Marker Gene nptII Research Articles

Multiplex Real-Time Loop-Mediated Isothermal Amplification (LAMP) Based on the Annealing Curve Analysis: Toward an On-Site Multiplex Detection of Transgenic Sequences in Seeds and Food Products.

Monitoring of the introduction of unapproved genetically modified (GM) events is required because the approval status of a GM event may differ from country to country. The on-site methods such as loop-mediated isothermal amplification (LAMP) offer a technological answer for the rapid GM detection beyond the laboratories. Real-time LAMP assays detecting one GM target were reported earlier. To increase the efficiency of the assay, a multiplex real-time LAMP simultaneously targeting Figwort Mosaic Virus promoter (P-FMV) that constructs region between the Cauliflower Mosaic Virus 35S promoter and cry1Ac gene (p35S-cry1Ac) and neomycin phosphotransferase II (nptII) marker gene was developed. The assay could detect as low as 0.1% for each GM target within 45 min. To the best of our knowledge, multiplexing in real-time LAMP using the Genie II system with applicability in GM detection has been reported herein for the first time. The developed method provides rapid, on-site, and real-time GM detection in seeds and food products.

Development of methods for regeneration and genetic transformation of the commercial plum variety Startovaya from somatic tissues

An effi cient technique for regeneration of plants from somatic tissues of domestic plum (Prunus domestica L.) varieties, as well as selecting schemes using various reporter (uidA, gfp) and selective marker genes (nptII, hpt) have been developed. During the transformation of somatic tissues of the commercial plum variety, transgenic plants containing both reporter genes and genes of economically valuable traits were obtained. For the fi rst time, the eff ect of calcium pantothenate on the adventive regeneration of plum shoots was investigated. It was shown that the selective antibiotic hygromycin is more eff ective for the selection of plum transformants than kanamycin or geneticin, which was expressed in a higher frequency of transformation — 1.2-2.2 % versus 0.2-0.5 % in selection on kanamycin. When using geneticin, transgenic plants were not obtained. Optimization of conditions made it possible to increase the percentage of regeneration of shoots to 79.4 % for the variety Startovaya and up to 39.6 % for the variety Etude. Analysis of GFP expression in transgenic lines showed that the leader sequences of herbaceous plant species encoding signal peptides can be used to eff ectively cellular compartmentalization of heterologous gene expression products in the plum. Preliminary results also show a higher level of expression of the reticular form of GFP in comparison with the vacuolar and cytosolic forms. Nine transgenic lines containing the insertion of the target bar gene, which gives resistance to phosphinothricin-containing herbicides of the “Basta” type, were obtained, which was confi rmed by the presence in the genome of the sequence of the transferred gene and the resistance of the obtained transgenic plants to 3 times the fi eld concentration of the herbicide “Basta”.

Overexpressing Arabidopsis thaliana ACBP6 in transgenic rapid-cycling Brassica napus confers cold tolerance

BackgroundRapid-cycling Brassica napus (B. napus-RC) has potential as a rapid trait testing system for canola (B. napus) because its life cycle is completed within 2 months while canola usually takes 4 months, and it is susceptible to the same range of diseases and abiotic stress as canola. However, a rapid trait testing system for canola requires the development of an efficient transformation and tissue culture system for B. napus-RC. Furthermore, effectiveness of this system needs to be demonstrated by showing that a particular trait can be rapidly introduced into B. napus-RC plants.ResultsAn in-vitro regeneration protocol was developed for B. napus-RC using 4-day-old cotyledons as the explant. High regeneration percentages, exceeding 70%, were achieved when 1-naphthaleneacetic acid (0.10 mg/L), 6-benzylaminopurine (1.0 mg/L), gibberellic acid (0.01 mg/L) and the ethylene antagonist silver nitrate (5 mg/L) were included in the regeneration medium. An average transformation efficiency of 16.4% was obtained using Agrobacterium-mediated transformation of B. napus-RC cotyledons using Agrobacterium strain GV3101 harbouring a plasmid with an NPTII (kanamycin-selectable) marker gene and the Arabidopsis thaliana cDNA encoding ACYL-COA-BINDING PROTEIN6 (AtACBP6). Transgenic B. napus-RC overexpressing AtACBP6 displayed better tolerance to freezing/frost than the wild type, with enhanced recovery from cellular membrane damage at both vegetative and flowering stages. AtACBP6-overexpressing B. napus-RC plants also exhibited lower electrolyte leakage and improved recovery following frost treatment, resulting in higher yields than the wild type. Ovules from transgenic AtACBP6 lines were better protected from frost than those of the wild type, while the developing embryos of frost-treated AtACBP6-overexpressing plants showed less freezing injury than the wild type.ConclusionsThis study demonstrates that B. napus-RC can be successfully regenerated and transformed from cotyledon explants and has the potential to be an effective trait testing platform for canola. Additionally, AtACBP6 shows potential for enhancing cold tolerance in canola however, larger scale studies will be required to further confirm this outcome.

