scholarly journals Agrobacterium tumefaciens-mediated Transformation of Embryogenic Callus and Somatic Embryos of the Banana cv “Ambon Lumut” (Musa acuminata)

2017 ◽  
Vol 2 (6) ◽  
pp. 599 ◽  
Author(s):  
Tifa R. Kusumastuti ◽  
Rizkita R. Esyantia ◽  
Fenny M. Dwivany
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Embryogenic Callus ◽  
Somatic Embryos ◽  
Crop Improvement ◽  
Pcr Analysis ◽  
Abiotic Factor ◽  
Gfp Gene ◽  
Culture Transformation ◽  
Biotechnological Approach

Banana is one of the major fruit crops, though its conventional breeding has limitations, such as sterility and high polyploidy  levels.  Biotechnological  approach  using genetic  transformation  crop for improvement  offers  an alternative  solution.  In  this  study  a  protocol  was developed  for  establishing genetic  transformation  from embryogenic callus and somatic embryos of the banana cv Ambon Lumut . Embryogenic callus was obtained in ID4 medium (MS-based medium) supplemented with 1 mg L-1 IAA, 4 mg L-1 2,4D, and 0.03 g L-1 active charcoal. Embryogenic callus was transferred into liquid mediu m to establish somatic embryos. Embryogenic callus and somatic embryos were used for Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain A GL1, containing pART-TEST7 p lasmid with gfp gene as a reporter and CaM V35S as a promoter, was used for transformations. The embryogenic callus and somatic embryos were transformed using heat-shock method followed by centrifugation  (2000 rpm) and co-cult ivation in liquid medium containing acetosyringone (100 M) for 3 days. Results of the GFP analysis showed transient expression from gfp gene reporter in transformed embryogenic callus and somatic embryos. Transformation efficiency in somatic embryos (85,9%) was higher than  that in embryogenic callus (32.09%). PCR analysis using CaMV primer showed bands that compatible with CaMV35S promoter at 507 bp. This is a report showing establisment of embryogenic callus and somatic embryo culture transformation by using A. tumefaciens-mediated transformation protocol of the local banana cv Ambon Lumut. This study proved  the huge potential for genetic transformation of banana cv Ambon Lumut for crop improvement, such as pest or disease  resistance and abiotic factor stress tolerance. Keywords: banana; embryogenic callus; somatic embryos.

Improved Genetic Transformation of Sugarcane ( Saccharum spp.) Embryogenic Callus Mediated by Agrobacterium tumefaciens

2017 ◽  
Vol 2 (3) ◽  
pp. 221-239 ◽  
Author(s):  
Marcos Fernando Basso ◽  
Bárbara Andrade Dias Brito da Cunha ◽  
Ana Paula Ribeiro ◽  
Polyana Kelly Martins ◽  
Wagner Rodrigo Souza ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Embryogenic Callus ◽  
Saccharum Spp

scholarly journals Agrobacterium-mediated Genetic Transformation of Two Varieties of Jute (Corchorus capsularis L.)

1970 ◽  
Vol 18 (1) ◽  
pp. 7-16 ◽  
Author(s):  
Rakha Hari Sarker ◽  
G.M. Al-Amin ◽  
Fathi Hassan ◽  
M.I. Hoque
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Plant Tissue ◽  
Genomic Dna ◽  
Mature Embryo ◽  
Gus Expression ◽  
Pcr Analysis ◽  
Gus Gene ◽  
Corchorus Capsularis ◽  
Selection Of

Transformation experiments were carried out using different explants of two varieties of white jute (Corchorus capsularis L.), namely, CVL-1 and CVE-3 with Agrobacterium tumefaciens strain (LBA4404/pBI121) containing the GUS and nptII genes. Maximum transformation ability was obtained from petiole-attached cotyledons and mature embryo explants. Kanamycin at a concentration of 200 mg/l was found optimum for selection of transformed shoots developed frommature embryos. Histochemical assay revealed the stable expression of the GUS gene within the various tissues of transformed plantlets. Stable integration of GUS and nptII genes were confirmed by PCR analysis of genomic DNA isolated from these transformed shoots. Key words: Jute, Transformation, GUS expression, PCR analysis D.O.I. 10.3329/ptcb.v18i1.3245 Plant Tissue Cult. & Biotech. 18(1): 7-16, 2008 (June)

scholarly journals Genetic transformation of Paulownia elongata S. Y. Hu., mediated by Agrobacterium tumefaciens and biolistic system

