Optimization of Agrobacterium tumefaciens-Mediated Genetic Transformation of Maize

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.

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

Functional Plant Biology ◽

10.1071/pp96024 ◽

1997 ◽

Vol 24 (1) ◽

pp. 97 ◽

Cited By ~ 4

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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Agrobacterium tumefaciens-mediated genetic transformation and production of stable transgenic pearl millet (Pennisetum glaucum [L.] R. Br.)

In Vitro Cellular & Developmental Biology - Plant ◽

10.1007/s11627-013-9592-y ◽

2014 ◽

Vol 50 (4) ◽

pp. 392-400 ◽

Cited By ~ 11

Author(s):

R. Ramadevi ◽

K. V. Rao ◽

V. D. Reddy

Keyword(s):

Agrobacterium Tumefaciens ◽

Genetic Transformation ◽

Pearl Millet ◽

Pennisetum Glaucum ◽

Transgenic Pearl Millet

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Agrobacterium tumefaciens-mediated genetic transformation of barley (Hordeum vulgare L.)

Plant Science ◽

10.1016/j.plantsci.2006.09.005 ◽

2007 ◽

Vol 172 (2) ◽

pp. 281-290 ◽

Cited By ~ 48

Author(s):

Ashok K. Shrawat ◽

Dirk Becker ◽

Horst Lörz

Keyword(s):

Agrobacterium Tumefaciens ◽

Hordeum Vulgare ◽

Genetic Transformation ◽

Hordeum Vulgare L

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Agrobacterium tumefaciens-transient genetic transformation of Habanero pepper (Capsicum chinense Jacq.) leaf explants

Electronic Journal of Biotechnology ◽

10.2225/vol13-issue4-fulltext-10 ◽

2010 ◽

Vol 13 (4) ◽

Cited By ~ 3

Author(s):

Guadalupe Fabiola Arcos-Ortega ◽

Rafael Antonio Chan-Kuuk ◽

Wilma Aracely González-Kantún ◽

Ramón Souza-Perera ◽

Yumi Elena Nakazawa-Ueji ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Genetic Transformation ◽

Leaf Explants ◽

Capsicum Chinense ◽

Habanero Pepper

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

KnE Life Sciences ◽

10.18502/kls.v2i6.1082 ◽

2017 ◽

Vol 2 (6) ◽

pp. 599 ◽

Cited By ~ 1

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.

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