Shoot apical meristem: A sustainable explant for genetic transformation of cereal crops

Summary Immature zygotic embryo has been the widely used explant source to develop embryogenic callus lines, cell suspensions and protoplasts for transformation of cereal crops including maize, wheat, rice, oat, barley, sorghum, and millet. However, the lack of competence of immature embryos in certain elite lines is still a barrier to rontine production of transgenic cereal crops in certain commercial cultivars. In addition, a great deal of effort is required to produce immature embryos, manipulate cultures, of immature embryos or their cell suspensions, and cryoperserve cultures for further use. In addition, undifferentiated cells may have reduced regenerability after a few months, of in vitro culture. Alternative explants and regeneration systems for efficient transformation of cereal crops are needed to avoid or reduce the above limitations. During the past decade, scientists have successfully manipulated the shoot apical meristerms from seedlings of maize oat, sorghum, millet, wheat, and barley in an effort to develop a less genetype-dependent and efficient cereal regneration system that can be maintained in vitro for long pertiods of time without the need for cryopreservation. Furthermore, apical mesistem regeneration systems were used to stably transform maize, wheat, rice, oat, barley, sorghum, and millet.     

Contribution to the fungal flora of cereal grains in Egypt

30 genera and 77 species, in addition to 4 varieties were isolated from 25 samples of each of barley, wheat, maize and sorghum grains collected from different places in Egypt. The broadest spectrum of genera and species was recorded in wheat (25 genera and 59 species + 4 varieties) followed by barley (21 genera and 52 species + 2 varieties), sorghum (14 genera and 33 species + 2 varieties) and maize grains (11 genera and 29 species + 2 varieties). Aspergillus, Fusarium, Penicillium and Rhizopus were the most common genera in the four grains, except maize where Penicillium emerged in low frequency. Aspergillus was the main component of the fungal flora of the four grains and contributed 79–94.4% of the gross total count of fungi. From the preceding genera A. niger, A. flavus, A. fumigatus, F. oxysporum, P. chrysogenum, P. corylophilum, P. notatum and R. stolonifer were the most frequent.     

Host range,mating type and population structure of Magnaporthe sp. of a single barley field in São Paulo state,Brazil

Brazil is blast disease hot spot because severe epidemics have occurred among wheat, triticale, rye, barley and oat crops. Although the first outbreak of barley blast appeared in 1998, little information is available. Therefore, this study aimed to examine host range, mating type composition and population structure of Magnaporthe sp. from a single barley field in São Paulo, Brazil. To examine pathogenicity, 25 Magnaporthe isolates were inoculated on five, three, two and two cultivars of barley, wheat, oat and rice, respectively, and one cultivar each of rye, corn, sorghum, triticale and certain weeds (Cenchrus echinatus, Setaria geniculata, Brachiaria plantaginea and Eleusine indica). Mating type distribution of 33 isolates was investigated by molecular tools. The genotypic divergence of 41 barley and five wheat isolates was investigated by 15 random amplified polymorphic DNA primers and unweighted pair group method with arithmetic mean. The host range of the barley blast pathogen included wheat, oat, rye and triticale but not rice and weeds. Sexual reproduction appeared to not be involved in the high genotypic diversity because only a single isolate, MAT1‐2, was identified. The majority of barley isolates clustered together with wheat blast, except for four, suggesting a different origin.     

Concentration of ergosterol in small-grained naturally contaminated and inoculated cereals