2021 ◽  
Vol 14 (2) ◽  
Author(s):  
Carlos R. Castillo-Martínez ◽  
Ma. Alejandra Gutiérrez-Espinosa ◽  
Jorge Cadena-Iñiguez ◽  
Marco T. Buenrostro-Nava ◽  
VALERIA MARTÍNEZ SIAS
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Pcr Analysis ◽  
Optimum Dose ◽  
Indirect Organogenesis ◽  
Gus Reporter ◽  
Selection Gene ◽  
Transformation Systems ◽  
Paulownia Elongata

Objective: The most appropriate conditions for genetic transformation through direct (bioballistic) and indirect (Agrobacterium tumefaciens) transformation systems in Paulownia elongata were established. Design/methodology/approach: Starting from in vitro propagation through both direct and indirect organogenesis, internodal stem segments with 0.5 to 1 cm length were determined as the best explant. The optimum dose for selection media was determined to be 15 mg L-1 of kanamycin. It was possible to obtain transgenic plants under both transformation systems. In the case of Agrobacterium tumefaciens, two hours of incubation, 48 h of co-cultivation, and optical density of 0.9 were used; while for bioballistics, the best conditions were 120 PSI of shot pressure, shot height at level 6 (16 cm), and vacuum pressure of 22 Hg mm, with particle inflow gun system (PIG). Results: Both systems produced complete transformants, chimeras, as well as those confirmed by histochemical X-GLUC and PCR analysis, producing a total of 14 positive plants by A. tumefaciens transformation from 26 trials and ten positive plants by the bioballistic system from 30 trials; a construction with chitinase and glucanase, NPT II selection gene and the GUS reporter gene were used. Findings/conclusions: So far, this has been the first report including integration of chitinase and glucanase genes.

scholarly journals Creation of transgenic sugar beet lines containing synthetic gene cry1C

1970 ◽  
pp. 221-225
Author(s):  
V. V. Kurylo ◽  
E. N. Shysha ◽  
A. I. Yemets
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Sugar Beet ◽  
Genetic Transformation ◽  
Marker Gene ◽  
Leaf Explants ◽  
Plant Genome ◽  
Direct Regeneration ◽  
Pcr Analysis ◽  
Cry Genes ◽  
Sugar Beet Crop

Aim. Insect pest’s impact makes a significant limitation of the sugar beet crop yield. Integration of cry-genes of Bacillus thuringiensis into the plant genome is one of the promising strategies to ensure of plant resistance. The aim of this work was the production of sugar beet lines (based on the MM1/2 line) expressing cry1C genes. Methods. Genetic transformation of sugar beet was performed using the method of co-cultivation of leaf explants with Agrobacterium tumefaciens. Results. Sugar beet line MM1/2 was transformed by Agrobacterium-mediated method of transformation using binary vector pRD400-cry1CST, containing synthetic cry1C gene and selectable marker gene neomycin phosphotransferase II (nptII) conferring resistance to kanamycin. After the optimization protocol of genetic transformation and direct regeneration from leaf discs a transgenic sugarbeet lines were obtained. Conclusions. PCR analysis revealed integration of cry1C into the genome of transgenic lines of B. vulgaris.Keywords: genetic transformation, Agrobacterium tumefaciens, Beta vulgaris, cry-genes.

scholarly journals Comparative Analysis of Different Nutrient Media for Growth of Agrobacterium tumefaciens under Small Volume Cultures

2021 ◽  
pp. 27-33
Author(s):  
Atif Iqbal ◽  
Nitish Dave ◽  
Tarun Kant
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Plant Species ◽  
Yeast Extract ◽  
Growth Pattern ◽  
Crop Improvement ◽  
Fast Growth ◽  
Growth Media ◽  
Differential Growth ◽  
Nutrient Media

