Define Agrobacterium, Media Preparation for Agrobacterium-mediated Gene Transfer

Define Agrobacterium

Define Agrobacterium is a phytopathogen that infects plants through sores, causes crown gall disease, and remains one of the most popular plants transformation tools in agriculture to this day.

Agrobacterium tumefaciens is the soil pathogen that uses its type IV bacterial secretion system to transfer its transferred (T) -DNA to host cells.

The genus Agrobacterium consists of different species depending on the symptoms of the disease and host range. Some of the Agrobacterium species include A. radiobacter, A. vitis, A. rhizogenes, A. rubi, and A. tumefaciens.

Organisms of this genus are primarily known as plant transformation tools used in a variety of host cells.

The host spectrum of the bacteria is determined by various bacterial and plant factors. Bacterial factors include virulence genes and T-DNA oncogenes, while plant factors include genes necessary for tumor formation and transformation.

The natural diversity of bacteria is determined by the presence of the most important pathogenic determinant, the Ti / Ri plasmid.

Various strains of Agrobacterium can be isolated in a variety of host plants from around the world. Some of the common host plants are roses, poplars, weeping figs, chrysanthemums, and other fruit trees.

The presence of various plasmids and the ability of the organism to transfer a section of DNA with the tumor-inducing plasmid to the genome of the host cell is the main factor in the use of Agrobacterium in the transformation of plants.

Agrobacterium is a rod-shaped, gram-negative bacterium with a size of 1.5 to 3 µm in length and 0.6 to 1.0 µm in width. The bacterium is mobile with one or up to six flagella.

These do not form spores and are strictly aerobic organisms that live in the soil with clinical and biotechnological applications.

Factors influencing Agrobacterium-mediated gene transfer

  • There are several methods used to obtain transgenic plants, some of which include Agrobacterium-mediated transformation, particle bombardment, polyethylene glycol-mediated protoplasts, and liposome-mediated transformation.
  • Among all these methods, Agrobacterium-mediated transformation leads to single-copy transgenes that are expressed comparatively more stably than copies of multiple genes.
  • However, the process is influenced by various factors such as bacterial strains and cell density, plant species, plant growth regulators, and environmental factors.
  • In order to develop an efficient transformation protocol, it is necessary to find the right combination of these factors.

Listed below are some of the factors that affect Agrobacterium-mediated transformation;

Explants

  1. Explants are target material for Agrobacterium-mediated transformation, which can be embryonic cultures, immature embryos derived from mature seeds, leaf blades, and even stem segments.
  2. The explants should be selected in such a way that the entire transgenic plant can be obtained.
  3. Various studies have been carried out to identify the best explanations for an efficient transformation process.
  4. The embryonic callus obtained from mature seeds is considered to be one of the best explants for Agrobacterium-mediated transformation in certain plant species.
  5. It was discovered that one of the major factors enhancing transformation is the desiccation of the explants before or after infection by Agrobacterium.
  6. Differences in transformation efficiency in different plant tissues have been attributed to differences in the ability of bacteria to adhere to plant cells and differences in the mechanism of T-DNA transfer.

B. Explant the wound

  1. Wounding explants is necessary for efficient Agrobacterium-mediated transformation.
  2. The type and method of wounding can range from simple wounds during explant preparation to particle gun-mediated micro-wounds.
  3. There are other forms of wounds that include Agrobacterium-filled syringes and sonication.
  4. The transformation is also enhanced by the formation of micro-wounds on the surface and below the surface of the target tissue, resulting in the release of phenolic compounds.
  5. The efficiency of the conversion also depends on the use of additional phenolic substances.

C. Plant species and genotype

  1. Differences in the efficiency of Agrobacterium-mediated transformation in different plant species are due to differences in the inducer molecules.
  2. The success of the transformation depends on the chromosomal genomes and plasmids, which encode all the materials necessary for DNA binding and transfer.
  3. Different plants have different levels of vir gene expression in different hosts, which affects susceptibility to Agrobacterium infection.
  4. Even within the same species, different cultivars or ecotypes show different susceptibility to tumor development by Agrobacterium species.
  5. Most Agrobacterium-mediated transformations occur in dicotyledonous species, but more recently the frequency of gene transfer has increased in monocotyledonous species.

Antibiotics

  • During the transformation, the coculture is followed by the suppression of bacteria so as not to disturb the growth and development of the host plant.
  • The elimination of the bacteria is achieved by using one or more antibiotics in the culture medium.
  • Some of the most commonly used antibiotics are carbenicillin and cefotaxime. The type of antibiotics used also depends on the plant species and Agrobacterium strains.
  • Determining the correct antibiotic ratio is crucial to achieving antibiotic selection and an appropriate tumorigenesis rate.

