Hereditary Behavior of bar Gene Cassette is Complex in Rice Mediated by Particle Bombardment

Particle bombardment transformation using minimal gene cassette （containing the promoter, open reading frame and terminator） is the novel trend in plant genetic transformation, and its use helps to alleviate the undesirable effects of plasmid vector backbone sequences on transgenic plants. In the present article, studies related to the hereditary behavior of bar gene cassette in T1 to T3 generations of the transgenic rice （Oryza sativa L.） lines transformed by particle bombardment have been discussed. The selectable marker bar gene cassette that integrated with the rice genome had multiple copies and showed complex segregation behaviors including the presence of ‘false homozygotes’, with abnormal segregation ratios ranging from 35：1 to 144：1 （Basta-resistant： sensitive plants） in their progenies. In five out of ten original transgenic lines, bar gene can be stably transmitted as a dominant gene to self-pollinated T2 progeny. The homozygotes were obtained in three transgenic lines in T1 generation regardless of the multiple-copy integration patterns of bar gene. Southern blotting analysis showed that multiple copies of bar gene cassette were linked, which formed transgene arrays in the host rice genome. The authors also observed stable transmission of integration patterns of bar gene cassette, as obtained from Southern blotting analysis, in the regularly segregated transgenic rice lines and loss of gene in an irregularly segregated transgenic line. The segregation behavior varied among the transgenic progenies that exhibited similar Southern hybridization patterns of bar gene. On the basis of these results, the multiple-copy integration, gene lost, and gene expres- sion interaction were the major reasons for the complex segregation behaviors of bar gene cassette in transgenic rice plants.

Transformation of TrxS Gene into Barley by Particle Bombardment

The production of malting barley in China can＇t meet the demand of beer industries because of poor quality and it becomes a bottleneck problem in beer manufacture industry. In this paper, TrxS gene cloned from Phalaris coerulescens was transferred into barley cultivar Yupi 1 （YP1） via biolistic bombardment. 1206 immature embryos were bombarded and seven transgenic plants carrying TrxS gene were confirmed by PCR and PCR-Southern blotting analysis. TrxS gene was expressed in transgenic plants by RT-PCR analysis. The activity of Trxh and α-amylase of transgenic line were higher than that of non-transgenic line, which is helpful to improve malting quality of barley.

Genetic transformation of Platymonas (Tetraselmis) subcordiformis (Prasinophyceae, Chlorophyta) using particle bombardment and glass-bead agitation

Platymonas (Tetraselmis) subcordiformis is a unicellular marine green alga. It was found to be very sensitive to the herbicide Basta through a sensitivity test indicating it could be employed as a selective agent. The bar gene is a practicable and selectable marker gene. The vector containing the expression cassette of the bar gene was transferred to P. subcordiformis by both particle bombardment and glass-bead agitation and transformants were then selected using Basta. Finally, Southern blotting analysis indicated that the bar gene had been successfully integrated into the nuclear genome of P. subcordiformis using both of the transgenic techniques, with the transformation efficiency of the glass-bead method being slightly higher than that of particle bombardment. This is the first report on stable transformation of P. subcordiformis, and will improve fundamental research and enlarge application of this alga.

Conifer genetic engineering: Particle bombardment and Agrobacterium-mediated gene transfer and its application in future forests

Many important advances in forest biotechnology have been made. The use of genetic transformation and the ap-plications of transgenic trees in modern forestry is now an important field. Two basic methodologies particle bombardment and Agrobacterium-mediated transformation have been used on conifers. However, routine procedures exist for only a limited number of conifers. As a result only a few species have been successfully transformed into stable transgenic plants. The use of a particle bombardment has been more successful and transgenic plants have been produced in Picea abies, Picea glauca, Picea mariana, and Pinus radiata, although the level of production of stable transgenic plants is lower than that of Agrobacte-rium. At present, breeding programs have been directed toward improving bole shape, growth rate, wood properties, and quality, as well as toward improving root and shoot performance, pest resistance, stress tolerance, herbicide resistance, and ability to resist stresses, which will drive forestry to enter a new era of productivity and quality. This article provides a brief overview of the current state of knowledge on genetic transformation in conifers.

Transfer and Detection of barstar Gene to Maize Inbred Line 18-599 (White) by Particle Bombardment

In China, the purity of maize hybrid strain is discomforting to the development of seed industrialization. Finding a new method for reproduction of maize hybrid strain is necessary. In this study, using particle bombardment, barstar gene was transferred into maize inbred line 18-599 （White）, which is an antiviral and high quality maize inbred line. By molecular detection of the anther of transgenic maize, two plants transferred with barstar gene were gained in this study, which are two restorer lines. The two plants showed normal male spike, and lively microspores. But the capacity of the two restorer lines should be studied in the future. The aim of this study is to find a new method of reproduction of maize hybrid strain using engineering restorer lines and engineering sterility lines by gene engineering technology.

