Plant regeneration from protoplasts of durum wheat (Triticum durum Desf. cv. D6962)

Suspension cultures of durum wheat were established from embryogenic callus maintained in liquid medium for 30 months. Protoplasts were readily isolated from the suspension cultures with yields as high as 3 X 10⁷ protoplasts per g fresh weight suspension cells. When incubated in a modified MS medium containing half strength of the macroelements, 5 M 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.6 M glucose, protoplast-derived cells divided at frequencies ranging from 1.4 to 10.0 %. After transfer to a solid subculture medium, the protoplast-derived colonies formed embryogenic protuberances, from which green plants have been regenerated.     

Catalogue of alleles of gliadin-coding loci in durum wheat (Triticum durum Desf.)

Gliadins are seed storage proteins which are characterized by high intervarietal polymorphism and can be used as genetic markers. As a result of our work, a considerably extended catalogue of allelic variants of gliadin component blocks was compiled for durum wheat; 74 allelic variants for four gliadin-coding loci were identified for the first time. The extended catalogue includes a total of 131 allelic variants: 16 for locus Gli-A1(d), 19 for locus Gli-B1(d), 41 for locus Gli-A2(d), and 55 for locus Gli-B2(d). The electrophoretic pattern of the standard cultivar and a diagram are provided for every block identified. The number of alleles per family is quite small for loci Gli-A1(d) and Gli-B1(d) of durum wheat, as contrasted to loci Gli-A2(d) and Gli-B2(d) that are characterized by large families including many alleles. The presence of large block families determines a higher diversity of durum wheat for loci Gli-A2(d) and Gli-B2(d) as compared to Gli-A1(d) and Gli-B1(d). The catalogue of allelic variants of gliadin component blocks can be used by seed farmers to identify durum wheat cultivars and evaluate their purity; by breeders, to obtain homogenous cultivars and control the initial stages of selection; by gene bank experts, to preserve native varieties and the original biotypic composition of cultivars.     

Effect of salt and osmotic stresses on germination in durum wheat (Triticum durum Desf.)

In order to determine the relative importance of ionic toxicity versus the osmotic component of salt stress on germination in durum wheat (Triticum durum Desf.), seeds of three cultivars differing in their salt and drought resistance (Omrabi-5, drought-resistant; Belikh, salt-resistant and Cando, salt-sensitive) were incubated in various iso-osmotic solutions of NaCl, mannitol and polyethylene-glycol (PEG) (osmotic potential of –0.15 (control solution) –0.58, –1.05 or –1.57 MPa). Moderate stress intensities only delayed germination, whereas the highest concentration of NaCl and PEG reduced final germination percentages. PEG was the most detrimental solute, while mannitol had no effect on final germination percentages. All osmotica reduced endosperm starch and soluble sugars content as well as -amylase activities recorded after 48 h of treatment while -amylase activities were, in contrast, slightly stimulated in all cultivars. Deleterious effects of NaCl and PEG were higher on isolated embryos germinated onto an in vitro Linsmaier and Skoog (LS) medium comparatively to whole seeds. All PEG-treated embryos, however, recovered after the stress relief while NaCl-treated embryos exhibited a lower rate of recovery and some extent of abnormal germination after rinsing. It was concluded that stress inhibition of germination could not be attributed to an inhibition of mobilisation of reserves and that the main effect of PEG occurred via an inhibition of water uptake while detrimental effects of NaCl may be linked to long-term effects of accumulated toxic ions. The behaviour of the three cultivars during germination did not fully reflect their mean level of putative stress resistance in field conditions and germination is, therefore, not recommended as a reliable selection criterion for breeding purposes.     

The addition of Dasypyrum villosum (L.) Candargy chromosomes to durum wheat (Triticum durum Desf.)

