Comparative field performance over 3 years and two sites of transgenic wheat lines expressing HMW subunit transgenes

A series of transgenic wheat lines expressing additional high molecular weight (HMW) subunit genes and the corresponding control lines were grown in replicate field trials at two UK sites (Rothamsted Research, approximately 50 km north of London and Long Ashton, near Bristol) over 3 years (1998, 1999, 2000), with successive generations of the transgenic lines (T₃, T₄, T₅) being planted. Four plots from each site were used to determine grain dry weight, grain nitrogen, dough strength (measured as peak resistance by Mixograph analysis) and the expression levels of the endogenous and “added” subunits. Detailed statistical analyses showed that the transgenic and non-transgenic lines did not differ in terms of stability of HMW subunit gene expression or in stability of grain nitrogen, dry weight or dough strength, either between the 3 years or between sites and plots. These results indicate that the transgenic and control lines can be regarded as substantially equivalent in terms of stability of gene expression between generations and environments.     

Field evaluation and agronomic performance of transgenic wheat

Seven transgenic lines of wheat have been evaluated under field conditions during 2 agonomic years. Four lines contained the transgenes for beta-glucuronidase ( uidA), herbicide resistance ( bar) and for one high-molecular-weight (HMW) subunit, and three lines contained only one transgene for one HMW glutenin subunit and no marker genes. Agronomic traits and yield components were studied in transgenic lines and compared with the non-transgenic parent and null segregant lines. Although phenotypic differences for many traits have been found, only heading date and the number of spikelets per spike showed clear genotypic differences for both field trials. All transgenic lines had a longer heading date than parent lines whereas the number of spikelets per spike in transgenic lines was around that for L88-31 and higher for L88-6 than the corresponding parent lines. No differences were found between lines constitutively expressing the uidA and bar genes from those which only expressed the HMW genes. We conclude that differences between transgenic lines and their parents are small, and could be eliminated by backcrossing transgenic lines with their parents and selecting for the wanted genotype.     

Polyamines in humic acid and their effect on radical growth of lettuce seedlings

Linum usitatissimum, Sorghum bicolor and Triticum aestivum plants were further colonised by the arbuscular mycorrhizal fungus, Glomus mosseae, during a four week period of hydroponic culture after a pre-culture period of three weeks with the fungus in perlite substrate. The viability of mycorrhizal colonisation of T. aestivum was indicated by an initial experiment where G. mosseae from mycorrhizal plants colonised non-mycorrhizal plants when the plants were grown together in the same hydroponic container using modified Long Ashton nutrient solution. Intermittant aeration of the plant roots (2 h periods, four times per day) provided a compromise between adequate aeration and minimal disturbance of the fungus. In a second experiment, two nutrient media, modified Long Ashton and modified Knop plus Hoagland medium were compared for culturing G. mosseae on T. aestivum. A significantly higher root dry weight was found for the mycorrhizal versus the non-mycorrhizal wheat plants in modified Long Ashton nutrient medium, which contained 10 µM P and an organic buffer. Modified Knop plus Hoagland nutrient medium contained a high P concentration (0.9 mM) and did not produce viable cultures of mycorrhizal colonisation. In a third experiment, modified Long Ashton medium was used for hydroponic culture of mycorrhizal L. usitatissimum, S. bicolor and T. aestivum. The root colonisation percentages for T. aestivum (73%), S. bicolor (36%) and L. usitatissimum (65%) were within the range of colonisation rates obtained with solid substrate culture in perlite. Viability of the mycorrhizal structures in hydroponic culture was assessed by monitoring activity of fungal succinate dehydrogenase and found to be similar to cultures in perlite. No difference in the P concentration of mycorrhizal and non-mycorrhizal plants was observed, possibly owing to the lack of diffusion limits for P in hydroponic solution. This report describes a system for the viable culture of G. mosseae with different plant species where a high mycorrhizal colonisation rate was produced under conditions of a short culture period using intermittent aeration, a low concentration of P supply and an organic buffer.     

