, N fertilizer use efficiency in irrigated wheat must be improved, not only in view of its increasing contribution to the cost of wheat production but also because of detrimental environmental effects associated with improper N management and its excessive use.

This presentation attempts to illustrate how breeders and agronomists can work together to develop needed management strategies to enhance water and nitrogen use efficiency and then identify suitable genotypes to fit these new reduced-till management strategies. To do this, management by genotype interactions must be understood and utilized to identify the right genotypes.

A key part of crop management strategies that CIMMYT agronomists are using to improve both water and N-use efficiency uses furrow-irrigated, bed planting systems. Farmer trials/observations and station trials have indicated saving up to 25-50 % of irrigation water as compared to typical flood irrigation systems in Mexico, China, India, Pakistan, and Iran. This planting system allows new management opportunities for planting orientation on the beds as well as for N timing and placement. Opportune field access facilitates management operations by tracking in the furrows between the beds.

Irrigation strategies. Figure 1a presents 2-year (1998-99 and 1999-00) averaged yield results for seven bread and seven durum wheat genotypes grown with five (554 mm H2O applied) or four (392 mm H2O applied) irrigations. Performance of durum wheat lines over the two irrigation treatments was decidedly different from bread wheat lines. Average yield for the durums was not affected by reducing the irrigation whereas a small but significant yield reduction occurred in bread wheat. However, there were significant 'irrigation x crop' and 'irrigation x genotype' within crop interactions indicating differential performance patterns which can offer positive selection opportunities for breeders. Only small year alone or interactions of the other treatment factors with year were noted.

Figure 1b gives the results from a similar trial where two durum and two bread wheat genotypes were produced in the 1998-99 and 1999-00 crop cycles with either five irrigations (508 mm H2O applied) or four (392 mm H2O applied) irrigations. The genotypes were planted using either three rows/bed (20 cm between rows) or two rows/bed (40 cm between rows) on 80-cm beds (width from furrow center to furrow center). As can be observed, average grain yield was higher with two rows/bed using five irrigations, whereas yield for four irrigations was higher for three rows/bed. However, highly significant 'irrigation x genotype' and' row #/bed x genotype' interactions indicated that differential genotypic performance patterns must be carefully considered in order to be utilized in developing new lines that will provide higher water use efficiencies under the most feasible planting methodology.

N-management strategies. N rates that many farmers use for irrigated wheat tend to be markedly higher than those used by most rain-fed wheat producers because of higher yield potential expectations. However, this can be exorbitant, as in the Yaqui Valley of Sonora where the current average N application to wheat by farmers is over 275 kg/ha. As in most irrigated wheat situations, farmers in the Yaqui Valley tend to apply a large part of the N preplant or at planting (commonly between 50-80 % of the total N applied). Our research has consistently demonstrated that when there is a marked reduction in the amount of fertilizer N applied at or before planting combined with the bulk applied at near the 1st node growth stage, yield is normally enhanced and remarkable grain quality improvement occurs.

Figure 2a presents the yields for four durum wheat varieties grown for 2 years (1998-99 and 1999-00) at CIANO where 225 kg N/ha were applied using three different timing patterns. Altar 84 currently is the most widely grown durum wheat in the Yaqui Valley and can be considered as a check. Applying all N at planting (similar to farmer practice) was grossly inferior to the other two application strategies using split applications. A small 'year x genotype' interaction for yield was observed but no other interactions were significant.

Figure 2b presents the % flour protein for the same varieties and N management treatments and serves as a quality indicator. The figure clearly indicates the exceptional advantages of split applications in quality expression. There were large yield and quality differences between the varieties. Concerning the split application treatments, applying 1/3 N at planting and two-thirds at 1st node provided the highest yields and increases in % flour protein compared to applying all N at planting. Applying two-thirds at 1st node and one-third at boot stage provided an intermediate yield increase but a greater increase in % flour protein. There was a significant 'N management x variety' interaction while all other interactions were not significant.

Figure 3a and Fig. 3b give similar information for a series of bread wheat genotypes grown during the 1999-00 crop cycle at CIANO where 225 kg N/ha was applied with different timing. Rayon 89 is currently the most widely grown bread wheat in the Yaqui Valley and is the check. Also included are the mean yields for four genotypes obtained from the rust resistance program and five from the bread wheat program.

Yield performance (Fig. 3a) also indicates the inferiority of applying all N at planting. Yields were higher for the two split-application treatments, which were at par. The splits were one-third at planting and two-thirds at 1st node versus one-third at planting, one-third at 1st node, and one-third at boot stage. Large genotypic differences occurred but there was a significant 'N management x genotype' interaction.

Figure 3b presents the % grain protein values for the same N-management and genotypes. As observed for durum wheat, split applications not only increased bread wheat yield but markedly increased grain protein content as compared to applying all N at planting. All genotypes responded in a similar manner for protein content although there were large genotypic differences. The 3-way split was better for both yield and protein for all genotypes except for the yield of Rayon 89. The 'N management x genotype' interaction for protein was not significant.

The three examples given above indicate the sharp differences in crop and genotype performances that can be obtained with different crop management strategies. Furthermore, they illustrate differential management and genotype interactions that can occur and could be exploited in variety development. Breeders and agronomists have not worked closely enough to exploit these kinds of elements to more efficiently develop the varieties farmers need. This is especially true when faced with new technologies like reduced/zero till planting systems with residue retention, bed-planting systems, or the inevitable constraints imposed by less available irrigation water or more costly fertilizers. The CIMMYT wheat program breeders and agronomists are trying to improve how we develop better germ plasm and to provide a purposeful example for our NARS colleagues.

Potential of Aegilops geniculata genetic resources for wheat improvement. [p. 102-103]

M. Zaharieva, A. Cortéz, V. Rosas, S. Cano, J. Sanchez, L. Juarez, R. Delgado, and A. Mujeeb-Kazi.

Aegilops geniculata is an annual, self-fertile, allotetraploid species (2n = 4x = 28, MU genomes), belonging to tribe Triticeae Dumort., subtribe Triticinae Griseb. Ae. geniculata has a wide distribution around the Mediterranean Sea region, showing adaptations to a large range of environmental constraints. Among the 22 species of the Aegilops genus, it is particularly interesting as a source of resistance to various diseases and pests, drought, and salinity (Gill et al. 1985), suggesting that this species could represent a valuable reservoir of genes for improving wheat resistance to biotic and abiotic stresses. A complete set of T. aestivum-Ae. geniculata chromosome addition lines were developed and described by Friebe et al. (1999).

