Analysis of Molecular and Morphological Characteristics of Plants Transformed (original) (raw)
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ANALYSIS OF MOLECULAR AND MORPHOLOGICAL CHARACTERISTICS OF PLANTS TRANSFORMED WITH ANTIFUNGAL GENE
2007
A defensin gene isolated from chickpea (Cicer arietinum L.), Ca-AFP in the background of pCAMBIA1301 was transferred into tobacco (Nicotiana tabaccum L. var. petit havana) genome following transformation using Agrobacterium tumefaciens strain LBA4404. Although all the explants showed GUS activity after co-cultivation, transgenic tobacco shoots were regenerated only from those explants cultured in presence of bacteriostatic antibiotic carbenicillin under hygromycin selection. Presence of the antifungal gene in the regenerated plants was confirmed by PCR, while integration of the whole T-DNA was demonstrated by southern blot hybridization. Furthermore, southern blot hybridization revealed that most of the transgenics contained single copy of the T-DNA while few were found to have multiple copies. Expression of the GUS and Ca-AFP genes in the transgenic plants was observed. Morphological analysis demonstrated that presence of the transgenes produced no morphological abnormality or yield discrepancy in the transgenics.
Agrobacterium tumefaciens -mediated transformation of tobacco (Nicotiana tabaccum)
International Journal Of Applied Research and Studies, 2017
The present study involves the development of genetically engineered tobacco plants with annexin gene. The plasmid DNA of pUC 19 vector having the desired Annexin gene (3Kbp) and the plasmid DNA of the binary vector pGPTV (13 Kbp) were isolated from E.coli (DH5α strain) by alkaline lysis method. The purified pUC 19/Annexin and pGPTV plasmid were restriction digested using the restriction enzymes EcoRI and XbaI as a linearised band was eluted from the gel. The digested plasmid shown in the band pattern in the gel were cut by gel elution technique and purified from other reaction mixture. Then the dot spot test was done to calculate the concentration of pGPTV and Annexin gene. The recombinant PGPTV plasmid with the annexin gene in Agrobacterium tumefaciens MTCC 431 was mobilized and transferred to plant system through the mobilization helper plasmid pRK2013. The kanamycin resistance gene (NPT II) was used as a selective marker. The calli used for isolating the genomic DNA which was th...
High-Efficiency Agrobacterium-Mediated Transformation of Tobacco (Nicotiana tabacum)
2018
To improve Agrobacterium-mediated transformation of tobacco, factors influencing gene delivery, including genotype of the plant, bacterial strain, and Agrobacterium transformation procedure, were tested via direct somatic embryogenesis. Leaf tissue of three different tobacco genotypes (Nicotiana tabacum L. cvs. Samsun, and Xanthi, and N. benthamiana) were used as explant. Leaf explants were transformed using three Agrobacterium tumefaciens strains (EHA105, GV3101, and LBA4404) harboring the binary vector pCAMBIA1304 using three different types of transformation methods as named Agro-inoculation, Agro-infection and Agro-injection. Selection of hygromycin resistant shoots was conducted on MS medium containing 3.0 mgL-1 BAP and 0.2 mgL-1 IAA, 250 mgL-1 cefotaxime and 30 mgL-1 hygromycin. Hygromycin resistant shoots were then rooted on MS medium supplemented with 250 mgL-1 cefotaxime and 15 mgL-1 hygromycin. The results indicated that A. tumefaciens strain LBA4404 was more effective in ...
American Journal of Plant Sciences, 2016
Studies on Agrobacterium tumefaciens-mediated transformation of wild tobaccos Nicotiana debneyi, Nicotiana clevelandii, and Nicotiana glutinosa were conducted. Leaf disks were infected and co-cultivated with A. tumefaciens strain EHA105 carrying the binary vector pBISN1 with an intron interrupted β-glucuronidase (GUS) reporter gene (gusA) and the neomycin phosphotransferase gene (nptII). Selection and regeneration of kanamycin resistant shoots were conducted on regeneration medium containing 8.88 µM 6-benzylaminopurine (BAP), 0.57 µM indole-3-acetic acid (IAA), 50 mg•L −1 kanamycin and 250 mg•L −1 timentin. Kanamycin resistant shoots were rooted Murashige and Skoog (MS) medium containing 100 mg•L −1 kanamycin and 250 mg•L −1 timentin. Using this protocol, kanamycin-resistant plants were obtained from all three wild tobaccos at frequencies of 75.6% for N. debneyi, 25.0% for N. clevelandii, and 2.8% for N. glutinosa. Transcripts of nptII and gusA were detected in kanamycin-resistant T 0 transformants (i.e., 2 for N. glutinosa and 5 for each of the N. debneyi and N. clevelandii) by the reverse transcript polymerase chain reaction (RT-PCR), and histochemical GUS assays confirmed expression of gusA in both T 0 plants and T 1 seedlings. The results indicate that the protocols are efficient for transformation of wild tobacco N. debneyi and N. clevelandii.
Genetics and Molecular Research, 2018
Plant transformation is a widely used procedure for obtaining transgenic plants and to develop plant models to understand gene function. Plant models such as Nicotiana tabacum are widely used for understanding gene responses to external influences. An important tool in such studies is genetic transformation through infection with Agrobacterium tumefaciens. However, this transformation is often inefficient. Consequently, development and optimization of techniques to promote high rates of seedling regeneration of transgenic tobacco is imperative. The methods tested for infection of tobacco explants consisted of injecting 10 μl of the bacterial culture directly into anodal segment using an insulin syringe (1 mL); bacterial co-cultivation with nodal segments and micro-sectioned leaf disks. Infection through punctures made with a syringe in nodal segments of tobacco and no co-cultivation period was the most efficient in the regeneration process and in obtaining genetically transformed plants, with 88 and 75% success rates, respectively. We obtained an increase of 50% in the transformation rates when compared to previous studies using N. tabacum.
2003
The mature embryo axes and cotyledonary nodes of chickpea (Cicer arietinum L.) were evaluated for Agrobacterium-mediated transformation and the production of stable transgenic plants expressing the reporter gene GUS was documented. The major limitation in delivering the T-DNA in these explants of grain legume has been the low frequency and inconsistency. With manipulation of co-cultivation conditions and preparation of excised explants with exposed regenerative cells in L2 and L3 layers followed by the stringent screening on selection pressure of 100150 mg C1, kanamycin exhibited significantly higher transformation frequency as indicated in the transient GUS expression. Amongst various strains ofAgrobacteria, the strain, LBA 4404 was found to be most suitable for maximal transfer of T-DNA and minimum induction of hypersensitive response into the excised explants of chickpea. Kanamycin resistant chickpea shoots selected after 3-4 cycles of kanamycin screening were grafted onto 8-day-...
Emirates Journal of Food and Agriculture, 2013
Agrobacterium mediated transformation has been widely used for research in plant molecular biology and for genetic improvement of crops exploiting its tremendous ability to transfer foreign DNA to plants. In this study, the transformation efficiency of five Agrobacterium tumefaciens strains GV2260, LBA4404, AGL1, EHA105, and C58C1 was evaluated in Nicotiana tabacum L. cultivar Samsun. The Agrobacterium strains contained the recombinant binary vector pBin19 harboring beta-glucuronidase uidA gene under 35S promoter. Neomycin phosphotransferase (nptII) gene was used as a selectable marker at a concentration of 100 mg L-1 kanamycin. The expression of uidA gene in regenerated T0 plants was firstly analyzed by GUS histochemical analyses and later on confirmation of presence of the nptII and uidA genes in regenerated plants was determined by PCR. The highest transformation rate (20%) was obtained with the Agrobacterium strain LBA4404, followed by EHA105, GV2260, C58C1 and AGL1. The higher transformation efficiency achieved in our studies make LBA4404 Agrobacterium strain optimal for functional genomics and biotechnological applications in tobacco plants.
Development of in Planta Transformation Methods using Agrobacterium tumefaciens
Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues Vol. II, 2006
This chapter describes simple and efficient in planta transformation methods using Agrobacterium tumefaciens for buckwheat (Fagopyrum esculentum M.), mulberry (Morus alba L.), kenaf (Hibiscus cannabinus L.) and rice (Oryza sativa L.). In the methods, meristems of either apical (buckwheat, kenaf and rice) or axillary bud (mulberry and kenaf) of young plants (buckwheat, kenaf, and mulberry) or soaked seeds (rice) were inoculated by A. tumefaciens after being pricked with a needle. The inoculated plants were grown to maturation in pots under nonsterile conditions. Transformation was demonstrated by several lines of evidence obtained by using mostly the progenies of the T1 generation; phenotypic inheritance from T0 plants to the plants of the following generation, detection of transgene and its transmission to the following generation, rescue of plasmids composed of T-DNA of the binary vector and flanking plant genomic DNA, detection of ȕ-glucuronidase activity in the transformants and resistance of seed germination of transformants to antibiotics (geneticin or hygromycin).
In this study, attempts were made to develop a protocol for regeneration of transgenic plants via Agrobacterium tumefaciens-mediated transformation of leaf segments from 'Valencia' sweet orange (Citrus sinensis L. Osbeck) using gfp (green fluorescence protein) as a vital marker. Sensitivity of the leaf segments regeneration to kanamycin was evaluated, which showed that 50 mg l -1 was the best among the tested concentrations. In addition, factors affecting the frequency of transient gfp expression were optimized, including leaf age, Agrobacterium concentration, infection time, and co-cultivation period. Adventitious shoots regenerated on medium containing Murashige and Tucker basal medium plus 0.1 mg l -1 anaphthaleneacetic acid (NAA), 0.5 mg l -1 6-benzyladenine (BA) and 0.5 mg l -1 kinetin (KT). The leaf segments from 3-month-old in vitro seedlings, Agrobacterium concentration at OD 600 of 0.6, 10-min immersion, and co-cultivation for 3 days yielded the highest frequency of transient gfp expression, shoots regeneration response and transformation efficiency. By applying these optimized parameters we recovered independent transformed plants at the transformation efficiency of 23.33% on selection medium (MT salts augmented with 0.5 mg l -1 BA, 0.5 mg l -1 KT, 0.1 mg l -1 NAA, 50 mg l -1 kanamycin and 250 mg l -1 cefotaxime). Expression of gfp in the leaf segments and regenerated shoots was confirmed using fluorescence microscope. Polymerase chain reaction (PCR) analysis using gfp and nptII gene-specific primers further confirmed the integration of the transgene in the independent transgenic plants. The transformation methodology described here may pave the way for generating transgenic plants using leaf segments as explants.