Deepak

An unrelated process called malignant transformation occurs spontaneously in the
progression of cancer.
In molecular biology transformation is the genetic alteration of a cell resulting from the
uptake, incorporation and expression of exogenous genetic material (exogenous DNA)
that is taken up through the cell membrane(s).[1] Transformation occurs most commonly
in bacteria and in some species occurs naturally. Transformation can also be effected by
artificial means. Bacteria that are capable of being transformed, whether naturally or
artificially, are called competent. Genetic material can also be transferred between cells
by conjugation or transduction. Conjugation involves the direct contact of two different
bacterial cells with the DNA being transferred from one cell to the other. In transduction,
viruses called bacteriophages inject the foreign DNA into their host. Introduction of
foreign DNA into eukaryotic cells is usually called "transfection".[2] Transformation is
also used to describe the insertion of new genetic material into nonbacterial cells
including animal and plant cells.
Contents
[hide]
• 1 History
• 2 Mechanisms
o 2.1 Bacteria
 2.1.1 Natural competence
 2.1.2 Artificial competence
 2.1.2.1 Plasmid transformation
o 2.2 Plants
o 2.3 Animals
• 3 References
• 4 External links
[edit] History
Transformation was first demonstrated in 1928 by Frederick Griffith, an English
bacteriologist searching for a vaccine against bacterial pneumonia. Griffith discovered
that a harmless strain of Streptococcus pneumoniae could be made virulent after being
exposed to heat-killed virulent strains. Griffith hypothesized that some "transforming
factor" from the heat-killed strain was responsible for making the harmless strain
virulent. In 1944 this "transforming factor" was identified as being genetic by Oswald
Avery, Colin MacLeod, and Maclyn McCarty. They isolated DNA from a virulent strain
of S. pneumoniae and using just this DNA were able to make a harmless strain virulent.
They called this uptake and incorporation of DNA by bacteria "transformation." See
Avery-MacLeod-McCarty experiment.
The results of Avery et al.'s experiments were at first sceptically received by the scientific
community and it was not until the development of genetic markers and the discovery of
other methods of genetic transfer (conjugation in 1947 and transduction in 1953) by
Joshua Lederberg that Avery's experiments were accepted.[3] Transformation did not
become routine procedure in laboratories until 1972 when Stanley Cohen, Annie Chang
and Leslie Hsu successfully transformed Escherichia coli by treating the bacteria with
calcium chloride.[4] This created an efficient and convenient procedure for transforming
bacteria and opened the way for molecular cloning in biotechnology and research.
Transformation using electroporation was developed in the late 1980s thus increasing the
efficiency and number of bacterial strains that could be transformed.[5] Transformation of
animal and plant cells was also investigated with the first transgenic mouse being created
by injecting a gene for a rat growth hormone into a mouse embryo in 1982.[6] In 1907 a
bacterium that caused plant tumors, Agrobacterium tumefaciens, was discovered and in
the early 1970s the tumor inducing agent was found to be a DNA plasmid called the Ti
plasmid.[7] By removing the genes in the plasmid that caused the cancer and adding in
novel genes researchers were able to infect plants with A. tumefaciens and let the bacteria
insert their chosen DNA into the genomes of the plants. Not all plant cells are susceptible
to infection by A. tumefaciens so other methods were developed including electroporation
and micro-injection.[8] Particle bombardment was made possible with the invention of the
Biolistic Particle Delivery System (gene gun) by John Sanford in 1990.[9]
[edit] Mechanisms
[edit] Bacteria
Bacteria transformation may be referred to as a stable genetic change brought about by

the uptake of naked DNA (DNA without associated cells or proteins) and competence
refers to the state of being able to take up exogenous DNA from the environment. Two
forms of competence exist: natural and artificial.
[edit] Natural competence
Main article: Competence (biology)
About 1% of bacterial species are capable of naturally taking up DNA under laboratory
conditions; many more are able to take it up in their natural environments. Such bacteria
carry sets of genes that provide the protein machinery to bring DNA across the cell
membrane(s).[10]
DNA material can be transferred between different strains of bacteria, in a process called
horizontal gene transfer.
[edit] Artificial competence

Artificial competence is induced by laboratory procedures and involves making the cell
passively permeable to DNA by exposing it to conditions that do not normally occur in
nature.[11]
Calcium chloride transformation is a method of promoting competence. Chilling cells in

the presence of divalent cations such as Ca2+ (in CaCl2) prepares the cell membrane to
become permeable to plasmid DNA. The cells are incubated on ice with the DNA and
then briefly heat shocked (e.g., 42°C for 30–120 seconds) thus allowing the DNA to enter
the cells. This method works very well for circular plasmid DNA. An excellent
preparation of competent cells will give ~108 colonies per microgram of plasmid. A poor
preparation will be about 104/μg or less. Good, non-commercial preparations should give
105 to 106 transformants per microgram of plasmid.{{[12]} The method, however, usually
does not work well for linear DNA, such as fragments of chromosomal DNA, probably
because the cell's native exonuclease enzymes rapidly degrade linear DNA. Interestingly,
cells that are naturally competent are usually transformed more efficiently with linear
DNA than with plasmid DNA.
Electroporation is another method of promoting competence. In the method the cells are
briefly shocked with an electric field of 10-20 kV/cm that creates holes in the cell
membrane through which the plasmid DNA enters. This method is amenable to the
uptake of large plasmid DNA.[13] After the electric shock the holes are rapidly closed by
the cell's membrane-repair mechanisms.
The efficiency with which a competent culture can take up exogenous DNA and express
its genes is known as Transformation efficiency.
[edit] Plasmid transformation
In order to be stably maintained in the cell a plasmid DNA molecule must contain an
origin of replication, which allows it to be replicated in the cell independent of the
replication of the cell's own chromosome. Because transformation usually produces a
mixture of relatively few transformed cells and an abundance of non-transformed cells a
method is needed to identify the cells that have acquired the plasmid. The method usually
consists of using a plasmid that contains a gene that gives the bacterial cells resistance to
an antibiotic that they are naturally sensitive to. The mixture of cells are then plated on
media that contains the antibiotic thus only the transformed cells are able to grow. Cells
that did not take up the plasmid are killed in the media.
Another selection method called blue-white screen uses a plasmid that contains an
antibiotic resistance gene and the lacZ gene. The lacZ gene codes for the lacZ-α subunit
of the enzyme β-galactosidase, a homo-tetramer with each monomer composed of one
lacZ-α subunit and one lacZ-ω subunit. The method also requires an E. coli strain that
possesses in its genome the code for only the lacZ-ω subunit and not the lacZ-α subunit.
One of the first steps in any transformation is the production of a recombined plasmid
obtained by the successful ligation of the gene of interest into its corresponding vector,
which in this method results in the disruption of lacZ because the gene of interest is
inserted within the lacZ code. A cell that takes up a recombined plasmid would thus not
be able to express the lacZ-α subunit and would, in turn, not be able to produce a
functional β-galactosidase. Conversely, a cell that has taken up non-recombined plasmid
(perhaps one formed by the ligation of the vector's own two ends) will express the lacZ-α
subunit and thus produce a functional β-galactosidase. A cell that does not take up any
plasmid is not conferred with antibiotic resistance and will die upon plating.
Consequently, the blue-white screen method allows for the ready detection of not just
transformed cells, but, most importantly, cells that have been transformed by a
successfully recombined plasmid. Selection occurs as a result of the action of β-
galactosidase on its substrate X-gal, which is included in the media along with the
appropriate antibiotic. X-gal is a colorless, modified galactose sugar whose hydrolysis by
β-galactosidase produces galactose and the pre-chromophore 5-bromo-4-chloro-3-
hydroxyindole. The latter is subsequently oxidized to 5,5'-dibromo-4,4'-dichloro-indigo,
an insoluble, blue product that is readily seen by the naked eye. Colonies of cells that
have been transformed by a successfully recombined plasmid will thus appear white
whereas those that have been transformed by non-recombined plasmid will appear blue.
[edit] Plants
A number of mechanisms are available to transfer DNA into plant cells:
• Agrobacterium mediated transformation is the easiest and most simple plant

transformation. Plant tissue (often leaves) are cut into small pieces, e.g.
10x10mm, and soaked for 10 minutes in a fluid containing suspended
Agrobacterium. Some cells along the cut will be transformed by the bacterium,
that inserts its DNA into the cell. Placed on selectable rooting and shooting
media, the plants will regrow. Some plants species can be transformed just by
dipping the flowers into suspension of Agrobacterium and then planting the seeds
in a selective medium. Unfortunately, many plants are not transformable by this
method.
• Particle bombardment: Particles of gold or tungsten are coated with DNA and
then shot into young plant cells or plant embryos. Some genetic material will stay
in the cells and transform them. This method also allows transformation of plant
plastids. The transformation efficiency is lower than in agribacterial mediated
transformation, but most plants can be transformed with this method.
• Electroporation: make transient holes in cell membranes using electric shock; this
allows DNA to enter as described above for Bacteria.
• Viral transformation (transduction): Package the desired genetic material into a
suitable plant virus and allow this modified virus to infect the plant. If the genetic
material is DNA, it can recombine with the chromosomes to produce transformant
cells. However genomes of most plant viruses consist of single stranded RNA
which replicates in the cytoplasm of infected cell. For such genomes this method
is a form of transfection and not a real transformation, since the inserted genes
never reach the nucleus of the cell and do not integrate into the host genome. The
progeny of the infected plants is virus free and also free of the inserted gene.
[edit] Animals
Introduction of DNA into animal cells is usually called transfection, and is discussed in
the corresponding article.
[edit] References
1. ^ bacterial transformation at Dorland's Medical Dictionary
2. ^ Alberts, Bruce; et al. (2002). Molecular Biology of the Cell. New York: Garland
Science. p. G:35. ISBN 9780815340720.
3. ^ Lederberg, Joshua (1994). The Transformation of Genetics by DNA: An
Anniversary Celebration of AVERY, MACLEOD and MCCARTY(1944) in
Anecdotal, Historical and Critical Commentaries on Genetics. The Rockfeller
University, New York, New York 10021-6399.
http://www.ncbi.nlm.nih.gov/pubmed/8150273.
4. ^ Cohen, Stanley; Chang, Annie and Hsu, Leslie (1972). "Nonchromosomal
Antibiotic Resistance in Bacteria: Genetic Transformation of Escherichia coli by
R-Factor DNA". Proceedings of the National Academy of Sciences 69 (8): 2110–
4. doi:10.1073/pnas.69.8.2110. PMID 4559594. PMC 426879.
http://www.pnas.org/content/69/8/2110.abstract.
5. ^ Wirth, Reinhard; Friesenegger, Anita and Fiedlerand, Stefan (1989).
"Transformation of various species of gram-negative bacteria belonging to 11
different genera by electroporation". Molecular and General Genetics MGG.
http://www.springerlink.com/content/xx826w544343jt8l/.
6. ^ Palmiter, Richard; Ralph L. Brinster, Robert E. Hammer, Myrna E. Trumbauer,
Michael G. Rosenfeld, Neal C. Birnberg & Ronald M. Evans (1982). "Dramatic
growth of mice that develop from eggs microinjected with
metallothionein−growth hormone fusion genes". Nature.
http://www.nature.com/nature/journal/v300/n5893/abs/300611a0.html.
7. ^ Nester, Eugene. "Agrobacterium: The Natural Genetic Engineer (100 Years
Later)". http://www.apsnet.org/online/feature/Agrobacterium/. Retrieved 28
January 2010.[dead link]
8. ^ Peters, Pamela. "Transforming Plants - Basic Genetic Engineering Techniques".
http://www.accessexcellence.org/RC/AB/BA/Transforming_Plants.php. Retrieved
28 January 2010.
9. ^ Voiland, Michael; McCandless, Linda. "DEVELOPMENT OF THE "GENE
GUN" AT CORNELL".
http://www.nysaes.cornell.edu/pubs/press/1999/genegun.html. Retrieved 28th
january 2010.
10. ^ Chen I, Dubnau D (2004). "DNA uptake during bacterial transformation". Nat.
Rev. Microbiol. 2 (3): 241–9. doi:10.1038/nrmicro844. PMID 15083159.
11. ^ Large-volume transformation with high-throughput efficiency chemically
competent cells. Focus 20:2 (1998).
12. ^ http://faculty.plattsburgh.edu/donald.slish/Transformation.html
13. ^ Transformation efficiency of "'E. coli'" electroporated with large plasmid DNA.
Focus 20:3 (1998).
[edit] External links
Electroporation
From Wikipedia, the free encyclopedia
Jump to: navigation, search
Diagram of the major components of an electroporator with cuvette loaded.
Dr.Eberhard Neumann - Electroporation Founder
Electroporation, or electropermeabilization, is a significant increase in the electrical

conductivity and permeability of the cell plasma membrane caused by an externally
applied electrical field. It is usually used in molecular biology as a way of introducing
some substance into a cell, such as loading it with a molecular probe, a drug that can
change the cell's function, or a piece of coding DNA.[1]
Electroporation is a dynamic phenomenon that depends on the local transmembrane

voltage at each point on the cell membrane. It is generally accepted that for a given pulse
duration and shape, a specific transmembrane voltage threshold exists for the
manifestation of the electroporation phenomenon (from 0.5 V to 1 V). This leads to the
definition of an electric field magnitude threshold for electroporation (Eth). That is, only
the cells within areas where E≧Eth are electroporated. If a second threshold (Eir) is
reached or surpassed, electroporation will compromise the viability of the cells, i.e.,
irreversible electroporation.[2]
In molecular biology, the process of electroporation is often used for the transformation
of bacteria, yeast, and plant protoplasts. In addition to the lipid membranes, bacteria also
have cell walls which are different from the lipid membranes and are made of
peptidoglycan and its derivatives. However, the walls are naturally porous and only act as
stiff shells that protect bacteria from severe environmental impacts. If bacteria and
plasmids are mixed together, the plasmids can be transferred into the cell after
electroporation. Several hundred volts across a distance of several millimeters are
typically used in this process. Afterwards, the cells have to be handled carefully until they
have had a chance to divide producing new cells that contain reproduced plasmids. This
process is approximately ten times as effective as chemical transformation.[1][3]
This procedure is also highly efficient for the introduction of foreign genes in tissue
culture cells, especially mammalian cells. For example, it is used in the process of
producing knockout mice, as well as in tumor treatment, gene therapy, and cell-based
therapy. The process of introducing foreign DNAs into eukaryotic cells is known as
transfection.
Contents
[hide]
• 1 Laboratory Practice
• 2 Electroporators
• 3 Medical Applications
• 4 Physical Mechanism
• 5 References
[edit] Laboratory Practice
Cuvettes for electroporation. These are plastic with aluminium electrodes and a blue lid.
They hold a maximum of 400 μl.
Electroporation is done with electroporators, appliances which create an electro-
magnetic field in the cell solution. The cell suspension is pipetted into a glass or plastic
cuvette which has two aluminum electrodes on its sides.
For bacterial electroporation, typically a suspension of around 50 microliters is used.