Management of Ae. geniculata genetic resources. A collection comprising nearly 160 Ae. geniculata accessions originating from different ecogeographical regions of southern Europe, northern Africa, and western Asia, and covering almost whole area of species distribution, was established. Genetic diversity analysis, based on morphological variation and DNA polymorphism (RAPD and RFLP) was performed (Zaharieva et al. 2001). Ae. geniculata exhibits a high level of intraspecific variability, principally distributed among accessions and geographically structured. This species presents also an important diversity for reaction to the major wheat diseases and pests. Promising accessions with resistance traits to BYDV, rusts, and cereal cyst nematodes already have been identified from the collection through a collaborative network. Evaluation was completed in CIMMYT for leaf rust and root lesion nematodes resistance. The collection also was studied for physiological traits related to drought and heat stress. Two major groups of accessions with different adaptive strategies were distinguished. Accessions originating from harsh environments (Jordan, Libya, Tunisia, and Cyprus) had high water use efficiency (low carbon isotope discrimination) low biomass, and low grain production. They were early, with small, thick leaves exhibiting low chlorophyll content, high surface temperature, and low epidermal transpiration, which could correspond to adaptation to strong heat and water stress conditions encountered in these countries. Accessions originating from regions with a mild Mediterranean climate (France, Greece, Croatia, Lebanon, Bulgaria, Italy, Portugal, Spain, and Turkey) were characterized by high carbon isotope discrimination, chlorophyll content, leaf area, biomass and grain production, and high relative water content. They are interesting sources of tolerance to moderate drought.

Intergeneric hybridization program. Promising Ae. geniculata accessions possessing resistance traits were selected for use in our wide hybridization program (Table 4). They have been crossed with susceptible high-yielding bread and durum wheat cultivars, with a priority currently given to transfers for BYDV resistance. Barley yellow dwarf virus has been recognized for many years as the most widespread and economically important viral disease of cereals worldwide. The absence of variability for BYDV resistance in T. aestivum and T. durum leads to use genes from alien species. The selected five Ae. geniculata accessions possess moderate resistance to BYDV, according to ELISA test values.

Table 4. Aegilops geniculata accessions resistant to barley yellow dwarf virus; stem, leaf and stripe rust; and cereal cyst nematode.

Biotic and abiotic stresses	Resistant accessions and origin
Barley yellow dwarf virus (Greece) *	MZ 20 (France), MZ 21 (France), MZ 97(Cyprus), MZ141 (Italy), MZ 149
Rusts **	MZ 6 (Bulgaria), MZ 27 (Morocco), MZ 48 (France), MZ 79 (Lebanon), MZ 96 (Cyprus)
Cereal cyst nematode (Spain) ***	MZ 1 (Bulgaria), MZ 61 (Tunisia), MZ 63 (Libya), MZ 77(Jordan), MZ 124
* Accessions with average optical density (OD) less than 2.5 times the OD of the resistant check TC14 after inoculation with PAV-Mex isolate of BYDV; test made in collaboration with Dr. M. Henry, CIMMYT (Mexico). ** Accessions with adult plant resistance for stem, leaf, and stripe rust tested for 3 years at ICARDA (Aleppo, Syria); adult plant and seedling resistance for leaf rust evaluated at IPGR (Sadovo, Bulgaria) and CIMMYT (Mexico) in collaboration with Drs. J. Valkoun and A. Dimov and R. Singh and J. Huerta, respectively. *** Accessions with resistance to several populations of H. avenae and one population of H. latipons; test made in collaboration with Dr. R. Rivoal (INRA-Rennes, France).

Strategy of introgression. F1 hybrids produced have been cytologically analyzed and validated to be 2n = 5x = 35 (ABDMU) or 2n = 4x = 28 (ABMU). For each cross, a few F1 hybrids were doubled by colchicine treated to produce amphiploids. The remaining F1 hybrids were backcrossed to their wheat parents for producing BC1 derivatives. A crossing program also is underway to hybridize Chinese Spring (phph) and Capelli (ph1c) with Ae. geniculata accessions to promote F1 homoeologous pairing. Current data suggests a lack of wheat genome/alien genome recombination. The mean chromosome association in hybrids of 'bread wheat/Ae. geniculata (2n = 5x = 35)' are 33.2 univalents + 0.9 rod bivalents and in durum 'wheat/Ae. geniculata' (2n = 4x = 28) are 27.6 univalents + 0.2 rod bivalents.

References.

• Friebe BR, Tuleen NA, and Gill BS. 1999. Development and identification of a complete set of Triticum aestivum-Aegilops geniculata chromosome additional lines. Genome 42:374-380.
• Gill BS, Sharma HC, Raupp WJ, Browder LE, Hatchett JH, Harvey TL, Moseman JG, and Waines JG. 1985. Evaluation of Aegilops species for resistance to wheat powdery mildew, wheat leaf rust, Hessian fly, and greenbug. Plant Dis 69:314-316.
• Zaharieva M, Gaulin E, Havaux M, Acevedo E, and Monneveux P. 2001. Drought and heat responses in the wild wheat relative Aegilops geniculata Roth: potential interest for wheat improvement. Crop Sci (in press).

Synthetic hexaploid wheats (2n = 6x = 42, AABBDD) and their salt tolerance potential. [p. 103-104]

D.J. Pritchard **, P.A. Hollington *, W.P. Davies *, J. Gorham *, J.L. Diaz de Leon ***, and A. Mujeeb-Kazi.
* Centre for Arid Zone Studies, University of Wales, Bangor LL57 2UW, Wales, UK;
** Royal Agricultural College, Cirencester, GL7 6JS, UK; and
*** Universidad Autonoma de Baja California Sur, Department of Agronomy, Apartado Postal 19-B, 23054 La Paz, B.C. S. Mexico.

Irrigation-induced salinity is a major constraint to crop production in many countries. K+/Na+ discrimination is a trait that enhances salinity tolerance in bread wheat compared to durum wheat, and is present in Ae. tauschii. A hydroponics experiment is described to assess K+/Na+ discrimination, and other traits, in a number of synthetic hexaploid wheat genotypes, produced by crossing Ae. tauschii with durum wheat. The durum parents of the synthetics also were used in the experiment.

K+/Na+ ratios were lower in the durum parents than in the elite synthetics, confirming that the trait was present in the synthetics, and demonstrating its successful transfer from Ae. tauschii to durum wheat. The 14 best-performing synthetics had similar K+/Na+ ratios to the tolerant check S24. There were highly significant correlations between K+/Na+ discrimination and fresh weight within the durum parents, and the elite synthetics

Material and methods. Experiments were carried out to assess K+/Na+ discrimination in a range of material that included 95 elite synthetic hexaploid genotypes, 35 of the tetraploid parental lines, and 13 genotypes from the standard CIMMYT salinity test set. The trial was conducted beginning in November 1999 at the University field station at Pen-y-Ffridd, in the glasshouse with heating and supplementary lighting. Four replications of a randomized block design were grown in plug trays over John Innes No. 1 compost and irrigated with saline nutrient solution. Salinity was gradually raised after germination to a final level of 100 mol/m3 NaCl, plus CaCl2 added to maintain an Na:Ca ratio of 20:1. Plants were grown for 21 days after attaining the final salinity level, a total of 35 days growth in saline conditions. Assessments were made of above-ground fresh weight, height and number of leaves, and between two and four fully expanded leaves per accession were removed and frozen. Sap was extracted using the methods of Gorham et al. (1984) and analyzed for K+Na+ and using a flame photometer.

