Kanamycin-resistant Calli Research Articles

Targeted gene exchange in plant cells mediated by a zinc finger nuclease double cut

Genome modification by homology-directed repair (HDR) is an attractive tool for the controlled genetic manipulation of plants. Here, we report the HDR-mediated gene exchange of expression cassettes in tobacco BY-2 cells using a designed zinc finger nuclease (ZFN). The target contained a 7-kb fragment flanked by two ZFN cutting sites. That fragment was replaced with a 4-kb donor cassette, which integrates gene markers for selection (kanamycin resistance) and for scoring targeting (red fluorescent protein, RFP). Candidates resulting from cassette exchange were identified by molecular analysis of calli generated by transformation via direct DNA delivery. The precision of HDR-mediated donor integration was evaluated by Southern blot analysis, sequencing of the integration locus and analysis of RFP fluorescence by flow cytometry. Screening of 1326 kanamycin-resistant calli yielded 18 HDR events, 16 of which had a perfect cassette exchange at the insert junction and 13 of which produced functional RFP. Our results demonstrate that ZFN-based HDR can be used for high frequency, precise, targeted exchange of fragments of sizes that are commercially relevant in plants.

Optimization of the conditions for Casuarina cunninghamiana Miq. genetic transformation mediated by Agrobacterium tumefaciens

Using epicotyl fragments of the actinorhizal tree Casuarinacunninghamiana and the disarmed strain of Agrobacterium tumefaciens C58C1 (pGV2260) containing the pBIN19-35S-GUSINT binary vector, a method for the genetic transformation of C. cunninghamiana was established. Transformed cells were initially selected for 2 weeks on nutrient medium supplemented with kanamycin 20 mg L−1 during callus induction, and then subcultured with 50 mg L−1 until adventitious bud and shoot differentiation. Different factors involved in the early stages of the T-DNA transfer process were studied. Agrobacterium-mediated DNA delivery was most successful when epicotyl fragments excised from 45-day-old seedlings were co-cultivated with an exponentially growing culture of A. tumefaciens at an OD600nm of 0.3, for 5 days in the presence of 50 μM of acetosyringone. Kanamycin resistant calli were observed on 88.89 % of the explants and transgenic rooted C. cunninghamiana plants were obtained in 6 months. Evidence of genetic transformation was demonstrated by s-glucuronidase histochemical assays and polymerase chain reaction analyses. The possibility to obtain transgenic nitrogen-fixing nodules after inoculation by the soil actinomycete Frankia was established.

Development of Insect Resistant Transgenic Lines Mediated by <i>Agrobacterium</i> in Cotton (<i>Gossypium hirsutum</i> L.)

In order to produce transgenic cotton resistant to insects, five months old embryogenic calli of Coker 310 were transformed with Agrobacterium tumefaciens strain LBA4404 harbouring the plasmid pBI 121 containing cry2Ab gene under the control of CaMv 35S promoter. Neomycin phosphotransferase (nptII) gene was used as a selectable marker. Infected embryogenic calli were incubated in dark at 24°C in co-cultivation medium containing 100mM acetosyringone for three days. Transformed calli were selected on selection medium containing 25 mg/L kanamycin and 500 mg/L cefotaxime. After four rounds of selection (each round with 15 days duration) the induction of kanamycin resistant embryogenic calli was at an average frequency of 37.0% and further two subcultures (each with one month duration) of the kanamycin resistant embryogenic calli in the same media resulted in the formation of somatic embryos at an average frequency of 23.7%. The matured cotyledonary stage embryos were germinated on the plantlet development medium without kanamycin selection. Out of 500 explants co-cultivated, 5 plants (1.0% transformation efficiency) showed the presence of genes nptII and cry2Ab through PCR analysis. Insect bioassay against Helicoverpa armigera performed with the five T 0 plants showed 33.0% to 67.0% larval mortality.