Summary Six monosomic addition lines were produced in which different Dasypyrum villosum (L.) Candargy chromosomes were added to the chromosome complement of Triticum durum Desf. cv. Creso. Each added alien chromosome was found to have a specific effect on plant morphology and fertility. Transmission rate varied widely (from 7.5 to 27.7%) among the six univalent chromosomes. Different monotelosomic addition plants derived by a relatively high frequency of chromosome misdivision were isolated. The addition lines should be useful for studying Dasypyrum chromosome homoeology and the introduction of alien variation into durum and common wheats.Research supported by a grant from the Italian Research Council for Finalized Project IPRA. Sub-project Plant Breeding, Paper No. 1095     

Biofortification of durum wheat (Triticum turgidum L. ssp. durum (Desf.) Husnot) grains with nutrients

Durum wheat (Triticum turgidum L. ssp. durum (Desf.) Husnot) was grown under conditions to promote mineral biofortification at the grain level. Along plant development, biomass accumulation and the kinetics of nutrients accumulation were assessed, identifying the nutrient fluxes of roots and shoots, and the timescale constraints of crop biofortification. Plants were grown under environmentally controlled conditions, submitted to four increasing concentrations of nutrient solutions (1-, 2-, 4- and 6-fold) of micro- (Fe, Zn, Cu and Mn) and macronutrients (Ca, K, P and Mg). The threshold of mineral toxicity was not reached as evaluated through plant biomass accumulation, but considering grain yield, the twofold nutrient concentration was the best treatment for biofortification. In the different treatments, the contents and the mineral unrests of roots uptake and shoots translocation varied, at different magnitudes and trends, before the onset of booting and from the physiological maturity onwards. Except for Cu, all mineral nutrients were mainly detected in the bran and embryo of the grains; therefore, the production of biofortified pasta for human consumption requires the use of integral semolina.     

Fertile wheat (Triticum aestivum L.) regenerants from protoplasts of embryogenic suspension culture

We report regeneration of fertile, green plants from wheat (Triticum aestivum L. cv. Aura) protoplasts isolated from an embryogenic suspension initiated from somatic early-embryogenic callus. The present approach combines the optimization of protoplast culture conditions with screening for responsive genotypes. In addition to the dominant effect of the culture media, the increase in fresh mass and the embryogenic potential of somatic callus cultures varied considerably between the various genotypes tested. Establishment of suspension cultures with the required characters for protoplast isolation was improved by reduction of the ratio between cells and medium and by less frequent (monthly) transfer into fresh medium. A new washing solution was introduced to avoid the aggregation of protoplasts. However, the influence of the culture medium on cell division was variable in the different genotypes. We could identify cultures from cultivar Aura that showed approximately a 9% cell division frequency and morphogenic response. The protoplast-derived microcolonies formed both early and late-embryogenic callus on regeneration medium and green fertile plants were obtained through somatic embryogenesis. The reproducibility of plant regeneration from protoplast culture based on the cultivar Aura was demonstrated by several independent experiments. The maintenance of regeneration potential in Aura suspension cultures required establishment of new cultures within a 9-month period.     

Effect of light and gibberellic acid on cell division in the first foliage leaf of durum wheat (Triticum durum Desf.)

The present paper is part of a research program which aims at a quantitative analysis of the effects of light and gibberellic acid (GA₃) on growth of the first foliage leaf in durum wheat (Triticum durum Desf.). Since leaf growth is the combined result of the increase in cell number (cell division) and cell enlargement, the influence of light and GA₃ treatment on cell division in the basal meristem of the first leaf in two cultivars, Cappelli and Creso, was investigated. Creso is a short-strawed cultivar carrying the Gai 1 gene which influences both plant height and insensitivity to applied GA₃. Cell division, as measured by mitotic index, was similar in darkness, continuous red light and dichromatic irradiation (far-red plus red), while lower mitotic rates were observed under continuous far-red light: this indicates that the response of cell division is modulated by a high-irradiance reaction of phytochrome in both cultivars. The two cultivars showed different responses to blue light. In Cappelli, blue light and dichromatic irradiation (blue plus red) gave lower mitotic indices than the dark control, indicating the action of a specific blue-light-absorbing photoreceptor, whereas in Creso the response kinetics to all light regimes which included blue light were more complex. On the basis also of the results obtained with GA₃ application in Cappelli, it appears that (i) the hormonal treatment is able to change the pattern of mitotic index only in the presence of the action of a blue-light receptor and (ii) the different responses of the two cultivars could be the result of different endogenous hormonal levels. The importance of the observations in relation to the data for first-leaf longitudinal growth reported in a previous paper (Baroncelli et al. 1984, Planta 160, 298–304) is discussed.Abbreviations BL blue light - D darkness - FR far-red light - GA gibberellin - GA₃ gibberellic acid - m.i. mitotic index - Norflurazon 4-chloro-5-(methylamino)-2-(,,,-trifluoro-m-totyl-3(2H)) pyridazinone - R red light - WL white light - phytochrome photoequilibrium     