Prior to electroporation it is mixed with the plasmid to be transformed. The mixture is
pipetted into the cuvette, the voltage and capacitance is set and the cuvette inserted into
the electroporator. Immediately after electroporation, one milliliter of liquid medium is
added to the bacteria (in the cuvette or in an eppendorf tube), and the tube is incubated at
the bacteria's optimal temperature for an hour or more to allow recovery of the cells and
expression of antibiotic resistance, followed by spreading on agar plates.
The success of the elecroporation depends greatly on the purity of the plasmid solution,
especially on its salt content. Solutions with high salt concentrations might cause an
electrical discharge (known as arcing), which often reduces the viability of the bacteria.
For a further detailed investigation of the process more attention should be paid to the
output impedance of the porator device and the input impedance of the cells suspension
(e.g. salt content). As the process needs direct electrical contact between the electrodes
and the suspension, and is inoperable with isolated electrodes, obviously the process
involves certain electrolytic effects, due to small currents and not only fields.
[edit] Electroporators
Electroporators come in two types: hand-held and bench-tops. Benchtop electroporators
are generally used as common lab equipment, residing atop a central bench or hood. They
offer the advantage of electroporating multiple samples at the same time. They can also
be set to different operating parameters depending on whether or not the cell has a cell
wall. In contrast, handheld electroporators are cordless, rechargeable and use disposable
pipectrodes, which combine elements of both cuvettes and pipettes. Their operating
parameters are preset to the optimal parameters for transforming either bacteria or
mammalian cells.
Both types of electoporators have been used on a wide range of cells - including E. coli
(for transformation) and mammalian cells such as neurons, astrocytes, neuroglia,
lymphocytes, monocytes, fibroblasts, epithelial and endothelial cells from humans, mice,
rats and monkeys (for transfection).
[edit] Medical Applications

A higher voltage of electroporation was found in pigs to irreversibly destroy target cells
within a narrow range while leaving neighboring cells unnaffected, and thus represents a
promising new treatment for cancer, heart disease and other disease states that require
removal of tissue.[4]
Electroporation can also be used to help deliver drugs or genes into the cell by applying
of short and intense electric pulses that transiently permeabilize cell membrane, thus
allowing transport of molecules otherwise not transported through a cellular membrane.
This procedure is referred to as electrochemotherapy when the molecules to be
transported is a chemotherapeutic agent or gene electrotransfer when the molecule to be
transported is DNA.
[edit] Physical Mechanism

Further information: Lipid bilayer mechanics
Electroporation allows cellular introduction of large highly charged molecules such as

DNA which would never passively diffuse across the hydrophobic bilayer core.[1] This
phenomenon indicates that the mechanism is the creation of nm-scale water-filled holes
in the membrane. Although electroporation and dielectric breakdown both result from
application of an electric field, the mechanisms involved are fundamentally different. In
dielectric breakdown the barrier material is ionized, creating a conductive pathway. The
material alteration is thus chemical in nature. In contrast, during electroporation the lipid
molecules are not chemically altered but simply shift position, opening up a pore which
acts as the conductive pathway through the bilayer as it is filled with water.
Schematic showing the theoretical arrangement of lipids in a hydrophobic pore (top) and
a hydrophilic pore (bottom).
Electroporation is a multi-step process with several distinct phases.[5] First, a short

electrical pulse must be applied. Typical parameters would be 300-400 mV for < 1 ms
across the membrane (note- the voltages used in cell experiments are typically much
larger because they are being applied across large distances to the bulk solution so the
resulting field across the actual membrane is only a small fraction of the applied bias).
Upon application of this potential the membrane charges like a capacitor through the
migration of ions from the surrounding solution. Once the critical field is achieved there
is a rapid localized rearrangement in lipid morphology. The resulting structure is believed
to be a “pre-pore” since it is not electrically conductive but leads rapidly to the creation
of a conductive pore. Evidence for the existence of such pre-pores comes mostly from the
“flickering” of pores, which suggests a transition between conductive and insulating
states.[6] It has been suggested that these pre-pores are small (~3 Å) hydrophobic defects.
If this theory is correct, then the transition to a conductive state could be explained by a
rearrangement at the pore edge, in which the lipid heads fold over to create a hydrophilic
interface. Finally, these conductive pores can either heal, resealing the bilayer or expand,
eventually rupturing it. The resultant fate depends on whether the critical defect size was
exceeded[7] which in turn depends on the applied field, local mechanical stress and bilayer
edge energy.
[edit] References
1. ^ a b c Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH (1982). "Gene
transfer into mouse lyoma cells by electroporation in high electric fields". EMBO
J. 1 (7): 841–5. PMID 6329708.
2. ^ Antoni Ivorra, Boris Rubinsky. "Gels with predetermined conductivity used in
electroporation of tissue USPTO Application #: 20080214986 - Class: 604 21
(USPTO)". http://www.freshpatents.com/Gels-with-predetermined-conductivity-
used-in-electroporation-of-tissue-dt20080904ptan20080214986.php.
3. ^ Sugar IP, Neumann E (1984). "Stochastic model for electric field-induced
membrane pores. Electroporation". Biophys. Chem. 19 (3): 211–25.
doi:10.1016/0301-4622(84)87003-9. PMID 6722274.
4. ^ Sarah Yang (2007-02-12). "New medical technique punches holes in cells,
could treat tumors".
http://www.berkeley.edu/news/media/releases/2007/02/12_IRE.shtml. Retrieved
2007-12-13.
5. ^ J. C. Weaver and Y. A. Chizmadzhev."Theory of electroporation: A review "
Biochemistry and Bioenergetics. 41. (1996) 135-160.
6. ^ K. C. Melikov, V. A. Frolov, A. Shcherbakov, A. V. Samsonov, Y. A.
Chizmadzhev and L. V. Chernomordik."Voltage-Induced Nonconductive Pre-
Pores and Metastable Single Pores in Unmodified Planar Lipid Bilayer "
Biophysical Journal. 80. (2001) 1829-1836.
7. ^ R. P. Joshi and K. H. Schoenbach."Electroporation dynamics in biological cells
subjected to ultrafast electrical pulses: A numerical simulation study." Physical
Review E. 62. (2000) 1025-1033.
INTRODUCTION
Plant transformation is the introduction of a foreign piece of DNA, confering a
specific trait, into host plant tissue. The foreign gene (termed the "transgene") is
incorporated into the host plant genome and stably inherited through future
generations. The correct regulatory sequences are added to the gene of interest i.e.
promoters and terminators, then the DNA is transferred to the plant cell culture
using an appropriate vector. The gene is attached to a selectable marker which
allows selection for the presence of the transgene. Genes conferring resistance to
a specific antibiotic are often used to serve this purpose. Once the plant tissue has
been transformed, the cells containing the transgene are selected and regeneration
back into whole plants is carried out. This is possible as plant cells are totipotent,
which means that they contain all the genetic information to control the
development of that cell into a potentially fertile plant. Therefore, the gene is
contained in every single plant cell, however, where it is switched on is
determined by the promoter which is controlling the gene. Plant transformation
can be carried out in a number of different ways depending on the species of plant
in question. This is discussed in the sections below. Plant transformation was
developed as an alternative to conventional breeding methods which are more
laborious than this fairly simple (now routine) laboratory proceedure.
WHY TRANSFORM PLANTS?

Plants are transormed in order to create transgenic plants that contain genes
conferring traits that are desirable to the plant breeder. This includes resistance to
specific plant diseases and pests, drought and saline tolerance. It also incudes
novel uses of plants, such as plantibody production and the production of edible
vaccines in plant tissue. Plants can be engineered to express sub-unit vaccines
which trigger oral immunity if plant tissues are consumed as food. Vaccines can
be created to a huge number of pathogens, such as bacteria that cause diarrhea;
responsible for 3 million infant deaths per year in the developing world.
(Arntzen, 1998)
Conventional breeding methods were seen to be slow and laborious, plant
transformation techniques were developed to overcome these problems. The
ability to introduce novel genes into plants, often across the species barrier,
provides many commercially viable applications to the plant breeder
Two of the major techniques used to transform plants are
:
AGROBACTERIUM-MEDIATED TRANFORMATION
A major method of DNA transfer in plants is Agrobacterium mediated transformation.
The natural living soil bacteria, Agrobacterium tumefaciens is capable of infecting a wide
range of plant species, causing Crown Gall diseases (see image below). It has natural
transformation abilities which can be exploited in plant biotechnology. When A.
tumefaciens infects as a cell, it transfers a copy of its T-DNA, which is a small section of
DNA carried on its Ti (Tumour Inducing) plasmid. The T-DNA is flanked by two
(imperfect) 25 base pair repeats. Any DNA contained within these borders will be
transferred to the host cell.(Zupan and Zambriski, 1995)
The T-DNA section on the Ti plasmid can be replaced by the transgene coding for the
desirable trait attached to the appropriate regulatory sequences. The recombinant bacteria
can then be used to infect both regenerating cell and protoplast cultures. (Protoplasts are
wall-less spehical plant cells)
Marker genes such as those coding for antibiotic resistance are attached to the transgene
so that it is possible to select those cells that have been transformed by the bacterium.
Cell to plant regeneration is carried out on the selected cells and transgene expression is
characterised i.e. it is necessary to check that the gene is expressed at the correct levels in
the correct tissues.
Agrobacterium tumefaciens has been used to transform many dicotyledonous plant
species with relative ease. However, it is, as yet, not possible to transform
monocotyledonous species, which include commercially viable cereal crops such as
maize and rice.
The image below shows a crown gall tumour on tomato. Tumours caused by
Agrobacterium tumefaciens are often apparent on the plant at ground level. The
picture of taken from
http://www.utoronto.ca/virology/bio351/plant/crngalltomato.jpg
BIOLISTICS
Biolistics (other wise known as Particle Bombardment) involves directly "shooting" a
piece of DNA into the recipient plant tissue. This is carried out using a gene gun.
Tungsten or gold beads (which are smaller than the plant cells themselves) are coated in
the gene of interest and fired through a stopping screen, accelerated by Helium, into the
plant tissue. The particles pass through the plant cells, leaving the DNA inside.
This method can be used on both monocotyledonous and dicotyledonous species

successfully. It is again a relatively simple laboratory proceedure. The transformed tissue
is selected using marker genes such as those that code for antibiotic resistance. Whole
plants are then regenerated from the totipotent transformed cells in culture, containing a
copy of the transgene in every single cell (Nottingham, 1998).
This picture was taken from http://www.agr.okstate.edu/ptf/chambergun.html - the

Oklahoma Plant Transformation Facility. The particles are fired through the gas
acceleration tube, through the rupture disk and into the target tissue.
Microinjection refers to the process of using a glass micropipette to insert substances at
a microscopic or borderline macroscopic level into a single living cell. It is a simple
mechanical process in which a needle roughly 0.5 to 5 micrometers in diameter
penetrates the cell membrane and/or the nuclear envelope. The desired contents are then
injected into the desired sub-cellular compartment and the needle is removed.
Microinjection is normally performed under a specialized optical microscope setup called
a micromanipulator. The process is frequently used as a vector in genetic engineering and
transgenics to insert genetic material into a single cell. Microinjection can also be used in
the cloning of organisms, and in the study of cell biology and viruses. Microcapillary and
microscopic devices are used to deliver DNA into a protoplast.
[edit] Examples
• Microinjection is used as a vector in transgenic plant production.
• Microinjection of genes into fertilized eggs is a common vector used in the
production of higher forms of transgenic animals.
• Microinjection of a gene knockdown reagent such as a morpholino oligo into eggs
or early zygotes is commonly used to probe the function of a gene during
development of embryos.
A gene gun or a biolistic particle delivery system, originally designed for plant
transformation, is a device for injecting cells with genetic information. The payload is an
elemental particle of a heavy metal coated with plasmid DNA. This technique is often
simply referred to as bioballistics or biolistics.
This device is able to transform almost any type of cell, including plants, and is not
limited to genetic material of the nucleus: it can also transform organelles, including
plastids.
Contents
[hide]
• 1 Design
• 2 Application
o 2.1 Plants
o 2.2 Humans and animals
• 3 References
• 4 Further reading
[edit] Design
The gene gun was originally a Crossman air pistol modified to fire dense tungsten
particles. The design was first used on onions to deliver particles coated with a marker
gene. Genetic transformation can then be proven when the onion tissue expresses the
gene.
The earliest custom manufactured geneguns (fabricated by Nelson Allen) used a 22

caliber nail gun cartridge to propel an extruded polyethylene cylinder (bullet) down a 22
cal. Douglas barrel. A droplet of the tungsten powder and genetic material was placed on
the bullet and shot down the barrel at a lexan "stopping" disk with a petri dish below. The
bullet welded to the disk and the genetic information blasted into the sample in the dish
with a doughnut effect (devastation in the middle, a ring of good transformation and little
around the edge). The gun was connected to a vacuum pump and was under vacuum
while firing. The early design was put into limited production by a Rumsey-Loomis (a
local machine shop then at Mecklenburg Rd in Ithaca, NY, USA). Later the design was
refined by removing the "surge tank" and changing to nonexplosive propellants. DuPont
added a plastic extrusion to the exterior to visually improve the machine for mass
production to the scientific community. Biorad contracted with Dupont to manufacture
and distribute the device. Improvements include the use of helium propellant and a multi-
disk-collision delivery mechanism. Other heavy metals such as gold and silver are also
used. Gold may be favored because it has better uniformity than tungsten and tungsten
can be toxic to cells, but its use may be limited due to availability and cost.
[edit] Application
Gene guns are so far mostly applied for plant cells. However, there is much potential use
in animals and humans as well.
[edit] Plants
The target of a gene gun is often a callus of undifferentiated plant cells growing on gel
medium in a petri dish. After the gold particles have impacted the dish, the gel and callus
are largely disrupted. However, some cells were not obliterated in the impact, and have
successfully enveloped a DNA coated tungsten particle, whose DNA eventually migrates
to and integrates into a plant chromosome.
Cells from the entire petri dish can be re-collected and selected for successful integration
and expression of new DNA using modern biochemical techniques, such as a using a
tandem selectable gene and northern blots.
Selected single cells from the callus can be treated with a series of plant hormones, such
as auxins and gibberellins, and each may divide and differentiate into the organized,
specialized, tissue cells of an entire plant. This capability of total re-generation is called
totipotency. The new plant that originated from a successfully shot cell may have new
genetic (heritable) traits.
The use of the gene gun may be contrasted with the use of Agrobacterium tumefaciens
and its Ti plasmid to insert genetic information into plant cells. See transformation for
different methods of transformation in different species.
[edit] Humans and animals
Gene guns have also been used to deliver DNA vaccines.
The delivery of plasmids into rat neurons through the use of a gene gun, specifically
DRG neurons, is also used as a pharmacological precursor in studying the effects of
neurodegenerative diseases such as Alzheimer's Disease.
The gene gun technique is also popularly used in an edible vaccine production technique,
where the nano-gold particles coated with plant genetic material under the high vacuum
pressurized chamber are transformed into suitable plant tissues.
The gene gun has become a common tool for labeling subsets of cells in cultured tissue.
In addition to being able to transfect cells with DNA plasmids coding for fluorescent
proteins, the gene gun can be adapted to deliver a wide variety of vital dyes to cells.[1]
Gene gun bombardment is also used to transform C. elegans, as an alternative to

microinjection.
[edit] References
1. ^ Gan W, Grutzendler J, Wong WT, Wong ROL, Lichtman JW, Multicolor
DiOlistic Labeling of the Nervous System Using Lipophilic Dye Combinations
Neuron Volume 27, Issue 2, 219-225
[edit] Further reading

John A O’Brien, Sarah C Lummis, Garth T. Whiteside, Ray W. Colburn and Michael H
Hastings. Modifications to the hand-held gene gun: Improvements for in-vitro Biolistic
transfection of neuronal tissue. J Neuroscience Methods 112:57-64 (2001).
Agrobacterium tumefaciens
A. tumefaciens attaching itself to a

carrot cell
Scientific classification
Kingdom: Bacteria
Phylum: Proteobacteria
Alpha
Class:
Proteobacteria
Order: Rhizobiales
Family: Rhizobiaceae
Genus: Agrobacterium
Species: A. tumefaciens
Binomial name
Smith & Townsend, 1907
Synonyms
Bacterium tumefaciens Smith
and Townsend 1907
Pseudomonas tumefaciens
(Smith and Townsend 1907) Duggar
1909
Phytomonas tumefaciens (Smith
and Townsend 1907) Bergey et al.
1923
Polymonas tumefaciens (Smith
and Townsend 1907) Lieske 1928
Agrobacterium tumefaciens (updated scientific name: Rhizobium radiobacter)[1][2] is the

causal agent of crown gall disease (the formation of tumours) in over 140 species of
dicot. It is a rod shaped, Gram negative soil bacterium (Smith et al., 1907). Symptoms are
caused by the insertion of a small segment of DNA (known as the T-DNA, for 'transfer
DNA') into the plant cell,[3] which is incorporated at a semi-random location into the plant
genome.
Agrobacterium tumefaciens (or A. tumefaciens) is an alphaproteobacterium of the family

Rhizobiaceae, which includes the nitrogen fixing legume symbionts. Unlike the nitrogen
fixing symbionts, tumor producing Agrobacterium are pathogenic and do not benefit the
plant. The wide variety of plants affected by Agrobacterium makes it of great concern to
the agriculture industry.[4]
Economically, A. tumefaciens is a serious pathogen of walnuts, grape vines, stone fruits,

nut trees, sugar beets, horse radish and rhubarb.
Contents
[hide]
• 1 Conjugation
• 2 Method of infection
o 2.1 Formation of the T-pilus
o 2.2 Transfer of T-DNA into the plant cell
• 3 Genes in the T-DNA
o 3.1 Hormones
o 3.2 Opines
• 4 Beneficial uses
• 5 See also
• 6 References
[edit] Conjugation
In order to be virulent, the bacterium must contain a tumour-inducing plasmid (Ti
plasmid or pTi), of 200 kb, which contains the T-DNA and all the genes necessary to
transfer it to the plant cell. Many strains of A. tumefaciens do not contain a pTi.
Since the Ti plasmid is essential to cause disease, pre-penetration events in the

rhizosphere occur to promote bacterial conjugation - exchange of plasmids amongst
bacteria. In the presence of opines, A. tumefaciens produces a diffusible conjugation
signal called 30C8HSL or the Agrobacterium autoinducer. This activates the transcription
factor TraR, positively regulating the transcription of genes required for conjugation.
[edit] Method of infection

