Comparison of Biolistic and Agrobacterium-mediated Transformation Methods on Transgene Copy Number and Rearrangement Frequency in Rice

Transferring foreign DNA into plant cells by biolistic and Agrobacterium _mediated methods may result in random integration of different copy numbers of the transgene, and different proportions of intact vs. rearranged copies of the transgene. This may, in turn, affect transgene expression levels. To test the above hypothesis, we first introduced the same plasmid, pAc1PG_CAM, into rice (Oryza sativa L.) calli separately by the biolistic method and by the Agrobacterium _mediated method. To show whether different plasmids may affect the results, we also introduced pTOK233 by the Agrobacterium _mediated method and pJPM44 by the biolistic method. Transgene expression of R0 plants was monitored by histochemical analysis of GUS activity. Transgene copy number was determined by Southern blot analysis after digesting genomic DNA with an enzyme that has a unique cutting site within the input plasmid. The total genomic DNA was also digested by a two_cut enzyme (the cuts are located at two sides of a given transgene expression cassette), followed by Southern blotting analysis, for determining the number of intact transgene expression cassettes. Our data showed that Agrobacterium _mediated transformation resulted in lower transgene copy number (average between 2.1 and 2.3) in transgenic rice plants, compared with those plants obtained by the biolistic method (average between 4.2 and 5.6). The frequency of DNA rearrangement in expression cassettes is lower in transgenic rice plants obtained by the Agrobacterium _ mediated method than those obtained by the biolistic method. The average rearrangement frequency is 0.07 to 0.106 for the Agrobacterium _mediated method, and 0.57 to 0.66 for the biolistic method. Our results suggest that it is better to compare the number of intact expression cassettes instead of the total copy number of the transgene in demonstrating their influence on the level of transgene expression. This is the first report on the frequency of expression cassette rearrangement in transgenic plants transformed with the same plasmid by two different transformation methods.

Studies of Improving the Frequency of Indica Rice Transformation by Biolistic Bombardment

In order to improve the frequency of indica rice transformation by biolistic bombardment, suitable culture conditions for embryonic calli,an optimal selection scheme for resistant calli and seedlings, and optimum bombardment parameters a investigated by using 14 commercially important indica rice cultivars. The main results show that the CC medium with 36g/L mannitol is a scheme subculture medium in which the browning of indica rice calli can be mitigated significantly; The concentration of 30～40mg/L Hyg or 150～200mg/L G418 or 10～20 mg/L Basta is suitable for selection of resistant calli; The transformation parameters of 100μg gold powder absorbing 0.2μg DNA per shot and 900 psi helium pressure and 6 cm bombardment distance and bombarded twice for each plate give the best result; Keeping the target calli on osmotic medium containing 60g/L mannitol from 12 ～24h before bombardment to 24～48h after it can increase the efficiencies of transformation . Furthermore, some transgenic indica rice plants are obtained using this optimized transformation system.

A transgenic wheat with a stilbene synthase gene resistant to powdery mildew obtained by biolistic method

Stilbene, a kind of phytoalexin, plays an important role in resistance to fungal and bacterial infection in plants. It strongly inhibits the growth of fungi and sprout of spore. Stilbene synthase gene (Vst1) obtained from grapevine has been transferred into common spring wheat Jinghong 5 by using the biolistic transformation method. Five transgenic plants (T0) were obtained from the bombarded 2014 immature embryos. One immune plantlet and 3 plantlets with mid-resistance to powdery mildew were identified from the transgenic plants of T3 generation which came from 2 T0 transgenic plants.

Biolistic Genetic Transformation of a Wide Range of Chinese Elite Wheat(Triticum aestivum L.)Varieties

Wheat （Triticum aestivum L.） is a major staple food crop worldwide. It is economically important because it can be grown in a wide range of climates and geographic regions, and it has made an enormous contribution to the increase in global food production over the past four decades （Dixon et al., 2009）. Wheat is produced on more than 18% of the arable land in the world, and is the most cultivated crop after maize and rice （FAOSTAT data, 2014）. Despite its global strategic significance, progress in genomic and genetic engineering research on wheat has lagged behind that on other major crops due to the difficulty of culturing tissues, and the complexity of its hexaploid genome. The first successful wheat trans- formation was achieved by particle bombardment （Vasil et al., 1992）. Since then additional transgenic wheat plants have been obtained by various transformation methods （Harwood, 2011）. Microprojectile bombardment is considered to be a promising method, since it is robust, versatile and relatively efficient in terms of gene delivery.

TRANSIENT EXPRESSION OF EXOGENOUS GUS GENE IN PORPHYRA YEZOENSIS (RHODOPHYTA)

Electroporation, PEG, PEG plus electroporation and Biolistics methods were tested ingene transformation of P. yezoensis. The exogenous gus was from plasmid of pBI121 and pCAMBI-A1301, both contain the CaMV35S promoter. The receptors included the prooplasts, tissues and free-liv-ing conchocelis filaments of P. yezoensis. Several factors, for example, the voltage, capacitance and bi-valent cations, etc., were studied. Results show that these four methods are all efficient for gene transfor-mation in P. yezoensis; and that PEG is the best one, with transformation efficiency of up to 4 ×10-5.GUS activity was detected 26 days after transfation by using PEG method.

