Tumefaciens Strain EHA105 Research Articles

Agrobacterium-mediated transformation and shoot regeneration in elite breeding lines of western shipper cantaloupe and honeydew melons (Cucumis melo L.)

A reliable Agrobacterium-mediated transformation and shoot regeneration protocol was developed for breeding lines of commercially important western-shipper cantaloupe and honeydew melons, ‘F39’ and ‘150’, respectively. Different media were tested to select a shoot regeneration system for each of three elite breeding lines ‘F39’, ‘141’ and ‘TMS’. Murashige & Skoog (MS) basal medium supplemented with 1 mg l−1 benzyladenine (BA), 0.26 mg l−1 abscisic acid (ABA) and 0.8 mg l−1 indole-3-acetic acid (IAA) was used for shoot regeneration from cotyledonary explants in ‘F39’ and ‘150’. Kanamycin sensitivity as well as Timentin™ and Clavamox® were evaluated using wild-type ‘F39’ and ‘150’ cotyledons. Kanamycin concentrations of 200 and 150 mg l−1 were chosen as the threshold levels for ‘F39’ and ‘150’, respectively. No significant differences were found between Timentin™ and Clavamox® in ‘F39’; however, Clavamox® reduced the incidence of vitrification and increased the frequency of shoot elongation in ‘150’. A. tumefaciens strain EHA105, harboring pCNL56 carrying neomycin phosphotransferase II (nptII) and gusA reporter genes, was selected to establish a transformation protocol for ‘F39’ and ‘150’. Putative transformants were evaluated using β-glucuronidase (GUS) histochemical assay, polymerase chain reaction (PCR) and Southern blot analyses. Based on these parameters, the transformation efficiency for cantaloupe ‘F39’ was 0.3% and that for honeydew ‘150’ was 0.5%.

Improved Agrobacterium-mediated transformation of cowpea via sonication and vacuum infiltration

An improved method of Agrobacterium-mediated transformation of cowpea was developed employing both sonication and vacuum infiltration treatments. 4 day-old cotyledonary nodes were used as explants for co-cultivation with Agrobacterium tumefaciens strain EHA105 harbouring the binary vector pSouv-cry1Ac. Among the different injury treatments, vacuum infiltration and their combination treatments tested, sonication for 20 s followed by vacuum infiltration for 5 min with A. tumefaciens resulted in highest transient GUS expression efficiency (93% explants expressing GUS at regenerating sites). After 3 days of co-cultivation, the explants were cultured in 150 mg/l kanamycin-containing selection medium and putative transformed plants were recovered. The presence, integration and expression of nptII and cry1Ac genes in T0 transgenic plants were confirmed by polymerase chain reaction (PCR), genomic Southern and qualitative reverse transcription (RT)-PCR analysis. Western blot hybridization and enzyme-linked immunosorbent assay (ELISA) detected and demonstrated the accumulation of Cry1Ac protein in transgenic plants. The cry1Ac gene transmitted in a Mendelian fashion. The stable transformation efficiency increased by 88.4% using both sonication-assisted Agrobacterium-mediated transformation (SAAT) and vacuum infiltration than simple Agrobacterium-mediated transformation in cowpea.

Agrobacterium tumefaciens-mediated transformation protocol of Jatropha curcas L. using leaf and hypocotyl segments

In the present study, Agrobacterium tumefaciens-mediated transformation protocol has been optimized for Jatropha curcas—an important biofuel plant using leaf segments (LS) and hypocotyl segments (HS) from in vitro-seedlings. Among different accessions used, ‘GJ-Ran-C2’ was selected as the best for transformation. A. tumefaciens strain EHA101, containing the binary vector pIG121Hm was used for transformation, along with the GUS reporter gene containing intron and CaMV 35S as promoter. Various parameters were optimized to get the highest transient transformation expression (TTE). Augmentin® was used in the medium to control A. tumefaciens. Selection of transgenic shoots was done in the presence of kanamycin. Integration of GUS gene was confirmed by PCR analysis, whereas the expression of GUS transgene was confirmed by RT-PCR, using actin gene as internal control. GUS histochemical analysis of explants just after cocultivation showed blue color in approximately all the LS and HS but stable transformation efficiency was 5% in case of LS, whereas 4% in HS.