Effects of supersonic fine particles bombarding on thermal barrier coatings after isothermal oxidation

This work was attempted to modify the current technology for thermal barrier coatings（TBCs） by adding an additional step of surface modification,namely,supersonic fine particles bombarding（SFPB） process,on bond coat before applying the topcoat.After isothermal oxidation at 1000 °C for different time,the surface state of the bond coat and its phase transformation were investigated using X-ray diffraction（XRD）,scanning electron microscopy（SEM） equipped with energy-dispersive X-ray spectrometry（EDS）,transmission electron microscopy（TEM） and Cr3＋ luminescence spectroscopy.The dislocation density significantly increases after SFPB process,which can generate a large number of diffusion channels in the area of the surface of the bond coat.At the initial stage of isothermal oxidation,the diffusion velocity of Al in the bond coat significantly increases,leading to the formation of a layer of stable α-Al2O3 phase.A great number of Cr3＋ positive ions can diffuse via diffusion channels during the transient state of isothermal oxidation,which can lead to the presence of（Al0.9Cr0.1）2O3 phase and accelerate the γ→θ→α phase transformation.Cr3＋ luminescence spectroscopy measurement shows that the residual stress increases at the initial stage of isothermal oxidation and then decreases.The residual stress after isothermal oxidation for 310 h reduces to 0.63 GPa compared with 0.93 GPa after isothermal oxidation for 26 h.In order to prolong the lifespan of TBCs,a layer of continuous,dense and pure α-Al2O3 with high oxidation resistance at the interface between topcoat and bond coat can be obtained due to additional SFPB process.

Effect of Supersonic Fine Particles Bombarding on the Service Life of Thermal Barrier Coating

Thermally Grown Oxide（TGO） is a dominating component in controlling the effectiveness of thermal barrier coating.During the growth of TGO,whether we could homogeneously distribute Al atom on the TGO and the intermediate metal layer will be the key factor in forming TGO with continuous,uniform and single-ingredient（Al2O3）.In this experiment,we bombarded particles on to the metallic bound layer.We studied the influence of supersonic particle bombardment on the diffusion of Al.We hope to control the growth of TGO by monitoring the diffusion of Al.Thermal barrier coating（TBC）,which consists of a NiCoCrAlY bond coat and a ZrO2-8Y2O3（wt.%） topcoat（TC）,is fabricated on the nickel-base superalloy by air plasma spray（APS）.NiCoCrAlY bond coat is treated by supersonic fine particles bombarding（SFPB）.The morphology,oxidation behavior of TBC and phase are characterized by scanning electron microscope（SEM） equipped with an energy dispersive spectromrter（EDS） and X-ray diffractometer（XRD）.The influence of supersonic fine particles bombarding technique on the service life of thermal barrier coating is studied.The results show that SFPB technique improves the flaw of excessive surface undulation in the as-sprayed bond coat.A continuous,uniform and single-ingredient（Al2O3） TGO can quickly form in the SFPB TBC during high temperature oxidation process.The thickening of TGO is relatively slow.These will effectively suppress the formation of other non-protective oxides.Therefore,SFPB technique reduces the growth stress level generated by the continuous growth of TGO,and also avoids the stress concentration induced by formation of the large particle spinal oxide.Thermal barrier coating still remains well after 350 thermal cycles.The service life of TBC is improved.The proposed research provides theoretical basis and technical references to further improve and enhance the SFPB technique.

Effects of supersonic fine particle bombarding on thermal cyclic failure lifetime of thermal barrier coating

Thermal barrier coating ( TBC) consisting of a NiCoCrAlY bond coat ( BC) and a ZrO2-8 wt. % Y2O3 topcoat ( TC) was fabricated on the nickel-base superalloy by air plasma spray ( APS) . The BC was treated by supersonic fine particle bombarding ( SFPB) . Thermal cyclic failure and residual stress in thermally grown oxide ( TGO) scale were studied by SEM with EDS and ruby fluorescence spectroscopy ( RFS) . As shown in the results,after treated by SFPB,thickening of TGO was relatively slow,which reduced the level of growth stress. The TBC with SFPB treatment was still remained well undergoing 350 times of thermal cycle. However,after thermal cycle with the same times,the separation of TC was observed in TBC without SFPB treatment. The residual stress analysis by RFS showed that the residual stress of SFPB-treated TBC increased with the increasing number of thermal cycle. The residual stress of conventional TBC reached a value of 650 MPa at 350 times of cycle and that of SFPB-treated TBC only reached 532 MPa at 400 times of cycle. The BC with SFPB treatment after 400 times of cycle was analyzed by RFS,the high stress value was not observed in local thickened region of TGO. Thermal cycling resistance of TBC can be improved by the SFPB technology.

