Dataset Information

An Improved Syringe Agroinfiltration Protocol to Enhance Transformation Efficiency by Combinative Use of 5-Azacytidine, Ascorbate Acid and Tween-20.

ABSTRACT: Syringe infiltration is an important transient transformation method that is widely used in many molecular studies. Owing to the wide use of syringe agroinfiltration, it is important and necessary to improve its transformation efficiency. Here, we studied the factors influencing the transformation efficiency of syringe agroinfiltration. The pCAMBIA1301 was transformed into Nicotiana benthamiana leaves for investigation. The effects of 5-azacytidine (AzaC), Ascorbate acid (ASC) and Tween-20 on transformation were studied. The ?-glucuronidase (GUS) expression and GUS activity were respectively measured to determine the transformation efficiency. AzaC, ASC and Tween-20 all significantly affected the transformation efficiency of agroinfiltration, and the optimal concentrations of AzaC, ASC and Tween-20 for the transgene expression were identified. Our results showed that 20 ?M AzaC, 0.56 mM ASC and 0.03% (v/v) Tween-20 is the optimal concentration that could significantly improve the transformation efficiency of agroinfiltration. Furthermore, a combined supplement of 20 ?M AzaC, 0.56 mM ASC and 0.03% Tween-20 improves the expression of transgene better than any one factor alone, increasing the transgene expression by more than 6-fold. Thus, an optimized syringe agroinfiltration was developed here, which might be a powerful method in transient transformation analysis.

SUBMITTER: Zhao H

PROVIDER: S-EPMC5371768 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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An Improved Syringe Agroinfiltration Protocol to Enhance Transformation Efficiency by Combinative Use of 5-Azacytidine, Ascorbate Acid and Tween-20.

Zhao Huimin H Tan Zilong Z Wen Xuejing X Wang Yucheng Y

Plants (Basel, Switzerland) 20170214 1

Syringe infiltration is an important transient transformation method that is widely used in many molecular studies. Owing to the wide use of syringe agroinfiltration, it is important and necessary to improve its transformation efficiency. Here, we studied the factors influencing the transformation efficiency of syringe agroinfiltration. The pCAMBIA1301 was transformed into <i>Nicotiana benthamiana</i> leaves for investigation. The effects of 5-azacytidine (AzaC), Ascorbate acid (ASC) and Tween-2 ...[more]

PMID: 28216553

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Project description:Polyethylene glycol sorbitan monoacylates (Tween) are detergents of widespread use in plant sciences. We show them, notably Tween 20, to cause a rapid and complex change in transcript abundance which bears all characteristics of a PAMP / elicitor-induced defense response, and they do so at concentrations which cause no detectable deleterious effects on plant cellular integrity. The activity does not reside in the intact Tween molecule itself, but is caused by medium-chain fatty acids, notably lauric acid (LA), which are efficiently released from the Tween-backbone by the plant. The Tween / LA-response is independent of the jasmonate signalling system. Medium-chain fatty acids are thus novel elicitors/regulators of plant pathogen defense. The results also have several practical implications: (i) The use of Tweens and, as we show, several other detergents, as solvating/wetting agents on intact plants causes profound physiological changes which may mask actual effects of test compounds; (ii) Tweens by themselves can be regarded (and probably used) as economical, non-toxic, and safe-to-apply elicitors of inducible plant immunity against pathogens. Experiment Overall Design: In preliminary Northern blot experiments using the Arabidopsis thaliana genes OPR1 / OPR2, strongest responses were observed between 1 h and 2.5 h after foliar treatment with 0.2 % Tween 20 solution (approx. 1.6 mM). These conditions were thus chosen for the microarray experiments. Three experiments were carried out, two 1 h after the onset of the Tween treatment (expt. IA and IB which are biological repetitions; GSM126247, GSM126248; GSM126990, GSM126991 files) and one after 2.5 h (expt. II; GSM126244, GSM126246 files, c and t respectively) to allow a distinction between fast, transient and slower, more permanent responses. Experiment Overall Design: All the experiments were conducted at the same day time in the morning. For every experiment a total of three independent replicas (33 Arabidopsis thaliana Col-0 plants per experiment) were done. The plants used were 28 days old, growing under sterile conditions in 0.5 L glass jars containing MS medium. The controls were sprayed with water and the samples with 0.2 % (w/v) Tween 20 detergent. The plant material was collected in a random way from the different glass jars, the samples were dried in tissue paper, weighted (1 g) and immediately frozen in liquid nitrogen. This complete procedure took place in less than 1 min. Total RNA extraction from every individual experiment was made with TRIzol. Once the RNA quality of every independent induction experiment was satisfactory, the RNA samples were mixed. Experiment Overall Design: RNA quality and quantity control, cRNA labeling, hybridizations, expression data normalization and analysis were performed in collaboration with Carsten MÃ¼ssig and Thomas Altmann (Max-Planck-Institute for Molecular Plant Physiology, Golm) according to the Affymetrix manual. Clustering was performed using the Access and the MAPMAN program. Experiment Overall Design: The levels of 118 transcripts were elevated (2-fold) after 1 h, i.e., in both, the IA and IB arrays, but only 12 levels were reduced (by 2-fold). In the 2.5 h array the corresponding numbers were 137 and 11. The transcript levels of 38 genes were found elevated in all three arrays. The validity of the array data was checked for 25 randomly-picked genes out of the 38 genes by RT-PCR.

Dataset Information

An Improved Syringe Agroinfiltration Protocol to Enhance Transformation Efficiency by Combinative Use of 5-Azacytidine, Ascorbate Acid and Tween-20.

Publications

An Improved Syringe Agroinfiltration Protocol to Enhance Transformation Efficiency by Combinative Use of 5-Azacytidine, Ascorbate Acid and Tween-20.

Similar Datasets

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