Agroinfiltration
Encyclopedia
Agroinfiltration is a method in plant biology to induce transient expression of gene
s in a plant
or to produce a desired protein
. In the method a suspension
of Agrobacterium
is injected into a plant leaf, where it transfers the desired gene to plant cell
s. The benefit of agroinfiltration when compared to traditional plant transformation
is speed and convenience.
First step of the protocol is to introduce a gene of interest to a strain
of Agrobacterium. Subsequently the strain is grown in a liquid culture and the resulting bacteria are washed and suspended into a buffer solution
. This solution is then placed in a syringe
(without a needle). The tip of the syringe is pressed against the underside of a leaf
while simultaneously applying gentle counterpressure to the other side of the leaf. The Agrobacterium solution is then injected into the airspaces inside the leaf through stomata, or sometimes through a tiny incision made to the underside of the leaf.
Vacuum infiltration is another way to penetrate Agrobacterium deep into plant tissue. In this procedure, leaf disks, leaves, or whole plants are submerged in a beaker containing the solution, and the beaker is placed in a vacuum chamber. The vacuum is then applied, forcing air out of the stomata. When the vacuum is released, the pressure difference forces solution through the stomata and into the mesophyll
.
Once inside the leaf the Agrobacterium remains in the intercellular space and transfers the gene of interest in high copy numbers into the plant cells. The gene is then transiently expressed (no selection for stable integration is performed). The plant can be monitored for a possible effect in the phenotype
, subjected to experimental conditions or harvested and used for purification of the protein
of interest. Many plant species can be processed using this method, but the most common ones are Nicotiana benthamiana
and Nicotiana tabacum
'.'
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
s in a plant
Plant
Plants are living organisms belonging to the kingdom Plantae. Precise definitions of the kingdom vary, but as the term is used here, plants include familiar organisms such as trees, flowers, herbs, bushes, grasses, vines, ferns, mosses, and green algae. The group is also called green plants or...
or to produce a desired protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
. In the method a suspension
Suspension (chemistry)
In chemistry, a suspension is a heterogeneous fluid containing solid particles that are sufficiently large for sedimentation. Usually they must be larger than 1 micrometer. The internal phase is dispersed throughout the external phase through mechanical agitation, with the use of certain...
of Agrobacterium
Agrobacterium
Agrobacterium is a genus of Gram-negative bacteria established by H. J. Conn that uses horizontal gene transfer to cause tumors in plants. Agrobacterium tumefaciens is the most commonly studied species in this genus...
is injected into a plant leaf, where it transfers the desired gene to plant cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
s. The benefit of agroinfiltration when compared to traditional plant transformation
Transformation (genetics)
In molecular biology transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material from its surroundings and taken up through the cell membrane. Transformation occurs naturally in some species of bacteria, but it can...
is speed and convenience.
First step of the protocol is to introduce a gene of interest to a strain
Strain (biology)
In biology, a strain is a low-level taxonomic rank used in three related ways.-Microbiology and virology:A strain is a genetic variant or subtype of a micro-organism . For example, a "flu strain" is a certain biological form of the influenza or "flu" virus...
of Agrobacterium. Subsequently the strain is grown in a liquid culture and the resulting bacteria are washed and suspended into a buffer solution
Buffer solution
A buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. It has the property that the pH of the solution changes very little when a small amount of strong acid or base is added to it. Buffer solutions are used as a...
. This solution is then placed in a syringe
Syringe
A syringe is a simple pump consisting of a plunger that fits tightly in a tube. The plunger can be pulled and pushed along inside a cylindrical tube , allowing the syringe to take in and expel a liquid or gas through an orifice at the open end of the tube...
(without a needle). The tip of the syringe is pressed against the underside of a leaf
Leaf
A leaf is an organ of a vascular plant, as defined in botanical terms, and in particular in plant morphology. Foliage is a mass noun that refers to leaves as a feature of plants....
while simultaneously applying gentle counterpressure to the other side of the leaf. The Agrobacterium solution is then injected into the airspaces inside the leaf through stomata, or sometimes through a tiny incision made to the underside of the leaf.
Vacuum infiltration is another way to penetrate Agrobacterium deep into plant tissue. In this procedure, leaf disks, leaves, or whole plants are submerged in a beaker containing the solution, and the beaker is placed in a vacuum chamber. The vacuum is then applied, forcing air out of the stomata. When the vacuum is released, the pressure difference forces solution through the stomata and into the mesophyll
Mesophyll
Mesophyll can refer to:* Mesophyll tissue, in plant anatomy, photosynthetic parenchyma cells that lie between the upper and lower epidermis layers of a leaf...
.
Once inside the leaf the Agrobacterium remains in the intercellular space and transfers the gene of interest in high copy numbers into the plant cells. The gene is then transiently expressed (no selection for stable integration is performed). The plant can be monitored for a possible effect in the phenotype
Phenotype
A phenotype is an organism's observable characteristics or traits: such as its morphology, development, biochemical or physiological properties, behavior, and products of behavior...
, subjected to experimental conditions or harvested and used for purification of the protein
Protein purification
Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest. The starting material is usually a biological tissue or...
of interest. Many plant species can be processed using this method, but the most common ones are Nicotiana benthamiana
Nicotiana benthamiana
Nicotiana benthamiana is a close relative of tobacco and species of Nicotiana indigenous to Australia.The herbaceous plant is found amongst rocks on hills and cliffs throughout the northern regions of Australia. Variable in height and habit, the species may be erect and up to 1.5 metres or...
and Nicotiana tabacum
Nicotiana tabacum
Nicotiana tabacum, or cultivated tobacco, is a perennial herbaceous plant. It is found only in cultivation, where it is the most commonly grown of all plants in the Nicotiana genus, and its leaves are commercially grown in many countries to be processed into tobacco. It grows to heights between 1...
'.'