Purple-leaved Ficus lyrata plants produced by overexpressing a grapevine VvMybA1 gene

This study established an efficient method of regenerating plants of Ficus lyrata and producing purple-leaved F. lyrata plants through genetic transformation using a VvMybA1 gene of grapevine. Ficus lyrata, a species with unique violin- or guitar-shaped leaves, was regenerated from leaf-derived calli cultured on Murashige and Skoog (MS) basal medium supplemented with 4.5 μM N-phenyl-N'-1, 2, 3-thiadiazol-5-yl urea (TDZ) and 0.5 μM α-naphthalene acetic acid (NAA). Leaf discs were inoculated with Agrobacterium tumefaciens strain EHA 105 harboring a binary vector DEAT that contains the VvMybA1 gene and neomycin phosphotransferase (npt II) gene and subsequently cultured on the established regeneration medium supplemented with 100 mg l(-1) kanamycin. Results showed that 87.5 % of the leaf discs produced kanamycin-resistant callus, and 68.8 % of them produced adventitious shoots. Transgenic plants with three leaf colors including green, green-purple, and purple were produced. Regular and quantitative real-time PCR analyses confirmed the integration of transgenes into the host genome. Semi-quantitative RT-PCR analysis indicated that the VvMybA1 gene was responsible for the purple-colored phenotype. Purple-leaved plants with strong color stability grew vigorously in a greenhouse. This study illustrated the feasibility of using a genetically engineered VvMybA1 gene for drastic modification of leaf color of an important woody ornamental plant.

English

  Cotton is the chief fibre crop of global importance. It plays a significant role in the national economy. Cotton crop is vulnerable to a number of insect species, especially to the larvae of lepidopteron pests. 60% insecticides sprayed on cotton are meant to control the damage caused by bollworm complex. Transgenic technology has become a popular option for the development of bollworm resistant cotton.Spodoptera litura is one of the notorious emerging pests in cotton. Introduction ofcry1EC gene into commercial variety have very significant importance. This research work was carried out to transform chimeric δ-endotoxin Cry1EC into cotton. The tobacco cutworm (Spodoptera litura) is a polyphagous foliage insect which is susceptible to the chimeric δ-endotoxin Cry1EC. Six month-old highly friable embryogenic calli derived from cotyledonary explants of Coker 310 were used for transformation using Agrobacterium tumefaciens strain LBA4404 harboring plasmid pBI101.1 carrying the marker gene neomycin phosphotransferase II (npt II) and a synthetic cry 1EC gene under a constitutive 35 S promoter. Agrobacterium treatedcalli were selected on MS medium containing, 50 mg/L kanamycin, 500 mg/L cefotaxime, 30 g/L maltose and 0.4% phytagel. Embryos developed on kanamycin resistant calli were maintained on the same medium till somatic embryos matured. The cotyledonary stage embryos (3 to 5 mm size) were germinated on MS basal slat with 0.1 mg/L GA3 + 1.0 mg/L IAA, 30 g/L sucrose and solidified with 0.4% phytagel. The regenerated putative transgenic plants were hardened and transferred to the transgenic green house. Transgenic plants were confirmed by polymerase chain reaction (PCR) amplification of 800 bp npt II fragment, and 578 bp amplification ofcry1Ec gene. Transgenic plant with single copy insertion of cry1EC was selected in T0 by southern blot hybridization. Insect bioassay using Spodoptera litura larvae of first instar stages on T0 plants showed 70% mortality. Not much data has been published on the toxicity of the endotoxins to S. litura, which is a common pest in warm and humid climates. Efforts are aimed at recovering more efficient transgenic plants through efficient transformation system and developing high resistant transgenic cotton against S. litura and will paved a way for promising future in cotton production.                                                 Key words: Agrobacterium transformation, cry 1EC, molecular analysis, insect bioassay.

