Inheritance of NmDef02 synthetic defensin gene and hpt selectable marker in four successive generations of transgenic rice (Oryza sativa, cv. J-104)

The evaluation of the inheritance and stability of the transgenes is essential for the application of transgenic plants in agriculture. In this work, we studied the inheritance of two transgenes in T1, T2, T3 and T4 rice progenies. Transgenic rice plants (cv. J-104) was obtained by biolistic method using a synthetic defensin gene (NmDef02) and hpt as selectable marker gene, co-transformed in a 4:1 proportion, respectively. Regenerated plants were acclimated under natural conditions. The study started from the primary transformants that fulfilled the agronomic characters reported by the experts for the J-104 rice cultivar in the maturation stage. The integration and relative expression of NmDef02 in T1 plants was verified by Southern blot and qRT-PCR, respectively. The inheritance of transgenes over four generations was analyzed by PCR. The following transgene combinations were identified: NmDef02(+)hpt(+), NmDef02(+)hpt(-) and NmDef02(-)hpt(-). The most advantageous combination was NmDef02(+)hpt(-), which corresponded to the marker-free plants harboring the gene of interest. The inheritance of NmDef02 was confirmed in T1 and T2 progenies, but in some T3 and T4 lines the loss of this gene was verified. This inheritance analysis provided information concerning the transgenic population, but the mechanisms that destabilize the inheritance of the gene of interest will be the goal of future research.

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Breeding of Selectable Marker-Free Transgenic Rice Lines Containing AP1 Gene with Enhanced Disease Resistance

Agricultural Sciences in China ◽

10.1016/s1671-2927(06)60128-4 ◽

2006 ◽

Vol 5 (11) ◽

pp. 805-811 ◽

Cited By ~ 8

Author(s):

Heng-xiu YU ◽

Qiao-quan LIU ◽

Ling WANG ◽

Zhi-peng ZHAO ◽

Li XU ◽

...

Keyword(s):

Disease Resistance ◽

Transgenic Rice ◽

Selectable Marker ◽

Marker Free

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Excision of a selectable marker in transgenic lily (Sorbonne) using the Cre/loxP DNA excision system

Canadian Journal of Plant Science ◽

10.4141/cjps2013-037 ◽

2013 ◽

Vol 93 (5) ◽

pp. 903-912

Author(s):

Sh. Li ◽

Y.-P. Du ◽

Zh.-Y. Wu ◽

C.-L. Huang ◽

X.-H. Zhang ◽

...

Keyword(s):

Southern Blotting ◽

Molecular Detection ◽

Selectable Marker ◽

Marker Gene ◽

Inducible Promoter ◽

Transformation Rate ◽

Site Specific ◽

Marker Free ◽

Drought And Salt Stresses ◽

Excision Rate

Li, S. H., Du, Y.-P., Wu, Z. H.-Y., Huang, C.-L., Zhang, X.-H., Wang, Z. H.-X. and Jia, G.-X. 2013. Excision of a selectable marker in transgenic lily (Sorbonne) using the Cre/loxP DNA excision system. Can. J. Plant Sci. 93: 903–912. To generate transgenic lily plants with no selectable marker and improved tolerance to abiotic stress, two vectors were co-transformed into the Lilium oriental hybrid Sorbonne by particle bombardment. The pKSB vector included the Cre/loxp-mediated site-specific cDNA excision system under control of the inducible promoter rd29A, and the pBPC-P5CS-F129A vector carried the P5CS gene, which we hypothesized would improve resistance to drought and salt stresses in transgenic lily plantlets. The presence of the two genes was simultaneously detected by PCR and Southern blotting in two resistant plantlets. The co-transformation rate was 0.16%. Subsequently, inducer expression was tested under varying conditions to optimize the deletion of marker gene. Results from molecular detection assays revealed that maintaining bases of bulblet scales at 4°C for 12 h resulted in an increase in the excision rate, reaching 60%. Expression of P5CS improved resistance to salt stress in transgenic lily plantlets. These results demonstrated the feasibility of using the Cre/loxP-based marker elimination system to generate marker-free transgenic plantlets with improved stress tolerance.

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Generation of selectable marker-free transgenic rice using double right-border (DRB) binary vectors

Functional Plant Biology ◽

10.1071/pp00129 ◽

2001 ◽

Vol 28 (3) ◽

pp. 241 ◽

Cited By ~ 8

Author(s):

Hui-Juan Lu ◽

Xue-Rong Zhou ◽

Zhu-Xun Gong ◽

Narayana M. Upadhyaya

Keyword(s):

Selectable Marker ◽

Binary Vector ◽

Marker Gene ◽

Marker Genes ◽

Binary Vectors ◽

Selectable Marker Genes ◽

Right Border ◽

Marker Free ◽

Dna Vectors ◽

Transgenic Progeny

Currently employed transformation systems require selectable marker genes encoding antibiotic or herbicide resistance, along with the gene of interest (GOI), to select transformed cells from among a large population of untransformed cells. The continued presence of these selectable markers, especially in food crops such as rice (Oryza sativa L.), is of increasing public concern. Techniques based on DNA recombination and Agrobacterium-mediated co-transformation with two binary vectors in a single or two different Agrobacterium strains, or with super-binary vectors carrying two sets of T-DNA border sequences (twin T-DNA vectors), have been employed by researchers to produce selectable marker-free (SMF) transgenic progeny. We have developed a double right-border (DRB) binary vector carrying two copies of T-DNA right-border (RB) sequences flanking a selectable marker gene, followed by a GOI and one copy of the left border sequence. Two types of T-DNA inserts, one initiated from the first RB containing both the selectable gene and the GOI, and the other from the second RB containing only the GOI, were expected to be produced and integrated into the genome. In the subsequent generation, these inserts could segregate away from each other, allowing the selection of the progeny with only the GOI. We tested this vector using two selectable marker genes and successfully obtained progeny plants in which the second selectable marker gene segregated away from the first. Using the DRB binary vector system, we recovered SMF transgenic lines containing a rice ragged stunt virus (RRSV)-derived synthetic resistance gene in the rice cultivars Jarrah and Xiu Shui. Approximately 36–64% of the primary transformants of these cultivars yielded SMF progeny. Among SMF Jarrah transgenic progeny <50% of plants contained the RRSV transgene. Thus, we have developed an efficient vector for producing SMF plants that allows straightforward cloning of any GOIs in comparison with the published ‘twin T-DNA’ vectors.

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Rapid Generation of Selectable Marker-Free Transgenic Rice with Three Target Genes by Co-Transformation and Anther Culture

Rice Science ◽

10.1016/s1672-6308(08)60001-3 ◽

2007 ◽

Vol 14 (4) ◽

pp. 239-246 ◽

Cited By ~ 8

Author(s):

Li ZHU ◽

Ya-ping FU ◽

Wen-zhen LIU ◽

Guo-cheng HU ◽

Hua-min SI ◽

...

Keyword(s):

Anther Culture ◽

Transgenic Rice ◽

Target Genes ◽

Selectable Marker ◽

Marker Free ◽

Rapid Generation

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Production of selectable marker gene-free Cavendish banana (Musa spp.) using a steroid-inducible recombinase platform

Transgenic Research ◽

10.1007/s11248-019-00179-6 ◽

2019 ◽

Vol 29 (1) ◽

pp. 81-93 ◽

Cited By ~ 1

Author(s):

Jennifer Kleidon ◽

Anthony Brinin ◽

Jean-Yves Paul ◽

Robert Harding ◽

James Dale ◽

...

Keyword(s):

Fluorescent Protein ◽

Selectable Marker ◽

Marker Gene ◽

Single Copy ◽

Selectable Marker Gene ◽

New Genes ◽

Cavendish Banana ◽

Marker Free ◽

Green Fluorescent ◽

Regulatory Acceptance

Abstract Genetic improvement of commercially accepted banana cultivars is strongly reliant on the ability to introduce genes that encode important agro-traits such as disease resistance. In most cases this can only be achieved using a transgenic approach. Public and regulatory acceptance of these events would greatly increase with “clean” single copy integration events free of the selectable marker gene and extraneous vector backbone. This would also allow for the successive addition of new genes and traits as they become available. In this study, we used the pMarker Free 1 (pMF1) vector containing the green fluorescent protein (gfp) reporter gene to assess the effectiveness of steroid-inducible recombination and positive/negative dual selection to regenerate transgenic Cavendish banana plants that were potentially free of the selectable marker gene. By examining the interaction of two different Agrobacterium strains with two different cultivars of Cavendish banana, namely Williams and Grand Naine, we describe a transformation and regeneration strategy that successfully produced marker-free, single transgene copy, gfp-expressing events. The system will provide a useful means of serially improving banana into the future.

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