Targeted modification of storage protein content resulting in improved amino acid composition of barley grain

C-hordein in barley and ω-gliadins in wheat are members of the prolamins protein families. Prolamins are the major component of cereal storage proteins and composed of non-essential amino acids (AA) such as proline and glutamine therefore have low nutritional value. Using double stranded RNAi silencing technology directed towards C-hordein we obtained transgenic barley lines with up to 94.7 % reduction in the levels of C-hordein protein relative to the parental line. The composition of the prolamin fraction of the barley parental line cv. Golden Promise was resolved using SDS-PAGE electrophoresis, the protein band were excised and the proteins identified by quadrupole-time-of-flight mass spectrometry. Subsequent SDS-PAGE separation and analysis of the prolamin fraction of the transgenic lines revealed a reduction in the amounts of C-hordeins and increases in the content of other hordein family members. Analysis of the AA composition of the transgenic lines showed that the level of essential amino acids increased with a concomitant reduction in proline and glutamine. Both the barley C-hordein and wheat ω-gliadin genes proved successful for RNAi-gene mediated suppression of barley C-hordein level. All transgenic lines that exhibited a reduction for C-hordein showed off-target effects: the lines exhibited increased level of B/γ-hordein while D-hordein level was reduced. Furthermore, the multicopy insertions correlated negatively with silencing.     

Investigations on Genetically Modified Maize (Bt-Maize) in Pig Nutrition: Chemical Composition and Nutritional Evaluation

The objective of the present study was to determine the composition and the nutritional value of parental and transgenic maize seeds fed to pigs. The parental maize line was genetically modified to incorporate a gene from Bacillus thuringiensis (Bt) expressing a toxin against the European corn borer (Ostrinia nubilalis). Both (parental and transgenic) maize lines were analyzed for crude nutrients, starch, sugar, non-starch polysaccharides (NSP), amino acids, fatty acids, as well as for selected minerals. Furthermore, four complete diets were mixed and were analyzed for the same nutrients and some selected ingredients. The diets contained 70% maize to attain a high effect level. To evaluate the feeding value of one variety of genetically modified maize (transgenic) compared to the feeding value of the unmodified maize (parental) line, a balance study with twelve pigs was designed. Three collecting periods were used for each maize line each with six animals. The collected faeces were analyzed for crude nutrients. All measured parameters were virtually the same ( e.g. crude protein 11.59% vs. 11.06% in DM), especially the digestibility of crude protein (85.8 ± 2.3% vs. 86.1 ± 1.8%), the amount of nitrogen-free-extract (92.8 ± 0.6% vs. 93.2 ± 0.6%) and the metabolizable energy (15.7 ± 0.2% vs. 15.8 ± 0.2% MJ/kg DM) for both maize lines. Compared to the parental line, the chemical composition and digestibility of crude nutrients and energy content were not significantly affected by the genetic modification of maize. Therefore, from the view of a nutritional assessment, the genetically modified maize can be regarded as substantially equivalent to the parental maize line.     

籼稻转反义蜡质基因后代的直链淀粉含量测定和纯系选育

通过根癌农杆菌介导将反义蜡质基因导入籼型雄性不育保持系龙特甫B中,获得30个PCR检测为阳性的转基因植株,其中,28个为Southern检测阳性。T1种子直链淀粉含量测定结果表明,有21个转基因植株比龙特甫B明显下降,下降幅度为3％-13％,并在部分转基因植株的种子中观察到蜡质状籽粒;对6个转基因植株进行了不同世代的直链淀粉含量测定,在L3和L5的T4代中,选择到直链淀粉含量分别为15．9％和8．4％的纯合株系,经凝胶电泳测定,WX蛋白量明显降低,并与直链淀粉含量下降表现一致。以L3-1-1-1(15．9％)和L5-8-2-1(8．4％)纯合株系为亲本,分别与龙特甫A进行成对杂交和回交,并测定了F1和B1F1种子直链淀粉含量,以L3-1-1-1作亲本的F1为21．4％,B1F1为17．1％;以L5-8-2-1作亲本的F1为13．6％,B1F1为9．3％,结果表明：在不育系的转育过程中,以中低直链淀粉含量的转基因纯合株系为亲本,能有效降低F1和B1F1的种子直链淀粉含量。     