Results. Results demonstrated the durum parents to be of significantly lower fresh weight (0.85 g) than the CIMMYT test set (1.35 g) and the elite synthetics (which were similar to each other; 1.20g). In terms of height, the durums were shorter (30 cm) than the tester set (37 cm) and the elite synthetics (39 cm). In terms of leaf Na+ content (mol/m3), there were significantly higher levels in the durum wheats (380), whereas the elites had a level similar to the CIMMYT test set (200-175). The elite synthetics showed much better K+/Na+ discrimination (2.10) than their durum parents (0.65).

These results clearly indicate that the trait for discrimination had been successfully transferred from into the tetraploid wheats, and in no case was the K+Na+ discrimination of the synthetic lines lower than the durum parent. This confirms the work of Gorham (1990), who concluded after examination of amphiploid hybrids that the trait was dominant in crosses of Ae. tauschii with species in which the trait was absent, including T. turgidum. He also found low leaf Na+ and high leaf K+ concentrations in synthetic hexaploids produced by crossing tetraploid Triticum species with Ae. tauschii, confirming the dominance of the trait in synthetic hexaploid wheats.

There were highly significant correlations on an overall basis (all genotype means) between K+/Na+ discrimination and fresh weight, indicating that at this early growth stage K+/Na+ discrimination was reflected in terms of salinity tolerance. Within the durum parents, the CIMMYT tester set, and the elite synthetics, there were also highly significant correlations.

We observed great variability among the elite synthetics for K+ discrimination with genotype mean values ranging from almost 5 to less than 0.5, with a mean of 2.04 and standard deviation of 1.79. This value compares well with the mean of the CIMMYT set of 2.26. Of the elite lines 'CETA/Ae. tauschii 895' and 'SCA/Ae. tauschii 409' also are tolerant to waterlogging (Villareal et al. 2001). However, the latter synthetic is susceptible to leaf and stripe rust. 'CETA/Ae. tauschii 1027' has multiple biotic stress resistance (Mujeeb-Kazi et al. 1999). Therefore, there appears to be considerable potential for these germ plasms in saline, and saline/waterlogged situations. Unfortunately, the genotypes are not yet fit agronomically, being difficult to thresh, and require further backcrossing with well-adapted varieties.

References.

• Gorham J. 1990. Salt tolerance in the Triticeae: K+/Na+ discrimination in Aegilops species. J Exp Bot 41:615-621.
• Gorham J, Wyn Jones RG, and McDonnel E. 1984. Pinitol and other solutes in salt-stressed Sesbania aculeata. Z Pflanzenphysiol 114:173-178
• Mujeeb-Kazi A, Fuentes-Davila G, Gilchrist LI, Velazquez C, and Delgado R. 1999. D-genome based synthetic hexaploids with multiple biotic stress resistances. Ann Wheat Newslet 45:105-106.
• Villareal RL, Sayre K, Bañuelos O, and Mujeeb-Kazi A. 2001. Registration of four synthetic hexaploid wheat (Triticum turgidum/Aegilops tauschii) germplasm lines tolerant to waterlogging. Crop Sci 41:274

Scab resistance in bread wheat/synthetic hexaploid derivatives. [p. 104-105]

A. Mujeeb-Kazi, R. Delgado, S. Cano, L. Juarez, and J. Sanchez.

One of the major biotic stress that affects wheat in the higher rainfall mega-environment is head scab induced by Fusarium species. Genetic diversity in conventional germ plasm for resistance is very narrow and this justifies evaluation of other Triticeae species as possible donors. The priority choice relates to the primary gene pool species Ae. tauschii because introgression of resistance from this source will yield products swiftly. Additionally, the species has several hundred accessions of diverse global distribution that provide a unique avenue to complement the current conventional gene resource available. Listed in Table 5 are the pedigrees and performance data of these lines as tested for type II (spread) scab resistance over several years of testing in Toluca, Mexico. Some phenology attributes are included. All lines have acceptable agronomic types and are free-threshing.

Three advanced lines (Table 5a) currently in use for the CIMMYT/U.S. Scab Initiative Project have superior Type II resistance. Independent evaluations by collaborators validate our type-II scores and also indicate that these lines possess type I (Penetration), type II (toxin content), and type IV (test-weight losses) mode of resistances.

The next category (Table 5b) not yet used in wheat breeding are F8 lines with unique Ae. tauschii diversity of accession numbers 190, 224, 437, 447, 498, and 895; and the superior type-II resistance scores from 2 to 3 years of tests in Toluca, Mexico. All lines are free threshing.

To facilitate breeding protocols sources with pyramided genes are advantageous. In order to address this aspect, resistant advanced BW/SH derivatives with diverse Ae. tauschii accessional origins in resistant SHs were intercrossed to yield F1 progeny (Table 6). Nine such combinations were made. Each F1 was crossed with maize to produce haploids, which subsequently yielded four to eleven double haploids from each combination. We anticipate these DHs to possess good Type II scores and are an additional resource for an efficient wheat improvement protocol. Though the mechanism is not clear at this stage, we have regularly observed that DHs tend to be shorter and possess earlier maturity than their parental materials when synthetics are involved in the pedigree.

T1RS·1AL and T5AS·5RL translocations in bread wheat and durum wheat. [p. 105-106]

A. Mujeeb-Kazi and A. Cortéz.

Wheat production has received a boost from some alien chromosome contributions not only for yield but also for biotic/abiotic stresses. The most significant of these exchanges are in terms of wheat/alien Robertsonian translocations where cultivars possessing them are cultivated on over 5 million hectares globally. These cultivars are comprised of the T1BL·1RS and T1AL·1RS translocations and predominant are cultivars with T1BL·1RS that in CIMMYTs germ plasm alone comprise of about 57 % of the entries. Additional translocations being exploited in wheat breeding are associated with BYDV resistance and the Lr19 germ plasm.

An extensive list of translocations has been published by Friebe et al. (1996), and these stocks have been made available to us. Our efforts, in part, are to transfer the translocations into spring wheat cultivars that will allow their evaluation under Mexican conditions, thus not be confounded by poor agronomic plant type and maturity constraints. A backcrossing protocol complimented by C-banding and FISH cytology at each backcross and after the final selfing stage is followed to obtain homozygous translocation products. Also involved are transfers to durum wheats where the contributions of the translocations have not received much attention.

Listed in Table 7 are the durum (4x) and bread wheat (6x) cultivars that have incorporated the T5AL·5RS and T1AL·1RS translocations. T5AL·5RS is reported to bestow copper efficiency in bread wheat and derivatives possess variable levels of pubescence (associated with 5RL, i.e., hairy neck) at the spike base. The five bread wheats, Ocoroni, Seri M82, Opata M85, Flycatcher, and Ciano 79, with the translocation have shown stability. Excess alien chromatin is not desirable in wheat breeding and hence minimum alien chromatin presence is preferred. Nevertheless, attempts to see the influence of double translocations have been contemplated by researchers. The cultivar Seri M82 is a T1BL·1RS type and was used as a recipient source for the T5AL·5RS chromosome. Fortuitously, one of the products obtained via backcrossing has maintained both translocation homozygotes and may be of interest in the future.

The T1AL·1RS of Amigo has been transferred to Pavon and Ciano 79 bread wheats. Each translocation also has been backcrossed into Altar 84.

