Genetic Transformation in Plant Cells using Ultrasonics

Sonication-Assisted Agrobacterium-Mediated Transformation (SAAT) is an efficient method to infect plant cells with foreign genes using Agrobacterium as transporter. Ultrasonic cavitation causes sonoporation, which can be described as targeted micro-wounding of plant tissue. Via these ultrasonically creates micro-wounds, DNA and DNA vectors can be efficiently transported into the cell matrix.

Sonoporation – Ultrasonically Enhanced Cell Transformation

Ultrasonicator UP100H is a lab homogeniser often used for sample preparation of cell culture plates.When low-frequency ultrasound (approx. 20kHz) is applied to cell suspensions, the effects of acoustic cavitation cause a transient membrane permeabilization on cell tissues. This ultrasonic effect is known as sonoporation and is used for gene transfer into cells or tissues.
Advantages of ultrasonication are based on its non-thermal mechanical working principle, which makes sonication often more versatile and less dependent on cell types. The versatile application of sonoporation opens the possibility to the utilization of transgenic plants, which have significant potential in the bioproduction of complex human therapeutic proteins. Such plant-based bioreactors can be easy genetically manipulated, prevent the potential contamination with human pathogens, do not damage transformation mediating bacteria (e.g. Agrobacterium), and are an inexpensive, efficacious method of bio-synthesis.

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The video shows the ultrasonic sample preparation system UIP400MTP, which allows for the reliable sample preparation of any standard multi-well plates using high-intensity ultrasound. Typical applications of the UIP400MTP include cell lysis, DNA, RNA, and chromatin shearing as well as protein extraction.

Ultrasonicator UIP400MTP for multi-well plate sonication

Ultrasonic-Assisted Cell Transformation

Probe-type ultrasonicators such as the UP200St are reliable tissue homogenizers and widely used for sample preparation in genetics, e.g., for Sonication-Assisted Agrobacterium-Mediated Transformation (SAAT).Sonication is a technique that applies low-frequency ultrasound waves to agitate particles in solution, to mix solutions, thereby increasing the rate of mass transfer and dissolution. Simultaneously, sonication can remove dissolved gases from liquids. In plant-transformation, sonication will cause the formation of microwounds on plant tissue and enhance the delivery of naked DNA into the plant protoplast.

For genetic transformation, the Sonication-Assisted Agrobacterium-mediated Transformation (SAAT) is the preferred method and has a significantly higher efficiency than sonication used to transfer naked DNA and DNA vectors directly into the protoplast. Numerous studies have demonstrated that the sonication assisted Agrobacterium-mediated transformation (SAAT) can used to induce mechanical disruption and formation of wounds on plant cells by ultrasound waves and the resulting acoustic cavitation. A short ultrasonic treatment creates micro-wounds on the surface of explants. As the wounded cells will allow the penetration of Agrobacterium into the deeper part of plant tissues, thus increasing the likelihood of plant cells being infected. Additionally, the secreted phenolic compounds enhance transformation. The ultrasonically generated micro-wounds make explant penetration by bacteria also more feasible. SAAT was successfully used for genetic transformation in plant species particularly considered as resistant to Agrobacterium.
Being a very simple and inexpensive method, as well as the significant enhancement of Agrobacterium-mediated gene transfer are the major advantages of SAAT. Apart from successful application of SAAT in the transformation of Chenopodium rubrum L. and Beta vulgaris L., this approach has also been applied in the production of recombinant Escherichia coli wild-type heat-labile holotoxin and Escherichia coli mutant LT vaccine adjuvants in Nicotiana tabacum, in which the highest systemic LT-B-specific IgG titres were detected in birds.
(cf. Laere et al., 2016; M. Klimek-Chodacka and R. Baranski, 2014)

The VialTweeter can be used for DNA infection of plan cells, e.g. using sonication-assisted Agrobacterium- mediated transformation (SAAT)

VialTweeter for the simultaneous sonication of multiple sample tubes, e.g. for sonication-assisted Agrobacterium- mediated transformation (SAAT)

Scientifically Proven Benefits of Sonication-Assisted Agrobacterium-Mediated Transformation (SAAT)

Sonication-Assisted Agrobacterium-Mediated Transformation (SAAT) has been applied to numerous plant species. A short and relatively mild ultrasonic treatment of plant cell cultures causes sonoporation, which subsequently allows a deep penetration of Agrobacterium as gene transporter. Below you can read exemplary studies demonstrating the beneficial effects of SAAT.