Ergosterol (ERG) is a major sterol constituent of most fungi. Its concentration is negligible in higher plants, but can be used as a chemical marker of the presence of fungal contaminations. In this study, ERG concentration was assessed in randomly collected samples of naturally contaminated grain (wheat, barley and oat) and in samples of grain (wheat, barley, triticale and oat) harvested after inoculation of heads with conidia of different Fusarium species. Wheat samples were analysed at three stages of grain development. The lowest ERG concentration was found in non-inoculated samples at the first stage of grain development. This concentration was increasing with grain ripening. In naturally contaminated samples collected after harvest, ERG concentration was lower in wheat than in barley and oat. ERG concentrations in inoculated samples varied significantly, but were always significantly higher than in naturally contaminated samples. In the above cereal samples it was much lower than the levels assayed in laboratory cultures inoculated with fungi from genus Fusarium. The content of ERG was also analyzed in milling products of small-grained cereals and other foodstuffs, where a considerable variation was observed. The lowest ERG amounts were assayed in flours with a high degree of purification, while the highest ones in case of flours and products with a low purification rate. The results indicate the potential application of HPLC combined with microwave-assisted extraction both when assaying samples with low ERG concentrations (naturally contaminated) and those characterized with high contents of fungal biomass (strongly infected, artificially inoculated). It also facilitates analyses of fungal biomass in technological processes, where results may be expected to vary considerably.     

Races of the Barley Root-Knot Nematode,Meloidogyne naasi. II. Developmental Rates

The developmental rates of the five newly designated races of Meloidogyne naasi were compared on barley, oat and sorghum. Races 1, 2, 3 and 4 developed and reproduced on both barley and oat but not on sorghum. Race 5 developed and reproduced readily on sorghum but poorly on oat. A more rapid rate of development of Race 5 on both barley and sorghum than that of other races on barley demonstrated that Race 5 has a shorter life cycle than do Races 1-4.     

Effects of root applications of gibberellic acid on photosynthesis and growth in C3 and C4 plants

The effects of root applications of gibberellic acid (GA₃) on growth and photosynthesis of 12 species of plants including C₃ monocots (Triticum aestivum L., wheat, Hordeum vulgare L., barley and Avena sativa L., oat), C₃ dicots (Vigna radiata L., mung bean, Cucurbita moschata L., squash and Capsicum annuum L., pepper), C₄ monocots (Zea mays L., corn, Sorghum vulgare L., sorghum and Panicum ramosum L., millet) and C₄ dicots (Amaranthus retroflexus L., pigweed, Kochia scoparis L., kochia and Gomphrena celosoides L., gomphrena) were evaluated. Relative growth rates (RGR) of barley, oat, squash, pepper, corn, sorghum, millet, pigweed and kochia were increased above the control by 12.7%, 9.9%, 11.3%, 10.7%, 19.2% 10.1%, 11.5%, 16.4% and 32.7% respectively, four days following optimum GA₃ treatments. There was no effect of GA₃ on RGR in wheat, mung bean, and gomphrena. Gibberellic acid decreased the chlorophyll content expressed on an area basis by 20.0%, 13.9%, 20.9%, 17.1%, 11.9% and 28.0% in barley, squash, pepper, sorghum, pigweed and kochia, respectively, while that of oat, wheat, mung bean, corn, millet and gomphrena remained unchanged. When photosynthetic rates were expressed per mg of chlorophyll, it showed that GA₃ could stimulate photosynthesis in barley, squash, pepper, sorghum, millet, pigweed and kochia by 20.4%, 20.6%, 16.5%, 17.4%, 10.4%, 24.2%, and 29.4%; while there was no effect in oat, wheat, mung bean, corn and gomphrena. An increase in leaf blade area and/or length of sheath were observed in GA₃ treated plants of oat, barley, mung bean, squash, pepper, corn, sorghum, millet and kochia. The transpiration rate remained unchanged following GA₃ treatment in all 12 species.This work was supported in part by the Fair Funds administered by the Pennsylvania Department of Agriculture. Contribution No. 39, Department of Horticulture, The Pennsylvania State University. Authorized for publications as paper no. 6886 in the journal series of the Pennsylvania Agricultual Experiment Station.Research assistant and assistant professor respectively.     