Additional wheat/alien translocations obtained from Kansas State University (B.S. Gill, B. Friebe, and J. Raupp) that are being currently transferred to bread wheat cultivar Prinia are T4DL·4Ai (TA5040), T6BL·6BS-6U (TA5523), T6BS·6BL-6U (TA5524), T7BL·7BS-6U (TA5525), T2DS·2DL-6UL (TA5521), T4BL·4BS-6U (TA5522), T2BS·2AL (TA5018), T6BS·6BL-6RL (TA5030), T4BS·4BL-6RL (TA5031), T4AS·4AL-6RL-4RL (TA5033), T6BS·6RL (TA5041), and T2AS-2RS·2RL (TA5543).

Their products in the elite spring wheat cultivar Prinia will enable satisfactory screening of the germ plasm under our conditions and form a suitable bridge to selectively move into other cultivars if an identified trait is of interest for wheat improvement.

Reference.

• Friebe B, Jiang J, Raupp WJ, McIntosh RA, and Gill BS. 1996. Characterization of wheat-alien translocation conferring resistance to diseases and pests: current status. Euphytica 91:59-87.
  

Cytogenetic manipulation to promote wheat/alien chromosomal association: our current strategy. [p. 107-108]

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

In general, upon meiotic analysis, hybrids between bread wheat and tertiary gene pool alien species have a lack of wheat/alien chromosomal associations. This precludes the opportunity of transferring the beneficial traits of the alien donor species into wheat by recombination at the F1 stage.

Classical cytogenetic protocols take substantial time and effort to yield the final introgressed product. Some alternatives to the classical route exist in the use of the monosomic 5B stocks, the ph mutant, the PhI genetic stock, irradiation, or callus culture. The potential of using wheat homoeologous group monosomics crossed with their respective addition lines to eventually select translocation products from the selfed progeny also exists.

At CIMMYT, we have focused upon a modified integrated protocol where use is made of the ph stock, maize haploid induction, and PCR diagnostics culminating in C-banding and FISH mitotic cytology. These steps are illustrated in the schematic of Fig. 6.

The choice of the 'bread wheat/Th. bessarabicum' combination was made because the species is ideal as a source for salinity tolerance and head scab resistance. The amphiploid (2n = 8x = 56) was already available, and the backcross to the ph stock was a swift way to advance this combination. This procedure could be applicable to other amphiploids, and we are suggesting this route to be actively explored, since our initial observations support the generation of translocations (Table 8).

The plants identified with translocations range from 42 to 50 chromosomes. The 42-chromosome plants are expected to be stable whereas those with higher chromosome numbers possess complete additional Th. bessarabicum chromosomes. We are making backcrosses with an elite CIMMYT spring wheat cultivar (Prinia) and shall cytologically advance the progeny to identify 42 chromosome derivatives homozygous for the translocations observed in Table 8.

A decade of progress in evaluating germ plasm for Karnal bunt resistance. [p. 108-109]

G. Fuentes-Davila, M. van Ginkel, and A. Mujeeb-Kazi.

Karnal Bunt is a fungal disease of wheat that affects the grain. In affected spikes, some grains appear dark as teliospore masses substitute partially the endosperm. Control of Karnal bunt is difficult because teliospores of the fungus are resistant to physical and chemical treatments. Chemical control can be accomplished with fungicide applications during flowering. However, this measure is not economically feasible for commercial use. Therefore, resistant cultivars are the most useful option for control. The availability of resistant cultivars will reduce the threat that introduction of Karnal bunt represents to other wheat production areas, because they can provide excellent management of the disease. At CIMMYT, breeding for genetic resistance to T. indica is based on the identification of sources of resistance, hybridization in order to incorporate resistance genes into agronomically suitable genotypes, and evaluation and selection of plant progenies to develop advanced lines. Among the sources evaluated are T. aestivum, T. turgidum, Triticosecale (Fuentes-Dávila et al. 1992), and synthetics of 'Ae. tauschii/T. turgidum'. Since the 1980s, this program has been conducted at CIANO (Centro de Investigaciones Agricolas del Noroeste) located in the Yaqui Valley, Sonora, Mexico (27°20'N, 105°55W, elevation 39 masl).

Methodology. Artificial field inoculation of experimental germ plasm has been necessary because of the erratic occurrence of Karnal bunt and quarantine regulations established in Mexico. Following the method of Chona et al. (1961), we have improved the technique as follows. To assure a genetically heterogenous composite of the fungus population, inoculum is prepared from 1-year-old teliospores from naturally infected wheat grains collected from various locations in the Yaqui Valley. To isolate teliospores, grains are shaken using a vortex shaker for about 15 seconds in a test tube containing water plus Tween 20 solution (one drop/l), and sieved through a 60 µm microsieve to remove debris. Teliospores are kept in the solution for 24 h at 18-22°C to enhance germination. Thereafter, they are briefly surface sterilized with 0.5 % sodium hypochlorite while centrifuging at 3,000 rpm (about 2 min), rinsed twice in sterile distilled water, and plated on water agar (1.5 % w/v agar). Plates are incubated at 18-22°C, and teliospore germination is assessed after 5 days. Pieces of agar with the fungus are inverted onto the lids of plates of potato-dextrose-agar (PDA) for ballistospore release and further multiplication.

To increase the inoculum, sterile water is added to fungal colonies, scraped, and transferred to fresh PDA plates. After 8-10 days, small pieces of the agar colonized by the fungus are inverted onto the lids of sterile, glass petri plates containing a small amount of sterile water. Allantoid sporidia discharged into the water are collected daily, counted with a hemacytometer, and adjusted to a concentration of 10,000 sporidia/ml. One ml of the sporidial suspension is injected into 10 heads primarily from main tillers at boot stage (Zadoks et al. 1974, stages 48-49) in the late afternoon. Inoculated heads are identified with color-coded plastic pieces. An overhead irrigation (misting) system that produces fine droplets of moisture is used after inoculation. Moisture is applied 3-5 times/day for 8 min to provide high relative humidity to help assure good levels of infection. Experimental nurseries of wheat lines are sown in 1-m long, double rows, on beds of 90-cm wide in the field. Experimental lines are sown on three dates, 8, 18, and 28 November, and the susceptible cultivar WL-711 is used as a control. At maturity, inoculated heads from each line and the susceptible checks are separately harvested and threshed by hand to calculate the percentage of infection by counting the number of healthy and infected grains.

Progress. Although the inoculation method is severe, some wheat lines have consistently shown low levels of infection through repeated testing. Using this method, some wheat lines that might present field resistance are discarded; however, the target has been to select highly resistant germ plasm. Sources of resistance from China, India, Brazil, and some CIMMYT wheats have been identified. The success of the breeding program to identify sources of resistance and to transfer resistance genes into suitable genotypes can be measured by comparing the results of inoculation of advanced bread wheat germ plasm from the nursery PC optimum environment (PCOE) in 1989 (Fig. 7), and those from the EPCME1KB in 1999 (Fig. 8). The PCOE had 2.8 % of lines within the 0-5 % infection level, whereas the EPC had 37 %. Of the lines in the PCOE, 87.5 % showed infection levels above 10 % and 40 % in the EPC, 35.8 % of lines from the PCOE had infection levels above 30 %, whereas the EPC had only 4 %. By this continuous effort, resistant bread wheat cultivars and other diverse germ plasm (synthetic hexaploids, 2n = 6x = 42, AABBDD) are now available for wheat breeding objectives. The modified methodology developed has proved to be an efficient tool for breeding for Karnal bunt resistance for the CIMMYT wheat program.

