PLANT BIOTECHNOLOGY & GENETICS
(3 CREDIT HOURS)
LECTURE 13
INCORPORATION OF TRANSGENES INTO CROPS,
METHODOLOGY OF PRODUCING TRANGENIC CROPS,
CONFIRMATION OF PUTATIVE TRANSGENIC PLANTS & TRANSFORMATION
EFFICIENCY
POLYMERASE CHAIN REACTION
SOUTHERN BLOTTING, NORTHERN BLOTTING, WESTERN BLOTTING,
FUNCTIONAL ASSAY,
PROGENY TEST,
CROP SPECIES AMENABLE TO TRANSFORMATION
INCORPORATION OF TRANSGENES INTO
CROPS
• Most of the methods currently used for plant transformation employ a
technique for delivering the DNA into the cell without regard to its ultimate
intracellular location. Once inside the appropriate cellular location by a
chance process, the DNA is integrated into the chromosomal (or organellar)
DNA, usually by a nonspecific recombination process. The exception is
Agrobacterium-mediated transformation, which delivers the DNA specifically
into the nuclear compartment with high efficiency and also provides a
mechanism for its integration.
• In plant transformation, two physical barriers prevent the entry of DNA into a
nucleus, namely the cell wall and the plasma membrane. Most plant
transformation methods overcome these barriers using physical and/or
chemical approaches. In protoplast-mediated transformation, the cell wall is
digested with a mixture of enzymes which attack cell wall components,
yielding individual protoplasts that can be maintained intact using
appropriate osmoticum in the medium. Entry of DNA is then facilitated by
the addition of permeabilizing agents, such as polyethylene glycol, that allow
DNA uptake, presumably by coating the negatively charged DNA inside the
cell and various cellular compartments in a random process.
INCORPORATION OF TRANSGENES INTO
CROPS
• A second method, often referred to as biolistic transformation, utilizes fine metal
particles (typically tungsten or gold) coated with DNA that are usually accelerated
with helium gas under pressure. Other methods, such as microinjection,
sonication, and electroporation cause transient microwounds in the cell wall and
the plasma membrane, allowing the DNA in the medium to enter the cytoplasm
before repair or fusion of the damaged cellular structures. However, many of
these methods are tedious and result in variable transformation efficiencies.
• In all transformation techniques, the desired transgene is placed under the
control of a promoter which produces high-level constitutive or inducible
expression of the gene in specific or all tissues. In addition, another gene that
allows detection or selection of the transformed cells is introduced in the same or
different vector DNA. The presence of a screenable or selectable marker gene
greatly simplifies the identification of transformed plants and increases the
efficiency of recovery of transgenic plants. Typical screenable markers are the gus
gene, encoding a β-glucuronidase, and the gfp gene encoding a green fluorescent
protein. Selectable markers that have been most useful are those conferring
resistance to antibiotics such as kanamycin, paromomycin, and hygromycin, or to
herbicides such as bialaphos, glyphosate, or cyanamide.
METHODOLOGY OF PRODUCING
TRANSGENIC PLANTS
• Most of the important crop species have been successfully transformed, at
least in the laboratory. Two major approaches have been widely used to
produce transgenic crop plants, both monocots and dicots. One is biolistic
bombardment and the other is Agrobacterium-mediated transformation. In
the biolistic protocol, the primary delivering system is the helium-powered
gun. The transgene and the selectable marker are inserted in the vector
between two unique sequences, called the left border and the right border,
which are utilized in insertion of the T-DNA into the host chromosomal
DNA.
• Parameters involved in the biolistic gun include pressure (ranging from 900
to 1300 psi), particle size (0.6 to 1.1 µm), and type of material (gold and
tungsten), target distance (7.5 to 10 cm), and target material (cell
suspension, callus, meristem, protoplast, immature embryo). These
parameters vary somewhat with the crop involved. Disadvantages of using
the biolistic gun are low success frequency, high copy numbers that often
are correlated with gene silencing, patent issues and cost.