Cloning and characterization of a cDNA encoding the wheat (Triticum durum Desf.) CM16 protein

A Triticum durum cDNA library prepared from developing endosperm (22 days after flowering (DAF)) was screened using synthetic oligonucleotide probes covering part of the CM3 and CM16 N-terminal protein sequences. A full-length cDNA clone (pTd78) encoding the CM16 protein (chloroform/methanol-soluble protein) was isolated and characterized. To our knowledge this is the first characterization of a clone coding for a wheat CM protein. The CM16 protein is synthesized as a preprotein with a signal peptide of 24 residues, the molecular weight of the mature protein being 13 438 Da. As other members of the cereal trypsin/-amylase inhibitor family, the CM16 protein contains 10 cysteine residues, their position being well conserved. In developing endosperm the highest level of CM16 mRNA was detected at mid-maturation.     

Somatic embryogenesis and plant regeneration from protoplasts isolated from embryogenic cell suspensions of wheat (Triticum aestivum L.)

We describe the early formation of somatic embryos followed by plant regeneration from protoplasts isolated from an embryogenic wheat cell suspension, which was initiated from small granular (0.2 to 1 mm in size) embryogenic calli. These granular calli formed embryogenic cell suspensions within 20 days in liquid culture, and were selected gradually from young inflorescence-derived nodular embryogenic calli of the winter wheat cv. Kehong 1041. The division frequency of protoplasts was 11 to 16%, and the frequency of differentiation into plants was about 0.001% (number of plants formed divided by the total number of protoplasts plated). About 20% of somatic embryos present in the culture formed directly from protoplast-derived cells within 15 days of cultures.     

Ethylene production and involvement during the first steps of durum wheat (Triticum durum) anther culture

The role of ethylene in anther culture of durum wheat ( Triticum durum Desf. cv. Ardente) was analyzed by testing the effects of 2-chloroethylphosphonic acid (ethrel) silver thiosulfate (Ag⁺), a -aminooxyacetic acid (AOA) and 1-aminocyclopropane-l-carboxylic acid (ACC) on microspore division observed after 21 days of culture and on development of calli estimated at day 45. The use of ethrel and Ag⁺ indicated a positive effect of ethylene on microspore division, whereas the use of AOA, and to a lesser extent ACC, snowed a negative effect. In contrast, the addition of ethrel or Ag⁺ indicated that ethylene inhibits the development of microspore-derived calli. AOA gave contradictory results. Ethylene production by anthers was about 7 pl anther⁻¹h⁻¹ and decreased during culture. ACC content in the anthers was maximal at day 9, whereas malonyl ACC (MACC) increased sharply from day 0 to day 3 and then decreased. The addition of AOA or ACC to the culture medium decreased or increased, respectively, ethylene production of anthers and the ACC and/or MACC content, but at concentrations higher than those that modified the formation of calli. This formation seems to occur in two successive phases: induction and initiation of microspore division, which was promoted by ethylene, followed by callus development, which was inhibited by ethylene.     

Shoot tips of wheat as an alternative source for regenerable embryogenic callus cultures

Shoot tips of Triticum aestivum L. cvs. Turbo and Nandu, both summer wheat varieties, were excised from 4 and 10 day-old seedlings, and used for induction of embryogenic callus. A modified L3 medium, supplemented with 10 M 2,4-dichlorophenoxyacetic acid (2,4-d) for culture initiation, and 5 M 2,4-d for subculturing, was optimal; 90% of 4 day-old Turbo seedlings formed embryogenic callus. Optimal plant regeneration was achieved from callus incubated on a modified MS medium without 2,4-d, but supplemented with 2.22 M 6-benzylaminopurine and 0.27 M naphthaleneacetic acid. Plantlets formed via embryogenesis from all embryogenic Turbo calli initiated from 4 day-old explants, with a mean number of 8 regenerants per explant. Regeneration occured via embryogenesis only. Results obtained using Nandu were within the same range.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid     

Molecular analysis of plants regenerated from embryogenic cultures of wheat (Triticum aestivum L.)