References.

• Chona BL, Munjal RL, and Adlakha KL. 1961. A method for screening wheat plants for resistance to Neovossia indica. Ind Phytopath 14:99-101.
• Fuentes-Dávila G. 1992. Karnal bunt and durum wheat. In: Durum wheat challenges and opportunity, Centro Internacional de Mejoramiento de Maiz y Trigo (CIMMYT).
• Zadoks JC, Chang TT, and Konzak CF. 1974. A decimal code for the growth stages of cereals. Weed Res 14:415-421.
  

Septoria tritici resistance in synthetic hexaploids and their advanced derivatives from bread wheat crosses. [p. 109-110]

R. Delgado and A. Mujeeb-Kazi.

Ten bread wheat germ plasms resistant to Septoria tritici leaf blotch were recently registered (Mujeeb-Kazi et al. 2000). All were based upon D-genome synthetic hexaploids in which Ae. tauschii accessions 191, 205, 213, and 224 were involved with BW cultivars Kauz, Borlaug M95, Seri M82, Papago M86, Opata M85, Yaco, and Bagula. Because a pathogenic system is involved in biotic stress resistance, additional genetic diversity plays a significant role in ensuring durability of the crops performance. Keeping this in focus, we have continued to produce D-genome SHs, screen them for resistance to leaf blotch, and introgress the resistance into elite spring bread wheats. Attempts are to incorporate substantial new diversity from these Ae. tauschii accessions.

The lines reported here were derived from S. tritici-resistant SHs (T. turgidum/Ae. tauschii) that were crossed with the S. tritici-susceptible wheat cultivars Flycatcher, Opata M85, Siren, and Bacanora.

Segregating generations of the crosses were advanced by the pedigree breeding method. Ratings for S. tritici resistance were based upon leaf damage recorded at progressive growth stages (81 to 132 days from planting in Toluca), using a double digit modified scale (Eyal et al. 1987). All lines had the euploid 2n = 6x = 42 chromosome number with predominantly normal bivalent meiosis. The disease ratings of each of the eight germ plasms indicated their superior resistance over the four bread wheat cultivars and their durum parents (Table 9). In addition, we observed that these germ plasms possessed resistance to leaf, stem, and yellow rusts. All germ plasms had a good agronomic plant type.

Reference.

• Eyal Z, Scharen AL, Prescott JM, and van Ginkel M. 1987. The Septoria diseases of wheat: concepts and methods of disease management. Mexico, D.F., CIMMYT. pp. 1-46.

Current status and potential use of A-genome hexaploid germ plasm (2n = 6x = 42, AAAABB). [p. 111-113]

A. Mujeeb-Kazi, R. Delgado, S. Cano, V. Rosas, and A. Cortéz.

Triticum turgidum improvement has predominantly been accomplished through conventional plant breeding methodologies and this approach shall continue to be the predominant procedure in the future. Novel approaches that complement plant breeding have emerged and are attracting research interest. Presumably one approach for exploiting alien genetic variability would be to separate the practical gains objectives into short- and long-term time frames. The short-term benefits hold a high potential with lesser constraints. For this to materialize, interspecific hybridization stands as a priority with emphasis assigned to Ae. tauschii because of its genetic proximity to the D genome of wheat. So far, major emphasis has been given to bread wheat. Constraints for durum wheat improvement also exist but have received lesser attention. The contribution of the D genome for improving durum wheats through D- and A-genome exchange is prevalent and needs exploitation. The variation within the diploid species of the A genome and the Sitopsis group also holds tremendous potential for durum improvement. One mechanism, of a few that exist for exploiting the A-genome diversity, is via bridge crosses where 'T. turgidum/A-genome species' hybrids (2n = 3x = 21, AAB) lead to generation of amphiploids (2n = 6x = 42, AAAABB) upon colchicine treatment.

As one option, we have is initially emphasized indiscriminate hybridization of elite T. turgidum cultivars with several A-genome accessions to produce amphiploids, establish some descriptors, and use them in durum breeding.

Materials and methods. Germ plasm. Triticum monococcum subsp. monococcum and aegilopoides and T. urartu accessions were obtained from the CIMMYT germ plasm bank in Mexico (El Batán) and from researchers in the U.S.A. (B.S. Gill, Kansas State University, Manhattan; J.G. Waines, University of California, Riverside, and H. Bockelman, National Small Grains Collection, Aberdeen, ID). A total of 1,110 accessions were acquired and increased for seed quantity by incorporating a seedling vernalization procedure of 8°C and 8 h light for 8 weeks. After the seed increase, some accessions were similarly vernalized and transplanted to the field in the Mexico location of Ciudad Obregon during November to May over 5 consecutive years for hybridization to T. turgidum cultivars.

Hybridization, embryo rescue, and plantlet regeneration. Forty-eight, elite durum wheat cultivars were planted over four dates at 10-day intervals over each of the 5 years (1990-95) in order to synchronize with the availability of A-genome pollen. Emasculation, pollination, embryo rescue, and regeneration procedures were similar to those reported earlier (Mujeeb-Kazi et al. 1987).

Cytology of hybrids, colchicine doubling, and cytology of amphiploids. Each hybrid plantlet was validated for hybridity. The hybrid plants were treated with 0.1 % colchicine + 2.0 % dimethyl-sulfoxide for 6 hours via an aerated root-treatment for doubling the chromosome number in order to obtain fertile amphiploids (2n = 6x = 42, AAAABB) which were cytologically documented, seed increased, and screened for some stresses.

The A-genome diploid species represent wild relatives of the primary gene pool of wheat. Genes within this gene pool are transferred by homologous chromosome recombination, backcross, and selection. The mean meiotic metaphase associations suggest this recombination trend and are inferred as the A-genome relationships in each of the three F1 hybrids of durum wheat with the T. monococcum subspecies and T. urartu. The entire F1 set (2n = 3x = 21, AAB) that produced the 194 AAAABB C-O amphiploids was meiotically analyzed. The mean meiotic relationships in Table 10 are a reflection of several diverse 'durum/A-genome diploid accession' combinations. The total mean bivalent range for the three combinations over all F1 hybrids was 5.5 to 6.0/meiocyte. These pairing trends are similar and consistent with earlier reports for such AAB hybrids.

All the 194 AAAABB C-O amphiploids were cytologically stable. Table 11 shows selected data for some combinations.

Conclusions. The amphiploids embody a wide array of genetic diversity from the A-genome accessions. They are all spring type in habit and offer an easier source for practical utilization, conservation, and global distribution of the germ plasm. The germ plasm further provides a unique gene pool for evaluating the A-genome response towards a wide range of biotic/abiotic stress conditions. International distribution of amphiploids has additional merit because following screening by national agricultural plus other research programs for different objectives, the variation can be readily incorporated into their local adapted germ plasm. Some highlights are summarized below.