Survival and duration of development of Oscinella spp. (Diptera: Chloropidae) on different Gramineae in the laboratory

The duration of development and survival of Oscinella frit, O. nitidissima and O. nigerrima at different growth stages of several plant species was investigated in the laboratory. The effect of the infestation on the growth of plants was also quantified. Survival of O. frit larvae was significantly lower (P < 0.01) on barley than on either oats or wheat, between which there was no significant difference. Survival was usually highest when plants were infested at the two-leaf stage and lowest when plants were infested at the five-leaf stage. The duration of development of both male and female O. frit was longer on barley than on either oats or wheat and generally increased when older plants were infested. The proportion of male flies was greatest when older plants were infested. Duration of development and survival of O. frit was similar on oats and Lolium multiflorum, but larvae did not survive on Dactylis glomerata. Of the other Oscinella species, O. nitidissima larvae did not survive on oats, wheat and barley and O. nigerrima larvae did not survive on either oats or barley. The main shoots of cereal plants infested at the two-, three- or four-leaf stages were rapidly killed by O. frit larvae whereas many of those infested at the five-leaf stage continued to grow slowly. In response to attack proportionately more tillers were produced by oat plants than by either wheat or barley plants. When cereals were infested with O. nigerrima larvae the central shoots were damaged, but most recovered and continued to grow. Damage symptoms were most obvious when cereals were infested at the two-leaf stage, oats being more severely affected than either wheat or barley.     

Isolation and Partial Characterization of a Tenuivirus from Wheat in Iran

A tenuivirus, tentatively designated Iranian wheat stripe virus (IWSV), was transmitted by the delphacid Unkanodes tanasijevici and induced dwarfing, striping and yellowing symptoms in wheat. It could also infect barley, oat, rice, rye, sorghum and a number of other gramineous species. The virus was purified from wheat by chloroform clarification and differential and density-gradient centrifugation. It formed several layers in density-gradient columns. Purified virus preparations had a UV absorption spectrum typical of nucleoproteins and contained flexuous supercoils of 8.7 nm and fine filaments of 4.3 nm width. The antiserum produced against the virus reacted with infected plant sap in agar-gel diffusion and enzyme-linked immunosorbent assay (ELISA). The virus could be readily detected in individual viruliferous planthoppers by ELISA. IWSV was serologically related to rice hoja blanca virus but not to maize stripe virus. The relationship of IWSV with other tenuiviruses is discussed.     

Targeted mutagenesis of a conserved anther‐expressed P450 gene confers male sterility in monocots

Targeted mutagenesis using programmable DNA endonucleases has broad applications for studying gene function in planta and developing approaches to improve crop yields. Recently, a genetic method that eliminates the need to emasculate the female inbred during hybrid seed production, referred to as Seed Production Technology, has been described. The foundation of this genetic system relied on classical methods to identify genes critical to anther and pollen development. One of these genes is a P450 gene which is expressed in the tapetum of anthers. Homozygous recessive mutants in this gene render maize and rice plants male sterile. While this P450 in maize corresponds to the male fertility gene Ms26, male fertility mutants have not been isolated in other monocots such as sorghum and wheat. In this report, a custom designed homing endonuclease, Ems26+, was used to generate in planta mutations in the rice, sorghum and wheat orthologs of maize Ms26. Similar to maize, homozygous mutations in this P450 gene in rice and sorghum prevent pollen formation resulting in male sterile plants and fertility was restored in sorghum using a transformed copy of maize Ms26. In contrast, allohexaploid wheat plants that carry similar homozygous nuclear mutations in only one, but not all three, of their single genomes were male fertile. Targeted mutagenesis and subsequent characterization of male fertility genes in sorghum and wheat is an important step for capturing heterosis and improving crop yields through hybrid seed.     

Early infection of maize roots bySporisorium reilianum f. sp.zeae

Summary A cytological study was carried out to describe the initial steps of infection of maize roots by the soil fungusSporisorium reilianum f. sp.zeae. Morphogenetic changes of the fungal cells were induced in the presence of maize roots. Extensive hyphal growth led to the formation of a thick fungal layer colonising the maize root surface. This structure is original in interactions of members of the family Ustilaginaceae with plants. In the thick fungal layer, we observed fimbriae inserted into the host cell wall, suggesting a direct role of these fibrillar structures in cell adhesion and infection processes. During infection, no reaction of host cells was observed. In this way, the fungus acts as a biotrophic endophyte during the initial steps of infection.     

Tissue specificity of the sugarcane bacilliform virus promoter in oat,barley and wheat

The tissue-specificity of the sugarcane bacilliform virus (SCBV) promoter was investigated in oat, barley, and wheat to determine whether its expression pattern in one species was predictive of promoter specificity in the other closely related Gramineae species. Progeny of transgenic plants produced using constructs containing the SCBV promoter driving gusA were sampled at different stages of plant development and stained for GUS activity using a histochemical assay. Overall, the GUS staining patterns were most similar between oat and barley. In all three species, similar GUS staining patterns were observed in mature endosperms, leaves, and floral bracts of developing infloresences. No GUS staining was detected in oat embryos whereas the entire barley embryo was stained, and GUS staining was confined to the scutellum of wheat embryos. Oat and barley stems exhibited GUS staining whereas no GUS staining was observed in stems of the transgenic wheat plants. The SCBV promoter conferred strong GUS staining intensity in most tissues of oat and barley but was generally weaker in wheat. These differences in SCBV promoter specificity indicate that promoter evaluation should be conducted in the target species of interest rather than by extrapolation from expression patterns in other species.     

Interaction Of Barley Yellow Dwarf Virus and Fusarium culmorum (W. G. Sm.) Sacc. in Winter Wheat

The barley yellow dwarf virus (BYDV) epidemics, which occurred predominantly in northern Germany in 1988–90 and caused unusual yield losses of wheat, prompted our study on interactions of BYDV and Fusarium culmorum. At the late stages of plant development (EC 55/65) infections with BYDV resulted in a lower yield reduction of wheat plants than infections with F. culmorum. Combined infections at flowering resulted in severer yield reduction, indicating additive effects of the two pathogens. However, if wheat infected by BYDV at stage EC 25/35 was secondarily inoculated with the fungus at EC 55/65 the yield was less reduced than in combined infections at EC 55/65. Our results proved that the susceptibility of wheat plants to F. culmorum is increased when infection by BYDV takes place during the late stages of growth. These results correspond to observations during 3 years of the epidemic in Germany. In these years BYDV was spread mainly during the late spring, resulting in a severe secondary infection by the fungus. From the results of these investigations it may be concluded that during the years of BYDV epidemic the yield of wheat was reduced to an economically important extent because of the fungal infection, which was favoured by the virus infection.     

Synchronization of cell division in root tips of seven major cereal species for high yields of metaphase chromosomes for flow-cytometric analysis and sorting

Plant tissues that yield large numbers of mitotic cells are useful for cytogenetic analyses and prerequisite to flow-cytometric analysis and sorting of plant chromosomes. Synchronization of cell division in samples from which chromosomes are to be isolated is necessary to ensure high yields of chromosomes. A method developed for synchronizing cell division in root tips ofVicia faba (Dolezel et al., 1992; Lucretti et al., 1993) has been modified, and parameters are presented for the effective synchronization of cell division in root tips ofAvena sativa (oat),Hordeum vulgare (barley),Oryza sativa (rice),Secale cereale (rye),Sorghum bicolor (sorghum),Triticum aestivum (wheat), andZea mays (maize). Optimum parameters for metaphase arrest and collection of chromosomes from synchronized samples are also presented. Using these parameters, the yield and quality of chromosome preparations are appropriate for flow-cytometric characterization and sorting.     

Hydrogen peroxide detoxifying enzymes show different activity patterns in host and non-host plant interactions with Magnaporthe oryzae Triticum pathotype

