Biotechnology Engineering

1. Genetic Transformation of
Plants
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Lecture- 36

2. Transformation methods in plants
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•Uptake of foreign DNA by seeds and
seedlings
•Uptake of DNA by pollen
•Transformation of protoplasts
•Agrobacterium-mediated transformation
•Gene gun method

3. Uptake of foreign DNA by seeds and seedlings
• Most of the early plant transformation experiments
were done with whole plants system such as seeds
and seedlings.
• It was shown that exogenous DNA was not only
taken up by plants but also integrated into the host
genome.
• The evidence for integration and replication of the
exogenous DNA was based mainly on isopynic
centrifugation of cesium chloride(CsCl).
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4. Experiments to testify Integration and
replication
• Radioactive labeled DNA of density different from
the host DNA was administered to the seedlings.
• DNA from seedlings (after a period of metabolism)
was isolated for analysis on CsCl gradients.
• Evidence for integration of the radioactive DNA was
ascertained by the occurrence of a radioactive band
approximately intermediate in density between the
host and donor DNA.
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5. • This band was further analyzed by sonication
and denaturation.
• Replication follows the same procedure but
differs only in respect of the donor DNA,
which is non-radioactive here.
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6. Uptake of DNA by pollen
• In 1976, Hess and co-workers incubated Nicotiana glauca
pollen with DNA isolated from Nicotiana langsdorffii and
used this pollen to pollinate emasculated N. glauca
flowers.
• This led to the production of the transformed plants which
developed tumors at the wound site of the stem.
• This is a characteristic feature exhibited by the sexual
hybrids arising between these two species.
• This suggests that pollen can act as a vehicle for the
transfer of foreign genes into plants. 6

7. Testifying uptake of DNA by pollen
• De la Pena et al. (1987) manually injected young floral
tillers of rye plant with a solution of plasmid DNA
carrying a chimeric gene consisting of T-DNA nopaline
synthase gene flanking coding sequence of the Tn5 neo
gene.
• Injected flowers were pollinated with normal pollen
and progeny seeds plated on kanamycin medium.
• Of the 300 seeds only two seedlings showing traits of
kanamycin as well as nos-neo gene were isolated.
• Thus it was concluded that the injected DNA was taken
up. 7

8. Transformation of protoplasts
• The isolated protoplasts system has proved
most promising for genetic modification of
cells.
• In the absence of a wall around the plasma
membrane, protoplasts are not only able to
fuse but also can take up chloroplasts, nuclei,
micro-organisms and isolated DNA.
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9. METHODS OF PROTOPLAST
TRANSFORMATION
UPTAKE OF ORGANELLES
UPTAKE OF VIRUSES/MICROORGANISMS
TRANSFORMATION USING AGAROBACTERIUM SYSTEM
DIRECT DNA TRANSFER METHODS
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10. Uptake of organelles
Chloroplast
• Uptake of chloroplasts by albino protoplasts
isolated from Petunia, tobacco and carrot
plants was reported in during 1973-74.
• This required exposing a mixture of albino
protoplasts and chloroplasts to PEG in a
manner similar to protoplast fusion.
• EM studies revealed that PEG caused the
fusion of two external membranes of the
plastids and protoplasts. 10

11. Mitochondria
• No evidence supports the direct uptake of isolated
mitochondria by protoplasts however there are
reports on incorporation of mitochondrial DNA
through protoplasts fusion.
• This leads to the formation of cybrids.
• It may also be possible to transfer traits such as
toxin sensitivity of Texas male sterile cytoplasm
encoded by mitochondrial DNA in maize.
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12. Nucleus
• Potrykus and Hoffman reported uptake of
isolated nuclei by mesophyll protoplasts of
Petunia, tobacco and maize.
• Transplantation success improved from 0.5% to
5% in the presence of PEG and Ca++.
• The uptake of isolated chromosome instead of
complete nuclei is yet to be explored.
• Transfer of micronuclei into protoplasts has
also been attempted. 12

13. Uptake of viruses/microorganisms
• Viruses have been used as vectors for genetic
modification.
• Experiments on uptake of tobacco mosaic virus (TMV)
by isolated protoplasts were initiated in 1969.
• Most plant viruses have encapsulated genomes and it
is assumed that no specific virus receptor site exists on
the exterior of the plasma membrane.
• Factors to be considered while selecting progenitor
(virus) are : 1. amenability to genetic engineering
techniques, 2. packaging constraints in the particle and
3. development of a system in which expression can be
studied.
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14. Contd…
• The cauliflower mosaic virus (CaMV) is the
most amenable and widely chosen progenitor.
• The ssDNA genome in CaMV has appropriate
restriction sites compatible with the
methodology developed for A. tumefaciens
plasmid.
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15. Transformation using Agrobacterium
system
• Agrobacterium tumefaciens is a soil bacterium,
has the ability to infect most dicotyledonous
plants usually at a wounded site.
• The tissue around the infected wound develops a
neoplastic growth known as gall tumor.
• The tumor tissue excised from the plant is
capable of growing on hormone free medium.
• In culture, the crown gall tissue produces a set of
metabolites termed as opines
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16. Contd…
• Agrobacterium harbors the Ti Plasmid, which is directly
responsible for tumor induction.
• The transfer of small DNA segments from this plasmid
and their integration into the genome of a host cell
induces tumor formation in the plants.
• Agrobacterium rhizogenes is another bacterium used in
plant genetic manipulations.
• The infections by this bacterium causes hairy roots
which are of clonal origin.
• The tumor-inducing properties of rhizogenes strains
are also carried on a large Ri plasmid DNA(Ri T-DNA).
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17. Infection of plant cells with Agrobacterium
• Agrobacterium engineered with a desired foreign gene
is directly inoculated at the wound site after removing
the top leaves and apical meristem of plant.
• Non-oncogenic vectors do not induce tumour
formation and instead a wound callus proliferates in 3-
4 weeks.
• The transformed callus tissue is excised and cultured in
the medium supplemented with an appropriate
combination of growth regulators to regenerate
transformed fertile plant.
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18. Other approaches
Co-cultivation with protoplast
• Protoplasts are co-cultivated with Agrobacterium for few days,
washed and then cultured in antibiotic containing medium (to
eliminate bacteria).
• These protoplasts are regenerated in a suitable medium enabling
the development of transformed callus, shoots and plants.
Leaf disc infection method
• A leaf disc of a plant is inoculated with A. tumefaciens strain having
modified tumour inducing plasmid and cultured for 2 days.
• The leaf disc which develops infection is transferred to a selection
media enriched only with kanamycin and further cultured to form
plants.
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19. Contd…
Floral dip method
• Used for genetic transformation of Arabidopsis thaliana
plants.
• Primary and secondary inflorescence shoots are cut at
their bases and inoculated with A. tumefaciens cell
suspension, carrying binary vector with an insert, at the
wound sites.
• After 3 successive inoculations at weekly intervals, treated
plants are grown to maturity and seeds are harvested.
• Progeny raised from these seeds is screened for
transformants on a selective medium.
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