Enhanced Iron and Zinc Accumulation in Genetically Engineered Pineapple Plants Using Soybean Ferritin Gene

Pineapple (Ananas comosus L. Merr., cv. "Queen") leaf bases were transformed with Agrobacterium tumefaciens strain EHA 105 harboring the pSF and pEFESF plasmids with soybean ferritin cDNA. Four to eight percent of the co-cultivated leaf bases produced multiple shoots 6weeks after transfer to Murashige and Skoog's medium supplemented with α-naphthalene acetic acid 1.8mg/l, indole-3-butyric acid 2.0mg/l, kinetin 2.0mg/l, cefotaxime 400mg/l, and kanamycin 50mg/l. Putatively transformed shoots (1-2cm) were selected and multiplied on medium of the same composition and elongated shoots (5cm) were rooted on liquid rooting medium supplemented with cefotaxime 400mg/l and kanamycin 100mg/l. The rooted plants were analyzed through PCR, genomic Southern analysis, and reverse transcription PCR. The results clearly confirmed the integration and expression of soybean ferritin gene in the transformed plants. Atomic absorption spectroscopic analysis carried out with six independently transformed lines of pSF and pEFE-SF revealed a maximum of 5.03-fold increase in iron and 2.44-fold increase in zinc accumulation in the leaves of pSF-transformed plants. In pEFE-SF-transformed plants, a 3.65-fold increase in iron and 2.05-fold increase in zinc levels was observed. Few of the transgenic plants were hardened in the greenhouse and are being grown to maturity to determine the enhanced iron and zinc accumulation in the fruits. To the best of our knowledge this is the first report on the transformation of pineapple with soybean ferritin for enhanced accumulation of iron and zinc content in the transgenic plants.

Transgenic sweetpotato plants expressing an LOS5 gene are tolerant to salt stress

Transgenic sweetpotato (cv. Lizixiang) plants exhibiting enhanced salt tolerance were developed using LOW OSMOTIC STRESS 5 (LOS5) with Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harbors the pCAMBIA1300 binary vector with the LOS5 and hygromycin phosphotransferase II (hptII) genes. Selection culture was conducted using 25 mg l−1 hygromycin. A total of 26 plants were produced from the inoculated 200 cell aggregates of Lizixiang via somatic embryogenesis. PCR analysis showed that 23 of the 26 regenerated plants were transgenic plants. All of the transgenic plants exhibited higher salt tolerance compared to the untransformed control plants by in vitro assay for salt tolerance with 86 mM NaCl. When plants were exposed to 86 mM NaCl, 16 transgenic plants had significantly higher levels of superoxide dismutase (SOD), proline, and abscisic acid (ABA) and significantly lower malonaldehyde (MDA) contents than those in untransformed control plants. Salt tolerance of these 16 plants was further evaluated with Hoagland solution containing 86 mM NaCl in a greenhouse. Four of the sixteen had significantly better growth and rooting ability than the remaining 12 plants and control plants. Stable integration of the LOS5 gene into the genome of the 4 salt-tolerant transgenic plants was confirmed by Southern blot analysis, and the copy number of integrated LOS5 gene ranged from 1 to 3. High level of LOS5 gene expression in the 4 salt-tolerant transgenic plants was demonstrated by real-time quantitative PCR analysis. This study provides an important approach for improving salt tolerance of sweetpotato.

Genetic transformation and regeneration of transgenic plants from protocorm-like bodies of vanilla (Vanilla planifolia Andrews) using Agrobacterium tumefaciens

An efficient transformation protocol was developed for vanilla (Vanilla planifolia) using protocorm-like bodies (PLBs) derived from shoot tips as explants. Of the ten media tested, Murashige and Skoog (MS) medium containing 0.45 μM thidiazuron (TDZ) produced maximum PLBs per shoot tip. Genetic fidelity of PLB-derived plantlets was confirmed by random amplified polymorphic DNA (RAPD) using 23 random primers. PLBs were co-cultured with Agrobacterium tumefaciens strain EHA105 harbouring the binary vector pBI121 containing the β-glucuronidase (gusA) and neomycin phosphotransferase II (npt II) genes for 3 days in MS medium supplemented with acetosyringone and transferred to selective regeneration medium containing 4.43 μM benzyladenine (BA), 2.68 μM naphthalene acetic acid (NAA) supplemented with 50 mg l−1kanamycin and 250 mg l−1 cefotaxime. After 15 days of culture, the surviving explants were transferred to the same regeneration medium but with a higher concentration of kanamycin (75 mg l−1). Finally, explants surviving after 30 days were subjected to more stringent selection in the regeneration medium supplemented with 100 mg l−1 kanamycin. Strong β glucuronidase activity was detected in the transformed plantlets by histochemical assay. Integration of T-DNA into the nuclear genome of transgenic plants was confirmed by polymerase chain reaction and Southern hybridization, while expression of transgene was confirmed by northern hybridization. This protocol allows effective and high frequency transformation of vanilla.