Tetracycline-Based Binary Ti Vectors pLSU with Efficient Cloning by the Gateway Technology for <i>Agrobacterium tumefaciens</i>-Mediated Transformation of Higher Plants

We constructed small high-yielding binary Ti vectors with a bacterial tetracycline resistance gene to facilitate efficient cloning afforded by the Gateway Technology (Invitrogen) for Agrobacterium tumefaciens-mediated transformation of higher plants. The Gateway Technology vectors are kanamycin-based, thus tetracycline-based destination and expression vectors are easily selected for the antibiotic resistance in the Escherichia coli media. We reduced the size of the tetracycline resistance gene TetC from pBR322 to 1468 bp containing 1191 bp of the coding region, 93 bp of 5’-upstream, and 184 bp 3’-downstream region. The final size of binary Ti vector skeleton pLSU11 is 5034 bp. pLSU12 and 13 have the kanamycin resistance NPTII gene as a plant-selectable marker. pLSU14 and 15 contain the hygromycin resistance HPH gene as a selection marker. pLSU13 and 15 also have the β-glucuronidase (GUS) reporter gene in addition to the plant selection marker. We also constructed a mobilizable version of tetracycline-based binary Ti vector pLSU16 in which the mob function of ColE1 replicon was maintained for mobilization of the binary vector from E. coli to A. tumefaciens by tri-parental mating. The final size of binary Ti vector skeleton pLSU16 is 5580 bp. New tetracycline-based binary Ti vectors pLSU12 were found as effective as kanamycin-based vector pLSU2 in promoting a 10-fold increase in fresh weight yield of kanamycin-resistant calli after A. tumefaciens-mediated transformation of tobacco leaf discs. Using the Gateway Technology we introduced the plant-expressible GUSgene to the T-DNA of binary Ti vector pLSU12. Expression of the β-glucuronidase enzyme activity was demonstrated by histochemical staining of the GUS activity in transformed tobacco leaf discs.

Establishment of an efficient Agrobacterium tumefaciens-mediated leaf disc transformation of spine gourd (Momordica dioica Roxb. ex Willd)

Spine gourd ( Momordica dioica Roxb. ex Willd) is a medicinally and economically important plant and also used as vegetable. In this study, we established an Agrobacterium tumefaciens -mediated transformation procedure for M. dioica . Leaf explants were incubated with A. tumefaciens strain LBA4404 containing a binary vector pBAL2 carrying the reporter gene β-glucuronidase intron (GUS-INT) and the marker gene neomycin phosphotransferase (NPTII). Following co-cultivation, leaf explants were cultured on Murashige and Skoog (MS) + Gamborg et al., (B 5 ) medium supplemented with 6.6 μM 2,4- dichlorophenoxyacetic acid (2,4-D) combined with 3.3 μM 6-benzylaminopurine (BAP) containing 100 mg L –1 kanamycin and 300 mg L -1 carbenicillin. Kanamycin-resistant calluses were induced from the leaf explants after three weeks. Shoot regeneration was achieved after transferring the calluses onto fresh selection medium 8.8 μM BAP and 2.2 μM 2,4-D. Transgenic shoots were excised from callus and elongated in MS medium fortified with 3.0 μM gibberellic acid (GA 3 ), 100 mg L -1 kanamycin and 300 mg L -1 carbenicillin. Finally, the shoots were rooted on MS basal medium supplemented with 3.0 μM indole 3-butyric acid (IBA) and 100 mg L -1 kanamycin. High transformation frequency was achieved by using three-day-old precultured leaf explants. Furthermore, the presence of acetosyringone (200 μM), infection of explants for 30 min and three days of cultivation proved to be critical factors for greatly improving the transformation efficiency. Incorporation and expression of the transgenes confirmed by polymerase chain reaction (PCR), Southern blot analysis, reverse transcription (RT)-PCR and GUS histochemical assay. Using this protocol, transgenic M. dioica plants can be obtained in approximately three months with a high transformation frequency of 9%. Key words : Agrobacterium tumefaciens, acetosyringone, growth regulators, GUS, genetic transformation, Momordica dioica .

Evaluation of key factors influencing Agrobacterium-mediated transformation of somatic embryos of avocado (Persea americana Mill.)