The Agrobacterium tumefaciens infects the plant through its Ti plasmid. The Ti plasmid
integrates a segment of its DNA, known as T-DNA, into the chromosomal DNA of its
host plant cells.
A. tumefaciens have flagella that allow them to swim through the soil towards
photoassimilates that accumulate in the rhizosphere around roots. Some strains may
chemotactically move towards chemical exudates from plants, such as acetosyringone
and sugars. The former is recognised by the VirA protein, a transmembrane protein
encoded in the virA gene on the Ti plasmid. Sugars are recognised by the chvE protein, a
chromosomal gene-encoded protein located in the periplasmic space.[5].
At least 25 vir genes on the Ti plasmid are necessary for tumor induction. In addition to
their perception role, virA and chvE induce other vir genes. The virA protein has kinase
activity: it phosphorylates itself on a histidine residue. Then the virA protein
phosphorylates the virG protein on its aspartate residue. The virG protein is a cytoplasmic
protein produced from the virG Ti plasmid gene. It is a transcription factor, inducing the
transcription of the vir operons. The chvE protein regulates the second mechanism of the
vir genes' activation. It increases VirA protein sensibility to phenolic compounds.[5]
Attachment is a two-step process. Following an initial weak and reversible attachment,

the bacteria synthesize cellulose fibrils that anchor them to the wounded plant cell to
which they were attracted. Four main genes are involved in this process: chvA, chvB,
pscA and att. It appears that the products of the first three genes are involved in the actual
synthesis of the cellulose fibrils. These fibrils also anchor the bacteria to each other,
helping to form a microcolony.
After production of cellulose fibrils a calcium-dependent outer membrane protein called

rhicadhesin is produced, which also aids in sticking the bacteria to the cell wall.
Homologues of this protein can be found in other Rhizobia species.
Possible plant compounds that initiate Agrobacterium to infect plant cells:[6]
• Acetosyringone: Phenolic compound

• alpha-Hydroxyacetosyringone
• Catechol
• Ferulic acid
• Gallic acid
• p-Hydroxybenzoic acid
• Protocatechuic acid
• Pyrogallic acid
• Resorcylic acid
• Sinapinic acid
• Syringic acid
• Vanillin
[edit] Formation of the T-pilus

In order to transfer the T-DNA into the plant cell A. tumefaciens uses a Type IV secretion
mechanism, involving the production of a T-pilus. When acetosyringone and other
substances are detected, a signal transduction event activates the expression of 11 genes
within the VirB operon which are responsible for the formation of the T-pilus.
The pro-pilin is formed first. This is a polypeptide of 121 amino acids which requires
processing by the removal of 47 residues to form a T-pilus subunit. The subunit is
circularized by the formation of a peptide bond between the two ends of the polypeptide.
Products of the other VirB genes are used to transfer the subunits across the plasma
membrane. Yeast two-hybrid studies provide evidence that VirB6, VirB7, VirB8, VirB9
and VirB10 may all encode components of the transporter. An ATPase for the active
transport of the subunits would also be required.
[edit] Transfer of T-DNA into the plant cell
A: Agrobacterium tumefaciens
B: Agrobacterium genome
C: Ti Plasmid : a: T-DNA , b: Vir genes , c: Replication origin , d: Opines catabolism
genes
D: Plant cell
E: Mitochondria
F: Chloroplast
G: Nucleus
The T-DNA must be cut out of the circular plasmid. A VirD1/D2 complex nicks the
DNA at the left and right border sequences. The VirD2 protein is covalently attached to
the 5' end. VirD2 contains a motif that leads to the nucleoprotein complex being targeted
to the type IV secretion system (T4SS).
In the cytoplasm of the recipient cell, the T-DNA complex becomes coated with VirE2
proteins, which are exported through the T4SS independently from the T-DNA complex.
Nuclear localization signals, or NLS, located on the VirE2 and VirD2 are recognised by
the importin alpha protein, which then associates with importin beta and the nuclear pore
complex to transfer the T-DNA into the nucleus. VIP1 also appears to be an important
protein in the process, possibly acting as an adapter to bring the VirE2 to the importin.
Once inside the nucleus, VIP2 may target the T-DNA to areas of chromatin that are being
actively transcribed, so that the T-DNA can integrate into the host genome.
[edit] Genes in the T-DNA

[edit] Hormones
In order to cause gall formation, the T-DNA encodes genes for the production of auxin or
indole-3-acetic acid via the IAM pathway. This biosynthetic pathway is not used in many
plants for the production of auxin, so it means the plant has no molecular means of
regulating it and auxin will be produced constitutively. Genes for the production of
cytokinins are also expressed. This stimulates cell proliferation and gall formation.
[edit] Opines
The T-DNA contains genes for encoding enzymes that cause the plant to create
specialized amino acids which the bacteria can metabolize, called opines.[7] Opines are a
class of chemicals that serve as a source of nitrogen for A. tumefaciens, but not for most
other organisms. The specific type of opine produced by A. tumefaciens C58 infected
plants is nopaline (Escobar et al., 2003).
Two nopaline type Ti plasmids, pTi-SAKURA and pTiC58, were fully sequenced. A.
tumefaciens C58, the first fully sequenced pathovar, was first isolated from a cherry tree
crown gall. The genome was simultaneously sequenced by Goodner et al.[8] and Wood et
al.[9] in 2001. The genome of A. tumefaciens C58 consists of a circular chromosome, two
plasmids, and a linear chromosome. The presence of a covalently bonded circular
chromosome is common to Bacteria, with few exceptions. However, the presence of both
a single circular chromosome and single linear chromosome is unique to a group in this
genus. The two plasmids are pTiC58, responsible for the processes involved in virulence,
and pAtC58, coined the “cryptic” plasmid.[8][9]
The pAtC58 plasmid has been shown to be involved in the metabolism of opines and to
conjugate with other bacteria in the absence of the pTiC58 plasmid.[10] If the pTi plasmid
is removed, the tumor growth that is the means of classifying this species of bacteria does
not occur.
[edit] Beneficial uses

Plants that have undergone transformation with Agrobacterium.
The DNA transmission capabilities of Agrobacterium have been extensively exploited in

biotechnology as a means of inserting foreign genes into plants. Marc Van Montagu and
Jeff Schell, (University of Ghent and Plant Genetic Systems, Belgium) discovered the
gene transfer mechanism between Agrobacterium and plants, which resulted in the
development of methods to alter the bacterium into an efficient delivery system for
genetic engineering in plants.[11] The plasmid T-DNA that is transferred to the plant is an
ideal vehicle for genetic engineering.[12] This is done by cloning a desired gene sequence
into the T-DNA that will be inserted into the host DNA. This process has been performed
using firefly luciferase gene to produce glowing plants. This luminescence has been a
useful device in the study of plant chloroplast function and as a reporter gene.[13] It is also
possible to transform Arabidopsis thaliana by dipping their flowers into a broth of
Agrobacterium: the seed produced will be transgenic. Under laboratory conditions the T-
DNA has also been transferred to human cells, demonstrating the diversity of insertion
application.[14]
The mechanism by which Agrobacterium inserts materials into the host cell by a type IV
secretion system is very similar to mechanisms used by pathogens to insert materials
(usually proteins) into human cells by type III secretion. It also employs a type of
signaling conserved in many Gram-negative bacteria called quorum sensing. This makes
Agrobacterium an important topic of medical research as well.
[edit] See also

• SuhB
[edit] References
1. ^ "Rhizobium radiobacter (Agrobacterium tumefaciens) (Agrobacterium
radiobacter)". UniProt Taxonomy. http://pir.uniprot.org/taxonomy/358. Retrieved
2010-06-30.
2. ^ Young J.M., Kuykendall L.D., Martínez-Romero E., Kerr A. and Sawada H.
2001. A revision of Rhizobium Frank 1889, with an emended description of the
genus, and the inclusion of all species of Agrobacterium Conn 1942 and
Allorhizobium undicola de Lajudie et al. 1998 as new combinations: Rhizobium
radiobacter, R. rhizogenes, R. rubi, R. undicola and R. vitis. International
Journal of Systematic and Evolutionary Microbiology, 51:89-103
3. ^ Chilton MD, Drummond MH, Merio DJ, Sciaky D, Montoya AL, Gordon MP,
Nester EW., Stable incorporation of plasmid DNA into higher plant cells: the
molecular basis of crown gall tumorigenesis, Cell. 1977 Jun;11(2):263-71.
4. ^ Moore LW, Chilton WS, Canfield ML. 1997. Diversity of Opines and Opine-
Catabolizing Bacteria Isolated from Naturally Occurring Crown Gall Tumors.
App. Environ. Microbiol. 63:201-207.
5. ^ a b Stanton B. Gelvin,Department of Biological Sciences, Purdue University,
West Lafayette, Indiana 47907-1392, Agrobacterium-Mediated Plant
Transformation: the Biology behind the “Gene-Jockeying” Tool,
http://mmbr.asm.org/cgi/reprint/67/1/16
6. ^ U.S. Patent 6483013
7. ^ Zupan J, Muth TR, Draper O, Zambryski P. 2000. The transfer of DNA from
Agrobacterium tumefaciens into plants: a feast of fundamental insights. Plant J.
23:11-28.
8. ^ a b Goodner B, Hinkle G, Gattung S, Miller N, et al. 2001. Genome Sequence of
the Plant Pathogen and Biotechnology Agent Agrobacterium tumefaciens C58.
Science. 294:2323-2328.
9. ^ a b Wood DW, Setubal JC, Kaul R, Monks DE, et al. 2001. The Genome of the
Natural Genetic Engineer Agrobacterium tumefaciens C58. Science. 294:2317-
2323.
10. ^ Vaudequin-Dransart V, Petit A, Chilton WS, Dessaux Y. 1998. The cryptic
plasmid of Agrobacterium tumefaciens cointegrates with the Ti plasmid and
cooperates for opine degradation. Molec. Plant-microbe Interact. 11:583-591.
11. ^ Schell J, Van Montagu M., The Ti-plasmid of Agrobacterium tumefaciens, a
natural vector for the introduction of nif genes in plants?, Basic Life Sci.
1977;9:159-79.
12. ^ Zambryski P. et al. 1983. Ti plasmid vector for introduction of DNA into plant
cells without alteration of their normal regeneration capacity. EMBO J. 2:2143-
2150.
13. ^ Root M. 1988. Glow in the dark biotechnology. Bioscience. 38:745-747.
14. ^ Kunik T, Tzfira T, Kapulnik Y, Gafni Y, Dingwall C, Citovsky V. 2001.
Genetic transformation of HeLa cells by Agrobacterium. Proc. Natl. Acad. Sci.
98:1871-1876.
• Dickinson, M. (2003). Molecular Plant Pathology. BIOS Scientific Publishers.

• Lal, Erh-Min and Kado, Clarence I. (2000). The T-Pilus of Agrobacterium
tumefaciens. Trends in Microbiology, Vol. 8, Issue 8.
• Ward, Doyle V., Zupan, John R and Zambryski, Patricia C. (2002).
Agrobacterium VirE2 gtes the VIP1 treatment in plant nuclear import. Trends in
Plant Science, Vol. 7 Issue 1.
Ti plasmid
The structure of the Ti plasmid
Ti plasmid is a circular plasmid that often, but not always, is a part of the genetic
equipment that Agrobacterium tumefaciens and Agrobacterium rhizogenes use to
transduce its genetic material to plants. The Ti plasmid is lost when Agrobacterium is
grown above 28°C. Such cured bacteria do not induce crown galls, i.e. they become
avirulent. pTi and pRi share little sequence homology but are functionally rather similar.
The Ti plasmids are classified into different types based on the type of opine produced by
their genes. The different opines specified by pTi are octopine, nopaline, succinamopine
and leucinopine.
The plasmid has 196 genes that code for 195 proteins. There is no one structural RNA.
The plasmid is 206,479 nucleotides long, the GC content is 56% and 81% of the material
is coding genes. There are no pseudogenes.
The modification of this plasmid is very important in the creation of transgenic plants,
but only in dicotyledon plants.
Contents
[hide]
• 1 Virulence Region
• 2 References
• 3 See also
[edit] Virulence Region
Genes in the virulence region are grouped into the operons virABCDEFG, which code for
the enzymes responsible for mediating transduction of T-DNA to plant cells.[1]
• virA codes for a receptor which reacts to the presence of phenolic compounds
such as acetosyringone,[2], syringealdehyde or acetovanillone[3] which leak out of
damaged plant tissues.[4]
• virB encodes proteins which produce a pore/pilus-like structure.[2]
• virC binds the overdrive sequence.[2]
• virD1 and virD2 produce endonucleases which target the direct repeat borders of
the T-DNA segment,[2] beginning with the right border.[4]
• virG activates vir-gene expression after binding to a consensus sequence,[2] once it
has been phosphorylated by virA.[4]
[edit] References
• Schell J, Van Montagu M., The Ti-plasmid of Agrobacterium tumefaciens, a
natural vector for the introduction of nif genes in plants?, Basic Life Sci.
1977;9:159-79.
• B.D.Singh, genetic engineering and cloning.
1. ^ Scott E.Stachelt, Eugene W.Nester (1986). "The genetic and transcriptional

organization of the vir region of the A6 Ti plasmid of Agrobacterium
tumefaciens". The EMBO Journal 5 (7): 1445–1454.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1166964/.
2. ^ a b c d e Schrammeijer, B., Beijersbergen, A., Idler, K.B., Melchers, L.S.,
Thompson, D.V., Hooykaas, P.J.J. (2000). "Sequence analysis of the vir-region
from Agrobacterium tumefaciens octopine Ti plasmid pTi15955". Journal of
Experimental Botany 51 (347): 1167–1169.
http://jxb.oxfordjournals.org/cgi/content/full/51/347/1167.
3. ^ Reconstitution of Acetosyringone-Mediated Agrobacterium tumefaciens
Virulence Gene Expression in the Heterologous Host Escherichia coli. Scott M.
Lohrke, Hongjiang Yang, and Shouguang Jin, J Bacteriol. 2001 June; 183(12):
3704–3711, doi:10.1128/JB.183.12.3704-3711.2001
4. ^ a b c Alexander N. Glazer, Hiroshi Nikaidō (2007). Microbial biotechnology:
fundamentals of applied microbiology. Cambridge University Press.
ISBN 0521842107.
Calcium chloride transformation
Calcium chloride (CaCl2) transformation is a laboratory technique in prokaryotic

(bacterial) cell biology. It increases the ability of a prokaryotic cell to incorporate plasmid
DNA allowing them to be genetically transformed.[1] The addition of calcium chloride to
a cell suspension promotes the binding of plasmid DNA to the cell surface, which can
then pass into the cell. CaCl2 transformation is also accompanied by heat shock, where
cells are cooled to a low temperature (+4 degrees Celsius) and then heated to a high
temperature (+42 degrees Celsius) for a short time.
[edit] References
1. ^ Dagert, M.; Ehrlich, S. (1979). "Prolonged incubation in calcium chloride
improves the competence of Escherichia coli cells". Gene 6 (1): 23–28.
doi:10.1016/0378-1119(79)90082-9. PMID 383576. edit
lternative methods of plant transformation--a short

review.
Rakoczy-Trojanowska M.
Department of Plant Genetics, Breeding and Biotechnology, Warsaw Agricultural

University, Nowoursynowska 166, 02-787 Warszawa, Poland.
RAKOCZY@ALPHA.SGGW.WAW.PL
Abstract
Several methods of transformation are currently available for delivering exogenous DNA
to plant cells. Agrobacterium-mediated transformation, microprojectile bombardment and
direct protoplast transformation are routinely used today. However, each of them has
certain disadvantages, which led to research into the development of novel alternative
systems such as infiltration, electroporation of cells and tissues, electrophoresis of
embryos, microinjection, pollen-tube pathway, silicon carbide- and liposome-mediated
transformation. The low efficiency of transformation is considered to be the main reason
for the limited popularity of the alternative transformation methods, other than infiltration
and silicon carbide-mediated transformation, which seem to be the most promising ones
for practice.
Abstract
Background
In plant transformation, method compliance is critical for success. Transformation

methods are complicated and tend to evolve over time. Until the complete method is
published, method details are often partially orally transmitted and thus bound to a few
people. Their documentation in text files are often a mixture of material and method
description with many references to other sources especially to media description. These
media are complex and often composed from several commercially available mixtures
plus individually prepared stocks. The actual transformation experiment is generally
documented in lab books, in which deviations from the methods and results are reported.
Additionally, work schedules are planned in diaries. Both paper-based sources lack
backup copies and miss unambiguous links to method descriptions and media recipes.
Description
To solve the problem, we devised a standard-operation-procedure system based on a

