Development of Transgenic Papaya throughAgrobacterium-Mediated Transformation

Transgenic papaya plants were regenerated from hypocotyls and immature zygotic embryo after cocultivation withAgrobacterium tumefaciensLBA-4404 carrying a binary plasmid vector system containing neomycin phosphotransferase (nptII) gene as the selectable marker andβ-glucuronidase (GUS) as the reporter gene. The explants were co-cultivated withAgrobacterium tumefacienson regeneration medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime for one week. The cocultivated explants were transferred into the final selection medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime + 50 mg/L kanamycin for callus induction as well as plant regeneration. The callus derived from the hypocotyls ofCarica papayacv. Shahi showed the highest positive GUS activities compared toCarica papayacv. Ranchi. The transformed callus grew vigorously and formed embryos followed by transgenic plantlets successfully. The result of this study showed that the hypocotyls ofC. papayacv. Shahi andC. papayacv. Ranchi are better explants for genetic transformation compared to immature embryos. The transformedC. papayacv. Shahi also showed the maximum number of plant regeneration compared to that ofC. papayacv. Ranchi.

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

Journal of Genetic Engineering and Biotechnology ◽

10.1186/s43141-021-00173-8 ◽

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.

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INTRODUCTION OF β-GLUCURONIDASE GENE INTO GINSENG (PANAX CINSENG) BY A TI-PLASMID VECTOR SYSTEM AND ITS EXPRESSION

HortScience ◽

10.21273/hortsci.27.6.621e ◽

1992 ◽

Vol 27 (6) ◽

pp. 621e-621

Author(s):

Jang R. Liu ◽

Haeng S. Lee ◽

Suk W. Kim ◽

Hyo W. Lee

Keyword(s):

Somatic Embryos ◽

Binary Vector ◽

Zygotic Embryo ◽

Plasmid Vector ◽

Vector System ◽

Selection Marker ◽

35S Promoter ◽

Dichlorophenoxyacetic Acid ◽

Gus Gene ◽

2,4 Dichlorophenoxyacetic Acid

β-Glucuronidase (GUS) gene of Escherichia coli was introduced into ginseng cells by an Agrobacterium binary vector system and expressed in somatic embryos derived from the cells. A binary vector pBI121 carrying CaMV 35S promoter-GUS gene fusion and a neomycin phosphotransferase gene as selection marker was transferred into Agrobacterium tumefaciens LBA4404. Zygotic embryo cotyledonary segments were co-cultivated with A. tumefaciens and transferred to the medium containg 1 mg 2,4-dichlorophenoxyacetic acid/liter, 0.5 mg kinetin/liter, and 100 mg kanamycin/liter. Kanamycin-resistant calli were formed after 3 to 4 weeks of culture. Southern analysis confirmed the resistant calli were transformed with GUS gene. High GUS activities were detected in somatic embryos developed from the calli.

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Somatic Embryogenesis and Plant Regeneration from Zygotic Embryo in <i>Carica papaya</i> L., cv. Red-Lady

Plant ◽

10.11648/j.plant.20160406.11 ◽

2016 ◽

Vol 4 (6) ◽

pp. 45

Author(s):

Md. Humayun Kabir

Keyword(s):

Somatic Embryogenesis ◽

Plant Regeneration ◽

Carica Papaya ◽

Zygotic Embryo

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Effects of antibiotics on the elimination of Agrobacterium tumefaciens from loblolly pine (Pinus taeda) zygotic embryo explants and on transgenic plant regeneration

Plant Cell Tissue and Organ Culture (PCTOC) ◽

10.1007/s11240-004-4657-6 ◽

2004 ◽

Vol 79 (1) ◽

pp. 71-81 ◽

Cited By ~ 15

Author(s):

Wei Tang ◽

Hongsong Luo ◽

Ronald J. Newton

Keyword(s):

Agrobacterium Tumefaciens ◽

Plant Regeneration ◽

Transgenic Plant ◽

Pinus Taeda ◽

Loblolly Pine ◽

Zygotic Embryo

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Faculty Opinions recommendation of Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1007570.95457 ◽

2002 ◽

Author(s):

Elizabeth Hood

Keyword(s):

Agrobacterium Tumefaciens ◽

Binary Vector ◽

Vector System ◽

Maize Embryos

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Comparison of intron-dependent and intron-independent gene expression

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4395-4405.1988 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4395-4405

Author(s):

A R Buchman ◽

P Berg

Keyword(s):

Simian Virus 40 ◽

Plasmid Vector ◽

Fold Increase ◽

Simian Virus ◽

Transcription Unit ◽

Vector System ◽

Beta Globin ◽

Animal Cells ◽

Transcriptional Enhancer ◽

Kinase Gene

Recombinant simian virus 40 viruses carrying rabbit beta-globin cDNA failed to express the beta-globin sequence unless an intron was included in the transcription unit. The addition of either beta-globin IVS1 or IVS2 caused a 400-fold increase in RNA production. Stable beta-globin RNA production required sequences in IVS2 that were very close to the splice sites and that coincided with those needed for mRNA splicing. In addition to the recombinant viruses, intron-dependent expression was observed with both replicating and nonreplicating plasmid vectors in short-term transfections of cultured animal cells. Unlike transcriptional enhancer elements, IVS2 failed to increase stable RNA production when it was placed downstream of the polyadenylation site. Using a plasmid vector system to survey different inserted sequences for their dependence on introns for expression, we found that the presence of IVS2 stimulated the expression of these sequences 2- to 500-fold. Sequences from the transcribed region of the herpes simplex virus thymidine kinase gene, a gene that lacks an intervening sequence, permitted substantial intron-independent expression (greater than 100-fold increase) in the plasmid vector system.

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