E. coli chromosomal DNA in a transgene locus created by microprojectile bombardment in tobacco

Transgenic Research -     

基因枪法向小麦导入几丁质酶基因的研究

利用基因枪法,以菜豆几丁质酶基因转化小麦幼胚愈伤组织。在轰击压力1300psi,轰击距离6cm、100μg金粉/枪和轰击距离9cm、150μg金粉/枪的2种处理条件下,获得4株春小麦东农7742转化植株,转化频率分别为0.36％和0.56％。经PCR和PCR-Southern杂交分析,证实菜豆几丁质酶基因已整合到T0和T1小麦基因组中。采用氨基葡萄糖法测定几丁质酶活力,结果表明,转基因小麦的几丁质酶活力明显高于对照株;转基因植株对白粉病症状减缓,并获得一株赤霉菌接种未扩展的转基因T1植株。     

Factors affecting the genetic engineering of plants by microprojectile bombardment

Since its development in the mid-1980s, microprojectile bombardment has been widely employed as a method for direct gene transfer into a wide range of plants, including the previously difficult-to-transform monocotyledonous species. Although the numerous instruments available for microprojectile-mediated gene delivery and their applications have been widely discussed, less attention has been paid to the critical factors which affect the efficiency of this method of gene delivery. In this review we do not wish to describe the array of devices used for microprojectile delivery or their uses which have already been definitively described, but instead wish to report on research developments investigating the factors which affect microprojectile-mediated transformation of plants.     

Analysis of a large number of independent transgenic rice plants produced by the biolistic method

Summary Over 500 independent transgenic rice plants have been obtained by the biolistic method with an average transformation frequency of 9.7% for japonica variety Taipei 309. A tight selection procedure using 50 mg/l of hygromycin B successfully prevented the growth of nontransformed tissues. Analysis of the T0 transgenic rice plants revealed that more than 97% of the transgenic plants were morphologically normal and more than 80% were at least partially fertile. The hyg^r trait was inherited as a dominant trait in a Mendelian manner in 8 out of 11 transgenic events assayed. Thirty-seven out of fifty transgenic plants were estimated to contain no more than five copies of the transgenes. In six out of seven transformation events, unlinked, co-transformed genes co-segregated in the T1 generation. The hyg^r trait has been stably inherited to the T4 generation. No chimerical transgenic plant has been found in an intensive search. Novel phenomena observed in transgenic rice plants are also reported.     

Transformation of maize using microprojectile bombardment: An update and perspective

Summary Using microprojectile bombardment of maize suspension cultures and bialaphos selection, transformed embryogenic calli have been recovered in numerous independent experiments. Fertile transgenic plants have been regenerated from several transformed callus lines. Stable inheritance and expression ofbar and functional activity of the enzyme phosphinothricin acetyl transferase were observed in three subsequent generations of transformed plants. Evidence to date indicates that the transformation process and the presence of the foreign gene per se do not detrimentally influence either plant vigor or fertility. This represents a practical method for introducing foreign genes into maize, which may be applicable to other monocot species. Presented in the Session-in-Depth Genetic Transformation and Genetic Analysis Using Microprojectile Bombardment at the Annual Meeting of the Tissue Culture Association, Houston, Texas, June 10–13, 1990.     

Wounding by bombardment yields highly efficient Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus L.)

Highly efficient Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus L.) was obtained by first wounding stem explants via microprojectile bombardment. When this was followed by cocultivation with disarmed Agrobacterium in the dark, the transformation frequency-based on transient GUS expression-increased to over 10-fold that of explants wounded by other means and cocultivated under constant light. Two cycles of regeneration/selection on kanamycin were employed to generate stably transformed carnation plants and eliminate chimeras: first, plantlets were regenerated from inoculated stem explants and then leaves from these plantlets were used to generate transgenes in a second selection cycle of adventitious shoot regeneration. Agrobacterium strain AGLO, carrying the binary vector pCGN7001 containing uidA and nptII genes, was used in the stable transformation experiments. The combination of wounding via bombardment, cocultivation in the dark and two cycles of kanamycin selection yielded an overall transformation efficiency of 1–2 transgenes per 10 stem explants for the three carnation varieties analyzed. Histochemical and molecular analyses of marker genes in T₀ and T₁ generations confirmed the transgenic nature of the selected plants.     