Wheat blast caused by the hemibiotroph fungal pathogen Magnaporthe oryzae Triticum (MoT) pathotype is a destructive disease of wheat in South America, Bangladesh and Zambia. This study aimed to determine and compare the activities of antioxidant enzymes in susceptible (wheat, maize, barley and swamp rice grass) and resistant (rice) plants when interacting with MoT. The activities of reactive oxygen species-detoxifying enzymes; catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX), glutathione S-transferase (GST), peroxidase (POX) were increased in all plants in response to MoT inoculation with a few exceptions. Interestingly, an early and very high activity of CAT was observed within 24 h after inoculation in wheat, barley, maize and swamp rice grass with lower H₂O₂ concentration. In contrast, an early and high accumulation of H₂O₂ was observed in rice at 48 hai with little CAT activity only at a later stage of MoT inoculation. The activities of APX, GST and POD were also high at an early stage of infection in rice. However, these enzymes activities were very high at a later stage in wheat, barley, maize and swamp rice grass. The activity of GPX gradually decreased with the increase of time in rice. Taken together, our results suggest that late and early inductions of most of the antioxidant enzyme activities occurs in susceptible and resistant plants, respectively. This study demonstrates some insights into physiological responses of host and non-host plants when interacting with the devastating wheat blast fungus MoT, which could be useful for developing blast resistant wheat.     

Lytic bodies from cereals hydrolysing maltose and starch

Protein bodies and spherosomes from sorghum contained carbohydrase activity against maltose, starch and p-nitrophenyl-α-d-glucoside. Maltase activities in sorghum and also in maize lytic bodies were very high; carbohydrase activities of lytic bodies from whole wheat, whole barley, sorghum aleurone, sorghum embryo and maize embryo were considerably lower. The pH response of sorghum lytic bodies was bimodal with an optimum in the range of 3·4–4·2 and a minimum or a shoulder near pH 3·8. Protein bodies from sorghum, maize, wheat and barley reduced the iodine-colouring capacity of soluble starch to give a purple colour typical of a β-limit dextrin. With spherosomes colour reduction was usually more rapid, eventually taking the breakdown of starch beyond the achroic point. The lytic bodies produce both maltose and glucose from starch, except in the case of maize when only glucose was found. The data suggest that protein bodies contain a linked β-amylase-maltase system and that spherosomes contain a linked α-amylase-maltase system.     

Phytotoxicity of pathogenic fungi and their mycotoxins to cereal seedling viability

Aspergillus flavus, Alternaria alternata and Fusarium oxysporum were the pathogenic fungi most reduced cereal (barley, sorghum and wheat) seedlings. The pathogens have the ability to produce aflatoxin B₁ and G₁, diacetoxyscirpenol, kojic acid and tenuazonic acid that reduced seedling viability. The inhibition dose for 50% reduction (LD₅₀) was recorded by aflatoxins at 0.83 mg L^-1 for barley, 1.74 mg L^-1 for wheat and 2.75 mg L^-1 for sorghum. Diacetoxyscirpenol produced its inhibition at 1.26 mg L^-1 for barley, 3.98 mg L^-1 for wheat and 10 mg L^-1 for sorghum. Kojic acid induced 50% inhibition at 63 mg L^-1 for barley, 105 mg L^-1 for wheat and 251 mg L^-1 for sorghum. However, tenuazonic acid was less toxic where the toxicity ranged between 79–550 mg L^-1. The germination inhibition was more pronounced in barley followed by wheat and was negligible in sorghum for all tested mycotoxins. This inhibition was attributed to the reduction in the seedling amylase activity, where amylase was also reduced in the same trend: barley > wheat > sorghum. Grain treated with carboxin-captan and thiophanatemethyl-thiram at 1 g kg^-1 grain increased the seedlings vigour of wheat in sterilized soil by 45 and 22%, barley by 24 and 33% and sorghum by 15 and 30%, respectively. These fungicides also had a positive effect on cereal when the soil was inoculated with A. flavus, A. alternata and F. oxysporum, but the improvement was still below normal. This revised version was published online in June 2006 with corrections to the Cover Date.     