Bacterial populations in the rhizosphere of tobacco plants producing the quorum-sensing signals hexanoyl-homoserine lactone and 3-oxo-hexanoyl-homoserine lactone

A tobacco line genetically modified to produce two N-acyl homoserine lactones and its non-transformed parental line were grown in non-sterile soil. Microbial populations inhabiting the bulk soil, and those colonizing the root system of the two tobacco lines, were analyzed using cultivation-independent (phospholipid fatty acid and denaturing gradient gel electrophoresis) and cultivation-based assays. The cell density of total cultivable bacteria, fluorescent pseudomonads, sporulated, and thermotolerant bacteria was also determined in a time-course experiment (15 weeks). A possible "rhizosphere effect" related to the development of the plant was seen. However, no dissimilarities in cell population densities or population ratios of the microbial groups were detected in the rhizosphere of the two plant lines. Similarly, bacterial communities that either produced N-acyl homoserine lactone or degraded the signal hexanoyl homoserine lactone were enumerated from the two plant lines. No noticeable differences were evidenced from one plant genotype to the other. Whilst the transgenic plants released detectable amounts of the quorum-sensing signal molecules and efficiently cross-talked with the surrounding microbial populations, the bias generated by these signals in the reported experimental conditions therefore appears to remain weak, if not non-existent.     

Characterization of transgenic sulfonylurea-resistant flax (Linum usitatissimum)

Summary Fourteen transgenic flax (Linum usitatissimum) lines, carrying a mutant Arabidopsis acetolactate synthase (ALS) gene selected for resistance to chlorsulfuron, were characterized for resistance to two sulfonylurea herbicides. Progeny of 10 of the 14 lines segregated in a ratio of 3 resistant to 1 susceptible, indicating a single insertion. Progeny of 1 line segregated in a 151 ratio, indicating two insertions of the ALS gene at independent loci. Progeny from 3 lines did not segregate in a Mendelian fashion and were likely the products of chimeric shoots. Resistance to chlorsulfuron was stably inherited in all lines. At the enzyme level, the transgenic lines were 2.5 to more than 60 times more resistant to chlorsulfuron than the parental lines. The transgenic lines were 25–260 times more resistant to chlorsulfuron than the parental lines in root growth experiments and demonstrated resistance when grown in soil treated with 20 g ha^-1 chlorsulfuron. The lines demonstrated less resistance to metsulfuron methyl; in root growth experiments, the transgenic lines were only 1.6–4.8 times more resistant to metsulfuron methyl than the parental lines. Resistance was demonstrated in the field at half (2.25 g ha^-1) and full (4.5 g ha^-1) rates of metsulfuron methyl.     

Comparative study of transgenic Brachypodium distachyon expressing sucrose:fructan 6-fructosyltransferases from wheat and timothy grass with different enzymatic properties

Fructans can act as cryoprotectants and contribute to freezing tolerance in plant species, such as in members of the grass subfamily Pooideae that includes Triticeae species and forage grasses. To elucidate the relationship of freezing tolerance, carbohydrate composition and degree of polymerization (DP) of fructans, we generated transgenic plants in the model grass species Brachypodium distachyon that expressed cDNAs for sucrose:fructan 6-fructosyltransferases (6-SFTs) with different enzymatic properties: one cDNA encoded PpFT1 from timothy grass (Phleum pratense), an enzyme that produces high-DP levans; a second cDNA encoded wft1 from wheat (Triticum aestivum), an enzyme that produces low-DP levans. Transgenic lines expressing PpFT1 and wft1 showed retarded growth; this effect was particularly notable in the PpFT1 transgenic lines. When grown at 22 °C, both types of transgenic line showed little or no accumulation of fructans. However, after a cold treatment, wft1 transgenic plants accumulated fructans with DP = 3–40, whereas PpFT1 transgenic plants accumulated fructans with higher DPs (20 to the separation limit). The different compositions of the accumulated fructans in the two types of transgenic line were correlated with the differences in the enzymatic properties of the overexpressed 6-SFTs. Transgenic lines expressing PpFT1 accumulated greater amounts of mono- and disaccharides than wild type and wft1 expressing lines. Examination of leaf blades showed that after cold acclimation, PpFT1 overexpression increased tolerance to freezing; by contrast, the freezing tolerance of the wft1 expressing lines was the same as that of wild type plants. These results provide new insights into the relationship of the composition of water-soluble carbohydrates and the DP of fructans to freezing tolerance in plants.     