Key factors influencing the efficiency of transformation of embryogenic cultures, induced from immature zygotic embryos, of avocado cv. ‘Duke 7’ were evaluated. Initially, the sensitivity of somatic embryos to the antibiotics kanamycin, used for selection, carbenicillin, cefotaxime and timentin, all used for elimination of Agrobacterium cells, were evaluated. Isolated globular somatic embryos were more sensitive to kanamycin than embryogenic masses, and 25 mg l−1 kanamycin completely restricted callus proliferation. Cefotaxime at 500 mg l−1 partially inhibited proliferation of embryogenic cultures, while both carbenicillin and timentin did not affect callus growth. For genetic transformation, somatic embryos were infected with A. tumefaciens containing the pBINUbiGUSint plasmid. After 2 days, the embryos were transferred to selection medium supplemented with 50 mg l−1 kanamycin and 250 mg l−1 timentin for 2 months. Then, kanamycin level was increased to 100 mg l−1 for two additional months. The A. tumefaciens strain AGL1 yielded higher transformation rates, 6%, than EHA105 or LBA4404, 1.2%. The percentage of kanamycin resistant calli obtained was significantly influenced by the embryogenic line used as source of explants. Genetic transformation was confirmed by PCR and Southern blot analysis. A significant improvement in the germination rate was obtained when transgenic embryos were cultured in liquid MS medium with 4.44 μM BA and 2.89 μM GA3 for 3 days in a roller drum and later transferred to the same medium gelled with 7 g l−1 agar. Plants from five independent transgenic lines were acclimated and grown in the greenhouse, being phenotipically similar to control plants.

TRANSFORMATION AND CHARACTERIZATION OF TFGA2-OX2 GENE IN CELL SUSPENSION CULTURES OF 'BINGTANGCHENG' SWEET ORANGE (CITRUS SINENSIS L. OSBECK)

Gibberellins (GAs) are endogenous hormones that play a predominant role in regulating plant stature. In this work, TfGA2-ox2 gene was introduced into callus lines of ‘Bingtangcheng’ Sweet orange (Citrus sinensis L. Osbeck) by Agrobacterium-mediated transformation with the aim of decreasing the amounts of bioactive GA in these plants and thereby reducing their stature. To optimize the transformation conditions, several factors were assessed, including co-cultivation period, duration of pre-culture in darkness and the infection time of Agrobacterium tumefaciens. PCR analysis confirmed the successful integration of TfGA2-ox2 gene in kanamycin-resistant calluses. The average percentage of kanamycin-resistant calli on selection medium containing MT basal medium supplemented with kanamycin (50 mg/L) and cefotaxime (400 mg/L) was 11.71%. Maximum transformation percentage of resistant callus lines based on polymerase chain reaction detection was 69.23%. The resistant callus lines will be used for quantification of GA concentrations and are now subject to plantlet regeneration. This approach may provide an alternative to the application of chemical growth retardants used for reducing the stature of plants.

Factors affecting Agrobacterium-mediated genetic transformation of embryogenic callus of Parthenocissus tricuspidata Planch

A protocol for Agrobacterium-mediated transformation was developed for embryogenic callus of an excellent climber species, Parthenocissus tricuspidata. A. tumefaciens strain EHA105 or C58 harboring the pCAMBIA2301 binary vector with the neomycin phosphotransferase (nptII) and β-glucuronidase (uidA) gene was used. Factors affecting the transformation efficiency, including the Agrobacterium strains, co-cultivation time, Agrobacterium concentration, and infection time, were evaluated. Strain EHA105 proved to be significantly better than C58, and 4 days of co-culture was critical for transformation. An Agrobacterium suspension at a concentration of 0.5–0.7 × 108 cells ml−1 (OD600 = 0.5–0.7) and an infection time of 40 min was optimal for transformation. By applying these optimized parameters, we recovered six independent transformed shoots that were kanamycin-resistant and contained the nptII gene, as verified by polymerase chain reaction (PCR) analysis. Southern blot analysis confirmed that T-DNA was stably integrated into the genome of three out of six PCR-positive lines. Furthermore, histochemical GUS assay revealed the expression of the uidA gene in kanamycin-resistant calli, somatic embryos, and leaves of transgenic plants.