Microsoft Access database containing the interlinked modules 'Media', 'Methods' and
'Experiments'. The Media module contains all basic chemicals, stocks and complex
media. In this module, complex media are composed from other elements of the Media
module, thus mimicking the workflows of media preparation in the lab. The Media
module is made attractive to the user by functions that generate file cards and labels. The
Methods module describes each method stepwise and links the steps to the media. Copy
functions allow cloning of old methods to document method evolution without alteration
of the old methods. Activation and inactivation functions in the Media and the Methods
module remove outdated entries from active use. The Experiments module links the
method to experiment specific information. This module generates a lab-book like user
interface and a work schedule, and it contains a simple result section.
Conclusion
The system has been evolved and tested over several years in a transformation service
unit, where it increased efficiency. Additionally, the system provided rapid access to data
for quality control and decision making. The system can be easily modified for the use in
other research environments.
Background
In tissue-culture-based generation of genetically modified plants, minute method

compliance is critical for success. Plant transformation methods are complex multi-step
methods that incorporate parts of older methods (bits that worked) and thus contain
references to older sources. In published methods, this results in a chain of cross-
references. For example, a modern rice transformation method ([1] references to [2,3]
and [4], which references to [5]). The problem is not tissue-culture specific, but can be
found in many other research fields as well, to the inconvenience of the reader trying to
reproduce a method.
In laboratory practice, methods are often documented on paper or in text files. The link
between methods is ambiguously provided by method names and often orally transmitted.
The method text generally contains both, the method description and (some) information
on media composition as well as cross-references to media described elsewhere. The
cross-reference is again done by media names. Media names tend to change, usually
because their initial name becomes ambiguous or is too long for practical use. Thus, the
same medium may have different names sometimes even in the same lab. Furthermore,
tissue culture media are often very complex with 20 and more different compounds. They
are thus often composed stepwise from stocks and mixtures of stocks, including
commercially available mixtures. The source of the chemicals, the preparation procedure
of the stocks and the complex mixtures are deemed important to the efficiency of the
transformation experiment. Efficiency results from the required man power and the
success rate. To increase efficiency, media and methods are improved over time.
Methods are furthermore changed to adapt them to different cultivars. During these
evolutionary processes, the original method can be easily lost. Thus, the reproducibility
of plant transformation experiments is in jeopardy, especially if the method is
unpublished, has not been used for some time or has been developed by a researcher who
left the laboratory.
To address the problem, we designed a computer-based documentation system that

interlinks media, methods and actual experiments unambiguously. To make its use
attractive to the lab staff, we provided reporting tools that produce stock labels and 'file
cards' with media description. The reporting tool additionally generates work schedules
for the current experiments. The system was established and tested in a service unit that
generates transgenic plants for researchers.
Construction and content
The database system consists of the three modules Media, Methods and Experiments that
represent the workflows preparation and storage of media, standard operation procedures
(methods) and the conduction of experiments. The start page of the database (additional
file 1, for readers without access to MS Access: additional file 2: SupplementaryFigures -
'Startpage') provides direct access to the most important functions of these three modules.
Additional file 1. MSTransformation2003. MS Access 2003 file, enable macros for full
functionality.
Format: ZIP Size: 986KB Download file
Additional file 2. SupplementaryFigures. The file contains pdf-files with screenshots

on various forms of MSTransformation2003 to enable readers without access to MS-
Access to view the forms. The content of each screenshot is addressed in the manuscript.
Format: ZIP Size: 552KB Download file
Media module
The Media module contains information on all basic chemicals, stocks and complex
media. Data entry into the module is performed on forms (Figure 1), for which
customized versions (different languages, personalized forms) can be generated easily
without programming skills (s. additional file 1: Method form without (Media_E_1) or
with datasheet view (Media_E_2), alternatively additional file 2: Media_E_1 or
Media_E_2).
Figure 1. Media form. The form contains unique name and id of the
medium, preparation and storage information (A). The graphic entry field 'Code' contains
color codes for plates. A medium can be composed from many compounds that can be
selected from the list of existing media. Basic compounds purchased from external
suppliers do not contain any further compounds. The 'Show media' button of each
compound displays the composition of the selected compound (B). The 'print label'
function prints labels on a label printer, the function 'Print file card' prints a report on a
standard (A4) printer. The background color of the entry fields indicates the status of the
medium (white = new, blue = approved, grey = obsolete).
On entry, each medium receives a unique media id and a unique name. For each medium,
information on preparation (remark, solvents, and sterilization) and storage conditions are
provided. On the form, media can be generated by selecting other media or stocks from
the Media module. For example, the medium in Figure 1 is generated from three basic
compounds. The complex medium Id 404 (see additional file 1, alternatively additional
file 2: Media_E_1) is generated from other media and stocks. Stocks reference back to
one basic chemical (see additional file 1: Glucose 16%, Id 411 alternatively additional
file 2: Media_E_1, Id 411). Basic chemicals or ready-made mixtures purchased from
suppliers do not reference back to any other media entry (Example Glucose Id 413
alternatively additional file 2: Media_E_1, Id 413). With this 'self-referencing', the Media
module mimics the workflow of media preparation in the lab. The referencing between
the media is based on the unique media id. This system allows editing of media names to
correct typos. Furthermore, multi-language versions can be generated without losing the
connection between media or to the Methods module.
To facilitate lab work and thus make the system more attractive to users, file cards and
labels are generated automatically (Figure 1). File cards contain all information needed to
make up the media in the lab including the name, id and the correct storage condition.
Autoclave-proof labels display the id and the name of the medium. The print-out date on
the label facilitates tracking the age of stocks.
Media entries can be put into three different stages by the scientists responsible for the
database (so-called 'administrator'). Administrator functions are accessible on in the
experts' module (additional file 1, alternatively additional file 2: 'expertsmodule'). The
status of a medium is depicted by the background color of the entry fields. New media
entries can be edited by all users (white background). As soon an approved date has been
entered by the administrator (into the form media experts), the background turns blue.
Then, changes to the media are no longer possible to avoid accidental changes by a user.
When a date has been entered in the 'obsolete' field (grey background) the user receives a
warning when he tries to choose the outdated medium to compose a new medium.
Methods module
The Methods module describes each method stepwise (Figure 2, additional file 1: Method
form with (Method form_E_1) or without datasheet view (Method_form E_2),
alternatively additional file 2: 'Method form_E_1'). Each step represents a logical unit
performed at a time. The information when a step is to be performed is given relative to
the beginning of the experiment. In our example, the start was defined as the
transformation date. This method allows calculating the real date for each step of an
experiment (s. 'Experiment module') based on the experiment-specific transformation
date. Each step links to the media required for this step and the recommended containers
(tubes, plates). Furthermore, it lists incubation conditions.
Figure 2. Method form. The form contains unique name and id of the
method, information on the species, on which it can be used, and the selection. The
method is divided in distinct steps that are performed at different times. Media can be
linked to each step by choosing them from the media list. The function 'copy method'
copies the entire method including all steps and media linked to them. The function 'Copy
step' allows copying a step from another method into a method.
To facilitate the documentation of new methods, two copy functions were designed. The
'copy method' function copies the entire method including all steps. For each step, it
copies all media linked to it. The second copy function ('copy step') allows copying single
steps from existing methods into a new method to build new methods from well-
established parts of other methods. Both copy functions assigns new ids and new names
to the method and each step. Modified methods can thus be generated from existing
methods by altering a few details like the incubation conditions or the media
composition. If the new method replaces the old method, the administrator enters the
'obsolete' date for the old method (see additional file 1 Form 'Methods Experts',
alternatively additional file 2: 'MethodsExperts'). Outdated methods remain in the actual
database and are available for referencing. Thus, information on experiments performed
with older methods remains unaltered.
Experiments module
The Experiments module contains the information on an actual experiment, in our case a
plant transformation, and links it to the method (Form see Figure 3). The basic
information for a transformation experiment is the transformation date, the plasmid
information and the details on the parent plant that is to be transformed.
Figure 3. Experiment form. The form for the documentation of plant

transformations collects the information on the parent plant and the plasmid used for a
transformation experiment. The plasmid identifier links to a plasmid table, in which
relevant details (e.g. resistance marker genes, approval status) are documented. The
approval status of the construct is displayed as color code. The experiment is started by
choosing a method from the method list and entering the start date. Results are entered in
the lower section of the form. 'Details' generates a schedule for the experiment with a
short method description. 'Document' generates the final filing document. For 'Lab book'
function see Figure 4.
The plasmid information is stored in a separate table (construct, see additional file 1:
Experts' module; alternatively additional file 2: 'Construct'). Plasmid information links to
the experiment by the plasmid id. Information relevant for the experiment (namely
plasmid name and selection system) is displayed on the experiment form but cannot be
changed there. The plasmid entries can be approved by an expert, when the plasmid
check was positive. Depending on the status of the plasmid, the plasmid id field is red
(not approved) or green (approved). The plasmid information can be entered and changed
in the table construct that is accessible from the experts' page. In our case, the plasmid
information is read from an Oracle database of a laboratory information management
system (LIMS) to save the time for double entries in both systems.
The details on the parent plants are stored in a separate parent table, from which they can
be selected on the experiment form. Again, relevant information like species and variety
is displayed on the experiment form, but can only be altered in the parent table (experts
module screen: Parents experts).
Depending on the transformation method, namely ballistic or Agrobacterium tumefaciens

mediated transformation, different additional information is required. These differences
are catered for by designing modified forms that feed into the same table. The form for
ballistic transformation requests information on the plasmid concentration and the cannon
used (see additional file 1, 'Enter Ballistic transformation', alternatively additional file 2:
'BallisticTransformation'). The Experiment form for Agrobacterium mediated
transformation (Figure 3) collects the name of the Agrobacterium strain. On each form,
only the respective methods for ballistic or Agrobacterium mediated transformation are
displayed. As transformations are often performed by specialized researchers and
technicians (experts) for other researchers (scientist), information on both experts and
scientists are requested in the form.
When a transformation experiment is started, the method is selected and the

transformation date entered. Now, a method report containing the real due-dates for each
step and a short description can be generated (function 'Details'). The function 'Generate'
produces a laboratory book (Figure 4); this function can be run only once on each
experiment. In this computer-based lab book, the most frequent deviations from standard
methods namely date and media can be altered without changing the description of the
original method. The text field 'Remarks' provides space to describe further method
deviations or observations.
Figure 4. Lab book. For each transformation experiment, a laboratory

book can be generated once by activating the function 'Lab book'. The function calculates
the due dates for each step from the start date and the method. Deviations with respect to
timing and media can be entered without changing the original method.
Work schedules for the next 9 or 31 days are automatically generated for all active
experiments (s. additional file 1, Work overview 'Next 31 days', alternatively additional
file 2: 'Next 31 days'). The due dates are calculated from the transformation date and the
selected method. For the schedules, the due dates are filtered for the time between today
and today + 9 (31) days. For each day, the ids of the transformations that are due for
processing and a short description of each step are listed (Table 1).
Table 1. Work schedule shows an example of an automatically generated work schedule

listing the work for the next nine days.
The end of the experiment is documented by keywords (handover, withdrawn,

contaminated) and, if successful, with the number of selected plants. Finished
experiments are withdrawn from the work schedule. From these data, reports on success
rates and failure reasons can be easily generated (example see Table 2).
Table 2. Evaluation example shows success rates and failure rates for the transformation
of different species in four years.
Utility and discussion
In large research institutions and academic communities, documentation of resources and

results in databases is state of the art. Database systems are used to manage genetic
resources ([6-10]). Databases make scientific results available to the scientific
community, especially in the areas of transcriptomics [11], proteomics [12] and
metabolomics [13]. The general availability of these data in standardized formats enable
derived knowledge and integrative approaches [14-17]. The big exception from
standardized formats is the documentation of experimental methods that link the genetic
resources with the result and the knowledge. Well established, reliably repeatable
methods - so-called standard operation procedures (SOPs) - are a valuable part of the
success of any research institution. SOPs are, however, mainly documented in text style
as part of a publication or - in case of method evolution - several publications linked by
references. Alternatively, methods are made available as text files on web sites [18,19].
There are academic approaches to document SOPs in databases [20,21]. In these
databases, text files with the method description are stored and linked to the result. As
pointed out in the introduction, text-based documents have their drawbacks. Some
methods are better stored in a database, especially those that involve complicated media
preparation or many steps performed over a long period of time. Most users are, however,
more familiar with word processing programs than databases. The 'interface' problem can
be solved by so-called laboratory information management systems (LIMS). A LIMS
provides a user-friendly graphic user interface to a database. In these systems, methods
can be modeled as workflows. However, commercial LIMS systems are unavailable for
most academic institutions, because of the considerable investment to set up such a
system and to keep it running. For transcript profiling, an excellent open-source database
system allows to document parameters of transcript profiling experiments [22]. For plant
transformation, we devised a simple system to store standard operation procedures. The
system is based on the widespread database system MS-Access and thus can be adjusted
to specific requirements with a minimum of programming knowledge.
One of important features of our system is the 'self-referencing' Media module that allows
generating complex media stepwise from more simple stock solutions. Thus, the entire
process of making media from - at the bottom - commercially available compounds is
completely and easily documented. The second important feature of the Media module is
the reporting tool that provides printouts. These are indispensable in daily work at the
bench, as the computer screen is usually not close to the lab bench. The database-
generated printouts also facilitate the identification of the right medium for a step of a
method by the matching name and identifier number (id) in the method description and
on the media container. The identifier does not only increase safety by double-coding but
also makes multi-language versions of the method much easier. This is especially
important in international labs, in which English speaking scientist cooperate with local
staff less fluent in English. Media and method printouts are admittedly also risky, as it is
tempting to document changes in the material or method on the printout. Thus, staff
needs to be trained to use the database system as the primary data source and document
any deviation from the standard in the database.
An important feature of the system is its ability to cope with media and method evolution.
If the deviation from the standard becomes a new standard, the new medium or method
can be easily documented by duplicating the old method. The administrator in charge of
the database can then approve of the new method and prevent its further alteration. At the
same time, old methods can be taken out of use, but remain available for cross
referencing.
The Experiment module of our SOP system is the module, in which the user can
document deviations from the standard. This module automatically produces the plan for
the experiment from the standard method description and generates an electronic lab
book. In this lab book, any deviation from the plan concerning timing, media or method
can be documented. For long-term experiments, the automatic generation of work
schedules is another comfortable asset of the system. The work schedule allows early
detection of work-peaks and facilitates hand-over of work between technical staff for
times of absence.
In the beginning, the SOP system was run as a standalone solution at the MPI-MP and all
data were entered directly. However, some of the information required in an experiment
e.g. on plasmids and users were also present in the LIMS plant database system of the
institute [10], which resulted in double entries. To save time, we devised a read access for
expert users. The read function imports the information needed in the transformation
process from the LIMS into the SOP database.
Finally, the system documents the result (number of lines) of the experiment. Failures are
documented in a standardized way, thus problems are detected in an early state. Thus,
data are rapidly available for decision making, e.g. about the stop of a method because of
low efficiency.
Conclusion
We constructed a database system to document media, methods and experiments in a

transformation service unit. The system increased efficiency and reduced the risk of
information loss when coworkers leave. Additionally, the system provided rapid access to
data for quality control and decision making. Altogether, the introduction of the system
was thus worth the effort. The system can be easily modified for the use in other research
environments.
Availability and requirements
The documentation system was first implemented in MS Access 2003 (Microsoft) and
subsequently upgraded to MS Access 2007. A MS Access 2003 version is available as
additional file 1 (transformation2003.mdb). A short introduction is given in additional
file 3: readme. The database scheme can be displayed with the Access 'relationship'
function. For readers without access to the program MS Access, the entity relation
diagram (additional file 4: ERdiagramTransformation2003.pdf) and the database
definition file (additional file 5: Object Definition for Transformation2003.pdf) are
provided as supplemental material.
Additional file 3. Readme. The file contains a short introduction to the system. It is
assumed that the user is generally familiar with the software MS-Access. Thus, the
introduction repeats only the most important features in handling the software and
otherwise restricts itself to the features specific to our database.
Format: PDF Size: 79KB Download file