Superfluous Transgene Integration in Plants

Referee: Dr. Paul Hooykass, Institut of Molecular Plant Sciences, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333, Al Leiden, Netherlands Recent reports suggest the transfer of superfluous DNA sequences to plant genomes during transformation processes. This review investigates the evidence from the published literature for the prevalence of this phenomenon and highlights methods to limit or prevent DNA transfer and subsequent potentially detrimental evolutionary consequences. Evidence for superfluous foreign DNA transfer using both Agrobacterium-mediated transformation and direct DNA transfer methods such as microprojectile bombardment and PEG-mediated transformation of protoplasts is reported. In the case of Agrobacterium-mediated transformation, the lack of information on the integration of sequences from outside of the T-DNA borders has been due to the general belief by researchers that T-DNA processing is precise. This assumption was based on analysis of T-DNA in tumors and as a result the majority of T-DNA integration events have been identified exclusively using DNA probes, which are homologous only to DNA from within the T-DNA borders. Where direct gene transfer protocols are employed, any part of the transforming plasmid and indeed accompanying carrier DNA may become integrated into the plant genome. The main body of evidence proving that superfluous vector DNA sequences are present in plant genomes transformed using direct transfer methods is confined to the identification of plasmid concatamers integrated into plant genomes. The limited amount of recorded evidence pertaining to superfluous vector DNA integration in transgenic plants and transformed tissues makes it impossible to draw definitive conclusions as to the factors involved in promoting this phenomenon. However, there are methods available for removing superfluous sequences from transgenic plants. These have been developed for the removal of selectable marker genes, whose presence in transgenic plants has been a source of much controversy, but can equally be applied to other DNA sequences. Suggestions have been made in the review that might limit or prevent the integration of superfluous vector sequences during transformation procedures; however, these are not proven and further research is required.     

Complete sequence analysis of transgene loci from plants transformed via microprojectile bombardment

A substantial literature exists characterizing transgene locus structure from plants transformed via Agrobacterium and direct DNA delivery. However, there is little comprehensive sequence analysis of transgene loci available, especially from plants transformed by direct delivery methods. The goal of this study was to completely sequence transgene loci from two oat lines transformed via microprojectile bombardment that were shown to have simple transgene loci by Southern analysis. In line 3830, transformed with a single plasmid, one major and one of two minor loci were completely sequenced. Both loci exhibited rearranged delivered DNA and flanking genomic sequences. The minor locus contained only 296 bp of two non-contiguous fragments of the delivered DNA flanked by genomic (filler) DNA that did not originate from the integration target site. Predicted recognition sites for topoisomerase II and a MAR region were observed in the transgene integration target site for this non-functional minor locus. Line 11929, co-transformed with two different plasmids, had a single relatively simple transgene locus composed of truncated and rearranged sequences from both delivered DNAs. The transgene loci in both lines exhibited multiple transgene and genomic DNA rearrangements and regions of scrambling characteristic of complex transgene loci. The similar characteristics of recombined fragments and junctions in both transgenic oat lines implicate similar mechanisms of transgene integration and rearrangement regardless of the number of co-transformed plasmids and the level of transgene locus complexity.     

Transgenic tobacco plants and their progeny derived by microprojectile bombardment of tobacco leaves

Transgenic tobacco plants and progeny carrying coding sequences for neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) were recovered following microprojectile bombardment of tobacco leaves. Transgenic plants were regenerated from bombarded leaf pieces of tobacco cvs. Xanthi and Ky 17 which were cultured in the presence of 100 or 200 g/ml kanamycin for six to eight weeks. Among 160 putative transgenic plants from at least 16 independent transformation events 76% expressed NPTII, and 50% expressed GUS. Southern analysis of plants expressing either one or both of the enzymes indicated DNA in high molecular weight DNA in 8 of 9 independent transformants analyzed. Two independent transformants and their progeny were analyzed in detail. Analysis of progeny for quantitative enzyme levels of NPTII and GUS, and Southern analysis of parents and progeny clearly demonstrated that the genes were transmitted to progeny. One transformant demonstrated Mendelian ratios for seed germination on kanamycin-containing medium while the other transformant had non-Mendelian ratios. DNA analysis of progeny indicate complex integration of the plasmid DNA, and suggest that rearrangements of this DNA has occurred. These results are consistent with other methods of direct DNA uptake into cells, and verify that the microprojectile bombardment method is capable of DNA delivery into intact plant cells which can give rise to transgenic plants and progeny.     

Histology of,and physical factors affecting,transient GUS expression in pearl millet (Pennisetum glaucum (L.) R. Br.) embryos following microprojectile bombardment

Transient GUS (-glucuronidase) expression was visualized in whole and sectioned embryos of Pennisetum glaucum (L.) R. Br. (pearl millet) after microprojectile bombardment with pMON 8678 DNA. Strongest GUS expression occurred in cells located in the center of GUS positive spots with decreasing intensity in surrounding cells. GUS positive cells could be seen up to 12 cell layers beneath the epidermis. Needle-like crystals of the GUS assay product were found throughout the cytoplasm of GUS positive cells. The number of GUS positive spots was correlated to the microprojectile spread pattern on the medium surface. Shorter bombardment distances (6.6 and 9.8 cm) and the standard accelerator speed gave the best results for transient expression but also caused maximum tissue damage. The speed and distance, however, had little influence on the ability of bombarded embryos to form compact callus. The developmental stage of the bombarded immature embryos was the determining factor in the formation of compact callus, from which plants were regenerated.     