Aims: To assess the effects of different selective complex growth media: Yeast Extract Peptone (YEP), Yeast Extract Mannitol (YEM), and Luria Bertani (LB) on growth and multiplication of three different strains of Agrobacterium tumefaciens. Place and Duration of Study: Molecular Biology Laboratory, Genetics and Tree Improvement Division, Arid Forest Research Institute, Jodhpur, Rajasthan, India, between July and December 2020. Methodology: Three strains of Agrobacterium (EHA105, GV3101 and LBA4404) were inoculated to grow on three different complex nutrient media (YEP, YEM, LB) supplemented with respective antibiotics for a period of 30 hours. Optical Density (O.D) at 600 nm was measured at every 3-hour interval to analyze differential growth pattern. Results: Though all the three nutrient media supported the growth of bacteria, YEP medium supported the fast growth of all strains of bacteria, LB medium was a little less efficient but comparable to YEP media. However, YEM medium proved the least supportive for bacterial growth among the three media types. Conclusion: Agrobacterium is used for genetic transformation of plant species and crop improvement. knowledge of the growth pattern of bacteria is necessary for the effective infection of plant material. We have analyzed that three strains of Agrobacterium on three different complex media, among the three media YEP media was supporting fast growth. This study can help the researchers to gain knowledge of the growth pattern of Agrobacterium to be used in the genetic transformation of plant species accordingly.

Transient genetic transformation of embryogenic callus of Cocos nucifera

Biologia ◽  
2011 ◽  
Vol 66 (5) ◽  
Author(s):  
Antonio Andrade-Torres ◽  
Carlos Oropeza ◽  
Luis Sáenz ◽  
Tomás González-Estrada ◽  
José Ramírez-Benítez ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Embryogenic Callus ◽  
Cocos Nucifera ◽  
Reporter Genes ◽  
Embryogenic Calli ◽  
In Vitro Morphogenesis ◽  
Practical Applications ◽  
Palm Species

AbstractCoconut palm (Cocos nucifera) is a plant species recalcitrant to in vitro morphogenesis and no protocols for the genetic transformation of coconut tissues have been published. The present study aimed to develop a protocol for genetic transformation of this palm species; evaluating reporter genes, transformation methods, and conditions for the use of antibiotics to select transformed plant cells. The gene gusA was first used for Agrobacterium tumefaciens mediated transformation of coconut embryogenic calli. However, endogenous GUS-like activity was found in calli not co-cultured with bacteria. Then essays for Agrobacterium-mediated transformation were developed using green and red fluorescent genes. Both genes are suitable as reporter genes for coconut transformation. In order to establish a protocol for coconut genetic transformation, an approach was used that combined biobalistics to generate micro-wounds in explants, vacuum infiltration and co-culture with Agrobacterium tumefaciens (C58C1 + pER10W-35SRed containing the embryogenesis related gene WUSCHEL). Calli treated with the combined protocol showed red fluorescence with greater intensity and greater area than calli treated with either biobalistics or infiltration, followed by bacteria co-culture. PCR amplification of DNA extracts from transformed embryogenic callus produced a band with the expected size using WUSCHEL primers (862 bp). No band was obtained using the VirE2 primers. This is the first report of transient genetic transformation of C. nucifera and it is the first step toward a protocol that will be useful for the study of the role of genes of interest and for practical applications, such as the improvement of coconut micropropagation via somatic embryogenesis.

