Biolistic transfer of large DNA fragments to tobacco cells using YACs retrofitted for plant transformation

To determine whether large DNA molecules could be transferred and integrated intact into the genome of plant cells, we bombarded tobacco suspension cells with yeast DNA containing artificial chromosomes (YACs) having sizes of 80, 150, 210, or 550 kilobases (kb). Plant selectable markers were retrofitted on both YAC arms so that recovery of each arm in transgenic calli could be monitored. Stably transformed calli resistant to kanamycin (300 mg/L) were recovered for each size of YAC tested. Two of 12 kanamycin-resistant transformants for the 80 kb YAC and 8 of 29 kanamycin-resistant transformants for the 150 kb YAC also contained a functional hygromycin gene derived from the opposite YAC arm. Southern analyses using probes that spanned the entire 55 kb insert region of the 80 kb YAC confirmed that one of the two double-resistant lines had integrated a fully intact single copy of the YAC DNA while the other contained a major portion of the insert. Transgenic lines that contained only one selectable marker gene from the 80 kb YAC incorporated relatively small portions of the YAC insert DNA distal to the selectable marker. Our data suggest genomic DNA cloned in artificial chromosomes up to 150 kb in size have a reasonable likelihood of being transferred by biolistic methods and integrated intact into the genome of plant cells. Biolistic transfer of YAC DNA may accelerate the isolation of agronomically useful plant genes using map-based cloning strategies.     

Gene Control in Somatic Embryos of Digitalis lanata: Expression of the β-Glucuronidase Gene Fused to a Plastocyanin Promoter

Digitalis lanata was transformed by agrobacteria-mediated gene transfer with a chimeric reporter gene encoding for β-glucuronidase (CUS) from Escherichia coll under the control of the plastocyanin 3 (Pc3) promoter from Spinada oleracea (Pc3::uidA fusion gene). Transformed cell lines were regenerated to plants via somatic embryos. CUS activity was determined fluorometrically and histochemically. The Pc3::uidA fusion gene was expressed in the late globular and bipolar stages of somatic embryos. Expression started in globular embryos (stage-1-globules) in that part of the parenchymatic tissue which later on formed the cotyledons. No GUS activity was detectable in the parenchymatic tissue forming the root pole, in cells of the developing procambium or in epidermal cells. These tissues were free of GUS activity also in bipolar embryos. The parenchymatic cells of the cotyledons and the primary cortex of the hypocotyl of germinating embryos showed GUS activity, in contrast to the epidermal cells and the cells of the central cylinder.     

Irregular patterns of transgene silencing in allohexaploid oat

An irregular pattern of transgene silencing was revealed in expression and inheritance studies conducted over multiple generations following transgene introduction by microprojectile bombardment of allohexaploid cultivated oat (Avena sativa L.). Expression of two transgenes, bar and uidA, delivered on the same plasmid was investigated in 23 transgenic oat lines. Twenty-one transgenic lines, each derived from an independently selected transformed tissue culture, showed expression of both bar and uidA while two lines expressed only bar. The relationship of the transgenic phenotypes to the presence of the transgenes in the study was determined using (1) phenotypic scoring combined with Southern blot analyses of progeny, (2) coexpression of the two transgenic phenotypes since the two transgenes always cosegregated, and (3) reactivation of a transgenic phenotype in self-pollinated progenies of transgenic plants that did not exhibit a transgenic phenotype. Transgene silencing was observed in 19 of the 23 transgenic lines and resulted in distorted segregation of transgenic phenotypes in 10 lines. Silencing and inheritance distortions were irregular and unpredictable. They were often reversible in a subsequent generation of self-pollinated progeny and abnormally segregating progenies were as likely to trace back to parents that exhibited normal segregation in a previous generation as to parents showing segregation distortions. Possible causes of the irregular patterns of transgene silencing are discussed.     

Maize α-tubulin genes are expressed according to specific patterns of cell differentiation

In the past few years many - and -tubulin genes of different organisms have been cloned and studied, and in most systems studied they constitute multigene families. In plants, most studies have been done in Arabidopsis thaliana and Zea mays. In this paper, the study of mRNA accumulation by in situ hybridization and the activity of three maize -tubulin gene promoters (tua1, tua2 and tua3) in transgenic tobacco plants are described. In maize, the expression of these three tubulin isotypes differ in the root and shoot apex and is associated with different groups of cells throughout the distinct stages of cell differentiation. In transgenic tobacco plants the promoters of the genes, fused to the uidA reporter gene (GUS), direct expression to the same tissues observed by in situ hybridization experiments. The tua1 promoter is mainly active in cortex-producing meristematic cells and in pollen, whereas tua3 is active in cells which are differentiating to form vascular bundles in the root and shoot apices. The accumulation of tua2 mRNA is detected by RNA blot in a similar form as tua1, but at a very much low level. In situ hybridization indicates that the tua2 mRNA specifically accumulates in the maize root epidermis. No GUS staining was detected in transgenic tobacco plants with the tua2 promoter. The difference in expression of the specific genes may be linked to processes where microtubules have different functions, suggesting that in plants, as in animals, there are differences in the function of the tubulin isotypes.     