Transgene expression produced by biolistic-mediated, site-specific gene integration is consistently inherited by the subsequent generations

The efficient production of stable transgenic plants is important for both crop improvement and functional genomics. Site-specific integration of foreign genes into a designated genomic position is an attractive tool for minimizing expression variability between transgenic lines. Here, we studied the utility of a Cre-mediated, site-specific integration approach, facilitated by particle bombardment, for streamlining the production of stable transgenic plants, using rice as a model species. Using this method, we generated 18 different transgenic lines containing a precise integration of a single copy of beta-glucuronidase gene (gusA) into a designated genomic location. Eleven of these lines contained no illegitimate integration in the background (single-copy lines), and seven contained illegitimate integrations in addition to the site-specific integration (multicopy lines). We monitored gusA expression in these lines up to three to four successive generations. Each of the single-copy lines expressed the gusA gene at consistent levels and nearly doubled the expression level in the homozygous state. In contrast, multicopy lines displayed expression variation and gene silencing. In about half of the multicopy lines, however, expression of the site-specific integration locus could be reactivated and stabilized on segregation of the illegitimate integrations, whereas, in the remaining half, expression could not be restored, as they contained genetically linked illegitimate integrations. This study demonstrates that biolistic-mediated, site-specific gene integration is an efficient and reliable tool for streamlining the production of stable transgenic plants.     

Transgene integration,organization and interaction in plants

It has been appreciated for many years that the structure of a transgene locus can have a major influence on the level and stability of transgene expression. Until recently, however, it has been common practice to discard plant lines with poor or unstable expression levels in favor of those with practical uses. In the last few years, an increasing number of experiments have been carried out with the primary aim of characterizing transgene loci and studying the fundamental links between locus structure and expression. Cereals have been at the forefront of this research because molecular, genetic and cytogenetic analysis can be carried out in parallel to examine transgene loci in detail. This review discusses what is known about the structure and organization of transgene loci in cereals, both at the molecular and cytogenetic levels. In the latter case, important links are beginning to be revealed between higher order locus organization, nuclear architecture, chromatin structure and transgene expression.     

Analysis of gene regulation in growing pollen tubes of angiosperm and gymnosperm species using microprojectile bombardment

A microprojectile based transient expression assay was used to investigate the functional conservation of gene regulatory mechanisms in the male gametophytes of an angiosperm ( Nicotiana tabacum ) and two gymnospermous ( Picea abies and Pinus pinaster ) species. The activities of two angiosperm gene promoters, which have previously shown to be either preferentially expressed in the male gametophyte ( lat52 ) or highly expressed in both the sporophyte and male gametophyte ( Act I), were analysed. The results showed that in P. abies and P. pinaster , activity of the Act 1 promoter was significantly higher than the activity of the lat52 promoter, while the converse was observed in N. tabacum . Detailed analysis of lat52 5'promoter deletions demonstrated that although the minimal -67 bp lat52 core promoter was active at low levels in all three species, upstream regulatory elements conserved among several pollen-expressed genes, including the PBI element, were not functional in P. abies and P. pinaster . These results suggest that both taxa-specific and conserved regulatory mechanisms operate to control gene expression during pollen germination and tube growth.     

Recovery and characterization of transgenic plants from two spring wheat cultivars with low embryogenesis efficiencies by the bombardment of isolated scutella

Summary Two commercial wheat cultivars with low embryogenesis efficiencies, AC Karma and Hy417, were transformed by the bombardment of isolated scutella with two gene constructs. Three AC Karma plants (433, 436, and 437) carrying plasmid pRC62 containing a gus:npt fusion gene, and one Hy417 plant (438) carrying plasmid pBARGUS containing a bar gene and a gusA gene were recovered and characterized. Presence of transgenes in T0 and T1 plants was confirmed by both PCR and Southern hybridization. Copy number of transgenes varied from one to six in these four plants. The inheritance of transgenes in the progeny was characterized. The gusA gene and its activity in AC Karma plant 436 and bar gene and its activity in Hy417 plant 438 segregated in the selfed T1 progeny in a Mendelian 3:1 ratio, but gusA gene and its activity in AC Karma plants 433 and 437 segregated in selfed T1 progeny in a non-Mendelian 1:1 ratio. The gusA activity in all three AC Karma plants was stably transmitted to selfed T2 or T3 progenies. The levels of gusA and nptII activities in nine T1 plants from AC Karma plant 437 were also determined. A GusA fluorometric assay indicated that gusA activity in the nine T1 plants increased by 2.5–7.2-fold compared with the nontransformed control, while and NptII ELISA assay detected nptII activity only in two of the nine T1 plants, suggesting the nptII gene was silenced in the other seven T1 plants.     