METHODOLOGY OF PRODUCING
TRANSGENIC PLANTS
Agrobacterium-mediated transformation may correct some of the weaknesses
encountered with the biolistic approach and has been successful in rice, maize,
sorghum, and other crops; its effectiveness with other crops, especially wheat,
remains questionable. Further, some cultivar versus Agrobacterium strain
specificity may limit the range of cultivars that can be successfully
transformed. Development of reliable and efficient protocols are needed to
improve the efficiency and range of both approaches in transforming crop
plants.
METHODOLOGY OF PRODUCING
TRANSGENIC PLANTS
Transformation techniques not involving tissue culture are desirable for crop
species in which many cultivars do not respond to tissue culture. These
techniques include soaking and vacuum infiltration transformation of
Arabidopsis inflorescence with Agrobacterium, transformation via the pollen
tube pathway and pollen transformation via biolistics. The dipping method
adopted in Arabidopsis must be modified for cereal crops by altering the
plant stage of infiltration, the concentration of bacterial culture, and the
duration of treatment.
METHODOLOGY OF PRODUCING
TRANSGENIC PLANTS
Further, each tiller (for wheat, barley etc) of the same plant must be kept
separate. If this technique works, selection for transformants is made
directly from seed-derived seedlings and tissue culture is avoided. Thus,
genotypes that respond negatively to callus induction and plant
regeneration will not present a problem; however, it is likely some
genotypes will be more amenable to infiltration transformation than
others.
CONFIRMATION OF PUTATIVE TRANSGENIC
PLANTS & TRANSFORMATION EFFICIENCY
Commonly used methods to confirm the putative transgenic plants are
polymerase chain reaction, Southern blotting, Western blotting, Northern
blotting, functional assay (testing the presence of selectable marker and
the target gene), in situ hybridization, and progeny analysis (segregation of
the target gene). Not all transgenic plants produce the same amount of
protein from the target gene and selection based on the Western blot is
necessary. This is because a positive correlation usually exists between the
effectiveness of the gene in the bioassay and the amount of protein it
produces. For example, the level of rice chitinase accumulating in
transgenic sorghum plants with the chi11 gene was positively correlated
with resistance to sorghum stalk rot.
POLYMERASE CHAIN REACTION
PCR, a simple and rapid procedure, is utilized to confirm whether a
putatively transgenic plant that has survived selection is indeed transgenic.
Usually, two primers (one forward and one reverse) specific for the
selectable marker (bar gene, for example) are used in a PCR reaction with
genomic DNA extracted from the transgenic plants. A thermostable DNA
polymerase amplifies the region between the two primers during the
multiple amplification cycles of the PCR, which yields a DNA fragment of
predicted size (the length equal to the number of base pairs between the
two primers in the transgene). This fragment is easily detected on an
agarose gel by staining with ethidium bromide. PCR is a very sensitive and
rapid method for identification of transgenic plants in the seedling stage
and requires only a small amount of plant tissue.
SOUTHERN BLOTTING/ HYBRIDIZATION
ANALYSIS
In Southern blotting, DNA fragments from transgenic plants generated by
digestion with restriction enzyme(s) are first separated according to fragment
size by electrophoresis through an agarose gel. The DNA fragments then are
transferred to a solid support, such as a nylon membrane or a nitrocellulose
sheet. The transfer is affected by simple capillary action, sometimes assisted
by suction or electric current. The DNA binds to the solid support, usually
because the support has been treated to carry a net positive charge, or some
other means of binding such as inducing covalent binding of the DNA to the
support. DNA fragments maintain their original positions in the gel after
transfer to the membrane. Hence, larger fragments will be localized toward
the top of the membrane and smaller fragments toward the bottom. The
positions of specific fragments can then be determined by “probing” the
membrane. The probe consists of the DNA fragments of interest, such as a
cloned gene, which has been labeled with a radioactive isotope or some
other compound that allows its visual detection. Under the proper set of
conditions, the denatured single-stranded probe will hybridize to its
complementary single strands of genomic DNA affixed to the membrane.
