PLNT2530 Plant Biotechnology
2020
Unit 8b
Direct Transformation with DNA
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Transformation by direct
transfection of DNA into plants
•It has long been observed that cells have mechanisms for
incorporating free DNA into their chromosomes.
•This seems to be an effect of the naturally-occurring DNA
repair mechanisms.
•Foreign DNA will often be integrated at random
chromosomal sites
•Therefore, if you can find a way to get DNA into the cell,
you can probably transform the cell.
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Gene Gun
• Known as The "Gene Gun" method.
• Also referred to as micro-projectile bombardment or biolistics
(ballistics using biological components).
• This technique is used for in vivo transformation and has been
especially useful in transforming monocot species like corn and rice.
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A Gene Gun shoots genes into plant cells. DNA is coated onto small
particles of gold or tungsten approximately two microns in diameter.
The particles are placed in a vacuum chamber and the plant tissue is
placed below the chamber. The particles are propelled at high velocity
using a short pulse of high pressure Helium gas into any target cell or
tissue.
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Two types of Gene Guns
Fixed gun for cell culture Handheld gun for in-planta
transformation
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The real trick is to fire pellets with enough force to break into cell, but
not so much as to go right through tissue. Must optimize distance,
placement of tissue, dispersal of charge (don't want charge to all go
in one place).
Advantages of "Gene Gun"
no host range problems
can transform intact tissue, eliminating de-differentiation,
somaclonal variation problems associated with callose or protoplasts
Disadvantages of "Gene Gun"
Has to be optimized for each tissue type
Can only transform superficial cells
very hard to target the specific cell layer you want
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Protoplast Transformation
Advantages
Not limited to species infected by A.
tumefaciens
No need to clone constructs into T-
DNA based vector; No cocultivation
with A. t.
Disadvantages
Protoplasts very delicate; difficult to
obtain
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Antibiotic resistance assay
It is often possible to screen plantlets for Transgenic Untransformed
presence or absence of a transgene using KanR plants control plants
the kanamycin resistance gene for
selection. In transgenic B. napus,
transformed plants expressing KanR show
full root growth when plantlets are
geminated and grown on media containing
Kanamycin (left). Root growth of non-
transformed plants is almost completely
inhibited (right).
[Peijun Zhang, Ph.D. thesis, University of
Manitoba, 1998].
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Selection of homozygous transformed lines
For many practical purposes, lines single copy, homozygous for
the transgene are needed.
With multiple copy numbers, gene silencing often occurs. Gene
silencing often results from high expression levels of an mRNA
transcript.
When transgenic loci are not homozygous, progeny segregate for
the transgene. Progeny in the initial transformant generation, T0, are
hemizygous* Result: some segregating progeny in later generations
will not have the trait!
*hemizygous - only one of
the two copies of a chromosome
will get the transgene (G).
The other copy, referred to as the null
allele (0), will not.
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Determining transgene copy number by Southern blot
Most genes are smaller than the
average fragment size for a 6-cutter
restriction endonuclease (~ 4096 bp)
Therefore, if there is only one copy of
a gene, we expect to see a single band
on a Southern blot.
For each transgenic locus, there is
usually a single band
Therefore, the number of bands is
a good estimate of the transgene
copy number.
Southern blot of T1 transgenic Brassica napus
transformed for pea PR10.
Dept. of Plant Science, Univ. of Manitoba
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Identification of homozygous transformed lines
The conventional way to determine
If we self T0
homozygosity is through Mendelian
genetics.
The progeny of parents from T1 or G0 x G0
later generations can be screened for
segregation of the transgene.
GG : G0 : 00 G:0
If the plant has a single-copy T1 3 :1
1 : 2 : 1
insertion, homozygous parental lines
can be identified by lack of Genotype Phenotype
segregation for the transgene.
It is usually safest to assay directly
for the gene of interest (eg. by PCR)
rather than relying on the selective
marker (eg. KanR). This is because
one can often get partial T-DNA
insertions, missing the gene of
interest.
It’s easy to screen for G_ plants using PCR, and looking for presence of the gene.
But how do we find out which plants are GG and which are G0? 12
To identify individual plants that are
homozygous for the transgene:
1) Grow 10 - 20 single copy plants until several true leaves are
available, but well before flowering.
2) Extract DNA from a leaf taken from each individual,
3) Test for presence of the transgene by PCR. Use primers specific for
the transgene, not the vector.
4) Discard null segregants, which do not give a band, and bag
positive plants at flowering to prevent cross-pollination.
5) Collect seed from each parent and keep in separate envelopes.
6) For 5 positive parents, grow at least 15* plants until several true
leaves are available.
7) Extract DNA from each individual, and test for the presence of the
transgene by PCR. (You may save some work by freezing leaf tissue
for 5 sets of progeny, but only testing one individual at a time, until you
get a true homozygote).
If any line tests positive in all individuals, that line must be
homozygous. If some individuals test negative, it may be because of
bad DNA preps. It is sometimes worth re-doing the DNA preps and
doing PCR on the new DNA.
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*How many progeny need to be tested, for each
transgenic line, to choose a homozygous parent?
We want to eliminate the possibility that a parent testing positive for G is
heterozygous
2/3 of all positive parents will turn out to be heterozygous
For any heterozygous parent, the probability that any single offspring will
test positive is 0.75.
P(heterozygous parent | all N progeny test positive) = 2/3 (0.75 N)
N P
1 0.500
5 0.158
10 0.038
15 0.009
20 0.002
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Identification of homozygous lines by qPCR
Jos C Mieog*, Crispin A Howitt and Jean-Philippe Ral (2013) Fast-tracking development of
homozygous transgenic cereal lines using a simple and highly flexible real-time PCR assay.
BMC Plant Biology 2013, 13:71 doi:10.1186/1471-2229-13-71
http://www.biomedcentral.com/1471-2229/13/71
qPCR (quantitative PCR) - a method for
quatifying DNA or RNA by carefully-
controlled PCR reactions.
Amplification of DNA by PCR is not linear
with respect to cycles. Very little DNA is
produced in early cycles, but increase is
exponential over time. As reaction
components are exhausted, the
amplification curve reaches a shoulder, with
little additional amplification.
Quantitative PCR is routinely used to quantify input DNA. Quantitation reqires:
●control DNAs whose concentration is known precisely
●replicate amplifications to measure the standard error of the PCR reaction
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Identification of homozygous lines by qPCR
The more copies of a sequence, the
fewer cycles of PCR are required to
reach an arbitrary threshold
concentration of PCR product, CT.
We can quantify DNA relative to
controls by comparing the number of
cycles at which controls reach CT, with
the number of cycles at which unknowns
reach CT.
The copy number for a gene of interest can then be calculated by the formula:
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Identification of homozygous lines by qPCR
To accurately measure CT ,
samples of transgenic wheat were
amplified at 5 different
concentrations, three replicates
each.
primer pairs amplify:
NOS -transgene
ECA - EC* from A genome only
(single copy control)
ECABD - EC from A,B and D
genomes (3-copy control)
Rint9 - rice intron, found in
transgene
EC* - wheat Epsilon Cyclase, present
in A,B and D genomes
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Identification of homozygous lines by qPCR
T0 plant has 1 copy: T0 plant has 3 copies: T0 plant has 7 copies:
look for homozygous look for homozygous look for homozygous
segregants in T1 segregants in T2 segregants in T2
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