Protamine-mediated DNA coating remarkably improves bombardment transformation efficiency in plant cells

We have developed a method by which remarkably higher efficiencies of transient and stable transformation were achieved in bombardment transformation of plants. Over fivefold increase in transient gus gene expression was achieved when rice or maize suspension cells were bombarded with gold particles coated with plasmid DNA in the presence of protamine instead of the conventional spermidine. A 3.3-fold improvement in stable transformation efficiency was also observed using rice suspension cells with the new coating approach. The coated protamine-plasmid DNA complex resisted degradation by a DNase or by rice cell extract much longer than the spermidine-plasmid DNA complex. The results from this study suggest that protamine protects plasmid DNA longer than spermidine when being delivered inside the cells, probably by forming a nano-scale complex, and thus helps improve the efficiency of particle bombardment-mediated plant transformation.     

Stably transformed herbicide resistant callus of sugarcane via microprojectile bombardment of cell suspension cultures and electroporation of protoplasts

Stably transformed callus of a hybrid sugarcane cultivar (Saccharum species hybrid, CP72-1210) was achieved following high velocity microprojectile bombardment of suspension culture cells, and electroporation of protoplasts. A three-day old cell suspension culture (SC88) was bombarded with gold particles coated with pBARGUS plasmid DNA containing the ß-glucuronidase (GUS) reporter gene and the bar selectable gene that confers resistance to the herbicide basta. The pBARGUS plasmid was also electroporated into the protoplasts of another cell line (SCPP). Colonies resistant to basta were recovered from both sources. Stable integration of the bar gene in the resistant cell lines was confirmed by Southern analysis. In addition, phosphinothricin acetyltransf erase (PAT) activity was also demonstrated in the transformed cell lines.Abbreviations GUS ß-glucuronidase - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP benzylaminopurine - PMSF phenylmethylsulfonyl fluoride - MES 2[N-Morpholino]ethanesulfonic acid - HEPES [N-2-hydroxyethyl] piperazine-N-[2-ethane sulfonic acid] - PAT Phosphinothricin acetyltransferase - CTAB cetyltrimethylammonium bromide     

Transformation of oat and inheritance of bar gene expression

Fertile transgenic plants of oat (Avena sativa L. var. Melys) were produced following microprojectile bombardment of primary embryogenic calli from immature embryos with two plasmids containing the bar gene or the β-glucuronidase (uidA) gene, after selection with glufosinate ammonium. Eleven plants were regenerated from phosphinothricin resistant callus, with three of the eleven plants containing either intact or rearranged copies. No plants co-transformed with the non-selected uidA gene were detected. Stable transmission and expression of the bar gene in the T₁ inbred progenies occurred in a Mendelian manner in one line, which contained an intact bar gene, and in all six T₂ lines tested from this transformant. This revised version was published online in June 2006 with corrections to the Cover Date.     

Integration,expression and inheritance of transgenes in hexaploid oat (Avena sativa L.)

Two oat varieties, Melys (spring variety) and Bulwark (winter variety) were transformed by particle bombardment of primary embryogenic callus using either a ubi-bar-ubi-gus co-integration vector or co-transformed (Melys) with a ubi-bar plasmid together with one of three plasmids containing the beta-glucuronidase (gus) gene under the control of either a rice actin promoter, a CaMV35S promoter or a wheat high molecular weight glutenin promoter. Morphologically normal and fertile transgenic plants were regenerated following callus selection with glufosinate ammonium. Evidence for the integration and functioning of the selectable (bar) and reporter (gus) genes in T0 and T1 plants was confirmed by PCR, Southern hybridisation, fluorescence in situ hybridisation (FISH), histochemical assays, and by progeny analysis. Transformation rates varied from 0.2 to 5.0 lines/plate of callus bombarded, with co-transformation frequencies of 83 to 100%, and co-expression frequencies of 60 to 100%. Copy numbers for the bar and gus gene varied from 3 to 17 and from 2 to 20 respectively. Cell and tissue specific expression of the gus gene was evident from the different promoters, with the HMW glutenin promoter showing endosperm specific expression in T1 seed. No expression of the gus gene under the CaMV35S promoter was detected in any tissues. Progeny analysis provided evidence of Mendelian inheritance of the introduced genes suggesting either one or two unlinked integration sites. This was confirmed by fluorescence in situ hybridisation to chromosome spread preparations. No segregation of the gus gene from the bar gene was observed in any of the progeny derived from co-transformation.