Total DNAs of plants regenerated from immature embryo-derived 2-month-old embryogenic calli of wheat (cultivars Florida 302, Chris, Pavon, RH770019) were probed with six maize mitochondrial genes (atpA, atp6, apt9, coxI, coxII, rrn18-rrn5), three hypervariable wheat mitochondrial clones (K, K3, X2), five random pearl millet mitochondrial clones (4A9, 4D1, 4D12, 4E1, 4E11) and the often-used wheat Nor locus probe (pTA71), in order to assess the molecular changes induced in vitro. In addition, protoplast-derived plants, and 24-month-old embryogenic and non-embryogenic calli and cell suspension cultures of Florida 302 were also analyzed. No variation was revealed by the wheat or millet mitochondrial clones. Qualitative variation was detected in the nonembryogenic suspension culture by three maize mitochondrial genes (coxI, rrn18-rrn5, atp6). A callus-specific 3.8-kb Hind III fragment was detected in all four cultivars after hybridization with the coxI gene. The organization of the Nor locus of the plants regenerated from Florida 302 and Chris was stable when compared to their respective control plants and calli. The Nor locus in regenerants of Pavon and RH, on the other hand, was found to be variable. However, Nor locus variability was not observed in 14 individual seed-derived control plants from either Pavon or RH sources. In Pavon, a 3.6-kb Taq I or a 5.6-kb Bam HI+ Eco RI fragment was lost after regeneration. In one of the RH regenerants, which lost a fragment, an additional fragment was observed.     

Effect of light and gibberellic acid on coleoptile and first-foliage-leaf growth in durum wheat (Triticum durum Desf.)

A comparison has been made of the relative effectiveness of light quality and quantity and gibberellic acid (GA₃) treatment on the elongation growth of the coleoptile and the first foliage leaf in durum wheat (Triticum durum Desf. cvs. Cappelli and Creso). The cultivar Creso is a shortstrawed variety carrying the Gai 1 gene on chromosome 4A, which influences both plant height and insensitivity to applied gibberellins. The main conclusions are as follows: 1) coleoptile elongation growth appears to be modulated via the fluencerate-dependent action of a blue-light receptor and via a low energy response of phytochrome; 2) the inhibition of first-foliage-leaf growth depends on the operation of a single blue-light-responsive photoreceptor; 3) high energy blue light produces the same inhibitory effect on the two wheat cultivars, whereas at relatively low fluences of white and blue light, the cultivar Creso is more sensitive; 4) the insensitivity to applied GA₃ exerted by the gene Gai 1 in Creso is independent of light; 5) in Cappelli, the action of light on coleoptiles appears to be independent of the applied GA₃, whereas the hormone is able to change the pattern of growth inhibition of the first-foliage-leaf.Abbreviations BL blue light - FR far-red light - GA gibberellin - GA₃ gibberellic acid - R red light - WL white light     

Production of doubled haploids in durum wheat (Triticum turgidum L.) through isolated microspore culture

The objective of this work was to produce doubled haploid plants from durum wheat through the induction of androgenesis. A microspore culture technique was developed and used to produce fertile doubled haploid plants of agronomic interest. Five cultivars, one selected line, plus a collection of 20 F₁ crosses between different genotypes of high breeding value were used. Studies on several factors such as pre-treatments and media components were carried out in order to develop a protocol to regenerate green haploid plantlets. Anthers were pre-treated in 0.7 M mannitol. Microspores, from anther maceration, were plated on a C₁₇ induction culture medium with ovary co-culture. The optimum regeneration medium J25–8 was used. From 35 microspore isolations, 407 green plantlets were obtained. With this technique mature embryos were obtained. Green plants were regenerated from all genotypes used and approximately 67% of them were spontaneously doubled haploids. Some haploids and a very few polyploids plants were obtained. From the 407 plants, 275 were completely fertile and gave enough seeds to be assayed in the field. This protocol could be used complementary to or instead of the intergeneric crossing with maize as an economically feasible method to obtain doubled haploids from most durum wheat genotypes.     