Characterization of transgenic poplar with ectopic expression of pine cytosolic glutamine synthetase under conditions of varying nitrogen availability

The present study addresses the hypothesis that enhanced expression of glutamine synthetase (GS) in transgenic poplar, characterized by the ectopic expression of pine cytosolic GS, results in an enhanced efficiency of nitrogen (N) assimilation and enhanced growth. Transgenic and control poplar were supplied with low and high N levels and the role of ectopic expression of the pine GS in growth and N assimilation was assessed by using amino acid analysis, (15)N enrichment, biochemical analyses, and growth measurements. While leaves of transgenic poplar contained 85% less (P < 0.01) free ammonium than leaves of nontransgenic control plants, leaves of transgenics showed increases in the levels of free glutamine and total free amino acids. Transgenic poplar lines also displayed significant increases in growth parameters when compared with controls grown under both low (0.3 mm) and high (10 mm) nitrate conditions. Furthermore, (15)N-enrichment experiments showed that 27% more (P < 0.05) (15)N was incorporated into structural compounds in transgenic lines than in nontransgenic controls. Using the methods described here, we present direct evidence for increased N assimilation efficiency and growth in GS transgenic lines.     

Establishment of introduced antagonistic bacteria in the rhizosphere of transgenic potatoes and their effect on the bacterial community

In a field release experiment, rifampicin resistant mutants of two antagonistic plant-associated bacteria were used for seed tuber inoculation of transgenic T4 lysozyme expressing potatoes, transgenic control potatoes and non-transgenic parental potatoes. The T4 lysozyme tolerant Pseudomonas putida QC14-3-8 was originally isolated from the tuber surface (geocaulosphere) of T4 lysozyme producing plants and showed in vitro antibacterial activity to the bacterial pathogen Erwinia carotovora ssp. atroseptica. The T4 lysozyme sensitive Serratia grimesii L16-3-3 was originally isolated from the rhizosphere of parental potatoes and showed in vitro antagonism toward the plant pathogenic fungus Verticillium dahliae. The establishment of the inoculated bacteria in the rhizosphere and geocaulosphere of the different plant lines was monitored over one growing season to assess the effect of T4 lysozyme produced by transgenic potato plants on the survival of both inoculants. Both introduced isolates were able to colonize the rhizo- and geocaulosphere of transgenic plants and non-transgenic parental plants, and established in the rhizosphere at levels of ca. log(10) 5 colony forming units g(-1) fresh weight of root. During flowering of plants, significantly more colony counts of the T4 lysozyme tolerant P. putida were recovered from transgenic T4 lysozyme plants than from the transgenic control and the parental line. At this time, the highest level of T4 lysozyme (% of total soluble protein) was detected. Effects of the inoculants on the indigenous microbial community were monitored by analysis of PCR-amplified fragments of the 16S rRNA genes of the whole bacterial community after separation by denaturing gradient gel electrophoresis (DGGE). At any sampling time, the DGGE pattern of rhizosphere and geocaulosphere communities did not show differences between the inoculated and non-inoculated potatoes. Neither of the introduced strains became a dominant member of the bacterial community. This work was the first approach to assess the establishment of plant growth promoting rhizobacteria and potential biocontrol agents on transgenic plants.     

