The bar Gene as Selectable and Screenable Marker in Plant Engineering

D’Halluin, Kathleen; Block, Marc De; Denecke, Jürgen; Janssens, Jan; Leemans, J.; Reynaerts, A.; Botterman, Johan

doi:10.1016/b978-0-12-765561-1.50017-5

Cited by 39 publications

(55 citation statements)

References 28 publications

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“…The efficacy of PPT as a selection marker and bar as a resistance mechanism has resulted in bar being widely exploited in the construction and selection of transgenic plant lines. Similar to our observations, bar gene, which provides resistance to herbicide PPT, has been used successfully as a selectable marker in several plants [27] .…”

Section: The Impact Of Selection Agentsupporting

confidence: 87%

Transgenic Indian Cotton (Gossypium hirsutum) Harboring Rice Chitinase Gene (Chi II) Confers Resistance to Two Fungal Pathogens

Ganesan¹,

Bhanumathi²,

Kumari³

et al. 2009

American J. of Biochemistry and Biotechnology

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Problem statement: The present investigation described a simple and reproducible protocol for transgenic cotton regeneration and characterization of chitinase (Chi II) gene expression against two different fungal pathogens in cotton. Approach: Transgenic cotton (Gossypium hirsutum cv. SVPR2) plants were produced by pCambia-bar-Chi II (13.8 kb) under the control of the CaMV 35S promoter, harbored in the strain LBA 4404 Agrobacterium tumefaciens by using shoot tip explants. Results: Finally, from the 10 experiments, 21.8% of transformation frequency was recorded. Segregation ratio of 3:1 was recorded in the T 0 plant seeds. Polymerase chain reaction and southern blotting analysis were used to confirm the integration of Chi II transgene in the T 0 plants genome of putative transgenics. Quantitiave and qualitative (SDS-PAGE) analyses were also carried out to confirm the expression of chitinase enzyme in T 0 plants. Further, randomly selected transgenic plants (T 0 ) were analyzed for disease tolerance by evaluating them with spores of Fusarium oxysporum and Alternaria macrospora. All the selected PCR positive plants showed enhanced disease resistance against Fusarium wilt. The plants selected randomly showed an enhanced survival rate compared with the control when they were grown in earthen pots inoculated with 1×10 5 spores 100 −1 g of soil mixture.Another four randomly selected plantlets were sprayed with spores of Alternaria macrospora in order to test their tolerance to Alternaria leaf spot disease. After 20 days of culture, the number of lesions per leaf and the lesion length per leaf spot in non-transferred leaves increased. In the case of transgenic plantlets, lesion formation was completely absent. Conclusion: The disease resistance against Fusarium wilt and Alternaria leaf spot in cotton strains would serve as good breeding materials for producing fungal disease resistant cotton varieties.

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Section: The Impact Of Selection Agentsupporting

confidence: 87%

Transgenic Indian Cotton (Gossypium hirsutum) Harboring Rice Chitinase Gene (Chi II) Confers Resistance to Two Fungal Pathogens

Ganesan¹,

Bhanumathi²,

Kumari³

et al. 2009

American J. of Biochemistry and Biotechnology

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“…The coding region of a synthetic bar gene (D'Halluin et al, 1992) was ampli®ed by PCR with the primers PPT 1 (5¢-GTC ATG AGT CCG GAG AGG AGA CCA) and PPT 5(5¢-GCA GGA TCC ATC ATA TCT GGG TAA CTG) and cloned into the Sma 1 site of pUC 18. After sequence veri®cation and determination of the correct orientation, the bar gene was isolated as a Sac I/Hind III fragment, gel-puri®ed (Hansen et al, 1993) and ligated into the Sac I/Hind III site of the vector pHB 234 (nos terminator in pUC 18).…”