Clean gene technology to develop selectable marker-free pod borer-resistant transgenic pigeon pea events involving the constitutive expression of Cry1Ac.

The most crucial yield constraint of pigeon pea is susceptibility to the pod borer Helicoverpa armigera, which causes extensive damage and severe economic losses every year. The Agrobacterium-mediated plumular meristem transformation technique was applied for the development of cry1Ac transgenic pigeon pea. Bioactivity of the cry1Ac gene was compared based on integration and expression driven by two promoters, the constitutive CaMV35S promoter and the green-tissue-specific ats1A promoter, in those transgenic events. The transgenic events also contained the selectable marker gene nptII flanked by loxP sites. Independent transgenic events expressing the Cre recombinase gene along with a linked bar selection marker were also developed. Integration and expression patterns of both cry1Ac and cre were confirmed through Southern and western blot analysis of T1 events. The constitutive expression of the Cry1Ac protein was found to be more effective for conferring resistant activity against H. armigera larvae in comparison to green-tissue-specific expression. Constitutively expressing Cry1Ac T1 events were crossed with Cre recombinase expressing T1 events. The crossing-based Cre/lox-mediated marker gene elimination strategy was demonstrated to generate nptII-free Cry1Ac-expressing T2 events. These events were subsequently analyzed in the T3 generation for the segregation of cre and bar genes. Five Cry1Ac-expressing T3 transgenic pigeon pea events were devoid of the nptII marker as well as cre-bar genes. H. armigera larval mortality in those marker-free T3 events was found to be 80-100%. The development of such nptII selectable marker-free Cry1Ac-expressing pigeon pea transgenics for the first time would greatly support the sustainable biotechnological breeding program for pod borer resistance in pigeon pea. KEY POINTS: • Constitutive expression of Cry1Ac conferred complete resistance against Helicoverpa armigera • Green-tissue-specific expression of Cry1Ac conferred partial pest resistance • Cre/lox-mediated nptII elimination was successful in constitutively expressing Cry1Ac transgenic pigeon pea events.

Assessment of risk of GM contamination in flaxseed accessions imported from Canada: a case study to restrict the unauthorized GM events from entering India

AbstractIn India, the restriction of genetically modified (GM) crops and derived products not approved in the country necessitates surveillance for transgene(s) in plant material/products imported into the country. CDC Triffid expressing acetolactate synthase (ALS) conferring tolerance to sulphonylurea herbicide is the only GM flax event that has got approval in Canada in 1990s and subsequently deregistered in 2001. In spite of deregistration, the unexpected and unauthorized detection of traces of GM flax in the consignments imported from Canada to Europe has further necessitated the stringent monitoring of flax shipments from Canada for suspected GM incidents. This study reports on the detection of transgenic elements being present in GM flax employing polymerase chain reaction assays, in a set of 123 flaxseed accessions imported from Canada for research purpose. Based on the tests conducted, none of the transgenic elements, namely, nos promoter (P-nos), nos terminator (T-nos), nptII marker gene, ALS transgene, as present in the GM flax CDC Triffid were detected in any of the tested accessions. The well-known herbicide tolerance gene cp4-epsps, being employed in Roundup® Ready events of other crops, was also not detected in these samples. This case study has demonstrated the importance of monitoring the presence of transgene(s) in flaxseed imports, and such studies need to be carried out for the imported seeds from the country where GM events of respective crop are being approved whereas they have not been approved in the country of import as a part of precautionary approach.

Development of marker-free insect resistant transgenic okra (Abelmoschus esculentus L. Moench) expressing the cry1Ac gene and identification of vector backbone-free events.