Agrobacterium-mediated transformation of Dioscorea zingiberensis Wright, an important pharmaceutical crop

Dioscorea zingiberensis Wright has been cultivated as a pharmaceutical crop for production of diosgenin, a precursor for synthesis of various important steroid drugs. Because breeding of D. zingiberensis through sexual hybridization is difficult due to its unstable sexuality and differences in timing of flowering in male and female plants, gene transfer approaches may play a vital role in its genetic improvement. In this study, the Agrobacterium tumefaciens-mediated transformation of D. zingiberensis was investigated with leaves and calli as explants. The results showed that both leaf segments and callus pieces were sensitive to 30 mg/l hygromycin and 50–60 mg/l kanamycin, and using calli as explants and addition of acetosyringone (AS) in cocultivation medium were crucial for successful transformation. We first immersed callus explants in A. tumefaciens cells for 30 min and then transferred the explants onto a co-cultivation medium supplemented with 200 μM AS for 3 days. Three days after, we cultured the infected explants on a selective medium containing 50 mg/l kanamycin and 100 mg/l timentin for formation of kanamycin-resistant calli. After the kanamycin-resistant calli were produced, we transferred them onto fresh selective medium for shoot induction. Finally, the kanamycin resistant shoots were rooted and the stable incorporation of the transgene into the genome of D. zingiberensis plants was confirmed by GUS histochemical assay, PCR and Southern blot analyses. The method reported here can be used to produce transgenic D. zingiberensis plants in 5 months and the transformation frequency is 24.8% based on the numbers of independent transgenic plants regenerated from initial infected callus explants.

Silicon Carbide Whisker-Mediated Embryogenic Callus Transformation of Cotton (Gossypium hirsutum L.) and Regeneration of Salt Tolerant Plants

A silicon carbide whisker-mediated gene transfer system with recovery of fertile and stable transformants was developed for cotton (Gossypium hirsutum L.) cv. Coker-312. Two-month-old hypocotyl-derived embryogenic/non-embryogenic calli at different days after subculture were treated with silicon carbide whiskers for 2 min in order to deliver pGreen0029 encoding GUS gene and pRG229 AVP1 gene, encoding Arabidopsis vacuolar pyrophosphatase, having neomycin phosphotransferaseII (nptII) genes as plant-selectable markers. Three crucial transformation parameters, i.e., callus type, days after subculture and selection marker concentration for transformation of cotton calli were evaluated for optimum efficiency of cotton embryogenic callus transformation giving upto 94% transformation efficiency. Within six weeks, emergence of kanamycin-resistant (kmr) callus colonies was noted on selection medium. GUS and Southern blot analysis showed expression of intact and multiple transgene copies in the transformed tissues. Kanamycin wiping of leaves from T1, T2, and T3 progeny plants revealed that transgenes were inherited in a Mendelian fashion. Salt treatment of T1 AVP1 transgenic cotton plants showed significant enhancement in salt tolerance as compared to control plants. Thus far, this is first viable physical procedure after particle bombardment available for cotton that successfully can be used to generate fertile cotton transformants.

Transgenic superroots of Lotus corniculatus can be regenerated from superroot-derived leaves following Agrobacterium-mediated transformation

Super-growing roots (superroots; SR), which have been established in the legume species Lotus corniculatus, are a fast-growing root culture that allows continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely growth regulator-free culture conditions. These features are unique for non-hairy root cultures, and they are now stably expressed since the culture was isolated more than 10 years ago (1997). Attempts to achieve direct and stable transformation of SR turned out to be unsuccessful. Making use of the supple regeneration plasticity of SR, we are reporting here an indirect transformation protocol. Leaf explants, derived from plants regenerated from SR, were inoculated with Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pBI121, which contains the neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) genes as selectable and visual markers, respectively. After co-cultivation, the explants were selected on solidified MS medium with 0.5 mg/L benzylamino purine (BAP), 100 mg/L kanamycin and 250 mg/L cefotaxime. Kanamycin-resistant calli were transferred to liquid rooting medium. The newly regenerated, kanamycin-resistant roots were harvested and SR cultures re-established, which exhibited all the characteristics of the original SR. Furthermore, kanamycin-resistant roots cultured onto solidified MS medium supplemented with 0.5 mg/L BAP produced plants at the same rate as control SR. Six months after gene transfer, PCR analysis and histochemical locating indicated that the NPTII gene was integrated into the genome and that the GUS gene was regularly expressed in leaves, roots and nodules, respectively. The protocol makes it now possible to produce transformed SR and nodules as well as transgenic plants from transformed SR.