Introduksi Konstruk Over-Ekspresi Kandidat Gen OsWRKY76 melalui Agrobacterium tumefaciens pada Tanaman Padi Nipponbare

<p>Delivering of Over-Expression Construct OsWRKY76<br />Candidate Gene in Rice cv. Nipponbare through<br />Agrobacterium tumefaciens. Aniversari Apriana, Atmitri<br />Sisharmini, Wening Enggarini, Sudarsono, Nurul.<br />Khumaida, and Kurniawan R. Trijatmiko. Plant genetic<br />improvement can be done through classical breeding or<br />genetic engineering. WRKY is a transcription factor involved<br />in regulating plant defense responses. OsWRKY76 gene is<br />located in a narrow segment of chromosome 9 which is<br />identified previously to be related to wide spectrum<br />resistance in rice. A sequence of OsWRKY76 (+1.200 bp)<br />has available in the gene bank and it makes possible to<br />isolate, clone, and construct the gene into over-expression<br />vector. The aim of this research was to assemble an overexpression<br />construct of OsWRKY76 candidate gene and<br />introduce it into rice through Agrobacterium-mediated<br />transformation. A construct of pCAMBIA-<br />1301::35S::OsWRKY76 has been successfully assembled and<br />transformed into embryogenic calli of rice cv. Nipponbare<br />using A. tumefaciens strain Agl-1 and EHA 105. A number of<br />126 independent lines has been produced, in which Agl-1<br />showed 3.8 times more efficient than EHA 105. PCR analysis<br />of randomly selected 25 independent lines showed that all<br />of them positively contained hptII gene, a selectable marker<br />used in the over-expression construct of the OsWRKY76<br />candidate gene. Based on the result, it could be concluded<br />that the over-expression construct of OsWRKY76 candidate<br />gene have been successfully introduced into the tissue of<br />Nipponbare.</p>

Enhanced Stem Nematode Resistance of Transgenic Sweetpotato Plants Expressing Oryzacystatin-I Gene

Enhanced stem nematode resistance of transgenic sweetpotato ( cv. Lizixiang) was achieved using Oryzacystatin-I ( OCI) gene with Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harbors a binary vector pCAMBIA1301 with OCI gene, gusA gene and hptII gene. Selection culture was conducted using 25 mg L −1 hygromycin. A total of 1 715 plants were produced from the inoculated 1 450 cell aggregates of Lizixiang via somatic embryogenesis. GUS assay and PCR analysis of the putative transgenic plants randomly sampled showed that 90.54% of them were transgenic plants. Transgenic plants exhibited significantly enhanced resistance to stem nematodes compared to the untransformed control plants by the field evaluation with stem nematodes. Stable integration of the OCI gene into the genome of resistant transgenic plants was confirmed by Southern blot analysis, and the copy number of integrated OCI gene ranged from 1 to 4. Transgene overexpression in stem nematode-resistant plants was demonstrated by quantitative real-time PCR analysis. This study provides a way for improving stem nematode resistance in sweetpotato.

Genetic transformation of Indian isolate of Lemna minor mediated by Agrobacterium tumefaciens and recovery of transgenic plants

Transgenic plants of an Indian isolate of Lemna minor have been developed for the first time using Agrobacterium tumefaciens and hard nodular cell masses 'nodular calli' developed on the BAP - pretreated daughter frond explants in B5 medium containing sucrose (1.0%) with 2,4-D (5.0μM) and 2-iP (50.0μM) or 2,4-D (50.0μM) and TDZ (5.0μM) under light conditions. These calli were co-cultured with A. tumefaciens strain EHA105 harboring a binary vector that contained genes for β-glucuronidase with intron and neomycin phosphortransferase. Transformed cells selected on kanamycin selection medium were regenerated into fronds whose transgenic nature was confirmed by histochemical assay for GUS activity, PCR analysis and Southern hybridization. The frequency of transformation obtained was 3.8% and a period of 11-13weeks was required from initiation of cultures from explants to fully grown transgenic fronds. The pretreatment of daughter fronds with BAP, use of non-ionic surfactant, presence of acetosyringone in co-cultivation medium, co-culture duration of 3d and 16h photoperiod during culture were found crucial for callus induction, frond regeneration and transformation of L. minor. This transformation system can be used for the production of pharmaceutically important protein and in bioremediation.