Agrobacterium-mediated co-transformation method was used to generate marker-free insect resistant transgenic okraplants expressing the cry1Ac gene. The cry1Ac gene was borne on the T-DNA of one plasmid while nptII and uidA (GUS) marker genes were present on the T-DNA of a second plasmid. Putative transgenic plants were screened by histochemical GUS assay for expression of -glucuronidase and 32 transgenic events were positive for GUS in which 21 transgenic events were positive in ELISA for the presence of Cry1Ac protein. Out of 21 Cry1Ac positive T0 events, three events displayed Mendelian inheritance of the transgenes in (9:3:3:1 ratio) T1 generation for Cry1Ac and GUS. Selected events were chosen for further genetic and molecular analysis. The cry1Ac and marker genes were found to segregate independently, of each other in 10 events in T1 generation out of 11 Cry1Ac gene inheriting events analysed indicating that the two T-DNAs insertions were genetically unlinked and identification of marker-free plants were possible in these 10 events. The marker-free nature and vector backbone-free Bt events (clean T-DNA insertions carrying cry1Ac gene) were confirmed by Southern analysis using suitable probes. The plants from selected transgenic events were rigorously screened in whole plant insect bioassays using the larvae of shoot and fruit borer, Earias vittella, an important pest of okra. Insect bioassays indicated 100% larval mortality without any infestation in five of the transgenic events and two events showed 5 to 10 percent infestation establishing the insect resistant nature of the transgenic plants. Finally the events inheriting transgenes in Mendelian fashion were characterized further and marker-free and vector backbone-free events were identified showing complete protection from the target pest Earias vittella in whole-plant insect bioassays. Quantification of Cry1Ac protein levels in the plant parts of selected events (lines) wasconsistent with the results of bioassays. Further, two lines identified in this study met the criteria for inclusion in commercial breeding programs.

Screening brinjal (Solanum melongena) accessions conserved in the National Genebank collected from states adjoining Bangladesh for adventitious presence of EE1 event

Systematic management of Plant Genetic Resources (PGRs) is the key to sustainable agriculture for food and nutritional security and in mitigating climate change. The National Genebank (NGB) at ICAR-National Bureau of Plant Genetic Resources (NBPGR), New Delhi, acts as a repository of PGRs for future use. This study aimed at screening for the adventitious presence of transgenes in brinjal (Solanum melongena L.) accessions conserved in the NGB. The study targeted the collections made during 2007-2016 from areas adjacent to Bangladesh (Assam, Meghalaya, Mizoram, Tripura and West Bengal), where field trials of Bt brinjal event EE1 were conducted during 2005-2012 and commercial cultivation of this event was permitted in 2013.There could be an apprehension of both unintentional introgression and transboundary movement through borders. Adventitious presence of transgenes was checked in a set of 96 accessions of brinjal employing Polymerase Chain Reaction (PCR) and real-time PCR assays. As event EE1 carries cry1Ac gene for insect resistance with Cauliflower Mosaic Virus (CaMV) promoter (P-35S) and marker genes (nptII and aadA), so these genetic elements were targeted for qualitative GM testing. Based on the test results, transgenes were not detected in brinjal accessions conserved in NGB. Our study showed that brinjal and wild species collected from adjoining areas of Bangladesh, post field trials and release, do not contain the event EE1. The study presents an efficient and reliable method to ensure conservation of GM-free germplasm in the NGB.

Extending the cDNA microarray detection system to evaluate genetically modified soybean and traditional soy foods

Aggravating controversies of GM (genetically modified) foods on the social, ethical and health aspects have lead to efficient and reliable detection systems to safeguard homogeneity of foods and address legal disputes. Improvising on the existing detection systems, the present investigation demonstrates the advantages of using cDNA microarray as a detection system for GM food. We have extended the study for the detection of genetic modification in commercial GM soybean seeds and in home-made traditional foods derived from these seeds, such as tofu and dried tofu. We looked for common T-DNA regions such as CaMV35S promoter, NOS or 35S terminators, nptII, hph or pat selection marker genes, GUS or GFP marker genes. We also searched for specific traits such as Bt11, T25, CP4EPSPS and four plant internal control genes (invertase, legumin, tubulin, actin gene). Results indicate that our microarray detection system can identify GM soybean seeds as well as processed food made from these seeds, with 100% accuracy. Transformation events identified in the GM soybean seeds were also visible in the processed foods, thereby confirming the accuracy and reproducibility of this procedure to even processed foods. We believe that with more popularity if the cDNA microarray detection system will soon be implemented as a diagnostic kit.