Transformation of pasta wheat (Triticum turgidum L. var. durum) with high-molecular-weight glutenin subunit genes and modification of dough functionality

Particle bombardment has been used to transform three cultivars (L35, Ofanto, Svevo) and one breeding line (Latino × Lira) of durum wheat (Triticum turgidum L. var. durum). These varieties were co-transformed with plasmids containing selectable and scorable marker genes (bar and uidA) and plasmids containing one of two high-molecular-weight (HMW) glutenin subunit genes (encoding subunits 1Ax1 or 1Dx5). Ten independent transgenic lines were recovered from 1683 bombarded scutella (transformation efficiency thus 0.6%). Five lines expressed either subunit 1Dx5 or 1Ax1 at levels similar to those of endogenous subunits encoded on chromosome 1B. To identify the effects of the transgenes on the functional properties of grain, three lines showing segregation for transgene expression were used to isolate sibling T₂ plants which were null or positive for the transgene product. Analysis of these plants using a small-scale mixograph showed that expression of the additional subunits resulted in increased dough strength and stability, demonstrating that transformation can be used to modify the quality of durum wheat for bread and pasta making.     

Recovery and regeneration of embryogenic cultures from female flowers of False Horn Plantain

Somatic embryogenesis from immature male flowers in Musa is only suitable for genotypes with a male bud. Six friable embryogenic cultures were obtained from 28 cultured buds of female flowers of the AAB False Horn Plantains, ‘Curraré’ and ‘Curraré Enano’. Embryogenic suspensions were established from these embryogenic cultures. Somatic embryogenesis was demonstrated histologicaly. Regeneration of plants was obtained either from somatic embryos directly isolated from embryogenic cultures or from suspensions after plating on a semi-solid medium. This study demonstrates that somatic embryogenesis from immature flowers is suitable for genotypes of Musa with or without male buds. This revised version was published online in June 2006 with corrections to the Cover Date.     

High frequency regeneration of plantlets from the leaf-bases via somatic embryogenesis and comparison of polypeptide profiles from morphogenic and non-morphogenic calli in wheat (Triticum aestivum)

High frequency regeneration via somatic embryogenesis was achieved in the leaf-base cultures of wheat ( Triticum aestivum L. cv Sonalika) by optimizing the concentration of the hormone, 2,4-dichlorophenoxy acetic acid, and selecting for the appropriate part of the leaf base as explant. It was possible to distinguish compact (morphogenic) and friable (non-morphogenic) calli by the naked eye, after about 60 days of culture on medium enriched with 2,4-dichlorophenoxy acetic acid. The fact that the compact calli are morphogenic, while the friable ones are not, was evident from the observation that only the former formed plantlets after transfer to the basal medium. The morphogenic and non-morphogenic cultures showed substantial difference in soluble protein content on a fresh weight basis. A comparison of silver-stained profiles of soluble polypeptides from morphogenic and non-morphogenic calli revealed many polypeptides specifically associated with either type of calli.     

AgNO3对橡胶树花药愈伤组织形态及体胚发生的影响

橡胶树的花药愈伤组织在长期继代过程中,胚性易下降甚至丧失;而AgNO3作为乙烯活性抑制剂,被广泛应用于植物组织培养中.该研究以继代培养4 a以上的热研7-33-97花药愈伤组织为材料,在继代培养基中添加2.5 mg·L-1 AgNO3预培养35 d后,观察预培养前后愈伤组织表形及其细胞形态的变化,并设计不同浓度AgNO3及不同处理时间对其进行体胚诱导,90 d后分别统计胚状体总数和正常胚数.结果表明:浅黄色质地柔软的愈伤组织在含AgNO3的培养基上预培养后能转变成鲜黄色易碎愈伤组织,在倒置显微镜下前者大多表现为不规则多边形,细胞内含物较稀薄;而后者则呈圆形或椭圆形,细胞内含物丰富,属于典型的胚性细胞.在体胚诱导的第1个月添加5 mg·L-1 AgNO3能显著促进体胚的发生,AgNO3浓度升至10 mg·L-1时体胚发生受到抑制,且畸形胚的形成率显著增加;在含5 mg·L-1 AgNO3的培养基中培养2个月以上,体胚发育明显受阻,大部分形成畸形胚.该研究结果在一定程度上恢复了橡胶树长期继代花药愈伤组织的胚性能力,并提高了其体胚发生频率,为橡胶树花药胚性愈伤组织长期继代培养过程中胚性的保持提供了参考.