Agrobacterium-mediated transformation using embryogenic calli in Satsuma mandarin (Citrus unshiu Marc.) cv. Miyagawa wase

Agrobacterium-mediated transformation in Satsuma mandarin (Citrus unshiu Marc.) cv. Miyagawa wase was achieved with reasonable transformation efficiency of about 22%, which was the percentage of transgenic plantlets confirmed by genomic PCR (37 plantlets/168 hygromycin-resistant calli). Embryogenic calli of Miyagawa wase were infected with Agrobacterium tumefaciens strain EHA105 harboring binary vector pCAMBIA1300 that contained miraculin gene (a taste-modifying protein) and hygromycin as a selection marker. After 5 days of co-culture in the medium containing 100 μM acetosyringone, calli were transferred to the liquid half embryogenic cell culture medium (half concentration of Murashige and Tucker’s (MT) medium modified with the addition of 500 mg·L− malt extract, 50 g·L− sucrose and 1.55 g·L− glutamine) with 15 mg·L− hygromycin and 250 mg·L− cefotaxime, and were cultured for two weeks. Then, the calli were grown on the solid selection medium with 20 mg·L− hygromycin for four weeks and 25 mg·L− hygromycin for another four weeks. The resistant embryos were selected and transferred to the embryo maturation medium. After 3 weeks of culture, the heart shaped embryos were transferred to the MT medium containing 1.0 mg ·L− GA3, 20.0 mL·L− coconut water, 0.02 mg·L− NAA and 0.0146 mg·L− coumarin for embryo germination. Finally the germinated embryos were cultured on MT medium containing 3.0% sucrose and 0.8% agar for growing to the normal plant. Stable integration of the transgene in the plant genome was confirmed by PCR and Southern blot analysis.

Production of transgenic sweetpotato plants resistant to stem nematodes using oryzacystatin-I gene

Stem nematode ( Ditylenchus destructor Thorne) is one of most serious diseases limiting sweetpotato ( Ipomoea batatas (L.) Lam.) production, and it is urgent to develop sweetpotato varieties resistant to this disease. In present study, we have developed transgenic sweetpotato (cv. Xushu 18) plants resistant to stem nematodes using oryzacystatin-I ( OCI) gene with Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harbors a binary vector pCAMBIA1301 with OCI gene, uidA gene and hptII gene. Selection culture was conducted using 7 mg/l hygromycin. A total of 2119 plants were produced from the inoculated 1710 cell aggregates of Xushu 18 via somatic embryogenesis. GUS assay and PCR analysis of the regenerated plants randomly sampled showed that 92.8% of the regenerated plants were transgenic plants. Transgenic plants exhibited enhanced resistance to stem nematodes compared to the untransformed control plants by the field evaluation and the inoculation test with stem nematodes and stem nematode-resistant plants were selected from the transgenic plants. Stable integration of the OCI gene into the genome of resistant transgenic plants was confirmed by Southern blot analysis, and the copy number of integrated OCI gene ranged from 1 to 3. High level of OCI gene expression in the resistant transgenic plants was demonstrated by real-time quantitative PCR analysis. This study provides a way for improving stem nematode resistance of sweetpotato.