Investigations on Genetically Modified Maize (Bt-Maize) in Pig Nutrition: Fattening Performance and Slaughtering Results

A grower finisher performance trial with forty-eight pigs was designed to compare the growth performance of pigs fed diets containing either genetically modified (GM) Bt-maize (NX6262) or its parental maize (Prelude) line. During the experiment, the pigs were fed with a grower and a finisher diet both containing 70% maize investigated in a previously study which showed that they contained similar ME values and digestibility of crude nutrients. The pigs with an initial live weight of 23.9 ± 3.0kg were allotted to single boxes. During a 91 days growing period the pigs of both groups recorded equal performance in daily weight gain ( j W) 815 ± 93 vs. 804 ± 64g/d depending on equal amounts of feed intake 1.95 ± 0.15 vs . 1.94 ± 0.15kg/d (parental vs. transgenic). The results confirm equal performance among growing-finishing pigs fed parental or genetically modified maize containing diets. For slaughtering the pigs were divided into 4 groups with a different duration of the finishing period. After slaughtering the carcass characteristic were registered.     

Dose-dependent lung remodeling in transgenic mice expressing transforming growth factor-alpha

Transgenic mice overexpressing human transforming growth factor-alpha (TGF-alpha) develop emphysema and fibrosis during postnatal alveologenesis. To assess dose-related pulmonary alterations, four distinct transgenic lines expressing different amounts of TGF-alpha in the distal lung under control of the surfactant protein C (SP-C) promoter were characterized. Mean lung homogenate TGF-alpha levels ranged from 388 +/- 40 pg/ml in the lowest expressing line to 1,247 +/- 33 pg/ml in the highest expressing line. Histological assessment demonstrated progressive alveolar airspace size changes that were more severe in the higher expressing TGF-alpha lines. Pleural and parenchymal fibrosis were only detected in the highest expressing line (line 28), and increasing terminal airspace area was associated with increasing TGF-alpha expression. Hysteresis on pressure-volume curves was significantly reduced in line 28 mice compared with other lines of mice. There were no differences in bronchoalveolar lavage fluid cell count or differential that would indicate any evidence of lung inflammation among all transgenic lines. Proliferating cells were increased in line 28 without alterations of numbers of type II cells. We conclude that TGF-alpha lung remodeling in transgenic mice is dose dependent and is independent of pulmonary inflammation.     

The effect of atebrine and an acridine analog (BCMA) on the coenzyme fluorescence spectra of cultured melanoma and Ehrlich ascites (EL2) cells.

Coenzyme fluorescence spectra of single living cells are due to free pyridine nucleotides (folded configuration), bound pyridine nucleotides (unfolded configuration) and a third component, possibly a mixture or flavins. Such spectra can be used to recognize possible differences in coenzyme composition between cell lines or changes of metabolic pathways due to chemicals acting at levels below or above cytotoxicity, by high resolution spectrofluorometry. A study of spectra recorded from cultured Ehrlich ascites (EL2), and Harding Passey melanoma cells (HPM-67 and HPM-73 line) grown under comparable conditions, shows that free NAD(P)H predominates in HPM-67 and EL2, while this coenzyme is bound in HPM-73. The free/bound ratio may be profoundly modifed by chemicals, e.g. in the HPM-73 increase of free and decrease of bound NAD(P)H occurred upon treatment with 10(-6) oligomycin. When atebrine at levels (10(-6) M) below cytotoxicity was added, there was a decrease of the free NAD(P)H spectrum possibly through energy transfer from NAD(P)H to atebrine. Consideration of long range energy transfer i.e., excitation of atebrine by fluorescence of NAD(P)H vs. short range transfer of excitation energy from free NAD(P)H to atebrine, favors the latter mechanism. A transient (reversible) increase in atebrine fluorescence is seen following intracellular microinjection of substrate (e.g. glucose-6-P) leading to an increase in free NAD(P)H. At cytotoxic levels of atebrine (e.g 2 x 10(-5) M) an irreversible increase of atebrine fluorescence is seen. The microspectrofluorometric technique appears therefore well suited to study physiological processes at the level of intracellular coenzymes, as well as possible processes of intermolecular energy transfer in the microenvironment.     