Section: Cloning Of the Bar-constructsmentioning

confidence: 99%

Isolation and characterization of signal transduction mutants of Arabidopsis thaliana that constitutively activate the octadecanoid pathway and form necrotic microlesions

et al. 2001

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Summary Thionins are a group of antimicrobial polypeptides that form part of the plant's defense mechanism against pathogens. The Thi 2.1 thionin gene of Arabidopsis thaliana has been shown to be inducible by jasmonic acid (JA), an oxylipin‐like hormone derived from oxygenated linolenic acid and synthesized via the octadecanoid pathway. The JA‐dependent regulation of the Thi 2.1 gene has been exploited for setting up a genetic screen for the isolation of signal transduction mutants that constitutively express the Thi 2.1 gene. Ten cet‐mutants have been isolated which showed a constitutive expression of the thionin gene. Allelism tests revealed that they represent at least five different loci. Some mutants are dominant, others recessive, but all cet mutations behaved as monogenic traits when backcrossed with Thi 2.1‐GUS plants. Some of the mutants overproduce JA and its bioactive precursor 12‐oxophytodienoic acid (OPDA) up to 40‐fold while others have the same low levels as the control wildtype plants. Two of the mutants showed a strong induction of both the salicylic acid (SA)‐ and the JA‐dependent signaling pathways, while the majority seems to be affected only in the octadecanoid pathway. The Thi 2.1 thionin gene and the Pdf 1.2 defensin gene are activated independently, though both are regulated by JA. The cet‐mutants, except for one, also show a spontaneous leaf cell necrosis, a reaction often associated with the systemic acquired resistance (SAR) pathway.

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“…In general, Agrobacteriummediated transformation is simpler, more efficient, and less expensive compared to other systems and also results in a low copy number of insertions. Sugar beet is highly susceptible in vitro to A. tumefaciens transformation (Krens et al, 1988;Lindsey and Gallois, 1990;D'Halluin et al, 1992b;Jacq et al, 1993;Zakharchenko et al, 2000) and susceptibility can be improved by preculturing explants before inoculation (Krens et al, 1996). Attempts have been made to develop transgenic sugar beet plants resistant to fungi; for example, a chitinase gene from pumpkin was transferred into sugar beet and suppression of disease symptoms caused by R. solani was detected in the transgenic plants (Hashimoto and Shimamoto, 2001;Gurel et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Generation of transgenic sugar beet (Beta vulgarism L.) overexpressing the polygalacturonase inhibiting protein 1 of Phaseolus vulgaris (PvPGIP1) through Agrobacterium-mediated transformation

et al. 2015

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Numerous diseases caused by fungal pathogens influence the annual production of sugar beet. In order to obtain a plant resistant to fungi, genetic transformation has been applied to the sugar beet. To invade a plant tissue, phytopathogenic fungi produce several cell wall-degrading enzymes (CWDEs); polygalacturonases (PGs) are pathogenicity factors produced at the earlier stages of a fungal infection that depolymerize the homogalacturonan. One of the strategies used by plants to limit the degradation of the cell wall polysaccharides by fungal CWDEs is the production of proteinaceous inhibitors. Against fungal, microbial, and insect PGs, plants produce cell wall-associated polygalacturonase-inhibiting proteins (PGIPs). The overexpression of PGIPs improves the resistance to fungal and bacterial necrotrophs in different plants. In this research, the gene encoding the PGIP1 fused downstream of the leader sequence for secretion in the extracellular environment was isolated from Phaseolus vulgaris and cloned into the expression vector pBI121 for the Agrobacterium-mediated transformation of sugar beet. Modified transformation protocol and selection strategies were developed. In comparison with the preexisting methods, the transformation efficiency was increased and different cultivars were transformed, highlighting the general effectiveness of the method applied. The presence of the transgene and the activity of PvPGIP1 were confirmed by PCR and agarose diffusion assay analyses, respectively, and the present and copy number of the transgene in the T0 plants' genome were demonstrated by Southern blot.

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The bar Gene as Selectable and Screenable Marker in Plant Engineering

Cited by 39 publications

References 28 publications

Transgenic Indian Cotton (Gossypium hirsutum) Harboring Rice Chitinase Gene (Chi II) Confers Resistance to Two Fungal Pathogens

Transgenic Indian Cotton (Gossypium hirsutum) Harboring Rice Chitinase Gene (Chi II) Confers Resistance to Two Fungal Pathogens

Isolation and characterization of signal transduction mutants of Arabidopsis thaliana that constitutively activate the octadecanoid pathway and form necrotic microlesions

Generation of transgenic sugar beet (Beta vulgarism L.) overexpressing the polygalacturonase inhibiting protein 1 of Phaseolus vulgaris (PvPGIP1) through Agrobacterium-mediated transformation

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