SOUTHERN BLOTTING/ HYBRIDIZATION
ANALYSIS
In this way, the size of the fragment on which the probe resides in the
genomic DNA can be determined. In transgenic plant experiments, the
Southern blot often is used to determine whether an introduced gene is
indeed present in the plant DNA and whether multiple transgenic plants
carry the introduced gene on the same size of DNA fragment (suggesting
independent transformation events). The results of Southern blots also
indicate whether a single copy of the gene has been inserted or if
multiple copies are likely to be present.
NORTHERN BLOTTING
Northern blotting – the name was derived as a play on words from the
Southern blot – is very similar to the Southern blot, except that instead
of restriction enzyme-digested DNA, native RNA is separated according
to size by electrophoresis through an agarose gel and then transferred to
a solid support. The rest of the Northern blot procedure is very similar to
that of the Southern blot and it is used to determine whether the
introduced gene has been transcribed into messenger RNA and
accumulates in the transgenic plant.
WESTERN BLOTTING
The Western blotting procedure detects the protein of the transgene in
an extract of proteins prepared from various parts of the transgenic
plants and is, therefore, an assay for a functional transgene. In this
technique the proteins are first electrophoresed in an SDSpolyacrylamide gel and the proteins are then transferred to
nitrocellulose membrane by electrophoretic transfer. The membrane is
then treated with an antibody specific for the protein encoded by the
transgene followed by a second antibody coupled to an enzyme, which
can act on a chromogenic (or fluorogenic) substrate leading to
visualization of the transgene protein with increased sensitivity. The
expression level of the protein can be quantified using known amounts
of the transgenic-encoded protein.
FUNCTIONAL ASSAY
When the selectable markers used are antibiotic-resistant or herbicideresistant genes, a functional assay can be made by spraying antibiotics or
smearing herbicide on the leaves of those putative transgenic seedlings
or plants in later segregating populations. Sensitive plants typically will
turn brown and shrivel up whereas resistant (transgenic) plants will stay
healthy and green. Such an assay provides the initial screening of large
number of putative transgenic plants and reduces the work load by
eliminating escapes during selection.
PROGENY TEST
With stable transformed genes, progeny testing should show the
presence and activity of the selectable marker and target genes, such as
the gene gfp encoding green fluorescent protein, or bar and disease
resistance. However, segregation does not always follow the typical
Mendelian fashion. For example, among the progeny in the
Agrobacterium-mediated wheat transformation experiments reported,
segregation in the T1 generation had ratios of 32:0, 1:34, 0:40 and 74:0 in
addition to the expected 1:1, 3:1 or 15:1 ratios. This variability indicates
aberrant segregation. However, in other cases, segregation follows the
normal Mendelian pattern. For example, among six sorghum T0
transgenic plants produced by biolistic bombardment, all showed typical
3:1 segregation ratios in the T1 generation.
CROP SPECIES AMENABLE TO
TRANFORMATION
• Any crop that is able to produce calli from explants and is capable of callus
regeneration into plants with high efficiency is amenable to transformation
using biolistic bombardment and Agrobacterium tumefaciens. However, it is
known that response to tissue culture is highly genotype dependent.
Furthermore, somaclonal variation; spontaneous genetic variations
occurring in cells growing in vitro could occur during tissue culture
processes. Thus, to confirm that the improved phenotype of the transgenic
plants is due to the transgene, two controls should be included – one from
seed-derived plants and the other from non-transformed tissue culturederived plants.
• For those crops of genotypes that show a negative response to tissue
culture, a transformation procedure independent from tissue culture should
be considered and tested. It will be a great accomplishment to perfect a
procedure bypassing tissue culture, because many cultivars of wheat and
rice and inbred lines of maize and sorghum do not respond to tissue culture
operations.
THE END