Agrobacterium tumefaciens-mediated transformation of Eucalyptus urophylla clone BRS07-01

Bettencourt, Gisela Manuela de França; Soccol, Carlos Ricardo; Giovanella, Thais Salete; Franciscon, Luziane; Kestring, Daiane Rigoni; Gerhardt, Isabel Rodrigues; Degenhardt, Juliana

doi:10.1007/s11676-018-0777-4

Cited by 8 publications

(6 citation statements)

References 58 publications

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“…Eucalyptus species are recalcitrant to in vitro organogenesis, and even more to genetic transformation, so much effort was required to obtain a few events [ 3 ]. As eucalyptus explants are prone to browning during the tissue culture process, it is more challenging to implement eucalyptus transgenes that were derived from a tissue culture.…”

Section: Discussionmentioning

confidence: 99%

“…A successful transgenic breeding program begins with the screening of promoters with excellent transcription characteristics in eucalyptus cells to drive the expression of the target gene. As a woody plant, eucalyptus is difficult to obtain genetically modified plants that will be stable [ 3 ]. Therefore, the study of woody plant promoters was frequently conducted using model plants [ 4 ].…”

Section: Introductionmentioning

confidence: 99%

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Expression Activity of Artificial Promoters for Disease Resistance in Transgenic Eucalyptus urophylla

Huang

Fang

et al. 2022

Genes

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The transcriptional properties of artificial promoters are closely related to the type and arrangement position of cis-elements. GWSF (374-bp) was an effective SPIP with four cis-element dimers. There were four pathogen-inducible cis-elements in the GWSF promoter (GST1-boxes, W-boxes, S-boxes, and F-boxes) and a minimal cauliflower mosaic virus 35S promoter. V-element dimers were inserted into the upstream (VGWSF), midstream (GWVSF), and downstream (GWSFV) regions of the original GWSF promoter sequence to examine their affect on the position. The expression activity of promoters was analyzed and estimated using the histochemical staining of leaf discs of eucalyptus with transient expression, an image digitization method to extract the color features, and the induction treatment by a plant pathogenic microorganism/inducer and qPCR assays. The histochemical staining results of the adventitious buds indicated that the promoters had been successfully integrated into the E.urophylla genome and that they drove the expression of the gus gene. There was a noticeable difference in the intensity of color between the adventitious buds on the same callus block, as well as the intensity of color within the same adventitious bud. According to the established two-factor model of blue value, there was a greater difference between the levels of the genotype factor than the promoter factor in eucalyptus leaf discs. Further, the basal and inducible transcriptional levels of the three improved promoters were investigated by qPCR. With the basal transcriptional level of the GWSF promoter normalized to one, the relative basal levels of VGWSF, GWVSF, and GWSFV were 1.40, 1.45, and 4.15, respectively. The qPCR results were consistent with the staining results of GUS histochemical staining. The three improved promoters all had the properties of being induced by salicylic acid, Ralstonia solanacearum, and Phytophthora capsici. The three improved promoters demonstrated a significantly higher TMV induction activity: their induction activity from high to low was GWSFV > GWVSF > VGWSF. The findings will be beneficial to the construction and optimization of artificial promoters for transgenic plants.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Expression Activity of Artificial Promoters for Disease Resistance in Transgenic Eucalyptus urophylla

Huang

Fang

et al. 2022

Genes

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“…RAPD analysis has been widely used to characterize somaclonal variation in plantlets in vitro because it is affordable and useful for detecting variations in vegetable crops [ 31 ]. In the present study, the RAPD markers OPD-08 and OPA-13 allowed the amplification of 17 fragments from all genotypes analyzed, with sizes between 250 and 4000 bp.…”

Section: Main Textmentioning

confidence: 99%

Efficient direct shoot organogenesis and genetic stability in micropropagated sacha inchi (Plukenetia volubilis L.)

et al. 2020

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Objective It is necessary to improve biotech platforms based on in vitro cell tissue culture to support sacha inchi ( Plukenetia volubilis L.) research programs and draw on the nutritional value of the high polyunsaturated fatty acid content of its oilseed. Here, we developed a rapid and efficient method for induction and direct in vitro shoot development for this species. Results Shoots were generated from hypocotyl explants. The highest organogenic response was obtained in woody plant medium supplemented with 1 mg/L thidiazuron and 0.5 mg/L zeatin supplemented with L-glutamine, adenine hemisulfate, and L-arginine. Shoots obtained using this medium were transferred and subcultivated with different concentrations of indole-3-butyric acid and 1-naphthylacetic acid for rooting. For the first time, a histological analysis was performed supporting direct organogenic development in this species. The plantlets obtained were transferred ex vitro with a survival percentage of 80%. The genetic stability of the plants recovered was confirmed by randomly amplified polymorphic DNA analysis. All results indicate that it would be possible to stimulate direct shoot formation from hypocotyls to support the sustainable use of this species.