This file can be viewed with: Adobe Acrobat Reader
Additional file 4. ERdiagramTransformation2003. The file contains the entity

relationship diagram for the database MSTransformation2003.mdb.
Additional file 5. Object Definition for Transformation2003.pdf. The file contains the
object definition for the database MSTransformation2003.mdb
The actively used 'live' database is stored on a shared folder of the MS Windows XP
operation system. User access is regulated by the security settings to the folder.
Developments on the system are performed in a separate database and subsequently
imported into the live database. Backup copies are generated every ten minutes based on
the Windows XP backup system. Backup copies to an independent storage system are
produced every night.
For less experienced users, an entry page and MS Access custom groups were generated.
The entry page provides access to the most important features of the system. The custom
groups in the example database are 'Favorites', 'Media module', 'Methods module',
'Experiments module', 'Experts module' and 'Experts module administration'. The custom
groups contain links to the database objects relevant to the respective subject and thus
facilitate work.
Autoclave-proof labels are printed on a Datamax E4304 label printer (AISCI, Bad
Salzuflen, Germany).
Abbreviations
LIMS: laboratory information management system; SOP: standard operation procedure.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
KK designed the database scheme, implemented the database, tested the live system and
wrote the manuscript. JG programmed the visual basic code, suggested improvements
and implemented the printer system. All authors read and approved the final manuscript.
Acknowledgements
We thank Romy Baran and Brigitte Buchwald for their help in data entry and their
feedback during the tests of the system and Stephanie Ruf for helpful discussions and
suggestions for the manuscript. All authors and all persons mentioned in
acknowledgements are on contracts of the Max Planck Institute of Molecular Plant
Physiology and did not receive any third party funding for the project.
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webcite
Lecture 11. Plant Transformation

I Elements of transformation
!!!!!!!1. uptake of DNA into competent
host cells
!!!!!!!2. integration vs. transient
expression
II methods of delivery
1. Agrobacterium-mediated
transformation
!!!!!!!!! - vacuum infiltration (floral dip)
- wounded explants
- Cultured cells (tobacco)
can’t be applied to monocot, nor
organelle
2. direct methods
!!!!!!!!!!-protoplast polyethyleneglycol
(PEG) method
!!!!!!!!!!-protoplast electroporation
!!!!!!!!!!-protoplast microinjection
!!!!!!!!!!-particle bombardment
Agrobacteria: -carry Ti-plasmid