Regeneration and transformation via Agrobacterium tumefaciens of the strawberry cultivar Chandler

The effects of growth regulator balance and culture conditions on the morphogenetic response of leaf disks from greenhouse grown plants of the strawberry cultivar Chandler, have been studied. Best results were obtained in the presence of 2.46 μM IBA and 8.88 μM BA, where 47% of the cultures regenerated after 16 weeks with 2.9 shoot colonies per regenerating leaf disk. Optimum incubation conditions included two weeks in the dark with subsequent transfer to light (40 μmol m-2 s-1, 16 h). The regeneration protocol was also valuable when leaf disks from in vitro grown plants were used as explants. Transformation was attempted using Agrobacterium tumefaciens carrying the plasmid pBI121. Leaf disks from in vitro cultures proliferating in the presence of 2.21 μM kinetin were best explants for transformation. A 4.22% of inoculated explants showed kanamycin resistance after 16 weeks in a medium containing 25 mg l-1 of this antibiotic. The transgenic nature of several shoots was also confirmed by the GUS assay and PCR analysis. This revised version was published online in June 2006 with corrections to the Cover Date.     

枳壳外植体离体再生及农杆菌介导的遗传转化

以枳壳实生苗的上胚轴及茎段为材料,在附加有ＢＡ和ＭＴ培养基上进行培养,上胚轴出芽率普遍高于茎段,ＢＡ为１ｍｇ／Ｌ时,出芽率最高,ＢＡ浓度升高,出芽率随之下降。外植体在培养基上的接种方式,对出芽有一定影响,上胚轴切段 形态学下端垂直插入,出芽率高。     

The stimulating effect of a cold dark-pretreatment on the etioplast/chloroplast transformation of angiosperms. III. Involvement of the excision factor and of cold-retarded aging processes?

Our question is whether the stimulating effect of a cold dark-pretreatment on the process of de-etiolation in primary leaves of wheat seedlings under subsequent continuous white light is essentially mediated by the retarding effect of highly lowered temperatures on the following processes: aging and/or senescence, realization of the so-called excision factor in detached leaves, decrease of the cytokinin level in detached leaves. The strong stimulating effect of a cold dark-preatreatment remains inspite of progressive aging in parts of the leaves and a strong decrease of the capability of chlorophyll accumulation in detached in contrast to attached leaves. The strong stimulatory effect of a cold dark-pretreatment is not diminished by application of cytokinin or gibberellic acid. The stimulating effect of a cold dark-pretreatment is detectable over several days under continuous light, but it is lost during a warm dark-phase of a few hours duration between the cold dark-pretreatment and the white light phase.     

Enhanced in vitro and in vivo antioxidant activity and mobilization of free phenolic compounds of soybean flour fermented with different β-glucosidase-producing fungi

Aims:  To evaluate the soybean polyphenol glucosides bioconversion to aglycone forms by different β-glucosidases-producing filamentous fungi to enhance their antioxidant activity.
Methods and Results:  Soybean defatted flour was submitted to solid-state fermentation with Aspergillus niger , Aspergillus niveus and Aspergillus awamori . The fungi studied produced approximately the same β-glucosidase activity units amount when p- nitrophenyl-β- d -glucopyranoside was used as substrate for the assay. However, electrophoretic analysis, using 4-methylumbellipheryl-β- d -glucopyranoside as substrate, showed that β-glucosidase produced by A.   niveus was more active. Fermented methanolic extracts showed an increase in polyphenol and genistein contents and antioxidant activities. The highest genistein content was found in soybean fermented by A. niveus . Methanolic extracts of the soybean fermented by the different fungi showed a similar capacity of scavenging H₂O₂ generated in vivo by the tumour promoter 12- O- tetradecanoyl phorbol-13-acetate.
Conclusions:  A.   niveus synthesized a β-glucosidase with higher specificity to hydrolyse genistin β-glycosidic bond than those produced by A .  awamori and A. niger .
Significance and Impact of the Study:  The utilization of these β-glucosidases-producing fungi in soybean fermentation processes resulted in the obtaining of methanolic extracts with different antioxidant potentials that could be used either therapeutically or as an antioxidant in nonphysiological oxidative stress conditions, as the one induced in skin by UV radiation.     

Loss of decreased-rubisco phenotype between generations of wheat transformed with antisense and sense rbcS

The elite UK winter wheat cv. Riband was transformed with constructs containing rbcS in sense and antisense orientations driven by the maize ubiquitin promoter with a transformation efficiency of 1.2%. Of 77 primary transformants 31% of the sense-rbcS transformed lines and 78% of the antisense-rbcS transformed lines had decreased rubisco content compared to wild-type and marker-only controls, with decreases of up to 60%. However, in the T1 progeny which inherited the transgene, only 5% showed significantly decreased rubisco content and these effects were on the margins of significance. Five potential T2 homozygous lines from T1 parents which had transgene segregation consistent with a single locus were identified. There was no significant decrease in rubisco content relative to wild-type in any of these lines (LSD of 8% for P= 0.05). Expression of antisense rbcS transgenes in two of these T2 lines was low but was increased following exposure of the plants to 37°C for 48 h. However this did not induce a significant decrease in rubisco protein content relative to controls. Southern analysis of two antisense lines showed that they had low copy number and 1–2 insertion events. In one of the two lines there was increased methylation of the ubiquitin intron in T2 samples compared to the TO primary transformant. Further work is required to establish whether methylation occurred in all the lines which lost the phenotype, and therefore the likelihood of this being the cause. The disappearance of the decreased rubisco-content phenotype between generations may therefore be attributable to (1) greater activity of the ubiquitin promoter due to greater stress in the T0 generation plants and/or (2) increased methylation of the transgene promoter region between generations.