Genetic transformation of Ginkgo biloba by Agrobacterium tumefaciens

2000 ◽  
Vol 108 (4) ◽  
pp. 413-419
Author(s):  
Patricia Dupré ◽  
Jerôme Lacoux ◽  
Godfrey Neutelings ◽  
Dominique Mattar-Laurain ◽  
Marc-André Fliniaux ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Ginkgo Biloba

scholarly journals Plant regeneration and transformation of Trachyspermum ammi using Agrobacterium tumefaciens and zygotic embryos

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Masoumeh Nomani ◽  
Masoud Tohidfar
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plant Regeneration ◽  
Genetic Transformation ◽  
Zygotic Embryo ◽  
Plant Biotechnology ◽  
Hplc Method ◽  
Zygotic Embryos ◽  
Naphthaleneacetic Acid ◽  
Transformed Plants ◽  
Trachyspermum Ammi

Abstract Background Trachyspermum ammi is one of the key medicinal plant species with many beneficial properties. Thymol is the most important substance in the essential oil of this plant. Thymol is a natural monoterpene phenol with high anti-microbial, anti-bacterial, and anti-oxidant properties. Thymol in the latest research has a significant impact on slowing the progression of cancer cells in human. In this research, embryos were employed as convenient explants for the fast and effectual regeneration and transformation of T. ammi. To regenerate this plant, Murashige and Skoog (MS) and Gamborg's B5 (B5) media were supplemented with diverse concentrations of plant growth regulators, such as 6-benzyladenine (BA), 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and kinetin (kin). Transgenic Trachyspermum ammi plants were also obtained using Agrobacterium-mediated transformation and zygotic embryos explants. Moreover, two Agrobacterium tumefaciens strains (EHA101 and LBA4404) harboring pBI121-TPS2 were utilized for genetic transformation to Trachyspermum ammi. Results According to the obtained results, the highest plant-regeneration frequency was obtained with B5 medium supplemented with 0.5 mg/l BA and 1 mg/l NAA. The integrated gene was also approved using the PCR reaction and the Southern blot method. Results also showed that the EHA101 strain outperformed another strain in inoculation time (30 s) and co-cultivation period (1 day) (transformation efficiency 19.29%). Furthermore, HPLC method demonstrated that the transformed plants contained a higher thymol level than non-transformed plants. Conclusions In this research, a fast protocol was introduced for the regeneration and transformation of Trachyspermum ammi, using zygotic embryo explants in 25–35 days. Our findings confirmed the increase in the thymol in the aerial part of Trachyspermum ammi. We further presented an efficacious technique for enhancing thymol content in Trachyspermum ammi using Agrobacterium-mediated plant transformation system that can be beneficial in genetic transformation and other plant biotechnology techniques.

Agrobacterium tumefaciens-mediated Transformation of Double Haploid Canola (Brassica napus) Lines

1997 ◽  
Vol 24 (1) ◽  
pp. 97 ◽  
Author(s):  
K. Kazan ◽  
M. D. Curtis ◽  
K. C. Goulter ◽  
J. M. Manners
Keyword(s):  
Brassica Napus ◽  
Agrobacterium Tumefaciens ◽  
Transgenic Plants ◽  
Genetic Transformation ◽  
Double Haploid ◽  
Gene Promoter ◽  
Root Explants ◽  
Hypocotyl Explants ◽  
Peroxidase Gene ◽  
Hypocotyl Segments

Double haploid (DH) genotypes of canola (Brassica napus L.) have a high level of genetic uniformity but have not been previously tested for genetic transformation. Transgenic plants from three of four DH genotypes derived from cv. Westar were obtained by inoculation of either hypocotyl segments or root explants with Agrobacterium tumefaciens. For hypocotyl transformation, A. tumefaciens strain LBA4404 containing a binary plasmid with the neomycin phosphotransferase gene (nptII) and a CaMV 35S-peroxidase gene cassette was co-cultivated with hypocotyl segments taken from the 5–6-day-old seedlings. Transformation frequencies for hypocotyl explants of two DH genotypes were 0.3–3%. Direct evidence for genetic transformation of hypocotyl explants was obtained through molecular hybridisation analysis. Using this protocol, mature transformed plants were obtained within 4–6 months of co-cultivation. A method of root transformation was successfully modified for one DH genotype of canola and transgenic plants were obtained at a frequency of 2%. Using this protocol, a peroxidase gene promoter–GUS fusion construct was introduced into a DH genotype. Tissue specific GUS expression driven by the peroxidase gene promoter in transgenic plants was analysed by GUS staining. Transformation systems for double haploid canola lines will permit the assessment of introduced genes for their effect on agronomic and physiological traits.

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