Changes in glucosinolates during crop development in single- and double-low genotypes of winter oilseed rape (Brassica napus): I. Production and distribution in vegetative tissues and developing pods during development and potential role in the recycling of sulphur within the crop

The distributions of glucosinolates and sulphur were measured in the vegetative and reproductive tissues in a series of single- and double-low cultivars of oilseed rape (Bienvenu, Ariana, Cobra and Capricorn) grown on a sulphur-sufficient soil at Rothamsted in 1987/88, and in crops of the cv. Libravo grown with none or 40 kg/ha of sulphur on a sulphur-deficient soil at Woburn in 1990/91. The glucosinolate measurements demonstrated large differences in the abilities of single- and double-low cultivars to synthesise glucosinolates, and showed that the biosynthetic differences were associated more with the developing pods than the vegetative tissues. It indicated that potential contribution of intact glucosinolates from vegetative tissues to the seed was likely to be small, but did not preclude the possibility that the vegetative tissues were a source of glucosinolate precursors. The sulphur measurements showed that the glucosinolates contained only a small proportion of the crop's total sulphur and that they were unlikely to be a major source of recyclable sulphur, even under conditions of severe sulphur deficiency.     

The effect of reduced activity of phytoene synthase on isoprenoid levels in tomato pericarp during fruit development and ripening

Carotenoids, gibberellins (GAs), sterols, abscisic acid and -amyrins were analysed in tomato (Lycopersicon esculentum Mill.) pericarp during fruit development and ripening. The contents of these isoprenoids in wild-type (cv. Ailsa Craig) fruit were compared with those in fruit of the carotenoid-deficient R-mutant and a transgenic plant containing antisense RNA to a phytoene synthase gene. In both carotenoid-deficient genotypes, a 14-fold reduction in carotene and twofold decrease in xanthophyll content, compared to the wild type, was found in ripe fruit. Immature green fruit from wild type and R-mutant plants contained similar amounts of the C₁₉-GAs, GA₁, and GA₂₀, and their C₂₀ precursor, GA₁₉. Immature fruit from the transgenic plants contained three- to fivefold higher contents of these GAs. In wild-type fruit at the mature green stage the contents of these GAs had decreased to < 10% of the levels in immature fruit. A similar decrease in GA₁₉ content occurred in the other genotypes. However, the contents of GA₁ and GA₂₀ in fruit from phytoene synthase antisense plants decreased only to 30% between the immature and mature green stages and did not decrease at all in R-mutant fruit. At the breaker and ripe stages, the contents of each GA were much reduced for all genotypes. The amount of abscisic acid was the same in immature fruit from all three genotypes, but, on ripening, the levels of this hormone in antisense and R-mutant fruit were ca. 50% of those in the wild type. Quantitative differences in the amounts of the triterpenoid -amyrins, total sterols, as well as individual sterols, such as campesterol, stigmasterol and sitosterol, were apparent between all three genotypes during development. Amounts of free sterols of wild type and antisense fruit were greatest during development and decreased during ripening, whereas the opposite was found in the R-mutant. This genotype also possessed less free sterol and more bound sterol in comparison to the other varieties. These data provide experimental evidence to support the concept of an integrated metabolic relationship amongst the isoprenoids.Abbreviations ABA abscisic acid - dpb days post breaker - FDP farnesyl diphosphate - GA gibberellin - GGDP geranyl-geranyl diphosphate We thank Mr. Paul Gaskin (Long Ashton Research Station) for the qualitative GC-MS of triterpenoids and Dr. R. Horgan (University of Wales, Aberystwyth) for a gift of [6-³H₂]ABA. The work was supported by a research grant (No. PG111/617) to P.M.B. from the Agricultural and Food Research Council to whom we express our thanks.     

Age-specific bioassays and fecundity of Phthorimaea operculella (Lepidoptera: Gelechiidae) reared on cry1Ac -transgenic potato plants