Effect of Changes in Genome Ploidy on the Mosaic Character of nptII Gene Expression in Epialleles of the Transgenic Tobacco Line Nu21

The influence of changes in the genome ploidy and the gene dose on the mosaic pattern of expression of the nptII gene in epialleles of the Nu21 transgenic tobacco line was evaluated. The general preservation of the expression profile of the marker gene nptII is shown (high in the Nu5 epiallele and low in the Nu6 epiallele) in heteroploid crosses of diploid transgenic plants (2n = 48) with polyploid nontransgenic plants (3n = 72, 4n = 96), as well as in self-pollination of experimentally obtained tetraploid transgenic plants of the Nu21 line (4n = 96) and in their hybrids from crossing with nontransgenic diploid plants. It has been established that the stability of expression of the nptII marker gene is influenced by the initial epigenetic status of epialleles and the direction of crossing in hybridization of tetraploid nontransgenic tobacco plants with diploid transgenic plants of the Nu21 line.

Agrobacterium tumefaciens-mediated transformation of common bean (Phaseolus vulgaris) var. Brunca

Common bean is a crop recalcitrant to in vitro regeneration and therefore it lacks an efficient transformation protocol that can be reproduced using A. tumefaciens. The main goal of this study was to establish a protocol for A. tumefaciens mediated transformation of Phaseolus vulgaris var. Brunca by marker genes (gusA and nptII) together with the gene for trehalose-6-phosphate synthase from Saccharomyces cerevisiae (TPS1) used in other species to increase tolerance to abiotic stress. The β-glucuronidase activity was detected in 45 % of the LBA4404 ElectroMAX® pCAMBIA1301 infected explants. Transformed explants regenerated new shoots after four to five months period in a kanamycin rich media. Surviving plants were evaluated by PCR and presented an 0.5 % efficiency of transformation. The established protocol for genetic transformation of common bean has two additional advantages with respect to previous reports: (1) it allows for obtaining transformed regenerants and (2) the genetic transformation was stable for the selective gene.

New Phenotypes of Potato Co-induced by Mismatch Repair Deficiency and Somatic Hybridization.

As plants are sessile they need a very efficient system for repairing damage done by external or internal mutagens to their DNA. Mismatch repair (MMR) is one of the systems that maintain genome integrity and prevent homeologous recombination. In all eukaryotes mismatches are recognized by evolutionary conserved MSH proteins often acting as heterodimers, the constant component of which is MSH2. Changes affecting the function of MSH2 gene may induce a ‘mutator’ phenotype and microsatellite instability (MSI), as is demonstrated in MSH2 knock-out and silenced lines of Arabidopsis thaliana. The goal of this study was to screen for ‘mutator’ phenotypes in somatic hybrids between potato cvs. ‘Delikat’ and ‘Désirée’ and MMR deficient Solanum chacoense transformed using antisense (AS) or dominant negative mutant (DN) AtMSH2 genes. The results demonstrate that first generation fusion hybrids have a range of morphological abnormalities caused by uniparental MMR deficiency; these mutant phenotypes include: dwarf or gigantic plants; bushiness; curled, small, large or abnormal leaves; a deterioration in chloroplast structure; small deep-purple tubers and early dehiscent flowers. Forty percent of the viable somatic hybrids planted in a greenhouse, (10 out of 25 genotypes) had mutant phenotypes accompanied by MSI. The majority of the hybrids with ‘mutator’ phenotypes cultured on media containing kanamycin developed roots so sustaining the presence of selectable marker gene nptII, from the initial constructs. Here for the first time, MMR deficiency combined with somatic hybridization, are used to induce new phenotypes in plants, which supports the role of MMR deficiency in increasing introgressions between two related species.

Development of a Taqman real-time PCR method to quantify nptII in apple lines obtained with ‘established’ or ‘new breeding’ techniques of genetic modification

Cisgenic plants must be free of exogenous genetic elements such as antibiotic or herbicide resistance genes commonly used in the selection phase of a gene transfer protocol. However, the use of a selection marker is essential for the transformation of many fruit crops, including apple (Malus × domestica), for which the efficiency of DNA integration is very low. Currently, the approach with the highest chances of success relies on the removal of undesired exogenous genes by means of an inducible site-specific recombinase enzyme and its recognition sites. We developed a quantitative, rapid and cost-effective method based on real-time PCR to quantify the copy number of nptII marker gene in apple lines and to evaluate its elimination after the activation of the recombinase system. MdTOPO6 gene was chosen as endogenous reference gene for apple due to the single-copy presence in the haploid genome and to the species-specificity. A recombinant plasmid harboring specific sequences of both the reference gene and the target gene nptII was used as calibrator to build the standard curves. The limit of quantification of the method was evaluated, and precision and trueness of the quantification performances proved to be valid according to international reference criteria. Finally, this method was applied to characterize transgenic and cisgenic apple lines, and to investigate the possibility of removing an entire T-DNA cassette from the genome of edited apple plants.

Development of a Taqman real-time PCR method to quantify nptII in apple lines obtained with ‘established’ or ‘new breeding’ techniques of genetic modification

Cisgenic plants must be free of exogenous genetic elements such as antibiotic or herbicide resistance genes commonly used in the selection phase of a gene transfer protocol. However, the use of a selection marker is essential for the transformation of many fruit crops, including apple (Malus × domestica), for which the efficiency of DNA integration is very low. Currently, the approach with the highest chances of success relies on the removal of undesired exogenous genes by means of an inducible site-specific recombinase enzyme and its recognition sites. We developed a quantitative, rapid and cost-effective method based on real-time PCR to quantify the copy number of nptII marker gene in apple lines and to evaluate its elimination after the activation of the recombinase system. MdTOPO6 gene was chosen as endogenous reference gene for apple due to the single-copy presence in the haploid genome and to the species-specificity. A recombinant plasmid harboring specific sequences of both the reference gene and the target gene nptII was used as calibrator to build the standard curves. The limit of quantification of the method was evaluated, and precision and trueness of the quantification performances proved to be valid according to international reference criteria. Finally, this method was applied to characterize transgenic and cisgenic apple lines, and to investigate the possibility of removing an entire T-DNA cassette from the genome of edited apple plants.

An efficient method for Agrobacterium-mediated genetic transformation of chilli pepper (Capsicum annuum L.)

In this study, we have describe a highly efficient transformation protocol for two elite Indian cultivars of chilli pepper (Capsicum annuum L.), Pusa Sadabahar and Pusa Jwala. We used hypocotyls and cotyledons derived from young seedlings as explants, standardized regeneration medium for enhanced regeneration, and optimized parameters for promoting efficient transformation in these chilli cultivars. The optimized regeneration medium consisting of MS medium augmented with 1 mg l−1 indole-3-acetic acid and 5 mg l−1 6-benzyl amino purine induced profuse shoot regeneration from the hypocotyl and cotyledon explants, with a regeneration frequency of 81% in Pusa Sadabahar and 78% in Pusa Jwala. The transformation protocol was standardized using Agrobacterium tumefaciens strain LBA4404 harbouring pCAMBIA2301 construct with GUS reporter and NPT-II marker genes. The results demonstrated that the hypocotyl explants are more amenable to transformation than cotyledonary explants in both the cultivars studied. Further, co-cultivation of the hypocotyl explants with agrobacterial cells (OD600 = 0.2–0.5) for the duration of 72 h was found to be optimum for obtaining high transformation efficiency of about 30% in both the cultivars. The transgenic status of chilli transformants recovered in the selection medium having 30 mg l−1 kanamycin was confirmed by GUS activity, PCR, Southern and RT-PCR analyses. About 85–90% of rooted transgenic plants survived hardening and acclimatization processes. Using this protocol we produced several independent transgenic lines of each chilli cultivar.

"Cell grafting": a new approach for transferring cytoplasmic or nuclear genome between plants.

A new method based on mixing and wounding of callus tissue was used to transfer plastid or nuclear DNA between cells. Methods alternative to sexual hybridization can be powerful tools for crop improvement. We have developed a new hybridization technology based on wounding a mixed population of cells of two parents growing in vitro as callus ("cell grafting"), and have demonstrated the utility of this system for plastid or nuclear genome transfer. In our proof-of concept experiments, non-organized growing tissue (callus) from tobacco var. Samsun, carrying the nuclear marker genes nptII and uidA (GUS), and tobacco var. Petit Havana, carrying aadA and gfp genes in the plastid genome, were mixed together, wounded with a razor blade and placed for regeneration on selection medium containing both spectinomycin (aadA) and paromomycin (nptII). Plants with aadA and gfp positive plastids and nptII plus uidA positive nuclear background were produced. Molecular analysis confirmed the presence of all four genes in these plants. Morphology and ploidy level analysis confirmed the production of "diploid" plants similar to var. Samsun possessing transformed plastids from var. Petit Havana. Reciprocal crosses between the experimentally produced plants and wild type tobacco confirmed maternal inheritance of aadA and gfp and Mendelian inheritance of nptII and uidA. For transfer of nuclear traits between plants we used two nuclear-transformed parents with different selectable markers; one with nptII (paromomycin resistant), and another with aadA (spectinomycin resistant). Plants resistant to both antibiotics which also had different visible markers were produced.

Establishment of an in vitro propagation and transformation system of Balanites aegyptiaca

Balanites aegyptiaca (Balanitaceae) is a drought-tolerant but salt-sensitive tree species distributed in the tropical and arid lands in Africa and Asia. The tree contains many secondary metabolites and a high percentage of oil in the kernels that can be used for biodiesel production. This study aimed to establish an in vitro propagation system of two B. aegyptiaca provenances (El-Kharga and Wadi El-Alaqi) from nodal and cotyledon explants. The explants were placed on Murashige and Skoog medium supplemented with different concentrations of 6-benzyladenine (BA) and thidiazuron (TDZ) for shoot induction. BA was significantly more effective in shoot induction from nodal explants and treatment with BA also resulted in higher regeneration rates of about 40–60 % of adventitious shoots on cotyledon explants, whereas on TDZ-containing medium slightly higher shoot numbers per explant but a negative effect on shoot length were recorded. Rooting was achieved in 40–60 % of the shoots on medium containing between 1.2 and 4.8 µM indole-3-butyric acid. Three different Agrobacterium tumefaciens strains (EHA105, GV3101, and LBA4404) harboring the plasmid pCAMBIA2301 containing the nptII marker and gus reporter genes were used to establish a transformation system in B. aegyptiaca. Strain GV3101 resulted in the highest survival rates and highest number of explants positive in the GUS assay. This selected A. tumefaciens strain was used to introduce pBinAR containing the sequence encoding ERD10 (early responsive to dehydration 10) to produce salt-tolerant B. aegyptiaca plants. The presence of the ERD10 and the nptII gene were detected by PCR in transformed B. aegyptiaca plants.

Efficient seed-specifically regulated autoexcision of marker gene (nptII) with inducible expression of interest gene in transgenic Nicotiana tabacum

In this study, we developed the seed-specifically regulated autoexcision system based on Cre/loxP site-specific recombination for coelimination of nptII and cre genes from transgenic plants. To accomplish this, a seed-specific gene promoter, BcNA1, from Brassica napus was used to drive conditional expression of recombinase. NptII and recombinase cassettes were placed between two directly repeated loxP sites. The loxP-flanked DNA was located between the SP-DDEE synthetic pathogen inducible promoter and pr??omoterless β-glucuronidase (Gus) gene, as a model gene of interest. Upon seed-specific expression of cre recombinase, the excision event would eliminate loxP-flanked DNA and bring the gus reporter gene under the control of the inducible promoter. This Cre/loxP system was transformed into Nicotiana tabacum via Agrobacterium-??mediated transformation. The regenerated plants were obtained by selection on kanamycin medium and PCR screening. Based on seed germination assay on kanamycin-containing medium, the transgenic lines were subdivided into homogeneous and heterogeneous categories. Phenotypic and molecular analysis of T1 progeny plants indicated that completely NptII-free plants were obtained in homogeneous transgenic lines. Coelimination of NptII and recombinase cassettes were verified with PCR, sequencing, and histochemical Gus staining assay. Full excision efficiency (100%) demonstrates the effectiveness of this autoexcision system for the production of marker gene-free transgenic plants.

Genetic transformation and expression of transgenic lines of Populus x euramericana with insect-resistance and salt-tolerance genes.