Preliminary Study of Fat Oxidation in Sorghum and Maize Brewing

Superoxide dismutase (SOD) activity in the white sorghum farafara and ICRISAT sorghum variety – ICSV 400 was at a high activity in the embryo at about 8 SOD units/tissue. This activity was almost completely destroyed at 80 °C. Totox index of the brewing grains were 366 for ICSV 400, 312 for farafara, 112 for maize grits and 90 for barley malt. Worts from sorghum/maize and sorghum/barley malt brews all had hydroperoxy linoleic acid (15–19 μM) which remained undetected after wort boiling. Sorghum/maize brews formed very little trub (wort proteinous sediments) in the whirlpool and trub increased in sorghum/barley brews with increased usage of barley malt. Sorghum/maize brews had free fatty acids (FFA) at 22 mg/l in pitching wort but in sorghum/barley brew (50/50) only 9 mg/l. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparative mapping in grasses. Oat relationships

The development of RFLP linkage maps in hexaploid and diploid oat allows us to study genetic relationships of these species at the DNA level. In this report, we present the extension of a previously developed diploid oat map (Avena atlantica x A. hirtula) and its molecular-genetic relationships with wheat, rice and maize. Examination of 92–99% of the length of the oat genome map with probes common to Triticeae species, rice or maize showed that 84, 79 and 71%, respectively, was conserved between these species and oat. Generally, the orders of loci among chromosomes homoeologous to oat chromosomes A and D were the most conserved and those of chromosomes homoeologous to oat chromosome G were the least conserved. Conservation was observed for blocks ranging from whole chromosomes 101 cM long to small segments 2.5 cM long containing two loci. Comparison of the homoeologous segments of Triticeae, rice and maize relative to oat indicated that certain regions have been maintained in all four species. The relative positions of major genes governing traits such as seed storage proteins and resistance to leaf rusts have been conserved between cultivated oat and Triticeae species. Also, the locations of three vernalization/or photo-period response genes identified in hexaploid oat correspond to the locations of similar genes in homoeologous chromosomes of wheat, rice or maize. The locations of the centromeres for six of the seven oat chromosomes were estimated based on the homoeologous segments between oat and Triticeae chromosomes.     

Male Sterility and Anther Wall Structure in Copper-deficient Plants

Anther development and pollen sterility were followed in plantsof wheat, oat, barley, sweetcorn, sunflower, petunia and subterraneumclover grown at a range of copper supplies. Copper-deficientplants had increased pollen sterility. Lignified wall thickenings were reduced or absent in the endotheciaof anthers from Cu-deficient plants. Reduced seed set may resultboth from reduced pollen fertility or failure of the stomiato rupture due to decreased lignification of anther walls. Triticum aestivum L., wheat, Hordeum vulgare L., barley, Avena sativa L., oat, Zea mays L., corn, sweetcorn, maize, Helianthus annuus L., sunflower, Petunia hybrida L., Trifolium subterraneum L., subterranean clover, male sterility, anther development, copper deficiency     

Anchor probes for comparative mapping of grass genera

 Comparative mapping of cDNA clones provides an important foundation for examining structural conservation among the chromosomes of diverse genera and for establishing hypotheses about the relationship between gene structure and function in a wide range of organisms. In this study, “anchor probes” were selected from cDNA libraries developed from rice, oat, and barley that were informative for comparative mapping in the grass family. One thousand eight hundred probes were screened on garden blots containing DNA of rice, maize, sorghum, sugarcane, wheat, barley, and oat, and 152 of them were selected as “anchors” because (1) they hybridized to the majority of target grass species based on Southern analysis, (2) they appeared to be low or single copy in rice, and (3) they helped provide reasonably good genome coverage in all species. Probes were screened for polymorphism on mapping parents, and polymorphic markers were mapped onto existing species-specific linkage maps of rice, oat, maize, and wheat. In wheat, both polymorphic and monomorphic markers could be assigned to chromosomes or chromosome arms based on hybridization to nullitetrasomic and ditelosomic stocks. Linkage among anchored loci allowed the identification of homoeologous regions of these distantly related genomes. Anchor probes were sequenced from both ends, providing an average of 260 bp in each direction, and sequences were deposited in GenBank. BLAST was used to compare the sequences with each other and with a non-redundant protein sequence database maintained at the European Molecular Biology Laboratory (EMBL). Of the anchor probes identified in this study 78% showed significant similarity to protein sequences for known genes with BLASTX scores exceeding 100. Received: 27 June 1997 / Accepted: 17 July 1997