Variation in the susceptibility of lepidopterous pest larvae of different ages to transgenic crops and the potential for survivors to reproduce could have important consequences for the development of resistance in such pests. Experiments were undertaken in the laboratory to determine if larvae of the potato tuber moth, Phthorimaea operculella, of different ages (0 (< 1 day old), 3, 5, 7 days) varied in their susceptibility to cry1Ac9–transgenic potato (Solanum tuberosum) foliage grown in the glasshouse or field. The survival and fecundity of larvae reared on transgenic tubers was also determined in the laboratory. There were no apparent differences in susceptibility of larvae of different ages to transgenic foliage. Larvae fed glasshouse or field‐grown non‐transgenic foliage had significantly larger relative growth indices and more larvae pupated, than those fed transgenic foliage, regardless of larval age. Eggs from a laboratory colony were placed on transgenic or non‐transgenic tubers to measure survival and fecundity. Between 6% and 15% of eggs placed on transgenic tubers developed into pupae for three of the four transgenic potato lines tested. On one transgenic line, only six adults emerged from 1300 eggs. In contrast, between 71% and 97% of the eggs placed on non‐transgenic tubers developed into pupae. Male and female pupae from transgenic lines weighed less than those from non‐transgenic lines. The fecundity of females from two of four transgenic lines was lower than from the non‐transgenic parent cultivar. Although larvae of different ages did not exhibit any overall age‐dependent pattern of increasing or decreasing susceptibility to transgenic foliage of glasshouse or field‐grown plants, the ability of larvae to survive and reproduce on transgenic tubers suggests this pest has the ability to evolve resistance to the transgenic plants used in the present study.     

The effects of anther culture and plant genetic background on Agrobacterium rhizogenes-mediated transformation of commercial cultivars and derived doubled-haploid Brassica oleracea

The production of transgenic roots was scored for eight Brassica oleracea cultivars from broccoli, cabbage, cauliflower and kale following inoculation with an Agrobacterium rhizogenes cell line carrying a binary plasmid bearing the green fluorescence protein (gfp) gene in the T-DNA. Significant differences in the numbers of explants producing transgenic roots were observed between cultivars, ranging from 1.4% for Marathon F1 to 57.8% for the Green Duke F1. Three F1 cultivars were subjected to anther culture, and doubled-haploid (DH) lines were used for transformation. The DH lines produced showed considerable variation for transgenic root production with some lines showing increased efficiency compared to the parental F1 cultivar. Grouping of the DH lines into response classes with respect to transgenic root production allowed the development of potential genetic models to explain the variation in performance released from each F1 cultivar. No apparent segregation distortion for transgenic root production was observed in the DH lines following anther culture.     

Stably expressed <Emphasis Type="SmallCaps">d</Emphasis>-genome-derived HMW glutenin subunit genes transformed into different durum wheat genotypes change dough mixing properties

Durum wheat (Triticum turgidum L. var. durum) is traditionally used for the production of numerous types of pasta, and significant amounts are also used for bread-making, particularly in southern Italy. The research reported here centres on the glutenin subunits 1Dx5 and 1Dy10 encoded by chromosome 1D, and whose presence in hexaploid wheats is positively correlated with higher dough strength. In order to study the effects of stable expression of the 1Dx5 and 1Dy10 glutenin subunits in different durum wheat genotypes, four cultivars commonly grown in the Mediterranean area (‘Svevo’, ‘Creso’, ‘Varano’ and ‘Latino’) were co-transformed, via particle bombardment of cultured immature embryos, with the two wheat genes Glu-D1-1d and Glu-D1-2b encoding the glutenin subunits, and a third plasmid containing the bar gene as a selectable marker. Protein gel analyses of T₁ generation seed extracts showed expression of one or both glutenin genes in four different transformed durum wheat plants. One of these transgenic lines, DC2-65, showed co-suppression of all HMW-GS, including the endogenous ones. Transgene stability in the transgenic lines has been studied over four generations (T₁–T₄). Fluorescence in situ hybridization (FISH) analysis of metaphase chromosomes from T₄ plants showed that the integration of transgenes occurred in both telomeric and centromeric regions. The three plasmids were found inserted at a single locus in two lines and in two loci on the same chromosome arm in one line. The fourth line had two transgenic loci on different chromosomes: one with both glutenin plasmids and a different one containing only the construct with the gene encoding the 1Dy10 glutenin subunit. Segregation of these two loci in subsequent generations allowed establishment of two sublines, one containing both 1Dx5 and 1Dy10 and the other containing only 1Dy10. Small-scale quality tests showed that accumulation of Dx5, Dy10 or both in transgenic durum wheat seeds resulted in doughs with stronger mixing characteristics. A. Gadaleta and A. E. Blechl have contributed equally to this work.