Enhanced Agrobacterium-mediated Transformation of Embryogenic Calli of Upland Cotton via Efficient Selection and Timely Subculture of Somatic Embryos

Agrobacterium-tumefaciens-mediated transformation of cotton embryogenic calli (EC) was enhanced by choosing appropriate EC and improving efficiency of coculture, selection cultivation, and plant regeneration. After 48-h cocultivation, the number of β-glucuronidase (GUS)-positive calli characterized by yellow, loose, and fine-grained EC was twofold greater than that of gray, brown, and coarse-granule EC. It indicated that efficiency of transient transformation was affected by EC morphology. And transient transformation efficiency was also improved by cocultivation on the medium adding 50 mg l−1 acetosyringone at 19°C for 48 h. Subculturing EC on the selection medium with low cell density was beneficial to production of more kanamycin-resistant (Km-R) calli lines. From an original 0.3-g EC, an average of 20 Km-R calli lines were obtained from a selection dish and the GUS-positive rate of Km-R clones was 81.97%. A large number of normal plants were rapidly regenerated on the differentiation medium with dehydration treatments and the GUS-positive rate of regeneration plants was about 72.60%. Polymerase chain reaction analysis of GUS-positive plantlets revealed a 100% positive detection rate for neomycin phosphotransferase II gene and uidA. Southern blot of transgenic plants regenerated from different Km-R calli lines demonstrated that the target gene, mostly with the low copy number, has been integrated into the cotton genome.

An improved procedure for Agrobacterium-mediated transformation of trifoliate orange (Poncirus trifoliata L. Raf.) via indirect organogenesis

Highly efficient Agrobacterium-mediated transformation of trifoliate orange (Poncirus trifoliata (L.) Raf.) was achieved via indirect shoot organogenesis. Stable transformants were obtained from epicotyl segments infected with Agrobacterium strain EHA 105 harboring the binary vector pBI121, which contained the neomycin phosphotransferase gene (NPTII) as a selectable marker and the β-glucuronidase (GUS) gene as a reporter. The effects of regeneration and selection conditions on the transformation efficiency of P. trifoliata (L.) Raf. have been investigated. A 7-d cocultivation on a medium with 8.86 μM 6-benzylaminopurine (BA)+1.43 μM indole-3-acetic acid (IAA) was used to improve callus formation from epicotyl segments after transformation. A two-step selection strategy was developed to select kanamycin-resistant calluses and to improve rooting of transgenic shoots. Transgenic shoots were multiplied on shoot induction medium with 1.11 μM BA + 5.71 μM IAA. Using the optimized transformation procedure, transformation efficiency and rooting frequency reached 417% and 96%, respectively. Furthermore, the number of regenerated escape shoots was dramatically reduced. Stable integration of the transgenes into the genome of transgenic citrus plants was confirmed by GUS histochemical assay, PCR, and Southern blot analysis.