• From the initial acquisition of A-genome diploids, 194 AAAABB amphiploids have been produced.
• All F1 hybrids cytologically suggest A-genome homologous exchanges that are a characteristic of primary
  gene pool species.
• The amphiploids show stable meiosis, are fertile, and have the potential to contribute genes for cereal
  improvement, after stress descriptors are established.
• Currently, diversity has been identified for resistance to H. sativum and head scab.
• Of high priority is screening for leaf rust because of the change observed on cultivated durum germ plasm in
  Mexico. This germ plasm will be significant in now evaluating the diploid species response in a susceptible
  durum base.

Reference.

• Mujeeb-Kazi A, Roldan S, Suh DY, Sitch LA, and Farooq S. 1987. Production and cytogenetic analysis of hybrids between Triticum aestivum and some caespitose Agropyron species. Genome 29:537-553.

New tetraploid germ plasm combining Septoria tritici resistance of some A- and D-genome diploids. [p. 113-114]

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

Septoria tritici is a fungal disease that limits wheat production in high rainfall across 10.4 million hectares globally. Apart from desirable diversity in the conventional wheat germ plasm primary gene-pool diploid species accessions also possess high levels of resistance. The A-genome sources are T. monococcum subsp. monococcum and aegilopoides and T. urartu, and the D-genome diploid donor is Ae. tauschii. Both diploids have been combined with elite durum wheat cultivars to generate AAAABB and AABBDD hexaploids that upon screening for leaf blotch in Toluca, Mexico, have unequivocally demonstrated a 1-1 to 2-2 level of resistance (susceptible score being 9-9 on a double-digit scoring system (Zadoks 1974). We concluded that the diploid A- and D-genome accessions were contributing to the blotch resistance in the hexaploid genetic stocks. Either hexaploid source can be independently utilized for wheat improvement with transfers going to the A or D genomes of bread wheat. To facilitate efficient crop improvement efforts, gene pyramiding is advantageous, and one option is to provide stocks that already possess combined resistance sources; leading us to produce such germ plasm. We selected some A- and D-genome accessions that had contributed to superior leaf blotch resistance to their respective hexaploids. All had disease scores of 1-1 or 2-1. Hybrids were made between these A- and D-genome species where the F1 (2n = 2x = 14, AD) combinations has 14 univalents at meiosis (see Fig. 9b).

Crossability in the accessions was less than 1.0 %, but some genotypic variation existed giving frequencies up to 2.8 %. The standard procedures for F1 growth and colchicine treatment and the cytologic evaluation of the doubled plants were used. Figure 9 documents the spike morphology and the meiotic relationship of one A­D amphiploid combination. The various combinations made are listed in Table 12, where the leaf blotch-resistance score of each diploid also is indicated. Fertility of the tetraploids is satisfactory. These stocks will enable simultaneous transfers of leaf blotch-resistance genes into the A- and D-genome chromosomes of the recipient bread wheat cultivars, thus adding to efficiency of dual source transfers.

Table 12. A- and D-genome parental accessions used in the production of AADD tetraploids. Leaf blotch scores of each diploid indicated based upon contribution in AAAABB and AABBDD hexaploids. In the double digit disease score, the first digit indicates height of infection, where 5 = up to mid-plant and 9 = up to flag leaf; the second digit indicates disease severity on infected leaves, where 1 = low and 9 = total leaf destroyed. The accession numbers of the wild species in CIMMYTs wide crosses working collection are in parentheses.

A-genome diploid	Disease score	D-genome diploid	Disease score
T. monococcum subsp. monococcum (111)	1-1	Ae. tauschii (458)	2-1
T. monococcum subsp. monococcum (111)	1-1	Ae. tauschii (319)	1-1
T. monococcum subsp. monococcum (111)	1-1	Ae. tauschii (323)	1-1
T. monococcum subsp. monococcum (111)	1-1	Ae. tauschii (273)	1-1
T. monococcum subsp. monococcum (111)	1-1	Ae. tauschii (321)	1-1
T. monococcum subsp. monococcum (111)	1-1	Ae. tauschii (434)	2-1
T. monococcum subsp. monococcum (111)	1-1	Ae. tauschii (222)	1-1
T. monococcum subsp. monococcum (111)	1-1	Ae. tauschii (319)	1-1
T. monococcum subsp. aegilopoides (56)	1-1	Ae. tauschii (219)	1-1
T. monococcum subsp. aegilopoides (56)	1-1	Ae. tauschii (1029)	2-1

Reference.

• Zadoks JC, Chang TT, and Konzak CF. 1974. A decimal code for the growth stages of cereals. Weed Res 14:415-421.
  

A second, elite set of synthetic hexaploid wheats based upon multiple disease resistance. [p. 114-115]

A. Mujeeb-Kazi and R. Delgado.

The first, elite set of synthetic hexaploids (95 entries) was prepared from the initial 450 synthetics produced and was based upon performance in three Mexican locations; El Batan, Toluca and Cd. Obregon.

We have currently produced 800 synthetics and several different combinations that are not included in Elite I express superior agronomic traits. In addition, observations suggest that some synthetics also possess multiple stress resistances, which had not been an important criteria when the first set was assembled. Multiple resistances have significance in wheat improvement and also simplifies generation of mapping populations if the wheat cultivar involved also is susceptible to those stresses.

Beginning with 98 new synthetics in 2000, we have reduced the number to 33 and put some phenology descriptors in place (Table 13). In general, the entries possess leaf, stem and stripe rust resistance and are scored for scab (type II) and currently are being studied for S. tritici, BYDV, H. sativum, and Karnal bunt. Other criteria will be added gradually for a more complete descriptor profile and DNA fingerprinting using D-genome microsatellites also will be included.

The above characterizations of set II are expected to be completed by the end of 2002 when distribution will be possible via the wheat germ plasm bank of CIMMYT. Small seed samples also will be available from CIMMYT Wide Crosses Program. In the U.S., the Wheat Genetics Resource Center at Kansas State University will be the repository, as is the case for Elite Set I.

Maize mediated haploid production in bread wheat: current status, constraints, and modifications. [p. 116-117]

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

Polyhaploid production in wheat has relied heavily on anther culture and sexual crosses with H. bulbosum. The occurrence of somaclonal variation, aneuploidy, and genotypic specificity are some major limitations of anther culture. The homoeologous group-5 crossability loci (Kr) influence sexual crosses of wheat with H. bulbosum. Producing wheat haploids by crossing bread wheat with maize, pearl millet, or Tripsacum has become a significant procedure, because the production constraints of anther culture and H. bulbosum crosses are not present. Currently, this procedure is being routinely used by CIMMYT in wheat cytogenetics, wide crosses, wheat breeding, and genetic analyses, with extensions of the application into genetic engineering and molecular mapping. A recent advance in the technique enhances the efficiency of haploid production by utilizing detached tillers from selected plants. The culture of the tillers is in a nutrient solution including sulfurous acid to avoid contamination and hot water (43°C) immersion of the spikes for 3 min to effect emasculation. An hormonal treatment (2,4-dichlorophenoxy acetic acid; 100 ppm) is essential, as is embryo rescue approximately 15 days after pollination. The protocol is almost 100 % effective for all bread wheat cultivars. Mean frequencies of embryo excision estimated over long-term experiments are 25 %, plantlet differentiation 80 %, with a colchicine-induced doubling range from 80-95 %. Although the application is effective for all bread wheat cultivars that are of spring, winter, and facultative habit, the frequency of embryo/spike tends to vary. In general, 4-5 DH/spike are produced for responsive cultivars but often drops to 1-2 DH/spike. Regeneration and doubling works with lesser variability across all germ plasm according to our observations. Here we report the variation in embryo excision in a facultative wheat F3 population (Table 14) and mention some modifications that we have incorporated. Also tabulated is the status of the DH-mapping populations produced thus far for some biotic and abiotic stresses (Table 15). Those combinations where the target DH number has not been obtained, F1s presently are being utilized for additional DH production.

