Ultratovush yordamida o'simlik hujayralarida genetik transformatsiya

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

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.

Ultrasonic-Assisted Cell Transformation

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)

General Procedure for Gene Transfer via Sonoporation in Plant Cells

1. Preparation of Genetic Material: Begin by preparing the genetic material you want to introduce into the plant cells. This could be plasmid DNA, RNA, or other nucleic acids.
2. Plant Cell Isolation: Isolate the plant cells you want to target. Depending on your experiment, these cells may be isolated from plant tissues or cultures.
3. Cell Suspension: Suspend the plant cells in a suitable medium or buffer. This is essential to ensure that the cells are healthy and in a state conducive to gene uptake.
4. Setup your Sonicator: Prepare your probe-type sonicator by sonication pre-setting parameters such as amplitude, time, energy, and temperature. Immerse the ultrasonic probe into the cell suspension.
5. Sonication: Start the sonication procedure. The rapid oscillation of the probe tip generates cavitation bubbles in the liquid. These bubbles expand and collapse due to the ultrasonic waves, creating mechanical forces and microstreaming in the suspension.
6. Sonoporation: The mechanical forces and microstreaming generated by cavitation create temporarily pores and holes in the membranes of the plant cells. The genetic material present in the suspension can enter the plant cells through these pores.
7. Incubation: After the sonoporation treatment, incubate the plant cells to allow them to recover and stabilize their membranes. This is a crucial step to ensure cell survival and successful gene transfer.

Gene Transfer Via Agrobacterium or Liposomes

There are two common forms to transfect plant cell. They use either agrobacterium, a genus of Gram-negative bacteria, or liposomes as carrier of the genetic material.

• Agrobacterium-Mediated Sonoporation: Agrobacterium tumefaciens is a bacterium commonly used in plant genetic engineering. In this method, plasmid DNA containing the desired gene is introduced into Agrobacterium, which is then mixed with plant cells. The cell suspension is subjected to sonoporation using a probe-type sonicator. The ultrasonic energy enhances the transfer of the genetic material from Agrobacterium to the plant cells. This method is widely used for the genetic modification of plants.
• Liposome-Mediated Sonoporation: Liposomes are lipid-based vesicles that can carry genetic material. In this method, liposomes loaded with plasmid DNA or other nucleic acids are mixed with plant cells. Sonoporation using a probe-type sonicator is employed to facilitate the uptake of liposomes by plant cells. The ultrasound disrupts the lipid bilayers of the liposomes, releasing the genetic material into the plant cells. This approach is useful for transient gene expression studies in plant cells.

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.

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)

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.

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.
Quyidagi jadvalda ultrasonikatorlarimizning taxminiy qayta ishlash quvvati ko'rsatilgan: