Agrobacterium-mediated transformation of the medicinal plant Centaurea montana (original) (raw)
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Plant Cell Reports, 1992
The efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana was compared with different organs, Arabidopsis ecotypes, and Agrobacterium strains. Efficiency of shoot regeneration was examined using hypocotyl, cotyledon and root explants prepared from young seedlings. Hypocotyl explants had the highest regeneration efficiency in all of the four Arabidopsis ecotypes tested, when based on a tissue culture system of callus-inducing medium (CIM: Valvekens et al. 1988) and shoot-inducing medium (SIM: Feldmann and Marks 1986). Histochemical analysis using the 13glucuronidase (GUS) reporter gone showed that the gusA gone expression increased as the period of preincubation on CIM was extended, suggesting that dividing cells are susceptible to Agrobacterium infection. In order to obtain transgenic shoots, hypocotyl explants preincubated for 7 or 8 days on CIM were infected with Agrobacterium containing a binary vector which carries two drug-resistant genes as selection markers, and transferred to SIM for selection of transformed shoots. Of four Arabidopsis ecotypes and of three Agrobacterium strains examined, Wassilewskija ecotype and EHA101 strain showed the highest efficiency of regeneration of transformed shoots. By combining the most efficient factors of preincubation period, Arabidopsis ecotype, tissue, and bacterial strain, we obtained a transformation efficiency of about 80-90%. Southern analysis of 124 transgenic plants showed that 44% had one copy of inserted T-DNA while the others had more than one copy.
Establishment of Agrobacterium-mediated genetic transformation system in Dahlia
Plant Biotechnology, 2013
An efficient system for Agrobacterium-mediated transformation was established in dahlia (Dahlia×pinnata) 'Yamatohime'. Mass of shoot primordia (MSP) induced on MS medium supplemented with 10 mg l −1 TDZ were inoculated with Agrobacterium tumefaciens strain EHA101 (pIG121-Hm) harboring both β-glucuronidase (GUS) and hygromycin resistant genes. After 2 days of co-cultivation, the MSP were transferred to a selection medium containing hygromycin with meropenem for bacterial elimination. Shoots were successfully regenerated from survived MSP on hormone-free medium without hygromycin and they rooted on hormone-free medium containing hygromycin. The hygromycin-resistant plants thus obtained showed histochemical blue staining for GUS. Transformation of plants was confirmed by PCR and Southern blot analyses. Transgenic 'Kokucho' were also produced by using the same transformation procedure, suggesting wide applicability of this Agrobacterium-mediated transformation procedure for other dahlia cultivars.
Production of Transgenic Plants via Agrobacterium-Mediated Transformation in Liliaceous Ornamentals
Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues Vol. II, 2006
Studies on the Agrobacterium-mediated production of transgenic plants in several Liliaceous ornamentals, Lilium spp., Agapanthus spp., Muscari armeniacum and Tricyrtis hirta are described. Different strains of A. tumefaciens were used, all of which harbored the binary vector carrying the neomycin phosphotransferase II (NPTII) gene, the intron-containing ȕ-glucuronidase (GUS) gene, and the hygromycin phosphotransferase (HTP) gene in the T-DNA region. Utilization of organogenic or embryogenic calluses as a target material for transformation and acetosyringone (AS) treatment during inoculation and/or co-cultivation with Agrobacterium were found to be critical for successful production of transgenic plants in Liliaceous ornamentals. Following transfer of co-cultivated organogenic or embryogenic calluses onto hygromycin-containing media, several hygromycin-resistant (Hyg r) tissues were obtained, and complete plants were subsequently developed from these tissues. Most of the plants were verified to be transgenic plants by GUS histochemical assay and PCR analysis. For Lilium 'Acapulco', A. praecox ssp. orientalis 'Royal Purple Select' and M. armeniacum 'Blue Pearl', Southern blot or inverse PCR analysis revealed the integration of 1-5 copies of the transgene into the genome of transgenic plants, but most of them had 1 or 2 copies. Agrobacterium-mediated transformation systems thus established may be useful as a tool for molecular breeding as well as molecular biological studies.