돼지 유행성 설사병 바이러스 스파크 단백질 유전자 발현 형질전환 담배 배양세포

Porcine epidemic diarrhea virus (PEDV)는 돼지의 급성장염을 유발하여 설사 등의 증상을 일으키는 바이러스이다. 본 연구에서는 PEDV 항원단백질을 생산하는 담배 배양세포주를 개발하고자 하였다. PEDV에서 항원성이 알려진 스파크 단백질의 일부분을 암호화하는 유전자를 PCR로 합성하여 4종류의 형질전환 벡터를 제작하였다. 담배 배양세포 BY-2를 재료로 하여 Agrobacterium tumefaciens을 매개로 형질전환하였다. 선발배지 (MS salt, <TEX>$KH_2PO_4$</TEX> 370 mg/L, 2,4-D 0.18 mg/L, Thiamin HCl 1 mg/L, kanamycin 100 mg/L, 침랙무 400 mg/L)에서 캘러스를 3주 간격으로 3개월 동안 계대배양하여 카나마이신 저항성 캘러스를 선발하였다. 선발된 캘러스를 대상으로 PCR 분석한 결과 형질전환 효율은 75% 이상이었으며 벡터당 40 여개 이상의 형질전환 배양세포주를 얻었다. 형질전환 배양세포주를 대상으로 Southern blot 분석하여 PEDV 유전자가 고구마 식물체의 게놈으로 안정적으로 도입되었음을 확인하였다. Northern blot 분석 결과 PEDV 스파크 단백질 유전자가 높은 수준으로 발현함을 확인하였으며 dot blot으로 PEDV 스파크 단백질 고생산 배양세포주를 선발하였다. 형질전환 담배 배양세포로부터 생산된 PEDV 항원단백질을 BALB/c 마우스에 경구투여 하여 면역활성을 조사한 결과 형질전환 세포주인 35S::SP1-M, 35S::SP2-M, 35S::SP4-M 세포주에서 1:10의 희석배수까지 바이러스 억제효과가 관찰되었다. 제작된 형질전환 벡터는 고구마와 같은 경구용 사료작물에 활용할 수 있을 것이다. Porcine epidemic diarrhea virus (PEDV) is an infectious and highly contagious virus of swine. In order to develop the transgenic tobacco culture cells producing PEDV antigen protein, four vectors expressing PEDV spike protein (SP) gene under the control of a CaMV 35S promoter were constructed. Four fragments of the SP region of PEDV, SP1 (444 bp, 1487-1930 bp), SP2 (1.7 kb, 2300-3987 bp), SP3 (1.4 kb, 1559-2950 bp), and SP4 (2.6 kb, 9-2643 bp) were amplified by PCR and then C-MYC tag was fused to the end of each SP gene, respectively. These cassettes are inserted into the pCAMBIA2300 (named as 35S::SP1-M, 35S::SP2-M 35S::SP3-M, and 35S::SP4-M, respectively). Tobacco (cv. BY-2) cultured cells were transformed by co-cultivation with Agrobacterium tumefaciens harboring expression vector. We selected kanamycin-resistant calli and checked for the presence of the introduced SP gene using PCR, resulting 70% of them showed the foreign gene. We selected the lines with high-level expression of PEDV antigen protein based on dot blot analysis. Southern blot analysis confirmed that the PEDV SP gene was integrated into the genome of the tobacco cultured cells. Northern blot analysis showed that the introduced gene was highly expressed in transgenic cultured cells. Transgenic tobacco cultured cells-derived antigen induced immunogenicity in mice as determined by a plaque reduction neutralization assay. These results suggest that the vectors expressing PEDV spike protein gene in this study will be useful for the development of transgenic plants and cultured cells producing PEDV antigene protein.

Stable recombinase-mediated cassette exchange in Arabidopsis using Agrobacterium tumefaciens.

Site-specific integration is an attractive method for the improvement of current transformation technologies aimed at the production of stable transgenic plants. Here, we present a Cre-based targeting strategy in Arabidopsis (Arabidopsis thaliana) using recombinase-mediated cassette exchange (RMCE) of transferred DNA (T-DNA) delivered by Agrobacterium tumefaciens. The rationale for effective RMCE is the precise exchange of a genomic and a replacement cassette both flanked by two heterospecific lox sites that are incompatible with each other to prevent unwanted cassette deletion. We designed a strategy in which the coding region of a loxP/lox5171-flanked bialaphos resistance (bar) gene is exchanged for a loxP/lox5171-flanked T-DNA replacement cassette containing the neomycin phosphotransferase (nptII) coding region via loxP/loxP and lox5171/lox5171 directed recombination. The bar gene is driven by the strong 35S promoter, which is located outside the target cassette. This placement ensures preferential selection of RMCE events and not random integration events by expression of nptII from this same promoter. Using root transformation, during which Cre was provided on a cotransformed T-DNA, 50 kanamycin-resistant calli were selected. Forty-four percent contained a correctly exchanged cassette based on PCR analysis, indicating the stringency of the selection system. This was confirmed for the offspring of five analyzed events by Southern-blot analysis. In four of the five analyzed RMCE events, there were no additional T-DNA insertions or they easily segregated, resulting in high-efficiency single-copy RMCE events. Our approach enables simple and efficient selection of targeting events using the advantages of Agrobacterium-mediated transformation.