We characterized new transgenic varieties of poplar with multiple insect-resistant and salt stress tolerant genes. Two insect-resistant Bacillus thuringiensis (Bt) genes, Cry1Ac and Cry3A, and a salt-tolerant gene, Betaine aldehyde dehydrogenase (BADH) were inserted into a vector, p209-Cry1Ac-Cry3A-BADH. The clone of Populus x euramericana was transformed by the vector using the Agrobacterium-mediated method. Three transgenic lines were assessed using genetic detection and resistance expression analysis. PCR revealed that exogenous genes Cry1Ac, Cry3A, BADH and selective marker gene NPTII were present in three transgenic lines. Quantitative real-time PCR (qPCR) showed significant differences in the transcriptional abundance of three exogenous genes in different lines. Results of assays for Bt toxic proteins showed that the Cry1Ac and Cry3A toxic protein content of each line was 12.83-26.32 and 2108.91-2724.79 ng/g, respectively. The Cry1Ac toxic protein content of different lines was significantly different; the Cry3A toxic protein content was about 100 times higher than that of the Cry1Ac toxic protein. The insect-resistance test revealed the mortality rate of transgenic lines to Hyphantria cunea L1 larvae varied by 42.2-66.7%, which was significantly higher than non-transgenic lines. The mortality rate of L1 and L2 Plagiodera versicolora larvae was 100%. The insecticidal effect of transgenic lines to P. versicolora larvae was higher than that to H. cunea larvae. NaCl stress tolerance of three transgenic lines under 3-6% NaCl concentration was significantly higher than that of non-transgenic lines.

Agrobacterium-mediated genetic transformation in Brassica species

An efficient and reproducible protocol for the production of transgenic Brassica plants was developed by inoculating hypocotyl explants with Agrobacterium tumefaciens strain LBA4404 carrying a binary vector pBI121, which contains selectable marker gene nptII conferring resistance to kanamycin and the GUS reporter gene. The transformation experiment was performed by optimizing two important parameters: preculture period and co-cultivation period. Infection was most effective when explants were precultured for three days (68.75% GUS positive) and co-cultivated for three days (82.92% GUS positive) with Agrobacterium. Among the varieties, BARI sarisa-8 showed the highest response to GUS assay (64.38% GUS positive). Callus induction was highest in Rai-5 (10%) and three day period of preculture and co-cultivation (12.33 and 13.33%, respectively). Transformation percentage was also highest in Rai-5 (4.75%), and three day period of preculture and co-cultivation (7.50 and 5.42%, respectively). Highest percentage (33.33) of root induction from transgenic shoots was observed in ½ MS + 0.5 mgL -1 NAA + 50 mgL -1 kanamycin + 50 mgL -1 cefotaxime. Among the varieties, BARI sarisa-8 produced the highest percentage (37.78) of rooted shoots.

Selectable Marker Gene Removal and Expression of Transgene by Inducible Promoter Containing FFDD Cis-Acting elements in Transgenic Plants.

Selectable marker gene (SMG) systems are critical for generation of transgenic crops. Transgenic crop production without using SMG is not economically feasible. However, SMGs are non-essential once an intact transgenic plant has been established. Elimination of SMGs from transgenic crops both increases public acceptance of GM crops and prepares gene stacking possibility for improvement of complex traits. Synthetic inducible promoters provide an efficient and flexible strategy to regulate transgene expression. This study aimed to construct a transformation vector based on Cre/loxP recombination system to enhance efficiency of SMG-free transgenic plant production followed by post-excision expression of gene of interest in transgenic plants by a pathogen inducible promoter. In pG-IPFFDD-creint-gusint construct, cre recombinase and selectable marker gene (nptII) cassettes were placed between the two loxP recognition sites in direct orientation. Seed-specific Napin promoter was used for regulation of Cre expression in transgenic seeds. In the construct, loxP flanked sequence containing nptII and recombinase cassettes, located between a pathogen inducible promoter containing FFDD cis-acting elements and β-glucuronidase coding region. The cunstuct was transformed into Nicotiana tabaccum via Agrobacterium-mediated transformation. The results showed that both cre and nptII excision occurs in T1 progeny tobacco plants through seed-specific cre expression. The excisions were confirmed by methods activation of the gus gene, germination test on kanamycin-containing medium and molecular analysis. Inducibility of gus expression by FFDD-containing promoter in T1 leaf tissues was confirmed by histochemical Gus staining assay. The established system is not only an efficient tool for marker gene elimination but also provides possibility for inducible expression of the transgene.