Protocol modifications.

• We have shifted almost exclusively to the detached spike technique and use hand emasculation where each
  spike has 20 florets to be pollinated.
• The spikes are kept under greenhouse conditions in plastic jars. The temperature is 24°C day and 14°C night,
  approximately 65 % relative humidity, and natural lighting of about 14 hours.
• The plastic jars only have tap water. No Hoagland's solution or sulfurous acid is used.
• 2,4-D solution treatment is done for 6 days after pollination.

The rest of the protocol is similar to the previous report of Mujeeb-Kazi (2000).

Reference.

• Mujeeb-Kazi A. 2000. An analysis of the use of haploidy in wheat improvement. In: Application of Biotechnologies to Wheat Breeding (Kohli MM and Francis M eds). La Estanzuela, Uruguay. 19-20 November, 1998. p. 33-48.
  

Salt tolerant bread wheat germ plasm. [p. 117-118]

J.L. Díaz-de-León*, R. Escoppinichi*, E. Molina, J. López-Cesati, R. Delgado, and A. Mujeeb-Kazi.
*Universidad Autonoma de Baja California Sur, Department of Agronomy, Apartado Postal 19-B, 23054 La Paz, B.C. S. Mexico.

Diaz-de-León et al. (Ann Wheat Newslet 46:88-90) have suggested that a good cutoff point in screening for salt tolerance in wheat may be at about 12.0-12.5 dS/m. Generally, electrical conductivity levels of 10 and over are unsuitable for wheat production. At these levels, reductions in growth performance are initiated with only those possessing tolerance showing little to no reduced plant vigor. Supportive data that is an indicator of tolerance is K:Na discrimination with a ratio of around 1.0 suggesting susceptibility. This test was conducted in Mexico in hydroponics using protocol of Gorham et al. (1987) and Shah et al. (1987). A NaCl concentration of 50 mM is an ideal level for discrimination data. The levels of sodium and potassium and their ratios are reported in Table 16.

The susceptible durum and bread wheat cultivars (PDW 34 and Oasis) had K:Na levels of 1.0 and 1.4, respectively. The acceptable tolerant bread wheat cultivars Kharchia, Shorawaki, and Chinese Spring had ratios of 3.1, 6.4, and 9.6, respectively. The other entries of the tester set ranged in K:Na between 4.6 and 8.1, which makes them good candidates for the current tester set that is being globally distributed. Not included was Pasban 90 and the new Indian release KRL19.

To be added to this tester set will be selections from the four bread wheats Cochimi, Mepuchi, Pericu, and Calafia, which were evaluated in a field salinity test yield trial in Baja California (UABCS) against Kharchia, Shorawaki, and Oasis. The tests electric conductivity level was 12.0 dS/m and some parameters observed (Table 17) were leaf area, flowering, spike emergence, physiological maturity, total yield, plant height, spike length, grains/spike, and 1,000-kernel weight.

Table 17. Some field testing parameters of entries evaluated for release in Baja California Sur, Mexico, at 12.0 dS/M.

Cultivar	Leaf area (cm)		Days to flowering		Days to physiological maturity		Plant height (cm)	Spike length (cm)	Grain/spike	Total yield (kg/ha)
	1.5^1	12.0	1.5	12.0	1.5	12.0	12.0	12.0	12.0
Kharchia	15.10	9.68 *	67	64	95	95	76.46 *	7.4 *	32.2	3.42
Shorawaki	15.72	10.80 *	78	74 *	103	103	92.00 *	8.9 *	37.5	3.25
Cochimi	15.79	10.43 *	72	72	98	98	55.93	8.8	45.0 *	4.34 *
Mepuchi	16.61	11.68 *	66	66	65	95	55.93 *	8.4 *	35.6 *	4.58 *
Pericu	13.81	8.99 *	68	68	98	98	67.53	7.6	34.8	3.60 *
Calafia	14.99	10.35 *	70	70	97	97	67.53 *	8.5	48.0	4.54 *
Oasis	13.06	7.83 *	74	74	96	96	62.73	8.4	40.0	3.61 *
PDW 34	---	---	---	---	---	---	54.33	5.8	35.2	3.23
* EC levels of 1.5 dS/m and 12.0 dS/M.

References.

• Gorham J, Hardy C, Wyn Jones RG, Joppa L, and Law C. 1987. Chromosomal location of a K+/Na+ discrimination character in the D genome of wheat. Theor Appl Genet 74:584-588.
• Shah S, Gorham J, Forster B, and Wyn Jones RG. 1987. Salt tolerance in the Triticeae: the contribution of the D genome to cation selectivity in hexaploid wheat. J Exp Bot 38:254-269.
  

Scab resistance (Type II: spread) in synthetic hexaploid germ plasm. [p. 118-120]

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

Because of their diversity and global distribution, accessions of the primary gene-pool, diploid wheat relative Ae. tauschii constitute a unique source of novel genetic variability for bread wheat, providing among other things resistance to several factors that reduce the crop's productivity in developing countries. Because of the constraints of stress screening and the winter habit and tendency for grain shattering in Ae. tauschii, we have hybridized available accessions indiscriminately with elite T. turgidum cultivars, producing 800 SHs to date, with several involving a unique Ae. tauschii accession. Here, we report on the current status of scab resistance in these SH wheats, and our attempt to pyramid resistant genes by producing DHs from 'resistant SH/resistant SH' F1s crossed with maize.

Materials and methods. Eight hundred SH wheats derived from crosses of 51 T. turgidum cultivars and 438 of the 490 Ae. tauschii accessions in the wide crosses working collection at CIMMYT. The plants were produced and grown at the CIMMYT station in Toluca, Mexico (19°17'N, 99°39'W, 2,640 masl). Unreplicated hill plots except, for 'BW/SH' advanced derivatives, were planted in two 2.0-m rows spaced at 15 cm between rows in 90-cm beds.