-induce tumor formation (crown gall
tumor)
in wound site of plants
-produce opines (nopaline or octopine)
1. Agrobacteria tumefaciens
mediated plant transformation
Ti-plasmid has two parts:
- T-DNA the part of DNA that is exported
into the plant cell
and integrated into the plant genome
- vir region: encode proteins involved in
this transfer,
but stays within the bacteria
1040-2519/97/0601-0297$08.00
297
PBLIRACNHT TRANSFORMATION
Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997. 48:297–326
Copyright © 1997 by Annual Reviews Inc. All rights reserved
PLANT TRANSFORMATION:
Problems and Strategies for Practical
Application
R. G. Birch
Department of Botany, The University of Queensland, Brisbane, 4072, Australia
KEY WORDS: plant improvement, gene transfer, transgenic plants, transgene expression, genetic
engineering
ABSTRACT
Plant transformation is now a core research tool in plant biology and a practical
tool for cultivar improvement. There are verified methods for stable introduction
of novel genes into the nuclear genomes of over 120 diverse plant species. This
review examines the criteria to verify plant transformation; the biological and
practical requirements for transformation systems; the integration of tissue
culture, gene transfer, selection, and transgene expression strategies to achieve
transformation in recalcitrant species; and other constraints to plant transformation
including regulatory environment, public perceptions, intellectual property,
and economics. Because the costs of screening populations showing diverse
genetic changes can far exceed the costs of transformation, it is important to
distinguish absolute and useful transformation efficiencies. The major technical
challenge facing plant transformation biology is the development of methods
and constructs to produce a high proportion of plants showing predictable
transgene expression without collateral genetic damage. This will require answers
to a series of biological and technical questions, someof which are defined.
CONTENTS
INTRODUCTION.............................................................................................
....................... 298
DEFINITION AND VERIFICATION OF
TRANSFORMATION ......................................... 299
PURPOSES OF PLANT
TRANSFORMATION ..................................................................... 301
An Experimental Tool for Plant
Physiology........................................................................ 301
A Practical Tool for Plant
Improvement.. ......................................................................... 301
BIOLOGICAL REQUIREMENTS FOR
TRANSFORMATION............................................ 302
PRACTICAL REQUIREMENTS OF TRANSFORMATION SYSTEMS
FOR
PLANT
IMPROVEMENT..............................................................................................
. 302
RECALCITRANT SYSTEMS AND APPARENT
CONSTRAINTS ..................................... 304
STRATEGIES TO ACHIEVE
TRANSFORMATION............................................................ 305
Tissue Culture
Strategies.....................................................................................................
305
Gene Transfer
Strategies.....................................................................................................
308
Selection
Strategies...........................................................................................................
... 310
Transgene Expression Strategies
........................................................................................ 311
Integrating Components of Transformation
Strategies....................................................... 312
OTHER CONSTRAINTS TO RESEARCH AND DEVELOPMENT IN
PLANT
TRANSFORMATION......................................................................................
............... 314
Regulatory Environment and Public Perceptions
............................................................... 314
Intellectual
Property............................................................................................................
316
FUTURE NEEDS AND DIRECTIONS IN PLANT TRANSFORMATION
RESEARCH AND
DEVELOPMENT............................................................................. 317
Transformation Efficiency
................................................................................................... 317
Useful vs Absolute Transformation Efficiency
.................................................................... 318
Collateral Genetic
Damage................................................................................................. 319
Ideal and Model Transformation Systems
......................................................................... 319
Transformation, Breeding, and Genetic Diversity
.............................................................. 320
When Practical Means
Commercial.................................................................................... 320
INTRODUCTION
Plant transformation is at a threshold. Over 3000 field trials of transformed
plants are in progress or completed in at least 30 countries. These trials
involve
over 40 plant species modified for various economic traits (33, and updates
in
Genetic Technology News). We are emerging from a period of plant
transformation
research dominated by the need to develop proven genetic transformation
methods for the major experimental and economic plant species, into the
era of application of transformation as a core research tool in plant biology
and
a practical tool for cultivar improvement. Some of the most important issues
(problems and strategies) affecting these uses are very different from those
foremost in our thinking in recent years while the discipline focused on the
scientific understanding and technical development of reliable systems for
genetic transformation of a wide range of plant species.
The capacity to introduce and express diverse foreign genes in plants, first
described for tobacco in 1984 (37, 74, 120), has been extended to over 120
species in at least 35 families. Successes include most major economic
crops,
vegetables, ornamental, medicinal, fruit, tree, and pasture plants. The rapid
and
simultaneous developments in transformation technology and information
technology make tabulations of transformed species quickly out of date (41,
131), and it is advisable to use computer-based searches to locate references
to
298 BIRCH
current transformation methods for species of interest. The process of
diversification
and refinement of transformation techniques for greater convenience,
higher efficiency, broader genotype range, and desired molecular
characteristics
of transformants will continue to good effect for some time. However,
gene transfer and regeneration of transgenic plants are no longer the factors
limiting the development and application of practical transformation systems
for many plant species. Attention is increasingly being directed to achieving
the desired patterns of expression of introduced genes and to solving
economic
constraints on practical plant molecular improvement.
There are excellent recent reviews of the development of plant
transformation
systems using Agrobacterium (72, 146, 165), direct gene transfer into
protoplasts (49, 113, 114), or particle bombardment (14, 25, 26), and the
potential for their practical application (30, 73). These topics are now
comprehensively
addressed in recent texts (12, 54, 61, 90, 110) and methods manuals
(39, 53, 57, 127, 153), to which the reader is referred for background
information.
In this review I aim to identify key problems remaining in the development
of plant transformation systems, key issues to be resolved in the practical
application of these systems, and strategies by which we may overcome
these
limitations.
DEFINITION AND VERIFICATION OF TRANSFORMATION
This review is concerned with the stable incorporation and expression of
genes
introduced into plants by means other than fusion of gametes or other cells.
It
focuses on transformation involving integration of introduced genes in the
plant nuclear genome, although some issues are equally applicable to
transformed
plants in which introduced genes are expressed from an organelle
genome (21), or a replicating viral vector (139).
Ingo Potrykus in 1991 (126) offered a provocative but clarifying assessment
of plant transformation technologies based on a rigid definition of proof
of integrative transformation, requiring a combination of genetic,
phenotypic,
and physical data. Unfortunately the combination specified was not useful in
practice for verification of transformation in some plants. For example,
analysis
of sexual offspring populations is problematic in trees that are slow to
reproduce sexually, and in some vegetatively propagated crops such as
sugarcane
with complicated (polyploid, aneuploid) genetics, and many sexually
sterile cultivars. Similarly, a “tight” correlation between physical and
phenotypic
data is not a defining characteristic of gene transfer methods that result in
integration of multiple and often rearranged copies of the transferred DNA,
because many transformants have unexpressed copies of introduced se-
PLANT TRANSFORMATION 299
quences. Integrative transformation has nevertheless been unequivocally
verified
in such cases.
Most critical researchers would therefore accept a more generally applicable
subset of criteria as rigorous proof of integrative transformation:
1. Southern DNA hybridization analysis of multiple independent
transformants,
using a probe(s) for the introduced gene(s) and restriction enzymes
predicted to generate hybridizing fragments of different length at different
integration sites (for a graphic representation, see 80). It is important to
confirm that sizes of hybridizing fragments including flanking DNA at each
integration site are reproducible within a transformed line, and that they
differ between independently transformed lines. High molecular weight
signals in uncut DNA, PCR-generated bands, or signals from a single
putative transformed line are not acceptable substitutes, because it is more
difficult to exclude the possibility of artifacts in such data.
2. Phenotypic data showing sustained expression of the introduced gene(s)
exclusively in the cells of plant lines positive for the gene(s) by Southern
analysis. Unambiguous phenotypic data require: (a) negative results from
all untransformed controls (the tested control population size must be at
least equivalent to that yielding 10 independent transformants from a parallel
treated population), and (b) an assay revealing the product of transgene
expression within plant cells as distinct from contaminating microbial cells,
preferably from a transgene shown not to be expressed in bacterial cells.
Intron-GUS (149) or anthocyanin regulatory (16, 18) reporter systems are
suitable for such assays, as are in situ analyses for gene products without
simple visual assays (153). Survival of lines on “escape-free” selection is
not sufficient, because of the possibility of cross-protection by secreted
products of contaminating microbial cells, or selection of mutants resistant
to the selective agent. Enzyme assays on cell extracts are inadequate because
of the possibility of contaminating transformed microbial cells.
Of course, more detailed molecular, phenotypic, and genetic characterization
is likely to be undertaken on transformed lines produced for practical
purposes. In some cases, target gene silencing rather than transgene
expression
may provide an unambiguous phenotype (15, 103). Data on co-transmission
of
introduced gene copies and the resulting phenotype in sexual offspring
populations,
where available, provide compelling confirmation of transformation,
given suitable controls (126). Applicability in several independent
laboratories
is an important practical confirmation, because some techniques with
published
molecular evidence have never been repeatable.
300 BIRCH
PURPOSES OF PLANT TRANSFORMATION
An Experimental Tool for Plant Physiology
The capacity to introduce and express (or inactivate) specific genes in plants
provides a powerful new experimental tool, allowing direct testing of some
hypotheses in plant physiology that have been exceedingly difficult to
resolve
using other biochemical approaches (31). Exciting examples include the
molecular
genetic analysis of cellular signals controlling sexual reproduction and
plant-microbe interactions (116, 143); the roles of specific enzymes in
metabolic
processes determining partitioning of photosynthates, and thus harvestable
yield (67); and the roles of specific enzymes and hormones in plant
developmental
processes, including those affecting quality and storage life of marketed
plant products (109, 147).
A Practical Tool for Plant Improvement
Much of the support for plant transformation research (and more broadly for
plant molecular biology) has been provided because of expectations that this
approach could: (a) generate plants with useful phenotypes unachievable by
conventional plant breeding, (b) correct faults in cultivars more efficiently
than
conventional breeding, or (c) allow the commercial value of improved plant
lines to be captured by those investing in the research more fully than is
possible under intellectual property laws governing conventionally bred
plants.
The first of these expectations has been met, with production of the first
commercial plant lines expressing foreign genes conferring resistance to
viruses,
insects, herbicides, or post-harvest deterioration (54, 71, 138, 147), and
accumulation of usefully modified storage products (30, 145), including
several
cases where there was no source of the desired trait in the gene pool for
conventional breeding. The future prospects in this respect are also exciting,
with preliminary indications that novel genes can be introduced to generate
plant lines useful for production of materials ranging from pharmaceuticals
(65) to biodegradable plastics (112).
The extent to which the other practical or commercial expectations of plant
transformation can be met depends on the efficiency and predictability of
production of lines with the desired phenotype, and without undesired side
effects of the transformation process. As the sophistication of the
physiological
hypotheses to be tested by transformation increases, exactly the same
factors become limiting. This occurs because of the practical difficulty of
screening large numbers of plants for the desired expression pattern and the
need to avoid misleading results from unrelated physiological effects of
unintended
genetic changes during the transformation process.
BIOLOGICAL REQUIREMENTS FOR TRANSFORMATION
The essential requirements in a gene transfer system for production of
transgenic
plants are: (a) availability of a target tissue including cells competent for
plant regeneration, (b) a method to introduce DNA into those regenerable
cells, and (c) a procedure to select and regenerate transformed plants at a
satisfactory frequency.
One of the simplest available plant transformation systems involves
infiltration
of Agrobacterium cells into Arabidopsis plants before flowering, and
direct selection for rare transformants in the resulting seedling populations
(7,
23). Unfortunately, small plant size, rapid generation time, and high seed
yield
per plant are prerequisites for this method. These features are not shared by
any economically important plant species. Other approaches to
transformation
via plant gametes have not been successful in practice (126). Therefore, the
totipotency of some somatic plant cells underlies most plant transformation
systems. The efficiency with which such cells can be prepared as targets for
transformation is today the limiting factor in achievement of transformation
in
recalcitrant plant species.
Using either Agrobacterium or particle bombardment, it is now possible to
introduce DNA into virtually any regenerable plant cell type. Only a small
proportion of target cells typically receive the DNA during these treatments,
and only a small proportion of these cells survive the treatment and stably
integrate introduced DNA (47, 59). It is therefore generally essential to
efficiently
detect or select for transformed cells among a large excess of untransformed
cells (12), and to establish regeneration conditions allowing recovery
of intact plants derived from single transformed cells (156). Alternatively,
transformed cells must contribute to the germline so that nonchimeric
transformants
can be obtained in the progeny from sexual reproduction (28).
PRACTICAL REQUIREMENTS OF TRANSFORMATION
SYSTEMS FOR PLANT IMPROVEMENT
Beyond the biological requirements to achieve transformation and the
technical
requirements for verification of reproducible transformation, desired
characteristics
to consider in evaluating alternative techniques, or developing new
ones for cultivar improvement, include:
1. Ready availability of the target tissue. The resources required to maintain
a
continuous supply of explants such as immature embryos at the correct
developmental stage for transformation can be substantial.
302 BIRCH
2. Applicability to a range of cultivars. Genotype-specific techniques are of
lower value because of the added complexity of extended breeding work to
move desired genes into preferred cultivars.
3. High efficiency, economy, and reproducibility, to readily produce many
independent transformants for testing.
4. Safety to operators, avoiding procedures, or substances requiring
cumbersome
precautions to avoid a high hazard to operators (e.g. potential
carcinogenicity
of silicone carbide whiskers).
5. Technical simplicity, involving a minimum of demanding or inherently
variable manipulations, such as protoplast production and regeneration.
6. Frequent cotransformation with multiple genes, so that a high proportion
of
plant lines selected for marker gene expression will also incorporate
cotransformed useful genes.
7. Unequivocal selection or efficient screening to recover transgenic plants
from transformed cells.
8. Minimum time in tissue culture, to reduce associated costs and avoid
undesired somaclonal variation.
9. Stable, uniform (nonchimeric) transformants for vegetatively propagated
species, or fertile germline transformants for sexually propagated species.
10. Capacity to introduce defined DNA sequences without accompanying
vector
sequences not required for integration or expression of the introduced
genes.
11. Capacity to remove reporter genes or other sequences not required
following
selection of transformed lines.
12. Simple integration patterns and low copy number of introduced genes, to
minimize the probability of undesired gene disruption at insertion sites, or
multicopy associated transgene silencing.
13. Stable expression of introduced genes in the pattern expected from the
chosen gene control sequences, rather than patterns associated with the
state of the cells at the time of transformation (34), or the chance site of
integration (122).
14. Optionally applicable to transformation of organelle genomes.
15. Absence of valid patent claims on products.
When tested against the above criteria, most published techniques for gene
transfer into plant cells must be dismissed as either disproven, unproven, or
impractical for use in routine production of transgenic plants. The techniques
that have been proven to produce transgenic plants from a range of species,
and in many laboratories, are Agrobacterium-mediated transformation,
bombardment
with DNA-coated microprojectiles, and electroporation or PEG
treatment of protoplasts. Techniques requiring protoplasts are generally
avoided because of the associated inconvenience and time in culture. As a
result virtually all plant transformation work aimed at direct production of
improved cultivars currently uses either Agrobacterium or microprojectiles
for
gene transfer. Neither of these approaches is free of patent claims, however,
and there is continuing interest in development of alternative techniques
(141).
The stages and time-courses for typical transformation strategies using
Agrobacterium
or DNA-coated microprojectiles shown in Figure 1.
RECALCITRANT SYSTEMS AND APPARENT
CONSTRAINTS
Cereals, legumes, and woody plants are commonly categorized as
recalcitrant
to transformation, because these groups have included a disproportionate
Figure 1 Typical transformation regimens using Agrobacterium or DNA-
coated microprojectiles.
304 BIRCH
number of untransformed or difficult to transform species. However, the
generalization
is becoming less useful as one species after another from these
groups joins the list of plants with reliable transformation systems. The
hypothesis
that some plants lack the biological capacity to respond to essential
triggers for integrative transformation, or have cellular mechanisms
preventing
integrative transformation, can effectively be rejected.
It is a reasonable proposition that transgenic plants can be regenerated only
from cells competent for both regeneration and integrative transformation
(126). Preliminary evidence indicates that T-DNA integration may be the
limiting step in maize transformation (111), but there is no evidence that
actively dividing, regenerable cells are not competent to integrate introduced
DNA. Where tissue culture systems have been developed to produce
proliferating
and regenerable cells, into which DNA can be introduced at a high
frequency (as indicated by transient gene expression) without interfering
with
regenerability of the penetrated cells, previously recalcitrant species have
become
transformable. The transformation efficiency has been proportional to
the efficiency of the tissue culture and gene transfer systems (18, 70, 76, 93).
STRATEGIES TO ACHIEVE TRANSFORMATION
It is instructive to consider species such as rice, which was once considered
recalcitrant to transformation but can now be transformed via direct gene
transfer into protoplasts (140), particle bombardment of immature embryos
(27) or cell cultures (20), or Agrobacterium treatment of embryogenic callus
(70). In each case, success seems to have followed identification (or
production
through tissue culture) of explants with many regenerable cells, optimization
of parameters for gene transfer into those cells, and tailoring selection and
regeneration procedures to recover transgenic plants. A generalized
approach
is illustrated in Figure 2. The nearest transformed relatives of an
untransformed
species of interest are an obvious reference point in initial work to
develop suitable tissue culture, gene transfer, and selection regimens.
Tissue Culture Strategies
Tissue culture is not a theoretical prerequisite for plant transformation, but it
is
employed in almost all current practical transformation systems to achieve a
workable efficiency of gene transfer, selection, and regeneration of
transformants.
Detailed consideration of the options for and optimization of tissue
culture systems useful for plant transformation is beyond the scope of this
review, but the broad technology and detailed protocols are addressed in
recent
techniques manuals (52, 154).
In tissue culture systems for plant transformation, what is most important is
a large number of regenerable cells that are accessible to the gene transfer
treatment, and that will retain the capacity for regeneration for the duration
of
the necessary target preparation, cell proliferation, and selection treatments.
A
high multiplication ratio from a micropropagation system does not
necessarily
indicate a large number of regenerable cells accessible to gene transfer (97).
Figure 2 A generalized approach to development of transformation systems
for recalcitrant plant
species.
306 BIRCH
Gene transfer into potentially regenerable cells may not allow recovery of
transgenic plants if the capacity for efficient regeneration is short-lived
(132).
There seems to be no reason to prefer embryogenic or organogenic plant
regeneration. In the happy event that several tissue culture systems meeting
the
primary requirement above are available for a species of interest, the choice
can be made based on features affecting convenience or efficiency, including
ready availability of explants, and minimal time in tissue culture.
Somaclonal
variation, once considered a potentially useful source of genetic variation for
plant improvement, is now more a bane of gene transfer programs (81). In
some circumstances, particularly the direct introduction of genes for desired
commercial traits into elite vegetatively propagated cultivars, the need to
avoid
such random genetic change may become the overriding consideration in the
choice of tissue culture and gene transfer systems.
The desire to minimize somaclonal variation is one motivation for
eliminating
or minimizing the tissue culture phase, by gene transfer into intact tissue
explants and regeneration without substantial in vitro culture. In several
cereals,
it has been possible to dispense with tissue culture for target preparation,
by gene transfer into immature zygotic embryos, although embryogenic
callus
culture is still required to recover transgenic plants (27, 157, 159).
The goal of genetic transformation without tissue culture has been
approached
in soybean, cotton, bean, and peanut by particle bombardment into
meristematic tissue of excised embryonic axes, shoot proliferation to yield
some lines with transformation of germline cells, and screening for
transformed
sexual progeny (26). The limiting factors remain the ability to mechanically
prepare the explants, transfer genes into regenerable cells, and
select or screen for transformants at an efficiency sufficient for practical use
in
cultivar improvement. For example, the reported germline transformation
rate
for bean (0.06% of excised and bombarded apical meristems, 0.03% of
assayed
shoots) would make the process too expensive for many laboratories
(133). There remains unexplained cultivar specificity for transformation via
meristem bombardment within some plant species such as bean (133),
impracticality
of gene transfer into meristems of others such as rice (27), and uncertainty
about applicability in vegetatively propagated species such as sugarcane
(51).
While tissue culture remains an essential component of practical
transformation
systems for most plant species, research aimed at minimizing somaclonal
variation deserves a high priority. Curiously little of the published
research on somaclonal variation has been directed at this goal (81).
Gene Transfer Strategies
Suggested approaches to development or optimization of transformation
protocols
using particle bombardment (12) or Agrobacterium (152) have been
published,
based on practical experiences in laboratories working on recalcitrant
crops. For particle bombardment, it is generally most efficient to first
examine
the available tissue culture systems, determine the modes of regeneration
and
the location of the cells involved, optimize tissue culture conditions to
increase
the number or accessibility of such cells if necessary, and then develop
conditions
for nonlethal transfer of DNA into large numbers of such cells per
bombardment (12).
For Agrobacterium, it is considered more efficient to first establish the
conditions for gene transfer and then work on conditions for regeneration of
transformed cells (152). This contrast may be biologically well-founded,
because
of the greater complexity and lesser understanding of the biological
interaction preceding the gene transfer event from Agrobacterium.
Unfortunately,
there is no guarantee that a transformable plant cell type will prove
regenerable, even in the hands of the most successful tissue culturist.
If preliminary transformation experiments using techniques successful in
similar plant systems are not successful, my advice is to establish by
histological
studies the precise cellular origins and timing of events leading to plant
regeneration within the explants to be used as targets for gene transfer. This
is
likely to avoid much wasted time and frustration from optimizing gene
transfer
and regeneration within the same region of the explant, but potentially in
different cells, so that transformed plants are unlikely to result. Work in
sunflower is a fine example of the value of this relatively simple check to
explain and potentially overcome difficulty in transformation of a
recalcitrant
species (91).
Assays for transient expression of introduced reporter genes in plant cells
can provide unequivocal evidence of gene transfer. A great deal of time can
be
wasted unless this analysis is focused on regenerable cells, which often
comprise
a small and inaccessible fraction of the target tissue (12, 91). Exhaustive
experiments to maximize transient expression are also futile if they involve
conditions harmful to regeneration or molecular characteristics of
transformed
cells (8, 26). For example, particle bombardment conditions can now be
arranged
to give highly reproducible results in transient assays, by delivering a
large number of copies of potentially transcribable DNA into the nuclei of
target cells. However, a different form and concentration of DNA is likely to
be optimal for efficient production of low copy number transformants (12).
308 BIRCH
There is little information on forms of DNA or sequences that may increase
the
frequency of stable transformation (8, 19, 142).
There is considerable batch-to-batch variation in the frequency of transient
expression events following cocultivation with Agrobacterium (78). The
correlation
between transient expression and stable transformation has not been
thoroughly tested. Agrobacterium employs a highly evolved and still
incompletely
understood gene transfer and integration system that appears optimized
for efficient nuclear targeting and integration of a protein-complexed
singlestranded
DNA introduced as a small number of copies per cell (165). Therefore,
stable transformation may occur at a high frequency in cells without
detectable transient expression of the introduced DNA. Positive results from
transient assays for Agrobacterium-mediated gene transfer into regenerable
cells are encouraging (70, 76, 93), but until the parameters affecting such
expression are better understood, it is unwise to abandon hope too quickly
based on negative results from transient assays.
It may be necessary to introduce a considerable number of genes into plants
for some purposes. The limits of available gene transfer techniques have not
yet been defined. At least 12 separate plasmids and up to 600 kb of DNA
can
be introduced at once by particle bombardment (62), but the number of