Improved Transformation of Anthurium

Methods to increase transformation efficiency and yields of transgenic Anthurium andraeanum Linden ex. André hybrids were sought while effecting gene transfer for resistance to the two most important pests, bacterial blight (Xanthomonas axonopodis pv. dieffenbachiae) and nematodes (Radopholus similis and Meloidogyne javanica). Differentiated explant tissues, embryogenic calli, and comingled mixtures of the two were transformed with binary DNA plasmid constructs that contained a neomycin phosphotransferase II (nptII) selection gene with a nos promoter and terminator. Explants included ≈1-cm long laminae, petioles, internodes, nodes, and root sections from light- and dark-grown in vitro plants. Bacterial blight resistance genes were NPR1 from Arabidopsis, attacin from Hyalophora cecropia, and T4 lysozyme from the T4 bacteriophage. For nematode resistance, rice cystatin and cowpea trypsin inhibitor genes were used. Cocultivation with Agrobacterium tumefaciens strains EHA105, AGLØ, and LBA4404 ranged from 2 to 14 days. Over 700 independent, putatively transformed lines were selected with 5 and 20 mg·L−1 geneticin (G418) for cultivars Midori and Marian Seefurth, respectively. Putative transgenic lines were selected 1 to 11.5 months, but on average 5.2 to 8.4 months, after cocultivation depending on the tissue type transformed. Significantly more embryogenic calli (one line per 5 mg calli) produced transgenic lines than did explants (one line per 143 mg explants) (P < 0.004) from ≈30 mg of tissue. Calli grew selectively from all explant types, but the type of explant from which each selection was made was not recorded because root, internode, and petiole explants were difficult to discern by the time calli developed. Shoots formed 3 months after calli were transferred to light. Non-transgenic control and transgenic ‘Marian Seefurth’ formed flower buds in the greenhouse ≈28 months after cocultivation. The plants resembled commercially grown plants from a private nursery. No non-transformed escapes were detected among the selections screened for NPTII by enzyme-linked immunosorbent assay and polymerase chain reaction (PCR). The selections were positive for transgenes as assayed by PCR and Southern hybridizations. Southern blots showed single-copy insertions of the NPR1 regulatory gene. The ability to produce large quantities of independent transgenic lines from embryogenic calli in a relatively short time period should enable researchers to evaluate the effectiveness of any transgene by screening numerous anthurium lines for improved performance.

Development of disease-resistant marker-free tomato by R/RS site-specific recombination

The selection marker genes, imparting antibiotic or herbicide resistance, in the final transgenics have been criticized by the public and considered a hindrance in their commercialization. Multi-auto-transformation (MAT) vector system has been one of the strategies to produce marker-free transgenic plants without using selective chemicals and plant growth regulators (PGRs). In the study reported here, isopentenyltransferase (ipt) gene was used as a selection marker and wasabi defensin (WD) gene, isolated from Wasabia japonica as a target gene. WD was cloned from the binary vector, pEKH-WD to an ipt-type MAT vector, pMAT21 by gateway cloning and transferred to Agrobacterium tumefaciens strain EHA105. Infected cotyledons of tomato cv. Reiyo were cultured on PGR- and antibiotic-free MS medium. Adventitious shoots were developed by the explants infected with the pMAT21/wasabi defensin. The same PGR- and antibiotic-free MS medium was used in subcultures of the adventitious shoot lines (ASLs) to produce ipt and normal shoots. Approximately, 6months after infection morphologically normal shoots were produced. Molecular analyses of the developed shoots confirmed the integration of gene of interest (WD) and excision of the selection marker (ipt). Expression of WD was confirmed by Northern blot and Western blot analyses. The marker-free transgenic plants exhibited enhanced resistance against Botrytis cinerea (gray mold), Alternaria solani (early blight), Fusarium oxysporum (Fusarium wilt) and Erysiphe lycopersici (powdery mildew).

Genetic transformation and overexpression of a rice Hd3a induces early flowering in Saussurea involucrata Kar. et Kir. ex Maxim

Saussurea involucrata is a valuable traditional Chinese medicinal herb. This is the first report of a successful genetic transformation protocol for S. involucrata using Agrobacterium tumefaciens. Leaf explants were incubated with A. tumefaciens strain EHA105 harboring the binary vector pCAMBIA 1301, which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene as a reporter gene. Following co-cultivation, about 23.7% of the explants produced hygromycin-resistant calli on MS basal medium (Murashige and Skoog in Physiol Plant 15: 473–497, 1962) supplemented with 1 mg l−1 benzyladenine (BA), 0.1 mg l−1 α-naphthaleneacetic acid (NAA), 0.1 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D), 20 mg l−1 hygromycin, and 500 mg l−1 cefotaxime. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 1.5 mg l−1 BA, 0.1 mg l−1 NAA, 0.25 mg l−1 gibberellic acid (GA3), 20 mg l−1 hygromycin, and 250 mg l−1 cefotaxime, and about 67.5% of the resistant calli differentiated into shoots. Finally, 80% of the hygromycin-resistant shoots rooted on MS media supplemented with 0.2 mg l−1 NAA, 20 mg l−1 hygromycin, and 250 mg l−1 cefotaxime. The transgenic nature of the transformants was demonstrated by detection of β-glucuronidase activity in the primary transformants and by Southern blot hybridization analysis. About 16% of the total inoculated leaf explants produced transgenic plants after approximately 5 months. Using this optimized transformation system, a rice ortholog of the Arabidopsis FLOWERING LOCUS T gene, Hd3a, was transferred into S. involucrata. Introduction of this gene caused an early-flowering phenotype in S. involucrata.