Genetic transformation of Pinus taeda by particle bombardment

A protocol is presented for genetically engineering loblolly pine (Pinus taeda L.) using particle bombardment. This protocol enabled the routine transformation of loblolly pine plants that were previously difficult to transform. Mature zygotic embryos were used to be bombarded and to generate organogenic callus and transgenic regenerated plants. Plasmid pB48.215 DNA contained a synthetic Bacillus thuringiensis (B.t.) cryIAc coding sequence flanked by the double cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (Nos) terminator sequences, and the selectable marker gene, neomycin phosphotransferase II (nptII) controlled by the promoter of the nopaline synthase gene was introduced into loblolly pine tissues by particle bombardment. The transformed tissues were proliferated and selected by kanamycin resistance conferred by the introduced NPTII gene. Shoot regeneration was induced from the kanamycin-resistant callus, and transgenic plantlets were then produced. The presence of the introduced genes in the transgenic loblolly pine plants was confirmed by polymerase chain reactions (PCR) analysis, by Southern blot analysis, and insect feeding assays. The recovered transgenic plants were acclimatized and then established in soil.

Transient GUS and GFP Expression in Spanish Red Cedar(Cedrela odorata L.)Somatic Embryos.Optimization of Bombardment Conditions and Evaluation of Selective Agent Lethal Dose

Cedrela odorata is a tropical tree widely appreciated for its wood. Commercial plantations are frequently hampered by the attack of the meliacea borer, Hypsipyla grandella, and the lack of resistant varieties. C. odorata traditional breeding would consume very long periods of time, thus direct transfer of entomotoxic coding genes to generate resistant varieties is a promising alternative. There are two prerequisites for gene manipulation of this species: 1) to set the conditions for transgene delivery and 2) to have a way to select regenerating transformed plants. In this paper, we report the optimal biolistics conditions for transient expression of uidA and gfp reporter genes in C. odorata somatic embryos and the selective doses for kanamycin, spectinomycin, phosphinotrycin and hygromycin to screen transformed cells.

Current and future editing reagent delivery systems for plant genome editing

Many genome editing tools have been developed and new ones are anticipated; some have been extensively applied in plant genetics, biotechnology and breeding, especially the CRISPR/Cas9 system. These technologies have opened up a new era for crop improvement due to their precise editing of user-specified sequences related to agronomic traits. In this review, we will focus on an update of recent developments in the methodologies of editing reagent delivery, and consider the pros and cons of current delivery systems. Finally, we will reflect on possible future directions.

Inheritance and expression of multiple disease and insect resistance genes in transgenic rice

2-3 anti-fungal disease genes are coinserted with hygromycin phosphotransferase in the same vector. Two insecticidal genes and PPT acetyl transferase genes are placed in another one. The vectors are co-delivered to rice embryonic cellus tissue at a molar ratio of 1:1 using the particle gun method. 55 independent regenerated lines have been obtained through screening for hygromycin resistance. Of these, 70% transgenic plants harbor 6-7 foreign genes. The genes on the same vectors are always co-delivered to rice plant. Northern blot analysis has indicated that the multiple foreign genes give stable expression. In the 6 transgenic plants carrying 6-7 foreign genes, multiple foreign genes tend to integrate in 1 or 2 genetic loci. Progeny segregation is consistent with Mendel’s 3:1 segregation law. 8 homozy-gous R1 transgenic plants harboring 2-3 anti-fungal and 2 insecticidal genes are selected from large number of transgenic progeny screening for hygromycin and Basta resistance.

A Study on the Gas-Solid Particle Flows in a Needle-Free Drug Delivery Device

Different systems have been used over the years to deliver drug particles to the human skin for pharmaceutical effect. Research has been done to improve the performance and flexibility of these systems. In recent years a unique system called the transdermal drug delivery has been developed. Transdermal drug delivery opened a new door in the field of drug delivery as it is more flexible and offers better performance than the conventional systems. The principle of this system is to accelerate drug particles with a high speed gas flow. Among different transdermal drug delivery systems we will concentrate on the contour shock tube system in this paper. A contoured shock tube is consists of a rupture chamber, a shock tube and a supersonic nozzle section. The drug particles are retained between a set of bursting diaphragm. When the diaphragm is ruptured at a certain pressure, a high speed unsteady flow is initiated through the shock tube which accelerates the particles. Computational fluid dynamics is used to simulate and analyze the flow field. The DPM (discrete phase method) is used to model the particle flow. As an unsteady flow is initiated though the shock tube the drag correlation proposed by Igra et al is used other than the standard drag correlation. The particle velocities at different sections including the nozzle exit are investigated under different operating conditions. Static pressure histories in different sections in the shock tube are investigated to analyze the flow field. The important aspects of the gas and particle dynamics in the shock tube are discussed and analyzed in details.

Rice transformation with a senescence-inhibition chimeric gene

A senescence-inhibition chimeric gene containing the specific promoter of SAG 12 and IPT gene was transferred into rice with the biolistic method. Results of PCR, Dot blotting and Southern blotting indicated that the chimeric gene had been integrated into rice genome. Analyses of GUS activity and cytokinin content in transgenic plants of rice and the observation of T 1 generation plant at grain formation stage indicated that the foreign gene was expressed.