Agrobacterium -mediated transformation and regeneration of transgenic 'Chancellor' wine grape plants expressing the tfd A gene

The establishment of grape vineyards in the Midwestern USA is greatly hampered by the use of the herbicide 2,4-D in grain crop fields. The overall goal of this study was to transform 'Chancellor' with the plant expressible bacterial tfd A gene to make it tolerant to 2,4-D. Embryogenic callus was infected and cocultivated with an Agrobacterium construct (LBA4404: pAL4404:: tfd A); transformed callus was selected with kanamycin; kanamycin resistant callus was bulked and transformed cell lines identified by PCR. Three PCRpositive embryogenic callus lines were used to regenerate transgenic plants. Analysis of the plant lines for the presence of the tfd A gene by PCR and southern hybridization confirmed its stable integration in their genomes. Transgenic 'Chancellor' grape plants regenerated from these calli proved to be resistant to up to 10 kg·ha -1 of a commercial ester-formulation of 2,4-D, indicating positive expression of the tfd A gene and affording them protection from 2,4-D injury.

Identification of a novel elite genotype for in vitro culture and genetic transformation of cotton

Hypocotyls of cotton (Gossypium hirsutum L.) cultivars cv. YZ-1, Coker 312 and Coker 201 were inoculated on Murashige and Skoog callus induction medium. YZ-1 exhibited a very high regeneration potential, with 81.9 % of the explants inoculated differentiated into embryogenic callus within 8–10 weeks. During the process of callus maintenance (subculture for 1 to 3 years), the total embryos number in Coker 312 and Coker 201 calli dropped sharply, and the percentage of embryo germination decreased. On the contrary, the callus of YZ-1 consistently maintains a high frequency of plant regeneration after long-time subculture. Transgenic kanamycin-resistant calli of Coker 201 partially lost the ability of somatic embryogenesis and plant regeneration. The stress produced by the transformation procedure slightly affected somatic embryogenesis and plant regeneration of YZ-1, which showed minimum loss of plant regeneration ability.

Production and analysis of transgenic wheat (Triticum aestivum L.) with improved insect resistance by the introduction of cowpea trypsin inhibitor gene

The cowpea trypsin inhibitor gene (CpTI) and neomycin phosphotransferase gene (nptII) were introduced into the embryonic callus cells of immature embryos of wheat elite line Shannong 995604 using Agrobacterium-mediated gene transfer. Independent plantlets were regenerated from kanamycin-resistant calli. PCR and real time PCR analysis, PCR-Southern and Southern blot hybridization indicated that there were three independently-dervied transgenic plants viz. transformed-I, II and III (T-I, T-II and T-III). The segregation of CpTI in the transgenic wheat progenies of T-Iand T-III were consistent with Mendelian inheritance. Resistance to the storage insect pest of wheat viz. the grain moth (Sitotroga cerealella Olivier) was improved significantly in seeds of the three transgenic wheat T2 lines obtained from T1 PCR-positive plants. The frequency of moth-eaten seed from T-I, T-IIand T-III was reduced 66.76%, 62.48% and 43.59% respectively. The investigation of agronomic traits of the three transgenic wheat T1 PCR-positive plants revealed that the three transgenic lines had excellent agronomic traits. They provide good germplasm resource for wheat genetic improvement.