Effect of explants, bacterial cell density and overgrowth-control antibiotics on transformation efficiency in tomato (Solanum lycopersicum L.)

Pawar, B. D.; Jadhav, A. S.; Kale, A. A.; Chimote, V. P.; Pawar, S. V.

doi:10.37855/jah.2013.v15i02.17

Cited by 12 publications

(6 citation statements)

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“…Prior to the tomato transformation, 5 mL of LB broth with 50 μg/mL kanamycin and 50 μg/mL spectinomycin was inoculated with A. tumefaciens and incubated at 28°C on a shaker at 200 rpm for two days, then 1 mL was subcultured in 100 mL of LB broth containing the same antibiotics and incubated for about 24 h under the same conditions until OD 600 reached 1. The bacteria were pelleted and resuspended in liquid MS medium [44] to OD 600 = 0.2 [45] and used for the tomato transformation.…”

Section: Plasmid Construct and Agrobacterium Preparationmentioning

confidence: 99%

“…Tomato seeds were surface sterilized with 75% ethanol for 1 min and 15% hydrogen peroxide for 15 min, rinsed with water, germinated on MS medium (4.3 g/L MS salts (Sigma-Aldrich, USA), 3% sucrose, 0.8% agar, pH 5.8) and cultivated at 22°C in a 16 h light/8 h dark regimen for 20 days or until the cotyledons had opened completely. Three days before transformation the hypocotyls were cut into 7-10 mm explants and placed on preculture medium (MS salts, 3% sucrose, vitamins, 0.5 mg/ L indole-3-acetic acid (IAA), 1 mg/L benzylaminopurine (BAP), 0.7% agar, pH 5.8) [46] and incubated for 72 h in the dark at 27°C [44,45]. For transformation, all the explants were immersed in the Agrobacterium suspension and shaken for 20 min at room temperature, blotted dry and transferred to the co-cultivation medium (MS salts, vitamins, 3% sucrose, 0.7% agar, 0.5 mg/L IAA and 1 mg/L BAP) and incubated for two days in the dark at room temperature [44,46].…”

Section: Plant Tissue and Transformation By Agrobacteriummentioning

confidence: 99%

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Pinpointing regulatory protein phosphatase 2A subunits involved in beneficial symbiosis between plants and microbes

et al. 2021

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Background PROTEIN PHOSPHATASE 2A (PP2A) expression is crucial for the symbiotic association between plants and various microbes, and knowledge on these symbiotic processes is important for sustainable agriculture. Here we tested the hypothesis that PP2A regulatory subunits, especially B’φ and B’θ, are involved in signalling between plants and mycorrhizal fungi or plant-growth promoting bacteria. Results Treatment of tomato plants (Solanum lycopersicum) with the plant growth-promoting rhizobacteria (PGPR) Azospirillum brasilense and Pseudomonas simiae indicated a role for the PP2A B’θ subunit in responses to PGPR. Arbuscular mycorrhizal fungi influenced B’θ transcript levels in soil-grown plants with canonical arbuscular mycorrhizae. In plant roots, transcripts of B’φ were scarce under all conditions tested and at a lower level than all other PP2A subunit transcripts. In transformed tomato plants with 10-fold enhanced B’φ expression, mycorrhization frequency was decreased in vermiculite-grown plants. Furthermore, the high B’φ expression was related to abscisic acid and gibberellic acid responses known to be involved in plant growth and mycorrhization. B’φ overexpressor plants showed less vigorous growth, and although fruits were normal size, the number of seeds per fruit was reduced by 60% compared to the original cultivar. Conclusions Expression of the B’θ gene in tomato roots is strongly influenced by beneficial microbes. Analysis of B’φ overexpressor tomato plants and established tomato cultivars substantiated a function of B’φ in growth and development in addition to a role in mycorrhization.

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Section: Plasmid Construct and Agrobacterium Preparationmentioning

confidence: 99%

Section: Plant Tissue and Transformation By Agrobacteriummentioning

confidence: 99%

Pinpointing regulatory protein phosphatase 2A subunits involved in beneficial symbiosis between plants and microbes

et al. 2021

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show abstract

“…Prior to the tomato transformation, 5 mL of LB broth with 50 µg/mL kanamycin and 50 µg/mL spectinomycin was inoculated with A. tumefaciens and incubated at 28°C on a shaker at 200 rpm for two days, then1 mL of the culture was subcultured in 100 mL of LB broth containing the same antibiotics and incubated for about 24 h under the same conditions until OD 600 reached 1. The bacteria were pelleted and resuspended in liquid MS medium (44) to OD 600 = 0.2 (45) and used for the tomato transformation.…”