expressed
genes has not been tested. There are some indications that large plasmids
(>10 kb) may be subject to greater fragmentation during particle
bombardment
(12). Transposon-derived vectors have been shown to deliver an
increased proportion of intact, single-copy inserts of up to 10 kb following
direct gene transfer into protoplasts (92). Recent evidence indicates that use
of
a binary bacterial artificial chromosome vector, with helper plasmids
enhancing
production of VirG and VirE proteins, can allow efficient
Agrobacteriummediated
transfer of at least 150 kb of foreign DNA into the plant nuclear
genome. Furthermore, the transferred DNA appeared to be present as an
intact
single copy that was faithfully inherited in the progeny of several of the
characterized transformants (64).
Vectors overexpressing virG are also a component of the thoroughly verified
systems for Agrobacterium-mediated transformation of rice (70), maize
(76), and cassava (93), and deserve wider testing in recalcitrant plants. Other
key variables in Agrobacterium-mediated gene transfer include
Agrobacterium
and plant genotype, treatment with vir gene inducers such as acetosyringone,
wounded cell extracts, feeder cells or sugars; pH, temperature, cell
concentration,
light conditions, and duration of cocultivation; explant type, quality,
preculture (152), hormone treatment (50), wounding, or infiltration (11); and
use of appropriate antibacterial agents (95), antioxidants (124), ethylene
antagonists
(35), and/or methylation inhibitors (117) to reduce damage and/or
gene silencing in treated plant cells. With so many variables, and little
evidence
of combinations that are broadly applicable across plant species, it is
evident why transient expression assays are useful, if imperfect, as indicators
of suitable conditions for gene transfer (76), before more expensive studies
to
optimize stable transformation.
Selection Strategies
For transformation systems that generate substantial numbers of
nonchimeric
primary transformants, genes conferring resistance to a selective chemical
agent (161), genes conferring a phenotype allowing visual or physical
screening
(16, 18, 129), or even PCR screening to identify plants containing
transferred
genes (27, 83) can all be used to recover transformants. Transformation
systems that generate chimeric primary transformants including transformed
germline cells, as intermediates in the production of homogeneously
transformed
(R1) progeny plants, generally require screening rather than lethal
selection to reveal primary transformants (28, 83, 105).
Screening approaches are expensive unless the transformation efficiency is
high, and generally impractical if the proportion of transformants among
regenerated
lines is below 10−2 to 10−3. In our hands, the recovery of transformed
plants was 10-fold lower from visual screening compared with antibiotic
selection (18). This may occur because antibiotic selection provides a
continuous advantage to transformed cells, which may otherwise be
overgrown
by the far greater numbers of proliferating nontransformed cells (48,
78). Under these circumstances, antibiotic selection may allow a higher
proportion
of transformed cells to multiply and regenerate, in addition to facilitating
the recognition of transformants.
An excellent review has been published on selectable marker genes,
assayable
reporter genes, and criteria for their use in plant transformation studies
(16). Broadly applicable, simple, and robust selection regimens now exist
for
transgenic plants, requiring little experimentation with the timing and
concentration
of selective agents to match the target tissue and gene transfer system.
However, it is still important to consider the physiology of antibiotic action
and resistance mechanisms when choosing or modifying selection protocols
(36). There are also reports of interactions between selective agent and
subsequent
regenerability (137), and interactions between antibiotic and gelling
agents (101). Attention is increasingly being directed to introduction of
multiple
agronomically useful genes into plant lines, without having to pyramid
selectable genes in the process (27, 32, 155, 163).
310 BIRCH
Transgene Expression Strategies
The first attempts to express foreign genes failed because of the inability of
the
plant transcriptional machinery to recognize some foreign gene control
sequences,
particularly promoter sequences of many bacterial genes. This initial
hurdle was overcome by exploiting control sequences isolated from genes of
Agrobacterium and cauliflower mosaic virus, which were known to be
expressed
in plant cells as part of the process of molecular subversion of host cell
machinery by these pathogens. Continuing characterization of many plant
genes, and analyses of transient and stable expression of foreign gene
constructs
in plants, have contributed to a growing understanding of features
useful for the regulated expression of transgenes in plants. Features typically
considered in preparation of such constructs include: (a) appropriate
transcriptional
promoters and enhancers (9); (b) introns (98, 100); (c) transcriptional
terminators and 3′ enhancers (130); (d) polyadenylation signals (3, 162);
(e)
untranslated 5′ leader and 3′ trailer sequences (38, 56, 148); (f) codon
usage
(87, 125); (g) optimal sequence context around transcription and translation
start sites, including absence of spurious start codons (5, 55, 66, 99); (h)
transit
sequences for appropriate subcellular compartmentation and stability of the
gene product (58, 68, 82, 115); (i) absence of sequences such as cryptic
introns
(129) or polyadenylation signals resulting in inappropriate RNA processing
(87, 125); and (j) absence of sequences resulting in undesired glycosylation
or
lipid anchor sites (24, 46). Recent progress with chloroplast transformation
has
added the possibility of expression from the plastid or nuclear genome (21,
104).
This understanding has greatly improved the ability to tailor transgenes for
various strengths and patterns of expression essential for practical plant
genetic
engineering (10). Levels and patterns of expression generally vary to
some extent, even between independent single copy transformants. This
reflects
the influence of different sequences flanking the integration sites upon
expression of the transgene (122, 151). Although this variation may generate
transformed lines which by chance have new and useful expression patterns
(34), this is unlikely to occur at a frequency that is useful in practice, unless
we
can bias integration toward regions of the DNA that are preferentially
expressed
under specified conditions. Otherwise, random variation will greatly
increase the expense of the varietal improvement process, because rigorous
evaluation of many transformed lines will be needed to identify those with
the
desired phenotype. One attraction of transformation for cultivar
improvement
is the theoretical potential for very precise genetic change. If random
variation
around a desired phenotype in individual transformants necessitates
extensive
field characterization to select commercial lines, there may be no advantage
in
using transformation over conventional breeding. In these circumstances, the
value of plant transformation in cultivar improvement would be restricted to
traits not achievable by conventional breeding, particularly introduction of
new genes to the germplasm available to breeders.
Of even greater concern is unpredictable loss of the improved phenotype in
transformed cultivars because of silencing of the transgene. This
phenomenon
is discussed in several excellent recent reviews (45, 103, 107). In practical
terms, it is fortunate that transgene silencing has to date always been
detectable
soon after transformation, crossing, or field testing of transformants (see 45).
The problem would be much more serious if unstable lines could not be
reliably detected and eliminated during the routine screening and
propagation
of transgenic lines before commercial release, because of the potential
commercial
damage from loss of an essential trait such as disease resistance. There
is still much to be learned about the causes of transgene inactivation. For
example, multicopy transgenes have been identified as a probable cause (see
45), indicating a disadvantage of direct gene transfer techniques which result
in higher average copy numbers than Agrobacterium. However, our
experience
from several years of laboratory and field testing with hundreds of
transgenic
sugarcane lines is that some promoter-reporter gene constructs are silenced
at high frequency, whereas others are almost invariably stably expressed,
with no relationship to copy number (13). It will be important to
discover what sequences within these constructs trigger or inhibit silencing,
and whether matrix attachment regions (142), demethylation sequences (94),
or targeted integration systems (2, 118) can be developed to protect
susceptible
foreign sequences from silencing in transgenic plants.
Integrating Components of Transformation Strategies
Features of representative strategies for production of transgenic plants are
compared in Table 1. Other combinations exist, but the table illustrates that
the
choice of transformation strategy influences many secondary parameters
such
as time in tissue culture and number of plants processed per transformant,
which often determine the practicality of the system.
It is commonly generalized that Agrobacterium produces simpler integration
patterns than direct gene transfer, but both approaches result in a similar
range of integration events, including truncations, rearrangements, and
various
copy numbers and insertion sites. Furthermore, the frequency distributions
of
copy number and rearrangements vary with transformation parameters for
both
gene transfer methods (25, 60, 107, 150). More careful work is required to
312 BIRCH
optimize methods for simple integration patterns before any reliable
conclusions
are drawn about the relative potential of the techniques to deliver such
patterns at a satisfactory frequency.
Table 1 Features influencing practical application of successful plant
transformation strategies
Successful Transformation Strategies
Parameters In planta Vegetative
tissue
Meristem Embryo Callus
Gene transfer
method
Agrobacterium Agrobacterium Particle bombardment
Particle bombardment
Particle bombardment
Explant Flowering plant Any tissue
with regenerable
cells
Embryonic
axes or meristems
Intact or sectioned
embryos
Embryogenic
or organogenic
callus
Target cells for
effective gene
transfer
Germline cells
late in floral
development
Any regenerable
cells
Germline cells
in meristems
Epidermal
cells of
scutellum
Surface or subsurface
cells
Regeneration Flowering
shoot/zygotic
embryo
Organogenesis
or
embryogenesis
Shoot formation/
zygotic
embryo
Embryogenesis Embryogenesis
or
organogenesis
1° regenerant
chimeric
Few floral cells
transformed
No Germline transformed
No No
Sexual reproduction
Essential Unnecessary Probably
essential
Unnecessary Unnecessary
Selection Yes (R1
progeny)
Yes Not before R1
progeny
Yes Yes
Screenable
marker
Optional Optional Essential Optional Optional
Hormonal
treatment
Unnecessary Auxin +/or
cytokinin
Usually
cytokinin
Auxin+/−
cytokinin
Auxin+/−
cytokinin
Transformants
per treated
explant
1–10 1–10 0.001 0.01 0.01–1
Transformants
per tested regenerant
0.001 1 0.0003 1 1
Tissue culture
duration
Unnecessary 10 weeks 4–6 weeks 10 weeks 20 weeks
Time from
treatment of
non-chimeric
transformant
7–10 weeks 10 weeks One generation
time
10 weeks 10 weeks
Proven applicability
(e.g.)
Arabidopsis (7) Many spp. (59) Several spp. including
legumes
(26,
105)
Several cereals
(27, 77)
Many spp. (17,
26)
The apparent targeting of T-DNA integration into transcribed regions is
useful for gene and promoter tagging, and for transgene insertion into
regions
favoring subsequent expression (86). However, the observation that over
90%
of T-DNA insertions may disrupt transcriptional units (96), with 15–26% of
transformants showing visible mutant phenotypes resulting from T-DNA
insertions
(44), sounds an alarm for direct production of improved cultivars in
highly selected crops, where most phenotypic changes from random
mutations
are likely to be adverse. For such work, integration should ideally be
directed
to transcribed regions without disruption of existing plant genes. To achieve
this will require more research to bring gene targeting technology in plants
closer to the level achieved in model animals (79, 118). Whether DNA
introduced
into plant cells by direct gene transfer is also preferentially integrated
into transcribed regions or active genes has not been adequately tested (85).
OTHER CONSTRAINTS TO RESEARCH AND
DEVELOPMENT IN PLANT TRANSFORMATION
Regulatory Environment and Public Perceptions
In most countries, planned field releases and commercial development of
transgenic plants are first scrutinized and approved by regulatory authorities
established by the national government, to ensure that products are safe to
the
environment and consumers (33, 43, 158). This process can be important to
obtain the maximum social benefit from transgenic plant lines. For example,
in
several countries, release of transgenic insect-resistant plant varieties has
been
linked to mandatory programs of insect monitoring and industry responses to
avoid premature loss of useful insect control genes because of a build-up of
resistant insect populations (88).
Scrutiny by regulatory authorities is also an important mechanism to
reassure
the general public of the safety of a new technology that is not well
understood by most people. However, if the process is conducted
inefficiently
by the regulatory authorities, it can severely slow research. For example, it is
essential to characterize a substantial number of independent transformed
plant
lines in both physiological experiments and in selecting genetically
improved
cultivars. The availability of sufficient containment greenhouse space
rapidly
becomes limiting if the process of evaluation before approval of field
releases
is slow.
The conservative assumption underlying regulations in many countries is
that all transgenic plants are potentially hazardous. Scientific theory and
practical
experience show that this is not the case. The hazards relate to the genes
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transferred or the phenotype produced, not to the gene transfer method used.
There have been no reports of any harmful environmental effects or other
hazardous unforeseen behavior of transgenic plants in the thousands of field
trials conducted internationally to date (33). As public experience and
understanding
of plant transformation increase, it is to be hoped that regulatory
processes may be streamlined, with the focus on products rather than on
processes of plant genetic modification (108).
Consumer response to transgenic plant products has now been tested with
the commercial release of improved varieties in a range of crops (Table 2).
Table 2 Commercial releases of transgenic plant varieties
Trait Crop Name Company Product Status
Quality (vine-ripened
flavor,
shelf life
Tomato Flavr Savr Calgene Released 1994
Quality (vine-ripened
flavor,
shelf life)
Tomato Endless Summer DNA Plant
Technology
Blocked by
patent claims
Quality (paste
consistency)
Tomato — Zeneca Released 1995
Oil characteristics Canola Laurical Calgene Released 1994
Virus resistance Tobacco
Tomato
Capsicum
— (China) Released
1993–1994
Virus resistance Squash Freedom II Asgrow Released 1995
Insect resistance Cotton
Potato
Maize
Bollgard
NewLeaf
YieldGuard
Monsanto Released
1996–1997
Insect resistance Maize Maximizer Ciba Seeds Released 1996
Herbicide
resistance
Flax Triffid University of
Saskatchewan
Released 1995
Herbicide
resistance
Cotton BXN Calgene Released 1995
Herbicide
resistance
Canola
Corn
Innovator
Liberty Link
AgrEvo Released
1995–1996
Herbicide
resistance
Soybean
Canola
Cotton
Roundup
Ready
Monsanto Released
1995–1996
Herbicide
resistance
Soybean
Corn
Roundup
Ready, STS
Liberty Link
Pioneer Released
1996–1997
Male sterility
hybrid system
Canola — Plant Genetic
Systems
Approved 1996
(USA, FDA)
These releases have coincided with increasing dissemination of information
on
transgenic plants in forms accessible to the general public (158). In each
case,
consumers have responded positively to quality or price advantages, and in
several cases demand has outstripped supply in the first season. However, it
is
clear that continued work is important to provide the broad community with
information to support considered responses to emerging products (63).
Intellectual Property
As technical limitations are overcome, it is possible that commercial
limitations
will become more serious barriers to exploitation of genetic transformation.
New technologies developed in this area are effectively inventions and
are therefore eligible for patent protection (84, 123). For example, patents
have
already been issued on most established or promising plant genetic
transformation
strategies (29, 69, 89, 102, 119, 134, 136) and on many isolated genes,
promoters, and techniques for plant gene manipulation (see 121 and monthly
patent updates in Genetic Technology News). The patent literature has
become
an important source of information in plant transformation research, albeit
more difficult and expensive to search than the scientific literature (6, 40).
A patent provides the inventor or assignee with a period of exclusive
ownership, or formally a right to exclude others from making, using, or
selling
the invention. There is no statutory exclusion for infringment when patented
products or methods are used for research purposes. The widespread
misconception
that disclosure in the patent document allows researchers to practice
the invention in order to improve on it (73) possibly arises because patent
owners are generally reticent in instituting infringement proceedings until
the
level of damages that may accrue becomes commercially significant. There
is
no obligation to license and no constraint on royalty levels provided the
patent
holder makes active use of the intellectual property. Some patents make
extremely
broad claims, and patent holders are not required to develop all possible
manifestations of an invention to retain broad ownership (144). Penalties
for infringement of proprietary rights can be severe, so it is important to
determine whether the tools or topics of proposed research are already in the
public domain or subject to patent protection (160). This can be difficult to
establish without periodic patent searching, or even legal challenges (75).
The position may differ between countries. For example, particle
bombardment
for gene transfer into plant cells has been patented in the United States
(134) but not in Australia. Apparatus for particle bombardment involving a
macroprojectile and stop plate has been patented in Australia (135) but not
apparatus involving particle acceleration in a gas pulse. Patent coverage for
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many genes and promoters is similarly restricted. For example, the maize
ubiquitin promoter is the subject of granted patents or applications in
Europe,
Japan, and the United States (128) but apparently not in many other
countries,
including Australia.
Under these circumstances, a transgenic plant variety produced and used
commercially without infringing any patent rights in one country could
infringe
certain patent claims if used (even for research or other noncommercial
purposes) in another country. To complicate matters further, patent
applications
are not available publicly in some countries (notably the United States)
until the patent is granted, which can be years after the application is filed.
There are moves to harmonize international practice, e.g. by providing for
ownership for 20 years from the date of application and publishing 18
months
after application to eliminate the practice of “submarine” patent applications
that only surface after a competitor has independently made the same
invention
(42). The issues involved are complicated, and some important differences
in patent law between countries appear unlikely to be resolved in the near
future (4).
Patents are intended to encourage and reward useful invention and technical
innovation, and the new technology enters the public domain after a period
of
17 to 20 years (42, 84). In the interim, commercial restrictions can appear
quite
ruthless as patent holders adopt commercialization strategies to capture the
value of protected intellectual property (75). In an era of tight public sector
research funding and high research and development costs, the benefits of
corporate investment to develop transformation technologies outweigh the
inconvenience of patent restrictions. Debate continues on mechanisms to
balance
the competing interests (22).
FUTURE NEEDS AND DIRECTIONS IN PLANT
TRANSFORMATION RESEARCH AND DEVELOPMENT
Transformation Efficiency
The methods for gene transfer into plant cells, particularly Agrobacterium
and
particle bombardment, are now sufficiently developed to allow
transformation
of essentially any plant species in which regenerable cells can be identified.
Broadly applicable selection methods are well established. The key to
transformation
of recalcitrant species appears to be development of methods to expose
many regenerable cells to nondestructive gene transfer treatments.
What currently limits the practical transformation of many plant species is
the combination of a low frequency of transformation and a high frequency
of
undesired genetic change or unpredictable transgene expression. These
problems
necessitate expensive large-scale transformation and screening programs
to produce useful transformants. The first constraint may be addressed by
research into tissue culture systems to enrich for regenerable cells accessible
to
gene transfer. Contract transformation services (106) may implement
economies
of scale to afford robotic systems (1) for routine large scale target
preparation
and gene transfer treatments.
A clearer understanding of the events surrounding gene transfer by
Agrobacterium
is also required. Is transient expression a satisfactory test for Agrobacterium-
mediated gene transfer into plant cells, or can another convenient
test be developed to allow rapid detection and optimization of this key
event?
Does Agrobacterium select between cell types, and if so what features
determine
favored cells for gene transfer? Can these features be imparted to highly
regenerable cell types? Direct gene transfer experiments indicate that if
naked
DNA is transferred into many actively dividing and regenerable cells, a
proportion
will be stably transformed. Is the same true for cells receiving typically
lower doses of T-DNA, or are there additional physiological requirements
for
efficient T-DNA integration (111)? Is T-DNA integration targeted to
potentially
expressed regions of the genome, or to regions undergoing active
transcription?
Can the transcriptional status of target cells be manipulated to
achieve a high frequency of integration into regions suitable for subsequent
transgene expression, but a low frequency of insertional inactivation of
genes
influencing the phenotype of regenerated transformants?
There are at least as many relevant questions surrounding direct gene
transfer.
Is stable transformation efficiency as sensitive as transient expression to
decreased DNA concentration? Does DNA concentration affect mean copy
number or cotransformation frequency in resulting stable transformants? Is
integration targeted to potentially transcribed regions as appears to be the
case
for T-DNA from Agrobacterium? Can artificial T-DNA complexes be
manufactured,
and will they influence the efficiency or integration patterns available
from direct gene transfer?
Useful vs Absolute Transformation Efficiency
In the longer term, a more important goal than increased transformation
efficiency
is the development of transformation methods and constructs tailored
for predictable transgene expression, without collateral genetic damage. We
may conclude that much of the current effort in plant transformation directed
toward increased transformation frequencies is naive and misdirected. We
need to distinguish between absolute and useful transformation frequencies.
The limiting process in the application of plant transformation for more so-
318 BIRCH
phisticated studies of plant physiology or for cultivar improvement is
generally
not the production of transformants but the screening (or subsequent
breeding) required to eliminate transformants with collateral genetic damage
that would interfere with meaningful physiological analysis or commercial
use. Depending on the ratio of effort required for these processes a large
increase in absolute transformation efficiency may be futile if accompanied
by
even a small decrease in the proportion of useful transformants. Conversely,
a
large drop in absolute transformation frequency may be more than
compensated
by a smaller gain in the proportion of useful transformants. These ideas
are familiar to most practicing plant breeders but have understandably not
been
foremost in the minds of most transformation scientists while they struggled
to
develop reliable and efficient systems for gene transfer into target plant
species.
Collateral Genetic Damage
To achieve a high proportion of useful transformants, we need to understand
more clearly the factors contributing to undesired genetic change during the
transformation process. To what extent is such change associated with the
integration of single or multiple copies of foreign DNA, as distinct from the
processes of tissue culture, selection, and plant regeneration? Is genetic
change
induced or selected during such processes, or is it commonly the effect of
preexisting mutations in somatic cells that are simply detected when entire
plants are regenerated from single (transformed) cells? If change is induced,
which are the mutagenic stages in the protocols, and can they be avoided? If
mutations are preexisting, can the procedures be tailored to selectively
prevent
regeneration of mutated cells? Compared to adventitious shoot proliferation,
is
somatic embryogenesis disadvantageous because of longer duration in
culture,
or advantageous because the complexity of the embryogenic process acts as
a
filter to eliminate many cells with mutations? Does the approach of germline
transformation of uncultured explants followed by crossing to obtain
nonchimeric
transgenic progeny reduce the frequency of undesired genetic
change, or just mask such change in the background of genetic variation
from
sexual reproduction? The relative importance of these questions varies
between
plant species; vegetatively vs sexually propagated crops provide an
extreme example. Unfortunately, the answers to many of these questions
may
also be genotype specific.
Ideal and Model Transformation Systems
As the emphasis on useful transformation frequency increases, we may see a
trend toward minimization or elimination of tissue culture stages, targeted
integration of single copy transgenes, and direct (leaf-disc PCR) screening
for
transformants with useful genes to eliminate the need for reporter sequences.
As our understanding of the genetic basis of agronomic traits increases, it is
likely that this goal will be extended to the introduction of greater lengths of
DNA encoding multiple genes. We will need to determine the capacity of
available methods to introduce such lengths of DNA intact.
Although some of these questions may be answered and approaches
developed
with model plants, the features that make the models attractive for some
genetic studies (e.g. small genome, small plant size, rapid generation time
for
Arabidopsis) generally cannot be exploited in practical transformation
systems
for most economically important plants. We must be prepared to select the
models according to the questions, and test the answers for applicability to
the
practical targets.
Transformation, Breeding, and Genetic Diversity
As with conventional breeding, it is highly undesirable for plant
transformation
to lead to excessive genetic uniformity in current varieties of any crop.
Even a single gene in all varieties can create problems. For example, the
United States maize crop in 1970 was devastated because of disease
susceptibility
accompanying a cytoplasmic male sterility trait used to simplify hybrid
seed production (164). This is another reason to aim for the capacity to
transform
diverse genotypes within a species, to develop diverse genes for desired
phenotypes, and to eliminate unnecessary sequences from the transformation
process.
When Practical Means Commercial
Plant transformation is already sufficiently developed to allow the testing
and
even commercialization of plants with novel phenotypes under simple
genetic
control. For continuing practical benefits, it will be necessary to extend our
understanding of the biological basis for efficient plant transformation and
develop improved technologies for predictable transgene expression without
collateral genetic damage, at a pace matching the exciting scientific
advances
in gene cloning and characterization. This will require support from industry
for the underlying research. As transformation projects are increasingly
undertaken
with the possibility of generating commercially useful products,
transformation
scientists in turn must increasingly integrate social, legal, and economic
issues as well as technical issues from the earliest stages of project
design.
320 BIRCH
ACKNOWLEDGMENTS
I thank my graduate students, who have contributed to many elements of this
review through their critical enthusiasm for the science and technology of
plant transformation. Tanya Newton contributed the concept for Figure 2.
Visit the Annual Reviews home page at http://www.annurev.org.
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326 BIRCH
Animal and Plant Transformation: The
Application of Transgenic Organisms in
Agriculture
Matthew B. Wheeler, Stephen K. Farrand, and Jack M. Widholm
A transgenic organism carries in all its cells a foreign gene that was inserted
by laboratory techniques. Each transgenic organism is produced by
introducing cloned genes, composed of deoxyribonucleic acid (DNA) from microbes,
animals, or plants, into plant and animal cells. Transgenic technology affords methods
that allow the transfer of genes between different species.
Animal Transformation
Through transgenic animal transformation, new genetic information is introduced into an