In vitro flowering and viable seed setting of transgenic lettuce cultures

Production of transgenic lettuce seeds via in vitro flowering and fruit setting is reported here. Six days old cotyledons were co-cultivated with Agrobacterium tumefaciens strain EHA105 harbouring the binary vector pCAMBIA2301 carrying the reporter gene α-glucuronidase intron (GUS-INT) and the marker gene neomycin phosphotransferase (NPTII). Transgenic calluses and shoot buds were induced on MS medium augmented with 0.1 mg l−1 benzyladenine (BA), 0.1 mg l−1 α-naphtaleneacetic acid (NAA), 100 mg l−1 kanamycin and 500 mg l−1 timentin (ticarcillin clavulanate). After transferring the cultures onto MS basal medium augmented or not with kanamycin, in vitro flower induction was observed within 90 days and eventually matured and produced seed pods. Pollen grains obtained from flowers induced on basal medium without kanamycin were heterogeneous containing both GUS positive and negative pollen grains. In the corresponding seed population, a Mendelian ratio (3 : 1) of gusA transgene segregation was observed. On the other hand, flowers that were induced on basal medium under kanamycin selection, all the pollen grains were GUS positives.

Rapid Acquirement of Transgenic Rice Plants Derived from Callus of Mature Embryos Transformed by Agrobacterium Mediation

In this research we transformed calli of two elite germplasms, Taipei 309 and Nipponbare, derived from mature embryos using the construct of pCAMBIA0390 mediated by Agrobacterium tumefaciens strain EHA105. The expression plasmid pCAMBIA0390 was constructed with expressing elements of ubi:bar-Gus and ACT:Bt . Meanwhile We studied the effects of the factors on the regeneration rates of callus tissues. The results showed that the regeneration frequencies of Taipei 309 and Nipponbare increased up to 39% and 44% respectively, under the procedures as following: Callus was cultured 21days in the conditions of, 28℃, 2000 Lx, and 13 h/d illumination period. Infected callus air-dried 20 minutes and then co-cultured callus air-dried 20 minutes, finally, the callus desiccated using sterile filter paper of callus 8 hours. It shows that transformation cycle can shortened from 105 days to 60 days in absences of subculture stage. The GUS expression and PCR analysis indicated that the exogenous Bt gene has been integrated into the rice genome.

Production of marker-free disease-resistant potato using isopentenyl transferase gene as a positive selection marker

The use of antibiotic or herbicide resistant genes as selection markers for production of transgenic plants and their continuous presence in the final transgenics has been a serious problem for their public acceptance and commercialization. MAT (multi-auto-transformation) vector system has been one of the different strategies to excise the selection marker gene and produce marker-free transgenic plants. In the present study, ipt (isopentenyl transferase) gene was used as a selection marker gene. A chitinase gene, ChiC (isolated from Streptomyces griseus strain HUT 6037) was used as a gene of interest. ChiC gene was cloned from the binary vector, pEKH1 to an ipt-type MAT vector, pMAT21 by gateway cloning and transferred to Agrobacterium tumefaciens strain EHA105. The infected tuber discs of potato were cultured on hormone- and antibiotic-free MS medium. Seven of the 35 explants infected with the pMAT21/ChiC produced shoots. The same antibiotic- and hormones-free MS medium was used in subcultures of the shoots (ipt like and normal shoots). Molecular analyses of genomic DNA from transgenic plants confirmed the integration of gene of interest and excision of the selection marker in 3 of the 7 clones. Expression of ChiC gene was confirmed by Northern blot and western blot analyses. Disease-resistant assay of the marker-free transgenic, in vitro and greenhouse-grown plants exhibited enhanced resistance against Alternaria solani (early blight), Botrytis cinerea (gray mold) and Fusarium oxysporum (Fusarium wilt). From these results it could be concluded that ipt gene can be used as a selection marker to produce marker-free disease-resistant transgenic potato plants on PGR- and antibiotic-free MS medium.

A highly efficient in vitro plant regeneration and Agrobacterium-mediated transformation of Brassica oleracea var. botrytis

A reliable and efficient regeneration protocol from the cotyledons and hypocotyls is successfully established using three cauliflower genotypes. Thidiazuron (TDZ) is found to be an effective inducer of shoot formation. Both the hypocotyl and cotyledon segments are highly responsive (100%) to shoot formation and produce a maximum of 15.8 shoots per hypocotyl explant on MS salts with B5 vitamins medium containing TDZ at 0.2 mg L−1 and α-naphthaleneacetic acid at 0.01 mg L−1. Using this efficient regeneration protocol, the reporter β-glucuronidase (GUS) under the constitutive cauliflower mosaic virus (CaMV) 35S promoter is introduced into cauliflower cells of hypocotyls and cotyledons with Agrobacterium tumefaciens strain EHA105. By optimizing the parameters on the procedure of Agrobacterium-mediated transformation, a high transformation efficiency is demonstrated. The transformation efficiencies are 21.6% and 18.3% for cotyledons and hypocotyls, respectively. Integration of the transgenes in the plant genome is demonstrated by PCR and Southern hybridization analysis. Expression of the transgenes is confirmed by RT-PCR analysis and GUS staining. Compared with previous works, the present study provides higher regeneration and transformation efficiencies and establishes a highly efficient transformation system in cauliflower.

Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

The N-terminal proline-rich domain of γ-zein (Zera) plays an important role in protein body (PB) formation not only in the original host (maize seeds) but in a broad spectrum of eukaryotic cells. However, the elements within the Zera sequence that are involved in the biogenesis of PBs have not been clearly identified. Here, we focused on amino acid sequence motifs that could be involved in Zera oligomerization, leading to PB-like structures in Nicotiana benthamiana leaves. By using fusions of Zera with fluorescent proteins, we found that the lack of the repeat region (PPPVHL)(8) of Zera resulted in the secretion of the fusion protein but that this repeat by itself did not form PBs. Although the repeat region containing eight units was the most efficient for Zera self-assembly, shorter repeats of 4-6 units still formed small multimers. Based on site-directed mutagenesis of Zera cysteine residues and analysis of multimer formation, we conclude that the two N-terminal Cys residues of Zera (Cys(7) and Cys(9)) are critical for oligomerization. Immunoelectron microscopy and confocal studies on PB development over time revealed that early, small, Zera-derived oligomers were sequestered in buds along the rough ER and that the mature size of the PBs could be attained by both cross-linking of preformed multimers and the incorporation of new chains of Zera fusions synthesized by active membrane-bound ribosomes. Based on these results and on the behavior of the Zera structure determined by molecular dynamics simulation studies, we propose a model of Zera-induced PB biogenesis.

Enhanced resistance against bacterial wilt in transgenic tomato (Lycopersicon esculentum) lines expressing the Xa21 gene

To enhance bacterial wilt resistance in tomato plants and simplify the protocol of Agrobacterium tum- efaciens mediated gene transfer, parameters affecting transformation efficiency in tomato have been optimized. A. tumefaciens strain EHA101, harboring a recombinant binary expression vector pTCL5 containing the Xa21 gene under the control of the CaMV 35S promoter was used for transformation. Five cultivars of tomato (Rio Grande, Roma, Pusa Ruby Pant Bahr and Avinash) were tested for transformation. Transformation efficiency was highly dependent on preculture of the explants with acetosyrin- gone, acetosyringone in co-cultivation media, shoot regeneration medium and pre-selection after co-cultivation without selective agent. One week of pre-selection fol- lowing selection along with 400 lM acetosyringone resulted in 92.3% transient GUS expression efficiency in Rio Grande followed by 90.3% in Avinash. The presence and integration of the Xa21 gene in putative transgenic plants was confirmed by polymerase chain reaction (PCR) and Southern blot analyses with 4.5-42.12% PCR-positive shoots were obtained for Xa21 and hygromycin genes, respectively. Transgenic plants of the all lines showed resistance to bacterial wilt. T1 plants (resulting from self-