Section: Plasmid Construct and Agrobacterium Preparationmentioning

confidence: 99%

“…Tomato seeds were surface sterilized with 75% ethanol for 1 min and 15% hydrogen peroxide for 15 min, rinsed with water, germinated on MS medium (4.3 g/L MS salts (Sigma-Aldrich, USA), 3% sucrose, 0.8 % agar, pH 5.8) and cultivated at 22°C in a 16 h light/8 h dark regimen for 20 days or until the cotyledons had opened completely. Three days before transformation the hypocotyls were cut into 7-10 mm fragments (explants) and placed on pre-culture medium (MS salts, 3 % sucrose, vitamins, 0.5mg/L indole-3-acetic acid (IAA), 1 mg/L benzylaminopurine (BAP), 0.7% agar, pH 5.8) ( 46) and incubated for 72 h in the dark at 27°C (44,45). For transformation, all the explants were immersed in the Agrobacterium suspension and shaken for 20 min at room temperature, blotted dry and transferred to the co-cultivation medium (MS salts, vitamins, 3% sucrose, 0.7 % agar, 0.5 mg/L IAA and 1 mg/L BAP) and incubated for two days in the dark at room temperature (44,46).…”

Section: Plant Tissue and Transformation Byagrobacteriummentioning

confidence: 99%

Pinpointing Regulatory Protein Phosphatase 2A Subunits Involved in Beneficial Symbiosis Between Plants and Microbes

Averkina

Harris

Asare

et al. 2021

Preprint

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Background PROTEIN PHOSPHATASE 2A (PP2A) expression is crucial for the symbiotic association between plants and various microbes, and knowledge on these symbiotic processes is important for sustainable agriculture. Here we tested the hypothesis that PP2A regulatory subunits, especially B’φ, are involved in signalling between plants and mycorrhizal fungi or plant-growth promoting bacteria. Results Treatment of tomato plants (Solanum lycopersicum) with the plant growth-promoting rhizobacteria (PGPR) Azospirillum brasilense and Pseudomonas simiae indicated a role for the PP2A B’θ subunit in responses to PGPR. Arbuscular mycorrhizal fungi also influenced B’q transcript levels, but only in soil-grown plants with canonical arbuscular mycorrhizae, not in vermiculite-grown plants which had only vesicular mycorrhizae. In plant roots, transcripts of B’φ were scarce under all conditions tested and at a lower level than all other PP2A subunit transcripts. In transformed tomato plants with 10-fold enhanced B’φ expression, mycorrhization frequency was decreased in vermiculite-grown plants. Furthermore, the high B’φ expression was related to abscisic acid and gibberellic acid responses known to be involved in plant growth and mycorrhization. B’φ overexpressor plants showed less vigorous growth, and although fruits were normal size, the number of seeds per fruit was reduced by 60% compared to the original cultivar. Conclusions Expression of the B’θ gene in plant roots is strongly influenced by beneficial microbes. Expression analysis and phenotype observations of established tomato cultivars and B’φ overexpressor plants substantiate a function of B’φ in growth and development in addition to a role in mycorrhization.

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“…To determine the adequate number of A. tumefaciens cells for smearing on the floral buds, we investigated the transformation efficiency when a series of bacterial concentrations was used. For the leaf-disk method for tomato transformation, it has been reported that the concentration of A. tumefaciens cells may strongly affect the transformation efficiency, and that the highest efficiency was obtained when a cell suspension with OD 600 =0.2 was used (Pawar et al 2013). In this study, we determined the influence of concentration of A. tumefaciens cells on the floral dip treatment.…”

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confidence: 99%

A simple method for <i>in planta</i> tomato transformation by inoculating floral buds with a sticky <i>Agrobacterium tumefaciens</i> suspension

Honda

Ohkawa

Kusano

et al. 2021

Plant Biotechnology

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Tomato transformation is conventionally performed using Agrobacterium tumefaciens-infected cotyledons. Here, we propose a simple procedure for tomato transformation, by which A. tumefaciens cells were smeared onto floral buds of a tomato plant using a paintbrush. Sufficient numbers of fruits were obtained from them, although the smearing of an excess number of A. tumefaciens cells led to an adverse effect on the plant growth. Progeny plants were screened by growth on a kanamycin-containing selection medium plate. The nptII gene was detected in 10 plants among 1,599 progenies. These transformants were derived from fruits other than those obtained from the smeared buds. This suggested that A. tumefaciens cells moved to the buds located near the smeared buds and caused the transformation event. Our findings suggest that this procedure can be used for the introduction of a foreign gene into plant cells.

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Effect of explants, bacterial cell density and overgrowth-control antibiotics on transformation efficiency in tomato (Solanum lycopersicum L.)

Cited by 12 publications

References 0 publications

Pinpointing regulatory protein phosphatase 2A subunits involved in beneficial symbiosis between plants and microbes

Pinpointing regulatory protein phosphatase 2A subunits involved in beneficial symbiosis between plants and microbes

Pinpointing Regulatory Protein Phosphatase 2A Subunits Involved in Beneficial Symbiosis Between Plants and Microbes

A simple method for <i>in planta</i> tomato transformation by inoculating floral buds with a sticky <i>Agrobacterium tumefaciens</i> suspension

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