 Plant growth regulators (PGR)

  • Plant transformation also requires the addition of plant growth regulators, and the right choice of regulators is one of the most important factors influencing the process.
  • Competition and susceptibility to Agrobacterium infection in recalcitrant explants is low or absent without PGR treatments.
  • It is known that the presence of 2,4-D in the growth medium during the co-cultivation process improves the transformation efficiency.
  • The use of growth regulators facilitates cell division and differentiation in many tissues; however, regulators must be used at a certain stage in the plant cell cycle.

Light

  • Light is an important factor that affects the efficiency of Agrobacterium-mediated transformation, as light affects various physiological factors in the plant, such as plant hormone levels, cell proliferation, and the cell cycle stage.
  • It is also known that light increases the amount of the inducer of the phenolic vir gene, which affects the transformation process as it regulates the transfer of T-DNA.
  • Several Agrobacterium-mediated transformation processes use dark coculture conditions to improve the morphology of the explants.
  • Some studies have also shown that the effect of light on the process is primarily based on the photoperiod.

 Temperature

  • The first studies on the efficiency of Agrobacterium-mediated transformation indicate that high temperatures are detrimental to tumor development.
  • It is known that a temperature of about 32 ° C suppresses tumor development due to conformational changes in virA genes. The optimal temperature for T-DNA transfer was 19 ° C.
  • The optimal temperature for transmission may differ for different species, but the temperature range of 19 ° C to 22 ° C has been considered ideal for many plant species.

Agrobacterium strains

  • The ability to infect different Agrobacterium strains differs depending on the presence of different plasmids.
  • The most efficient group of bacteria used for transformation is the combination of a standard binary vector in a supervirulent strain and a super binary vector in a regular strain.
  • Different combinations of Agrobacterium strains can be used for different plant cell species.

Agrobacterium-mediated gene transfer principle

  1. Agrobacterium-mediated transformation is based on the organism’s ability to efficiently transfer its T-DNA to host cells.
  2. The biology of the process has two components; T-DNA consists of 25 bp repeats ending in the T region and the virulence region (vir), which consists of seven major loci.
  3. The Agrobacterium-mediated transformation mechanism is based on the transfer of a piece of plasmid by bacteria to plant cells during infection.
  4. The plasmid then integrates into the nuclear genome to express its own genes and affect the hormonal balance in the host cell.
  5. In addition to this, bacteria also produce a number of enzymes that are involved in the synthesis of opinions, which the bacteria then use as nutrients.
  6. Some of the essential components of the bacteria involved in the infection are the T-DNA, which is present in the plasmid called the Ti plasmid (tumor inducer), along with other functional components such as virulence (vir), conjugation (with), and origin of replication (ori ).
  7. The infection begins when the bacteria enter the injured areas. The attachment of bacteria to plant cells is increased by the release of phenolic acetosyringone (AS) from damaged plant cells.
  8. AS activates VirA proteins in bacteria, which activate VirG by phosphorylating its aspartate residue.
  9. The activated form of VirG then binds to other vir genes and induces their expression. VirD activated by this process stimulates the formation of the T-chain (a single-stranded copy of T-DNA).
  10. VirD2 is covalently linked to the 5 ‘end of the T-strand since the 5’ end is the leading end in transfer. Other factors such as VirE2 and VirB proteins also bind to the T strand and form a T complex.
  11. The complex is then delivered to the nucleus via nuclear target signals released by Vir proteins. The T-DNA strand randomly integrates into the plant genome as a single copy or as multiple copies.
  12. Integration usually takes place in the active or repetitive transcription regions of the genome through the process of recombination.
  13. While much is known about the molecular biology of T-DNA transfer in Agrobacterium cells, not much is known about the plant-encoded factors involved in this process.

Requirements (Materials and Reagents)

Materials/ Equipment

  • Sterile 50 ml plastic tubes
  • Autoclave
  • Controlled Tissue Culture Rooms at 25°C with 16/8 hr light/dark period
  • Shaker Incubator
  • Vacuum pump
  • Laminar hood for tissue culture
  • Glassware (Beakers, cylinders, Petri dishes, Duran bottles, and Flasks)
  • Filter paper
  • Parafilm
  • Forceps and Scalpel
  • Pipettes
  • Centrifuge
  • Spectrophotometer
  • Tissue culture vessels
  • Surgical blades

Reagents

  • Explant (Stems, embryo, cotyledons, or other tissues)
  • Agrobacterium strain
  • 13% Sodium hypochlorite
  • B5 Medium
  • Agar
  • Tryptone
  • Yeast Extract
  • Sodium Chloride
  • 35% Hydrochloric acid
  • Sterile distilled water
  • 75% Ethanol
  • Sucrose
  • Abscisic Acid
  • Rifampicin
  • Kanamycin monosulfate
  • Gellan gun powder
  • PCR primer star Mix
  • Carbenicillin disodium salt