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“…Many efforts have been made to establish regeneration systems by modulating concentrations of exogenous auxin and cytokinin ( Hajari et al., 2006 ; Girijashankar, 2012 ; Shwe and Leung, 2020 ). Thidiazuron, as an important cytokinin, has also been widely used in Eucalyptus regeneration ( Cid et al., 1999 ; Shabannejad Mamaghani et al., 2009 ; de França Bettencourt et al., 2020 ). Recently, N -phenyl- N’ -[6-(2-chlorobenzothiazol)-yl] urea (PBU) was attempted to promote regeneration in Eucalyptus ( Ouyang and Li, 2016 ).…”

Section: Introductionmentioning

confidence: 99%

“…Agrobacterium tumefaciens -mediated transformation was the most commonly utilized method in plants. This transformation protocol was also applied for various Eucalyptus species ( Prakash and Gurumurthi, 2009 ; Aggarwal et al., 2011 ; de Alcantara et al., 2011 ; Silva et al., 2011 ; de França Bettencourt et al., 2020 ). The transformation efficiencies were influenced by a variety of factors including Agrobacterium strain, bacterial concentration, infection time, and precultivation time.…”

Section: Introductionmentioning

confidence: 99%

Agrobacterium-mediated genetic transformation of the most widely cultivated superior clone Eucalyptus urophylla × Eucalyptus grandis DH32-29 in Southern China

et al. 2022

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Eucalyptus, as an economically important species for wood and paper industries, still remains a challenge to genetic improvement by transgenic technology owing to the deficiency of a highly efficient and stable genetic transformation system, especially in cultivated superior clones. Eucalyptus urophylla × Eucalyptus grandis clone DH32-29 is most widely planted in southern China, but it is relatively recalcitrant to adventitious bud regeneration, which blocks the establishment of a genetic transformation system. Here, an efficient adventitious bud regeneration and transformation system of Eucalyptus was established using E. urophylla × E. grandis DH32-29 as material. The in vitro leaves from microshoots that were subcultured for 20–25 days were immersed into liquid Woody Plant Medium supplemented with 0.02 mg·L−1 of α-naphthaleneacetic acid (NAA) and 0.24 mg·L−1 of forchlorfenuron (callus-inducing medium (CIM)). After 15 days, explants were transferred to a medium containing 0.10 mg·L−1 of NAA and 0.50 mg·L−1 of 6-benzyladenine (shoot-inducing medium (SIM)) for adventitious bud induction. The highest regeneration efficiency of adventitious buds was 76.5%. Therefore, an Agrobacterium tumefaciens-mediated genetic transformation system was optimized. The leaves were precultured for 7 days and infected for 30 min with A. tumefaciens strain EHA105 grown to a bacterial density of 0.3 (OD600). After 72 h of cocultivation in the dark, leaves were transferred to CIM supplemented with 100 mg·L−1 of cefotaxime (Cef), 100 mg·L−1 of timentin, and 15 mg·L−1 of kanamycin (Kan) for 15 days to induce calluses. Then, the explants were transferred to SIM supplemented with the same concentration of antibiotics, and the fresh medium was replaced every 15 days until resistant adventitious buds appeared. After induction of roots in root-inducing medium supplemented with 200 mg·L−1 of Cef and 75 mg·L−1 of Kan, completely transgenic plants were obtained. With the use of the aforementioned method, the transformation frequency can reach 1.9%. This provides a powerful approach for genetic improvement of E. urophylla × E. grandis DH32-29 and gene function analysis in Eucalyptus.

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Agrobacterium tumefaciens-mediated transformation of Eucalyptus urophylla clone BRS07-01

Cited by 8 publications

References 58 publications

Expression Activity of Artificial Promoters for Disease Resistance in Transgenic Eucalyptus urophylla

Expression Activity of Artificial Promoters for Disease Resistance in Transgenic Eucalyptus urophylla

Efficient direct shoot organogenesis and genetic stability in micropropagated sacha inchi (Plukenetia volubilis L.)

Agrobacterium-mediated genetic transformation of the most widely cultivated superior clone Eucalyptus urophylla × Eucalyptus grandis DH32-29 in Southern China

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