Studies on Genetic Transformation of NPR1 Gene into Maize by Microprojectile Bombardment

[Objective] This study aimed to explore the conditions of transformation of maize by microprojectile bombardment. [Method] Immature embryo-derived callus of maize inbred line 7239 was used as explants to study the effects of shoot distance, helium pressure, vacuum and bombardment frequency on the transformation efficien- cy in the particle bombardment system of maize. [Result] Considering the transfor- mation efficiency, particle bombardment with 100 μg/P of golden particles, at a shoot distance of 9 cm from the target cells, under helium pressure of 1 350 psi and vac- uum 25 inHg, and bombarding twice could achieve relatively ideal results. After se- lection on media supplemented with different concentration of hygromycin, some re- generated plants were obtained. The results of PCR and Southern blotting analysis demonstrated that the NPR1 gene had been integrated into the genome of trans- genic maize plants, with an average transformation efficiency of 1.76%. [Conclusion] The study laid the foundation for the cultivation and breeding of excellent resistant varieties of maize.

Study on the Obtaining of Transgenic Wheat with GNA Alien Gene by Biolistic Particle

The immature embryos of wheat plants, cv. Jing 411, 12 - 14 days after pollination, were cultured on SD2 medium for callus induction. After 10 days culture, 800 wheat calli were bombarded by biolistic particle coated with the DNA of plasmid pBI121-2 harboring both Galanthus nivalis agglutinin gene and bar gene. 67 green plants were finally regenerated from the bombardment calli on selection medium containing 4mg/L Basta. The results of bioassay by both inoculating wheat aphids onto the plants and applying Basta solution of 50 mg/L and 75 mg/L onto the wheat leaves in the field, and the molecular analysis, such as PCR and Southern blotting, indicated that 8 T2 plants contaning the target genes were obtained.

Transformation of Pea Lectin Gene and Parasponia Haemoglobin Gene into Rice and Their Expressions

Lectin and leghemoglobin in legumes play the important roles, respectively, in recognition of host plants to their rhizobial bacteria, and lowering the oxygen partial pressure around bacteroids and protecting nitrogenase from oxygen in symbiotic nitrogen-fixing nodules. In order to extend the host range of the rhizobial bacteria and to make them fix nitrogen in non-legumes, pea lectin gene (pl) and Parasponia hemoglobin gene ( phl,) have been constructed into a plant expression vector (pCBHUL) and the vector pCBHUL was introduced into rice calli from immature young embryos by particle bombardment. After the calli were regenerated into plantlets on the resistant-selecting media containing hygromycin, they were identified by PCR and Southern blot hybridization. It was indicated that the pi and phb genes were integrated into nucleic genome of the transformed rice plants. GUS activity and the product of the pi gene were determined by GUS staining, Western blot and in situ hybridization at translational level. Eighteen out of 40 plants resistant to hygromycin were positively identified by PCR analysis with the rate of 45%. The pi gene was expressed in 3 out of 18 plants with 17% and 7.5% in 40 plants. The results may provide a clue for exploring whether Rhizobium leguminosarum by. viceae could extend its host range and make the transgenic rice plants have the possibility of being symbiotic, or associative to nitrogen fixation.

Novel Techniques for Gene Delivery into Plants and Its Applications for Disease Resistance in Crops

From the early past to the present, biotechnologies have produced the ability to genetically transform a wide variety of plant species. The plant transformation technologies have changed the face of agriculture and plant biology. Plant genetic transformation is one of the key technologies for crop improvement in addition to emerging approach for producing recombinant proteins in plants. Both plastid genomes and plant nuclear can be genetically modified. Until now, essential functional differences between the prokaryotic-like genome of the plastid and the eukaryotic genome of the plant cell nucleus will have an impact on characteristics of transgenic organism. Thus, the main goals are to generate transgenic plants with the traits of interest as well as minimizing the amount of transgenic DNA in plants while maximizing stability of gene expression and trait performance. In this review, two broad groups of gene delivery methods will be discussed namely, (bilogical and physical methods) and subsequently there applications for improving disease resistance will be discussed.

Creation of Transgenic Bananas Expressing Human Lysozyme Gene for Panama Wilt Resistance

Human lysozyme （HL） inhibits Fusarium oxysporum （FocR4） growth in vitro. To obtain transgenic bananas （Musa spp.） that are resistant to Panama wilt （F. oxysporum）, we introduced an HL gene that is driven by a constitutive cauliflower mosaic virus 35S promoter into the banana via Agrobacteriummediated transformation. PCR confirmed that 51 transgenic plants were obtained. The development of Panama wilt symptoms were examined after the plants had been grown in pots. The non-transgenic plants developed typical fusarium symptoms 60 d after FocR4 inoculation, whereas 24 of 51 transgenic plants remained healthy. The transgenic banana plants that showed resistance to FocR4 in the pots were then planted in a field that was heavily infected with FocR4 for further investigation. Eleven of 24 plants devel- oped symptoms before bud emergence; another 11 plants showed symptoms after bud emergence and the remaining two plants, H-67 and H-144, remained healthy and were able to fruit. Northern blotting analysis demonstrated that H-67 and H-144, bearing the strongest resistance to Panama wilt, had the highest level of HL expression and that the expression of HL was well correlated with the FocR4 resistance of transgenic plants. We conclude that Agrobacterium-mediated transformation, with the assistance of particle bombardment, is a powerful approach for banana transformation and that a transgenic HL gene can cause resistance of the crop to FocR4 in the field.