Media Preparation for Agrobacterium-mediated Gene Transfer

  1. LB medium for Agrobacterium culture
  • 5 grams of yeast extract, 10 grams of tryptone, and 5 grams of sodium chloride are dissolved in 1 liter of distilled water.
  • To 50 ml of the LB medium, 50 µl of 100 mg/ml rifampicin stock and 50 µl of 50 mg/ml kanamycin stock are added.
  1. Murashige and Skoog medium for seed germination
  • 4.43 gram of Murashige and Skoog basal medium power and 3 gm of sucrose are added to 1 liter distilled water. 
  • To this, 2.5 gm gellan gun powder is added and autoclaved. 25 ml of this medium is poured on Petri plates under laminar flow.
  1. Cocultivation medium
  • To the Murashige and Skoog basal medium, 750 µl of 2 mg/ml BAP is added. Again, 500 µl of 2mg/ml ABA is added to the medium.
  • 25 ml of the medium is poured onto sterile Petri plates in the laminar flow.
  1. Shooting Medium
  • The shooting medium is prepared by adding 50 µl of 50 mg/ml kanamycin and 2.5 ml of 200 mg/ml carbenicillin to the cocultivation medium.
  1. Rooting Medium
  • Rooting medium is prepared by adding 50 µl of 50 mg/ml kanamycin and 1 ml of 200 mg/ml carbenicillin. 

Agrobacterium-mediated gene transfer method or protocol

The protocol or procedure for Agrobacterium-mediated transformation may vary depending on the type of explants selected for the procedure. The following is the protocol for Agrobacterium-mediated transformation in the case of an embryo;

a. Sterilization and germination of seeds.

The seeds are sterilized with Cl2 for 1-2 hours and then the seeds are immersed in water in a Petri dish for 2 hours at room temperature.

The seed coats are removed with seed forceps and further sterilized with 75% ethanol for 30 seconds. They are then rinsed with 20 ml of 3% sodium hypochlorite.

The sterilized seeds are placed in Petri dishes containing the seed germination medium and incubated at 28 ° C. for 2 days in the dark. Each plate can hold around 15-25 seeds.

The protocol or procedure for Agrobacterium-mediated transformation may vary depending on the type of explants selected for the procedure. The following is the protocol for Agrobacterium-mediated transformation in the case of an embryo;

a. Sterilization and germination of seeds.

The seeds are sterilized with Cl2 for 1-2 hours and then the seeds are immersed in water in a Petri dish for 2 hours at room temperature.

The seed coats are removed with seed forceps and sterilized for 30 seconds with 75% ethanol. They are then rinsed with 20 ml of 3% sodium hypochlorite.

The sterilized seeds are placed in Petri dishes containing the seed germination medium and incubated at 28 ° C. for 2 days in the dark. Each plate can hold around 15-25 seeds.

Applications of Agrobacterium-mediated Gene Transfer

The following are some of the important applications of Agrobacterium-mediated transformation;

  • The agrobacterium-mediated transformation has been used as a method of genetically modifying plants to produce various substances such as proteins, antibodies, and even vaccines.
  • Various plants have also been modified to make life-saving medicines such as blood thinners, human epidermal growth factors, and interferons.
  • Transgenic plants prepared with Agrobacterium serve as a biomonitor to detect toxic compounds in the environment and to detoxify contaminated soil and water.
  • The agrobacteria-mediated transformation has also significantly increased crop yield by modifying the lifespan and biosynthesis of plants.
  • Plants can be modified to improve tolerance to biotic and abiotic factors and to absorb nutrients with greater resistance to pests.
  • The agrobacterium-mediated transformation has been used to create insect-resistant crops by incorporating several toxic genes, such as Bt toxin genes.
  • Increased resistance to pests leads to a reduction in the use of harmful agrochemicals and herbicides.
  • Agrobacteria-mediated transformation is one of the less complicated genetic engineering techniques that have the potential to be improved for use with other organisms as well.

Limits of Agrobacterium-mediated Gene Transfer

Although Agrobacterium-mediated transformation has advanced with great success over the years, there are several problems and limitations associated with this technique. Some of the limitations and issues that are common with this technique include:

  1. The main limitation associated with this technique is the narrow host range as it is still restricted to certain plant species.
  2. While much is known about the mechanism of T-DNA transfer in bacteria, not much is known about the plant-encoded factors that affect the efficiency of this process.
  3. The technique is labor-intensive as it requires the development of detailed plant regeneration protocols and time-consuming processes. Many of these processes are susceptible to variations in vitro that lead to unfavorable results.
  4. The success of transformation in monocots depends on the use of embryos as explants; however, these are only available for a short time each year.
  5. Agrobacterium-mediated transformation cannot transfer large DNA molecules to more economically important plants, indicating a possible introduction of a powerful vector system.
https://microbenotes.com/agrobacterium-mediated-gene-transfer/

Definebiology.com

Define Agrobacterium, Media Preparation for Agrobacterium-mediated Gene Transfer

%d bloggers like this: