GENETIC MANIPULATION

IN PLANTS

Assoc. Prof. Dr. Asmah Awal

Faculty of Plantation & Agrotechnology
asmah138@uitm.edu.my

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Genetic Manipulation in Plants
3.1 The Phenomena of Gene Transfer
3.2 Recombinant DNA (rDNA) Technology – Direct
Methods, Agrobacterium-mediated,
Transient/Permanent Expression
3.3 Expression of Foreign Genes in Plants
3.4 Transgenic Plants
3.5 Application of Genetic Engineering in Agriculture –
Resistance to Disease, Pest Drought and Other Value-
added Crop Products
3.6 Bioethics – Regulations on the Use of Genetically
Manipulated and Transgenic Plants

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 GENETIC TRANSFER

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 GENE EXPRESSION

 Gene : certain sequence of nucleotides that controls a

characteristic of an organism
 Allele : a pair of genes (each from a parent) that control
a characteristic of an organism
 If both the genes in an allele are the same, i.e. AA or aa,
the genes are said to be homozygous
 If the genes in an allele are different, i.e. Aa, the genes
are said to be heterozygous

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 Punnett square
(all possible squares)

BB x BB BB x Bb Bb x Bb Bb x bb bb x bb

B B B B B b B b b b

B BB BB B BB bB b Bb bb
B BB BB b bb bb
b Bb Bb b Bb bb b Bb bb
B BB BB b bb bb

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 GENE EXPRESSION – cont.
 If the effect of one gene takes priority over the other gene, it is
said to be the dominant gene. The other gene is said to be
recessive gene. For example, if the red colour (A) is
dominant, the flower will be red although the plant contains
genes for both red (A) and white (a) flower. The flower will be
white in colour if the alleles are homozygous recessive, i.e. aa

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 GENE EXPRESSION – cont.

 Some genes are of equal

power and are said to be
codominant. In the previous
example, the plant that
contains Aa will have pink
colour flower.

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 GENE EXPRESSION – cont.

 Some characteristics
may be controlled by
several pairs of genes
(alleles) and not a
single allele, e.g. milk
production in cows is
controlled by several
alleles.

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 GENE
EXPRESSION
– cont.

Chromosome :
genes group
together on dense
bodies

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 GENE EXPRESSION – cont.

 Genotype : expression of genetic makeup

of an organism
 Phenotype : the characteristics brought
about by the environment and the genetic
code of an organism

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 Mendel’s Laws
Law of segregation : alleles responsible for the characteristics from
each parent are separated and then combined with alleles from the
other parent at fertilization

Law of independent assortment : alleles for certain characteristics

are passed from parent to the next generation separate from other
alleles that transmit other characteristics

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 Mendel’s Law of Segregation

                                                                                                                                

                                                                                                                 

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 Mendel’s Law of Segregation

  
                                                                                                                              

                                                                                                                          
                                                                                                                 

                                                                                     

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Mendel’s Law of Independent Assortment

                                                                                                           

                                                                                                                          
            

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Mendel’s Law of Independent Assortment

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 BREEDING OBJECTIVE

Specific
e.g. yield increase, disease
resistance, improved protein
content, herbicide tolerance etc.

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 CONVENTIONAL BREEDING
 there must exist somewhere
a plant with the desired
characteristic (natural
mutation)
 or, such a trait must be
induced, e.g. by mutation
(radioactive isotopes, e.g.
Co16 (left), X-ray, gamma ray,
ion beam, chemical e.g.
EMS, ethyl
methanesulfonate)

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 CONVENTIONAL BREEDING – cont.
 hybridization / crossing of two different plants
(individuals) (pollen grains from the desired
source are deposited on the stigma of a
receptive female plant; needs
emasculation = the female flower is rid of all
male organs or use male sterility gene)
 fertilization & development into an embryo
 selection procedure needs a large segregating population of plants
from which to search for the desired recombinant individual
 selection starts in F2
 lasts for several to many years

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 LIMITATIONS OF CONVENTIONAL BREEDING
 long duration : lasts for several to many years
 limited to crossing within species: the parents must be compatible &
belong to the same species for hybridization
 lower selection efficiency: the methods used to sort among the
enormous variation generated from a cross in the case of plants is
not precise
 large segregating population: for a high chance of identifying the
recombinant of interest; requires large amount of space, and thus
increases breeding expense
 only genes for traits expressed within a restricted gene pool are
accessible

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RECOMBINANT DNA TECHNOLOGY

 Genetically modified organism :

organism that have had certain
gene(s) inserted or deleted
 Transgenic organism : organism that
have had foreign gene(s) (from
another species) inserted
 The process is gene splicing /
genetic engineering

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 GENETIC ENGINEERING PROCESS

 Determine the desired characteristic

 Locate the gene(s) & controller gene(s)/sequences responsible for
the desired characteristic (e.g. use marker gene and run restriction
analysis / RFLP or
by using DNA probe)
Genetic markers

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 GENETIC ENGINEERING PROCESS – cont.

 Vector (e.g. plasmid, bacteria / Agrobacterium tumefaciens, virus,

bacteriophage) is used to carry the desired genetic material
(recombinant DNA) into the host plant
 Agrobacterium tumefaciens is commonly used as it naturally injects
its DNA into the plant cell and causes tumors/crown galls in plants.
 A plasmid is removed from the bacterium and the gene that causes
the crown gall is removed and replaced with the desired gene. The
plasmid is then placed back to bacterium and the bacterium
becomes a vector now.

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 GENETIC ENGINEERING PROCESS – cont.
 The transformed bacteria is then used as soil inoculation. Instead of
causing crown gall, the plant will express the desired characteristic.
 Works well with dicot but not with monocot
 Transformation can also be carried out on in vitro (tissue culture)
leaf discs or incubation of protoplasts in suspension of
Agrobacterium tumefaciens
 For in vitro transformation, media containing either an antibiotic or
chemical is used to select the transformant. Then, the whole plant
will be regenerated (transformed plant) based on the concept of
totipotency.

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 GENETIC ENGINEERING PROCESS - cont.
 Or insert the recombinant DNA into plant cell [microinjection (cell
wall must be removed), electroporation (cell wall must be removed),
biolistics / microprojectile bombardment technique]
 In microinjection (below), an extremely tiny, hollow glass needle is
used to open up a small hole in the cell membrane and the DNA is
then injected through the tube directly into the cell.

 In electroporation, protoplast (plant cell without cell

wall) is subjected to an electric charge that causes
the cell membrane to be permeable to the DNA.

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Electroporation

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 GENETIC ENGINEERING PROCESS – cont.

 Biolistic is commonly used as it can be used with intact tissues;

DNA is blasted into the cell using tiny bullets composed of tungsten
or gold particles with transformed DNA attached; .22 caliber gun
cartridge or compressed air is used to fire the particles into the cell
 Regeneration of transformed cell (tissue culture under controlled
environment with proper hormones, nutrients, light and temperature)
 Indentification & selection / isolation of transformed cell / organism

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 Biolistics technique

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 TRANSIENT GENE EXPRESSION

 Introduced genes which become integrated into the DNA of the

plant can become permanently established and sexually
transmitted.
 Some foreign genes are active soon after they have been inserted
into plant cells, but not on a long term basis. In these cases,
transient expression results from DNA which has not been fully
integrated and is eventually broken down.

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 ONLINE LECTURE

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