animal in one generation without compromising or limiting the overall pool of genetic
information. Transgenic animals are produced by inserting genes into embryos prior to
birth. Each transferred gene is assimilated by the genetic material or chromosomes of the
embryo and subsequently can be expressed in all tissues of the resulting animal. The
objective is to produce animals which possess the transferred gene in their germ cells
(sperm or ova). Such animals are able to act as "founder" stock to produce many
offspring that carry a desirable gene or genes.
Transgenic animals have been produced by three methods: microinjection of cloned

gene(s) into the pronucleus of a fertilized ovum, injection of embryonic stem cells into
embryos, and exposure to retroviruses. The third method is not discussed in this article.
The first method is the one that is most widely and successfully used for producing
transgenic mice. After microinjection, the recently fertilized single cell embryos are
removed from the animal. Micromanipulators on a specially equipped microscope are
used to grasp each embryo. A glass pipette drawn or pulled to a fine point immobilizes
the embryo on one side, as shown in the photos to the right. On the opposite side, the
foreign DNA is injected into the embryo's pronucleus--either of two nuclei (male or
female) containing half the chromosomes of a fertilized ovum--with a second finely
drawn injection needle. After the injection, the embryos are transferred back into the
hormonally prepared or pseudopregnant recipient females or foster mothers. The
recipients follow normal pregnancy and deliver full-term young. This method is presently
the most efficient for generating transgenic animal lines: about 1 to 4 percent of the
injected embryos result in a transgenic offspring.
The second method involves microinjection of embryonic stem (ES) cells derived from
the inner cell mass of blastocyst-stage embryos (about 7 days postfertilization) into
embryos to produce "hybrid" embryos of two or more distinct cell types. The ES cells are
able to produce all tissues of an individual. Once isolated, ES cells may be grown in the
lab for many generations to produce an unlimited number of identical cells capable of
developing into fully formed adults. These cells may then be altered genetically before
being used to produce embryos. When these transformed cells participate in the
formation of sperm and eggs, the offspring that are produced will be transgenic. Results
have shown this method to be promising for producing transgenic mice. Studies are
presently under way at the University of Illinois Department of Animal Sciences to
develop ES cell lines for livestock species such as swine, cattle, and sheep.
To produce transgenic mice, Matthew B. Wheeler microinjects DNA into the pronucleus of one-cell
embryos.
Injection of cloned DNA into embryos. One-cell embryo is positioned for micro-injection into the
pronucleus (left). The plasma membrane has been pierced, and the tip of the needle remains inside the
pronucleus, while DNA is expelled from the needle, causing the pronucleus to swell visibly.
These methods, which enable the insertion of foreign genes into embryos, have provided
the tools for producing new strains or breeds of animals that carry new, beneficial genetic
information. These technologies do not produce new species but work within the
established genetic framework of existing species to improve them. Some new strains
developed include leaner, more feed-efficient, faster-growing swine containing additional
copies of the growth hormone gene, and mice containing the regulatory elements of the
human immunodeficiency virus (HIV) genome. The latter are used as a noninfectious
animal model for the study of AIDS.
The scope of the information acquired from transgenic animal technology is pertinent to
virtually all areas of modern agriculture and biomedical science--cancer research;
immunology; developmental biology; gene expression and regulation; and models for
human genetic diseases such as muscular dystrophy, Lou Gehring's disease, and sickle
cell anemia. Potential applications for transgenic animals include manipulation of milk
composition, growth, disease resistance, reproductive performance, and production of
pharmaceutical proteins by livestock.
Plant Transformation
There has been much excitement in the last few years about our ability to genetically
engineer plants using the new techniques of gene isolation and insertion. Paired with
standard methodologies of plant tissue culture and plant regeneration, these new
techniques allow us to construct transgenic plants that contain and express a single, well-
defined gene from any source - microbe, animal, or other plant species. The transgenic
plants, usually normal in appearance and character, differ from the parent only with
respect to the function and influence of the inserted gene.
This directed genetic engineering of plants requires that genes of interest are available,
that the gene be introduced into plant cells capable of regenerating into intact plants, and
that the gene carries with it a selectable marker so that the transformed plant cells can be
isolated from a large population of untransformed, normal cells. Finally, the transformed
plant cell must retain its capacity to regenerate. Certain species such as tobacco and
petunia regenerate plants quite easily, making transgenic plants readily obtainable.
Although corn, soybean, and wheat--the primary agricultural crops of Illinois and the
Midwest--are more recalcitrant to these manipulations, progress is being made toward
routine transformation and regeneration of transgenic progeny of these species.
Several techniques can introduce genes into plant cells. Perhaps the most successful
method involves the pathogenic bacterium Agrobacterium tumefaciens, which has the
innate ability to transfer DNA to plant cells. In nature, this transfer results in formation of
plant tumors (crown galls) at the infection site. Molecular biologists, however, have
disarmed this bacterium and constructed domesticated strains that no longer cause tumors
but transfer any DNA of interest to plant cells. The major disadvantage of the highly
efficient Agrobacterium system is that it does not work with all plant species, most
notably the cereals.
Other techniques use physical or chemical agents to transfer DNA into plant cells.
Protoplasts, plant cells that have been stripped of their protective cell walls, will take up
pure DNA when treated with certain membrane-active agents or with electroporation, a
rapid pulse of high-voltage direct current. Once inside the cell, the DNA is integrated and
the foreign gene will express. These two techniques largely depend upon the
development of protoplast systems that retain the capacity to regenerate intact plants.
Transgenic corn, rice, and soybean have been produced with these techniques, especially
electroporation. Success rates, however, are low, and the techniques not very
reproducible.
DNA can also be microinjected into target plant cells using very thin glass needles in a
method similar to that used with animals. Microinjection, however, has produced only a
few transgenic plants. The technique is laborious, technically difficult, and limited to the
number of cells actually injected.
Biolistics, a new method, involves accelerating very small particles of tungsten or gold
coated with DNA into cells using an electrostatic pulse, air pressure, or gunpowder
percussion. As the particles pass through the cell, the DNA dissolves and becomes free to
integrate into the plant-cell genome. This improbable technique actually works quite well
and has become, along with electroporation, one of the methodologies of choice.
Biolistics has the advantage of being applicable to whole cells in suspension or to intact
or sliced plant tissues. For example, plant meristems or tissues capable of regeneration
can be targeted directly. Unlike transformation or electroporation, the technique does not
require protoplasts or even single-cell isolations. Using biolistics, transgenic corn and
soybean plants have been produced that contain heritable copies of the inserted gene.
Only a few genes of agronomic importance have been inserted into plants: genes
conferring resistance to certain insects and viruses and also those conferring tolerance to
broad-spectrum herbicides. The latter result in increased herbicide specificity, allowing
the farmer to use more effective, environmentally safe chemical agents. More recently, a
gene has been introduced into tomato that delays overripening and prolongs shelf life of
the fruit.
Other traits of interest include those associated with grain quality. Genes to increase the
content of amino acids such as lysine, methionine, and tryptophan in seed will increase
nutritional value, thereby decreasing the need for amending grains with costly feed
supplements.
All traits discussed here are associated with expression of single genes. But many
important agronomic traits such as yield and lodging are not well understood and are
controlled by many genes. Manipulating such polygenic traits by genetic engineering will
require further research and the development of techniques for isolating, reconstructing,
and transferring complex blocks of genes. Extensive and promising research is being
conducted about additive disease resistance and stress tolerance, important polygenic
traits. Plant genetic engineering is thus moving slowly but steadily from the laboratory
bench into the field.
Matthew B. Wheeler, assistant professor of animal sciences; Stephen K. Farrand,

professor of plant pathology and microbiology; and Jack M. Widholm, professor of plant
physiology, Department of Agronomy
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Methodology
Documentation system for plant

transformation service and research
Karin I Köhl and Jürgen Gremmels
Max-Planck-Institute of Molecular Plant Physiology, Am

Muehlenberg 1, 14476 Golm, Germany
author email corresponding author email

Plant Methods 2010, 6:4doi:10.1186/1746-4811-6-4
Published:27 January 2010
Abstract
Background
In plant transformation, method compliance is critical for success.

Transformation methods are complicated and tend to evolve over
time. Until the complete method is published, method details are
often partially orally transmitted and thus bound to a few people.
Their documentation in text files are often a mixture of material and
method description with many references to other sources especially
to media description. These media are complex and often composed
from several commercially available mixtures plus individually
prepared stocks. The actual transformation experiment is generally
documented in lab books, in which deviations from the methods and
results are reported. Additionally, work schedules are planned in
diaries. Both paper-based sources lack backup copies and miss
unambiguous links to method descriptions and media recipes.
Description
To solve the problem, we devised a standard-operation-procedure

system based on a Microsoft Access database containing the
interlinked modules 'Media', 'Methods' and 'Experiments'. The Media
module contains all basic chemicals, stocks and complex media. In
this module, complex media are composed from other elements of
the Media module, thus mimicking the workflows of media
preparation in the lab. The Media module is made attractive to the
user by functions that generate file cards and labels. The Methods
module describes each method stepwise and links the steps to the
media. Copy functions allow cloning of old methods to document
method evolution without alteration of the old methods. Activation
and inactivation functions in the Media and the Methods module
remove outdated entries from active use. The Experiments module
links the method to experiment specific information. This module
generates a lab-book like user interface and a work schedule, and it
contains a simple result section.
Conclusion
The system has been evolved and tested over several years in a
transformation service unit, where it increased efficiency.
Additionally, the system provided rapid access to data for quality
control and decision making. The system can be easily modified for
the use in other research environments.
the genus
Agro bacterium in nature, this bacterium infects plants and transfers some of its own
bacterial DNA into the plant. Through the action of proteins produced by the bacteria,
bacterial DNA is made to integrate permanently into the plant’s own genomic DNA.
Expression of the bacterial DNA in the plant causes the plant to produce unusual
quantities of plant hormones and other compounds, called opines, which provide food for
the bacteria. The unusual quantities of plant hormones around the infection site cause
the plant cells to grow abnormally, producing characteristic tumors. Scientists have
harnessed this pathogenic bacterium to insert genes into plants by deleting the bacterial
genes that cause tumors in the plant and then inserting desirable genes in their place.
When the modified Agrobacterium infects a plant, it transfers the desir able genes into
the plant genome instead of causing tumors. The desirable genes become a permanent
part of the plant genome, and expression of these genes in plant cells produces desirable
products.
One major drawback of the Agrobacterium method is that insertion of bacterial DNA into
the plant genome is essentially random. The gene may not be efficiently transcribed at its
location, or the insertion of bacterial DNA may knock out an important plant gene by
inserting in the middle of it or both may occur. Therefore, the fact that a cell is
genetically transformed does not guarantee that it will perform as desired.
Microparticle bombardment is the introduction of foreign DNA constructs into plant

cells by attaching the DNA to small metal particles and blasting the particles into plant
cells using either a compressed air gun or a gun powered by a 0.22 caliber gun cartridge.
This is truly a “brute force” method of introducing DNA into a cell that inadvertently
causes many lethal casualties among the bombarded plant cells. However, some plant
cells blasted with the DNA-containing metal particles will recover and survive. The plant
cells may express the DNA for only a short time (transient expression), because the DNA
does not readily integrate into the plant genome, but occasionally the foreign DNA may
spontaneously recombine into the plant genome and become permanent.
Other ways of introducing foreign DNA into plant cells include electro oration,
microinjection, sonication, and chemical treatment. These methods are not used
extensively, because they generally require the production of protoplasts (plant cells that
lack their cell walls) from plant cells before transformation. To create protoplasts, the
plant cell wall is removed by digestion with the enzymes cellulase and pectinase.
Protoplasts are fragile structures, but the absence of a cell wall is desirable because it
leaves only the plasma membrane as a barrier to foreign DNA entering a plant cell.
Electroporation uses very brief pulses of high voltage electrical energy to create
temporary holes in the plasma membrane through which the foreign DNA can pass.
Microinjection involves physically injecting a small amount of DNA into a plant cell
using a microscope and an extremely fine needle. Sonication uses ultrasonic waves to
punch temporary holes in the plasma membrane, this method is therefore similar to
electroporation. Chemical treatment involves the use of polyethylene glycol to render the
plasma membrane permeable to foreign DNA.
All the transformation procedures produce only a few transformed cells out of the
millions of cells in an explant, so selection of transformed cells is essential.

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An unrelated process called malignant transformation occurs spontaneously in the

Bacteria transformation may be referred to as a stable genetic change brought about by

[edit] Natural competence

Main article: Competence (biology)

[edit] Artificial competence

Calcium chloride transformation is a method of promoting competence. Chilling cells in

[edit] Plasmid transformation

A number of mechanisms are available to transfer DNA into plant cells:

• Agrobacterium mediated transformation is the easiest and most simple plant

Diagram of the major components of an electroporator with cuvette loaded.

Dr.Eberhard Neumann - Electroporation Founder

Electroporation, or electropermeabilization, is a significant increase in the electrical

Electroporation is a dynamic phenomenon that depends on the local transmembrane

[edit] Laboratory Practice

For bacterial electroporation, typically a suspension of around 50 microliters is used.

[edit] Medical Applications

[edit] Physical Mechanism

Electroporation allows cellular introduction of large highly charged molecules such as

Electroporation is a multi-step process with several distinct phases.[5] First, a short

WHY TRANSFORM PLANTS?

This method can be used on both monocotyledonous and dicotyledonous species

This picture was taken from http://www.agr.okstate.edu/ptf/chambergun.html - the

The earliest custom manufactured geneguns (fabricated by Nelson Allen) used a 22

[edit] Humans and animals

Gene guns have also been used to deliver DNA vaccines.

Gene gun bombardment is also used to transform C. elegans, as an alternative to

[edit] Further reading

A. tumefaciens attaching itself to a

Agrobacterium tumefaciens (updated scientific name: Rhizobium radiobacter)[1][2] is the

Agrobacterium tumefaciens (or A. tumefaciens) is an alphaproteobacterium of the family

Economically, A. tumefaciens is a serious pathogen of walnuts, grape vines, stone fruits,

Since the Ti plasmid is essential to cause disease, pre-penetration events in the

[edit] Method of infection

Attachment is a two-step process. Following an initial weak and reversible attachment,

After production of cellulose fibrils a calcium-dependent outer membrane protein called

Possible plant compounds that initiate Agrobacterium to infect plant cells:[6]

• Acetosyringone: Phenolic compound

[edit] Formation of the T-pilus

[edit] Transfer of T-DNA into the plant cell

[edit] Genes in the T-DNA

[edit] Beneficial uses

The DNA transmission capabilities of Agrobacterium have been extensively exploited in

[edit] See also

• Dickinson, M. (2003). Molecular Plant Pathology. BIOS Scientific Publishers.

The structure of the Ti plasmid

1. ^ Scott E.Stachelt, Eugene W.Nester (1986). "The genetic and transcriptional

Calcium chloride (CaCl2) transformation is a laboratory technique in prokaryotic

lternative methods of plant transformation--a short

Department of Plant Genetics, Breeding and Biotechnology, Warsaw Agricultural

In plant transformation, method compliance is critical for success. Transformation

To solve the problem, we devised a standard-operation-procedure system based on a

In tissue-culture-based generation of genetically modified plants, minute method

To address the problem, we designed a computer-based documentation system that

Construction and content

Format: ZIP Size: 986KB Download file

Additional file 2. SupplementaryFigures. The file contains pdf-files with screenshots

Figure 3. Experiment form. The form for the documentation of plant

Depending on the transformation method, namely ballistic or Agrobacterium tumefaciens

When a transformation experiment is started, the method is selected and the

Figure 4. Lab book. For each transformation experiment, a laboratory

Table 1. Work schedule shows an example of an automatically generated work schedule

The end of the experiment is documented by keywords (handover, withdrawn,