GrainGenes

Triticum monococcum: bread baking quality, amino acid composition, and toxicity in coeliac disease.

V. Vallega.

Recent studies suggest that diploid wheat, Triticum monococcum L. ("Einkorn"), may play a significant role in modern agriculture, both as a source of useful genes and as a crop in its own right. Some of its main agronomical, chemical, and technological characteristics have been discussed in previous issues of the AWN and in other publications. This note summarizes the results of further investigations, aimed at establishing einkorn's bread making potential (collaborative work with Dr. M.G. D'Egidio, I.S.C., Rome), amino acid composition (with Drs. M.G. D'Egidio and R. Acquistucci of the Istituto Nazionale della Nutrizione), and toxicity in coeliac disease (in collaboration with Drs. M. Frisoni, G.R. Corazza, and G. Gasbarrini of the University of Bologna).

Baking, farinograph, and mixograph tests were made to compare the quality of eight germplasm accessions of T. monococcum with that of two durum wheats (cvs. Creso and Latino) and two common wheats (cvs. Mec and Centauro). Doughs of monococcums were weak in regard to mixing requirements, but strong with respect to gas retention capacity. Einkorn doughs were also rather sticky and difficult to handle and characterized by a low mixing tolerance. Monococcums produced loaves of decidedly high volume (662-871 cc), analogous to those obtained from the polyploid wheat checks (mean = 837 cc). Moreover, bread made from monococcum flours exhibited a relatively fine and uniform texture, intermediate between that of the common and durum wheat controls. Crumb colour of the T. monococcum loaves was distinctly yellowish. On the whole, the quality level of the eight germplasm accessions of T. monococcum proved low, yet well within the range commonly encountered among commercial polyploid wheats and winter cereals utilized to produce leavened foodstuffs.

Grains of 15 strains of T. monococcum and of two modern cultivars of common and durum wheat were compared with regards to amino acid composition and protein content. Grain protein percentage of monococcums ranged from 14 % to 25 % (d.m.). Among diploid wheats, correlations between grain protein percentage and amino acid values were positive for glutamine and proline and negative for threonine, 1/2 cystine, valine, isoleucine, leucine, asparagine, serine, glycine, and alanine. Differences in amino acid composition between the three wheat species and between diploid wheat strains were minimal, especially after amino acid values were adjusted to a common protein level. However, one of the einkorn accessions presented a deviant amino acid profile. This strain may be of use for investigating the biosynthetic pathway of wheat proteins. High-lysine types were not identified.

Coeliac disease is a "gluten-induced" enteropathy, and treatment involves life-long avoidance of aliments made from the grains of wheat, barley, oats, and rye. Differences in toxicity between different wheat species are little studied. An organ culture system was utilized to compare the effects on coelial mucosa of peptic-tryptic digests of gluten extracted from the flours of three wheat varieties representative of the species T. aestivum L. (common wheat, genome AABBDD), T. durum Desf. (macaroni wheat, genome AABB); and T. monococcum L. (diploid wheat, genome AA). Intestinal biopsy specimens from four treated, adult, coeliac patients and from four healthy subjects were cultured for 24 hrs in a control medium (no digest added) and in the presence of each of the digests compared. After culture, enterocyte height was measured and taken as an index of histological damage. No significant change in enterocyte height was recorded in cultures from healthy subjects. In cultures from treated coeliac patients, the mean enterocyte height reduction observed following challenge with the common wheat digest was 4.5 FONT SIZE=2 FACE="WP MathA"Fm. Challenge with digests from the diploid and the macaroni wheats induced mean height reductions of 2.6 FONT SIZE=2 FACE="WP MathA"Fm and 2.2 FONT SIZE=2 FACE="WP MathA"Fm, respectively. The lower toxicity observed for the latter two wheat species is in accord with preliminary reports by other authors. Further research on the toxicity of T. monococcum appears to be of particular interest, in that the diploid nature of this species renders it ideal for identifying and directly exploiting mutants devoid of putative toxic protein fractions. The FONT SIZE=2 FACE="WP Greek Century""-gliadins presently are considered as the most probable (or only) toxic agents of coeliac disease. Monococcums lacking these proteins should be easy to identify using standard electrophoretical techniques.

Publications.

Vallega V. 1992. Agronomical performance and breeding value of selected strains of diploid wheat, Triticum monococcum. Euphytica 61:13-23.

D'Egidio MG, Nardi S, and Vallega V. 1993. Grain, flour and dough characteristics of selected strains of diploid wheat, Triticum monococcum L. Cereal Chem 70:298-303.

D'Egidio MG and Vallega V. 1994. Bread baking and dough mixing quality of diploid wheat Triticum monococcum L. Italian J Food Beverage Tech 4:6-9.

Acquistucci R, D'Egidio MG, and Vallega V. 1995. Amino acid composition of selected strains of diploid wheat, Triticum monococcum L. (In press).

Protein content and alveograph W of durum wheat.

M.G. D'Egidio, B.M. Mariani, and P. Novaro.

Protein content and alveograph W were determined for 21 durum wheat varieties grown in several location in Italy from 1990 to 1993. The effect of genotype, environment, and their interaction on protein content and alveograph W, the most important variables to predict pasta cooking quality, were studied. Protein content was determined essentially by the additive effects of environment, whereas alveograph W appeared more influenced by the additive effects of genotype. The genotype-environment interaction for both the variables was significant, but small in

magnitude, when compared with the additive effects of genotype and environment. The majority of varieties appeared stable, with a coefficient of regression equal to one and the deviation from regression mean square negligible. The predictions based on linear regression have considerable practical value.

Publications.

Mariani BM, D'Egidio MG, and Novaro P. 1995. Durum wheat quality evaluation: influence of genotype and environment. Cereal Chem (In press).

D'Egidio MG, Mariani BM, and Novaro P. 1994. Durum wheat grain proteins: effects of genotype, environment and drying technologies on pasta quality. Proc "Wheat kernel proteins" Meeting - S. Martino al Cimino (VT) Italy, September 28-30, 1994.

Experimental Institute for Cereal Research

20079 S. Angelo Lodigiano (Milano), Via Mulino, 3, Italy.

Storage protein composition in high quality diploid wheat accessions.

R. Castagna and R. Redaelli.

In the framework of a breeding program for T. monococcum, about 1,400 accessions of ssp. monococcum and ssp. boeoticum were screened by means of the SDS-sedimentation test. One hundred and forty accessions were chosen, comprising 125 lines with high sedimentation volumes (>60 ml) and 15 with low sedimentation volumes (15-40 ml).

These lines then were characterized from a genetic point of view through the analysis of the allelic variability of their storage proteins. The electrophoretic patterns of gliadins and high molecular weight and low molecular weight glutenin subunits are being described and classified for each line.

The relationships, if any, between protein composition and the technological quality level, also evaluated by breadmaking tests, will be investigated.

Progress in hexaploid wheat tissue culture and genetic transformation.

M. Cattaneo, E. Patrucco, G.M. Borrelli, and E. Lupotto.

The establishment of routine procedures for genetic transformation of the most important crop species represents the major goal of the last decade in the tissue culture field. Besides the techniques of gene transfer, either direct or mediated, a major need for the fruitful and consistent production of transgenic plants is represented by a highly regenerative system. This need is of extreme importance for cereal species in that, in all cases but rice, the unique tools for the introduction of foreign genes are direct, such as microbombardment, electroporation, and PEG-mediated uptake. In these cases, the major constraint for obtaining an agronomically valuable transgenic plant is represented by a high copy number of the gene inserted, often affected by gene rearrangements, deletions, inactivations, and so on. All these problems would be overcome partly by a high number of transgenics among which one could choose the best individuals for further use, after molecular and biochemical characterization.

Two major key factors limit the routine application of genetic transformation to cereal breeding: i) the type of transformation technique to be applied, and ii) the type of tissue culture used for the obtention of transgenic individuals.

During our research activity in 1994, two direct tools for wheat transformation were adopted: electroporation of proliferating immature embryos and microbombardment of cells, calli, and immature embryos. These methods allow the delivery of DNA to tissues with minimal damage of the tissue itself and offer the possibility of easy regeneration from the cultures treated, especially in the case of proliferating immature embryos. For the electroporation, a Bio-Rad Gene Pulser device was utilized, with capacitor extender discharging one electrical pulse for each sample at a field strength of 375-400 V cm^-1 at 960 FONT SIZE=2 FACE="WP MathA"FF capacitance. The size of the callus treated in each electroporation was about 80-150 mg fresh weight tissue, chosen from proliferating immature embryos of the hexaploid wheat cultivars Oderzo and Veery. We focused our work on these two cultivars because of their high regenerative capacity from in vitro cultured calli. The expression vector used for these studies was plasmid pAHC25 (a kind gift from P. Quail), a dual vector carrying the uid A (FONT SIZE=2 FACE="WP MultinationalA Roman"8-GUS), and the bar genes, both under the promoter of ubiquitin-1 of maize. The same plasmid was utilized successfully in several other transformation experiments in wheat (data from recent literature) and also was employed by us in maize (Lupotto unpublished, 1994). For microbombardment, a homemade Particle Inflow Gun was used, assembled by us according to Finer et al. (1992), and cell suspensions, immature embryos, young embryogenic calli, and established callus cultures of wheat were used as target tissues.

At the moment, we are characterizing at the molecular level events of transformation on cell suspension cultures of wheat cv. Veery. In order to apply, with routine success, the methodology to embryogenic young calli, constant subculture and selection are being performed on the above-mentioned cultivars and a series of other genotypes, in order to select for cell lines characterized by regenerative capability and embryogenic friable texture. From these selected callus lines, green plantlets were regenerated, although the regeneration efficiency was lower if compared to the same callus cultures of other cereal species (maize and rice). The regenerative capacity of the best-responding genotypes (Oderzo, Farneto, and Veery) was maintained for at least 12 months.

Because a common observation is that the longer the time of selection lasts, the less regenerative the cultures are, we are now trying to improve the whole procedure by shortening, as much as possible, the various steps, from the explant through transformation, selection, and regeneration. These studies also are being carried out with other cultivars of hexaploid wheat: Maestra, Centauro, and Eridano; and on the Triticale varieties: Magistral, Antares, and Boccale; all important cultivars of the Mediterranean basin. In particular, the two Triticale cultivars, Antares and Boccale, show an extremely prompt regeneration from the primary embryogenic calli and have been chosen as material for transformation via microbombardment. However, we should note that in the cases of durum wheat (Triticum durum Desf.) and Triticale, the selection of friable cell lines so far has been unsuccessful.

Relationships between molecular markers and heterosis in bread wheat hybrids.

M. Corbellini, M. Accerbi, P. Vaccino, M. Perenzin, and B. Borghi.

Both the RFLP and RAPD techniques were used to study a set of 40 parental varieties of about 100 hybrids.

With regard to RFLPs, 70 probes have been tested, among which only 21 (30 %) were polymorphic: all the cultivars were differentiated and clustered into two main groups, but the coefficient of genetic similarity (GS) and the main agronomic traits were not correlated significantly.

Limited to seven parents of a diallelic cross, the RAPD analysis was carried out. Fifty-nine of 116 primers (50 %) produced 181 polymorphic fragments. A significant correlation was found between GS and grain yield.

Work is in progress with the analysis of new primers in order to establish the genetic distances between cultivars and to confirm the correlation already found.

Response to selection for adaptation to Mediterranean climate after two cycles of recurrent selection in bread wheat.

P. Gavuzzi and B. Borghi.

In Italy, the yield potential of modern cultivars largely depends on their better adaptation to the unfavourable conditions of the Mediterranean environments, where in the final stages of grain filling, a gradual rise of temperature associated with a severe depletion of soil water resources is common.

A selection programme, based on the evaluation of agronomical traits and the response to physiological tests, has been carried on in the last few years in order to develop new lines more tolerant to the Mediterranean climate.

Thirteen cultivars selected from 150 accessions, evaluated for stress tolerance in field conditions in drought-prone areas or with physiological laboratory tests, were intercrossed in a diallel scheme. The 78 F₁ hybrids were submitted to a second cycle of random mating, and the lines derived from the C₀ population were selected for tolerance to stress as indicated for the parental cultivars. Twenty-eight C₀S₂ progenies were intercrossed, and the progenies submitted to a second cycle of selection.

In the year 1992-93, the original 13 cultivars, 13 C₀S₆ lines, and 13 C₁S₄ lines were evaluated in replicated plot trials in three locations and characterized by physiological laboratory tests. In the following season, the best C₀ and C₁ lines were compared again for their agronomical performance.

The results indicate a significative improvement in the C₀ and C₁ lines over the parental cultivars for several physiological parameters associated with tolerance to stress, such as cell membrane stability after heat shock water loss from detached leaves and tolerance to early desiccation. Average grain yield of the three groups did not differ significantly in the three locations; however, the C₀ and C₁ lines presented a significative improvement of the susceptibility index, suggesting that the new lines were more adapted than the parental cultivars to the stress conditions typical of the Mediterranean climate.