A Review: Agrobacterium-mediated Gene Transformation to Increase Plant Productivity
The Journal of Phytopharmacology
In genetics and molecular biology Gene transformation is a gene alteration technique that involves the introduction and expression of a foreign gene into the host organism. There are many gene transformation methods like particle bombardment electroporation micro-injection, (PEG), for different biotechnological experiments But Plant gene transformation is a widely used procedure for obtaining transgenic plants and plant models to understand gene functions. Agrobacterium tumefaciens is a natural genetic engineer which is rod-shaped gram-negative soil-born barteri. Initially Agrobacterium was utilized to transform only dicot plants but over the year’s modification in plant transformation protocol. It was now utilized in monocot plants as well as in fruits plants too. Agrobacterium tumefaciens inserts its (DNA), (Transfer DNA-T-DNA), into the host plant. The transmitted (DNA), is randomly integrated into the host cell's genetic material inside the infected plant cell nucleus. Alter...
Proceedings of the National Academy of Sciences, India, Section B: biological sciences, 2012
A reproducible and highly efficient protocol for genetic transformation mediated by Agrobacterium has been established for greengram (Vigna radiata L. Wilczek). Double cotyledonary node (DCN) explants were inoculated with Agrobacterium tumefaciens strain LBA 4404 harboring a binary vector pCAMBIA 2301 containing neomycin phosphotransferase (npt II) gene as selectable marker, β-glucuronidase (GUS) as a reporter (uidA) gene and annexin 1 bj gene. Important parameters like optical density of Agrobacterium culture, culture quantity, infection medium, infection and co-cultivation time and acetosyringone concentration were standardized to optimize the transformation frequency. Kanamycin at a concentration of 100 mg/l was used to select transformed cells. Transient and stable GUS expressions were studied in transformed explants and regenerated putative plants, respectively. Transformed shoot were produced on regeneration medium containing 100 mg/l kanamycin and 250 mg/l cefotaxime and rooted on ½ MS medium. Transient and constitutive GUS expression was observed in DCN explants and different tissues of T 0 and T 1 plants. Rooted T 0 and T 1 shoots confirming Polymerase Chain Reaction (PCR) positive for npt II and annexin 1bj genes were taken to maturity to collect the seeds. Integration of annexin gene into the greengram genome was confirmed by Southern blotting.
In vitro and in planta Agrobacterium tumefaciens madiated transformation of Arabidopsis thaliana
1998
Cell transformation of Arabidopsis thaliana (L.) Heynh. with Agrobacterium tumefaciens (LBA4404)was performed in two ways: in vitro - by the transformation of root and stem explants - and in planta - by the transformation of whole plants in the greenhouse. Agrobaacterium contained the binary plasmid pBI121 or its constructs with the fragments of carrot DC-8 promoter. The results show that transformation efficiency in vitro is influenced by the age and type of explant. It was not possible to induce transgenic calli on eexplants of 5 week old Arabidopsis roots, although it was possible on explants of 3 week old roots. Although green calli grew on Arabidopsis stem explants, transgenic shoots did not develop. When using traansformation in vitro, from seed to mature primary transformant it took 15 to 16 weeks and for in planta transformation it took 19 to 20 weeks.
Agrobacterium-mediated transformation of plants: Basic principles and influencing factors
African Journal of …, 2011
Transformation is an important topic in plant biology and transgenic plants have become a major focus in plant research and breeding programs. Agrobacterium-mediated transformation as a practical and common method for introducing specific DNA fragments into plant genomes is well established and the number of transgenic plants produced using this method is increasing. Despite the popularity of the method, low efficiency of transformation is a major challenge for scientists. Modification of different genetic and environmental aspects of transformation method may lead to better understanding of the system and result in high efficiency transformation. In this review, we deal with recent genetic findings as well as different environmental factors which potentially influence Agrobacterium-mediated transformation.
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).