Ultrasonically-Assisted Transformation of Ashwagandha

In order to improve the transformation efficiency in W. somnifera (known as ashwagandha or winter cherry), Dehdashti and colleagues (2016) investigated the use of acetosyringone (AS) and sonication.
Acetosyringone (AS) was added in three stages: Agrobacterium liquid culture, Agrobacterium infection and co-culture of explants with Agrobacterium. The addition of 75 μM AS to Agrobacterium liquid culture was found to be optimum for induction of vir genes.
The additional application of sonication (SAAT) resulted in highest gene expression. The gusA gene expression in hairy roots was found to be best when the leaves and shoot tips were sonicated for 10 and 20s, respectively. The transformation efficiency of the improved protocol was recorded 66.5 and 59.5% in the case of leaf and shoot tip explants, respectively. When compared with other protocols the transformation efficiency of this improved protocol was found to be 2.5 fold higher for leaves and 3.7 fold more for shoot tips. Southern blot analyses confirmed 1–2 copies of the gusA transgene in the lines W1-W4, while 1–4 transgene copies were detected in the line W5 generated by the improved protocol.

Sonication duration (time exposed to ultrasound) affects the sonication-assisted Agrobacterium- mediated transformation (SAAT) on transformation in W. somnifera

Effect of the duration of sonication-assisted Agrobacterium- mediated transformation (SAAT) on transformation frequency of W. somnifera leaf (a) and shoot tip (b) explants
(study and graphic: © Dehdashti et al., 2016)

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Ultrasonically-Assisted Transformation of Cotton

Hussain et al. (2007) demonstrate the beneficial effects of sonication-assisted cotton transformation. Acoustic cavitation caused by low-frequency ultrasound creates micro-wounds on and below the surface of plant tissue (sonoporation) and allows Agrobacterium to travel deeper and completely throughout the plant tissue. This wounding fashion increases the probability of infecting plant cells lying deeper in tissue. In order to evaluate the transformation efficiency of SAAT, GUS gene expression was measured. The GUS reporter system is a reporter gene system, particularly useful in plant molecular biology and microbiology. Adjusting various SAAT parameters, GUS transient expression in cotton using mature embryos as explant was significantly enhanced. GUS was first detected 24h following incubation of the explants and by 48h, GUS expression was very intense which served as a useful indicator of successful transformation of the cotton explant following sonication assisted Agrobacterium mediated transformation (SAAT). The comparison of various transformation techniques (namely biolistic, Agro, BAAT, SAAT), sonication-assisted Agrobacterium-mediated transformation (SAAT) showed by far the best results of transformation.

Sonication-assisted Agrobacterium-mediated transformation (SAAT) shows a significantly higher transient expression when compared to alternative infection methods.

Choice of transformation procedure on the basis of transient expression of GUS. Sonication-assisted Agrobacterium-mediated transformation (SAAT) shows a significantly higher transient expression.
(study and graphic: © Hussain et al., 2007)

High-Performance Ultrasonic Solutions for Sonoporation and SAAT

Hielscher Ultrasonics is long-time experienced in the development and manufacturing of high-performance ultrasonicators for laboratories, research facilities as well as industrial production with very high throughputs. For microbiology and life science, Hielscher offers various solutions to accommodate different requirements necessary for specific tissues and their treatments. For the simultaneous ultrasonication of numerous samples Hielscher offers the UIP400MTP for multiwell plates, the VialTweeter for the sonication of up to 10 vials (e.g., Eppendorf tubes) or the ultrasonic CupHorn. Probe-type ultrasonicators are available from 50 to 400 watts as lab homogenizers, while industrial systems cover the power range from 500 watts to 16kW.
Please get in touch with us and let us know about your application and process requirements. Our well-experienced staff will be glad to recommend the most suitable ultrasonicator for your biological process.
The table below gives you an indication of the approximate processing capacity of our ultrasonicators:

Batch Volume Flow Rate Recommended Devices
multiwell / microtiter plates n.a. UIP400MTP
up to 10 vials n.a. VialTweeter
up to 5 vials/tubes or 1 larger vessel n.a. CupHorn
1 to 500mL 10 to 200mL/min UP100H
10 to 2000mL 20 to 400mL/min UP200Ht, UP400St
0.1 to 20L 0.2 to 4L/min UIP2000hdT
10 to 100L 2 to 10L/min UIP4000hdT
n.a. 10 to 100L/min UIP16000
n.a. larger cluster of UIP16000

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Ultrasonic high-shear homogenizers are used in lab, bench-top, pilot and industrial processing.

Hielscher Ultrasonics manufactures high-performance ultrasonic homogenizers for mixing applications, dispersion, emulsification and extraction on lab, pilot and industrial scale.

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High performance ultrasonics! Hielscher's product range covers the full spectrum from the compact lab ultrasonicator over bench-top units to full-industrial ultrasonic systems.

Hielscher Ultrasonics manufactures high-performance ultrasonic homogenizers from lab to industrial size.