Fusarium head scab isolates were obtained from Toluca, Patzcuaro, and El Tigre, Mexico. A spore concentration of 50,000/ml of water and the cotton inoculation method were used (A tiny, inoculum-permeated tuft of cotton is placed in the floret by opening the glumes of a spikelet in the middle of the spike with a pair of tweezers. The spike is then covered with a glassine bag to prevent damage). Ten, randomly selected spikes of each entry were inoculated. Disease scoring for type-II (spread) Fusarium head scab infection was done 30­35 days after inoculation. The inoculated spikes were harvested, percentage of spikelets infected with scab evaluated, and scab scores of the inoculated spikes averaged. Selected resistant SHs were intercrossed to yield F1 progeny that were hybridized with maize and produced haploids, which upon doubling with colchicine were the source of DH stocks.

Results. Resistance in the SHs. The SH wheats most resistant (less than 15 % infection) to F. graminearum (type II) are given in Table 18. The resistant BW check Sumai 3 scored around 15 % or slightly less, whereas the moderately susceptible BW check Flycatcher always had over 20 % infection and the durum wheat Altar 84 over 40 %.

Gene pyramiding. Sixteen SH/SH combinations produced DH progeny and were a source of combining different Ae. tauschii accessions (see Table 19). These germ plasms are anticipated to provide greater accumulated diversity for wheat breeding programs targeted for scab resistance.

Table 19. Pyramiding of some resistant synthetic hexaploid germ plasm with superior Type-II scab resistance yielding double haploid derivatives as a result of the 'F1 / maize' protocol. Ae. tauschii accession number in wheat wide crosses working collection is given in parentheses.

Female SH Parent	Type II % score	Male SH Parent	Type II % score
YUK/Ae. tauschii (217)	11.4	68.111/Rgb-u//Ward/3/Fgo/4/Rabi/5/Ae. tauschii (890)	11.4
YUK/Ae. tauschii (217)	11.4	Gan/Ae. tauschii (180)	10.7
YUK/Ae. tauschii (217)	11.4	Doy1/Ae. tauschii (333)	11.1
YUK/Ae. tauschii (217)	11.4	Cpi/Gediz/3/Goo//Jo/Cra/4/Ae. tauschii (305)	10.3
YUK/Ae. tauschii (217)	11.4	Croc_1/Ae. tauschii (205)	10.1
68.111/RGB-u//Ward/3/Fgo/4/Rabi/5/Ae. tauschii (629)	11.9	68.111/Rgb-u//Ward/3/Fgo/4/Rabi/5/ Ae. tauschii (890)	11.4
68.111/RGB-u//Ward/3/Fgo/4/Rabi/5/Ae. tauschii (882)	11.1	68.111/Rgb-u//Ward/3/Fgo/4/Rabi/5/ Ae. tauschii (890)	11.4
YAR/Ae. tauschii (783)	12.3	68.111/Rgb-u//Ward/3/Fgo/4/Rabi/5/Ae. tauschii (878)	12.4
68.111/RGB-u//Ward/3/FGO/4/Rabi/5/Ae. tauschii (878)	12.4	Ceta/Ae. tauschii (172)	12.7
Sora/Ae. tauschii (884)	12.9	Lck59.61/Ae. tauschii (324)	12.5
Sora/Ae. tauschii (884)	12.9	Cpi/Gediz/3/Goo//Jo/Cra/4/Ae. tauschii (1038)	12.9
68.111/RGB-u//Ward/3/FGO/4/Rabi/5/Ae. tauschii (890)	11.4	Ceta/Ae. tauschii (895)	10.8
68.111/RGB-u//Ward/3/FGO/4/Rabi/5/Ae. tauschii (890)	11.4	Doy1/Ae. tauschii (333)	11.1
Ceta/Ae. tauschii (895)	10.8	Doy1/Ae. tauschii (333)	11.1
Lck59.61/Ae. tauschii (313)	12.5	Croc_1/Ae. tauschii (205)	10.1
Doy1/Ae. tauschii (333)	11.1	Cpi/Gediz/3/Goo//Jo/Cra/4/Ae. tauschii (305)	10.3

Conclusions.

• Ae. tauschii is a valuable source of genetic diversity for resistance to FHB in bread wheat. The resistance is distributed over several accessions.
• Synthetic hexaploid wheats derived from 'T. turgidum/Ae. tauschii' crosses express new levels of diversity for scab resistance that compliment the resistance levels in the best bread wheat cultivars.
• This resistance is being transferred to elite-but-susceptible bread wheat cultivars.
• Gene pyramiding attempts have led to the production of DH genetic stocks that combine the diversity found in the Ae. tauschii accessions.
  
  

Screening of a conventional bread wheat mapping population for Karnal bunt. [p. 120]

G. Fuentes-Davila, R. Delgado, and A. Mujeeb-Kazi.

Conventional bread wheat cultivars WL711 and HD29 are susceptible or resistant to Karnal bunt. F1 cross combinations were made as 'WL711/HD29' and 'HD29/WL711', and the F1 seed was used for producing respective DH mapping populations. There were 264 and 275 DHs/population. Artificial field inoculations at two planting dates were conducted in Obregon, Mexico, during the 1998-99 and 1999-00 crop cycles using a modified method based upon that of Chona et al. (1961).

Figures 11 and 12 elucidate the frequency of the DH plants over 0-5, 5.1-10, 10.1-30, and above 30 % KB infection categories. The trend in 1999-00 is presented and was the general distribution pattern for 1998-99. The cross direction had negligible effect in the infection categories. The mapping populations are currently being tested for molecular characteristics by the Kansas State University Wheat Genetics Resource Center.

Reference.

• Chona BL, Munjal RL, and Adlakha KL. 1961. A method for screening wheat plants for resistance to Neovossia indica. Ind Phytopath 14:99-101.

Advanced 'bread wheat/synthetic hexaploid' free-threshing derivatives resistant to Karnal bunt. [p. 120-122]

G. Fuentes-Davila and A. Mujeeb-Kazi.

Bridge crosses utilizing the D-genome SH (T. turgidum/Ae. tauschii) are a potent means of improving bread wheats for biotic and abiotic stresses. This new SH germ plasm enables incorporation of the genetic diversity of T. turgidum cultivars together with that contributed by the Ae. tauschii accessions. From the 800 SH wheats produced so far, several SHs have been identified that are resistant to Karnal bunt.

Resistance diversity is based upon the disease screening data over several years in a testing location within Mexico. Selected immune SHs from diverse Ae. tauschii accessions were crossed to elite but KB-susceptible bread wheat cultivars. Progeny was advanced by the pedigree method leading to selections of advanced derivatives that were free threshing and resistant to KB.

Evaluations were made under field conditions in Ciudad Obregon (27°20'N, 105°55'W, 39 masl), Sonora, Mexico, over four crop cycles. Ten spikes were boot-inoculated. The seed on these spikes was evaluated at maturity using procedures and scales as described by Warham et al. (1986).

Durum wheats with field resistance to KB were susceptible under greenhouse inoculation tests. Hence, resistant SH wheats were interpreted to be so because of the involvement of the respective Ae. tauschii accession. Resistance genes from the SHs have been transferred successfully to elite bread wheat cultivars (Table 20).

Reference.

• Warham EJ, Mujeeb-Kazi A, and Rosas V. 1986. Karnal bunt (Tilletia indica) resistance screening of Aegilops species and their practical utilization for Triticum aestivum improvement. Can J Pl Pathol 8:65-70.