Gene Transfer and its applications; Plant tissue culture and its application for production of secondary metabolites

1. GENE TRANSFER IN
PLANTS
Presented By: Varsha Srivastav
M. Pharm 2nd Semester
(Pharmacognosy)
Jamia Hamdard

2. What is Gene transfer?
Insertion of copies of a gene into living cells in order to induce synthes
is of the gene's product.
Why Gene transfer?
To add a desired trait to a crop, a foreign gene (transgene) encoding the
trait must be inserted into plant cells, along with a “cassette” of
additional genetic material. The cassette includes a DNA sequence
called a “promoter,” which determines where and when the foreign
gene is expressed in the host, and a “marker gene” that allows breeders
to determine which plants contain the inserted gene by screening or
selection.

3. Methods of Gene Transfer
Direct or Vector-less DNA TransferVector-Mediated Gene Transfer
Agrobacterium(Ti-
plasmid) mediated
Plant viral vectors Physical Chemical
 Crown gall
disease(A.tumefaciens)
 Hairy root
disease(A.rhizogenes)
 Caulimoviruses as
Vectors:Eg.Cauliflower
mosaic virus (CaMV)
 Gemini viruses
 RNA plant viruses
 Use of cDNA
 Electroporation
 Microprojectile(particle
bombardment)
 Microinjection
 Liposome fusion
 Silicon carbide fibres
 PEG mediated
 DEAE dextran
mediated

4. Agrobacterium tumefaciens mediated gene
transfer
A. tumefaciens is basically a bacterium which infects wounded or damaged plant tissues, which
induces the formation of a plant tumor called crown gall.
Phenolic compounds acetosyringone, hydroxyacetosyringone)released by the wounded sites.
Crown gall formation occurs when the bacterium releases its Ti plasmid (tumor- inducing
plasmid) into the plant cell cytoplasm.
A fragment (segment) of Ti plasmid, referred to as T-DNA, is actually transferred from the
bacterium into the host where it gets integrated into the plant cell chromosome (i.e. host
genome).
T-DNA carries genes that code for proteins involved in the biosynthesis of growth hormones
(auxin and cytokinin) and novel plant metabolites namely opines — amino acid derivatives
Entry of bacterium

5. Formation of
crown gall
tumor in a
plant infected
with
A.tumefaciens

6. A.rhizogenes mediated gene transfer
• Like A. tumefaciens, A. rhizogenes also infect plants.
• But, unlike A. tumefaciens, it causes infection in hairy root giving rise to Hairy
root disease.
• As A. tumefaciens is tumor inducing, A. rhizogenes is Root inducing hence the
plasmids isolated are referred to as Ri plasmids(Root inducing).
• Some of the Ri plasmid strains possess genes that are homologous to Ti plasmid
e.g. auxin biosynthetic genes.
• The products of these genes are involved in the metabolism of plant growth
regulators which gets sensitized to auxin and leads to root formation.

8. Virus mediated gene transfer
• Plant viruses are considered as efficient gene transfer agents as they can infect
the intact plants and amplify the transferred genes through viral genome
replication. Viruses are natural vectors for genetic engineering. They can
introduce the desirable genes into almost all the plant cells since the viral
infections are mostly systemic.
• Plant viruses are non-integrative vectors i.e. the viral genomes are suitably
modified by introducing desired foreign genes. These recombinant viruses are
transferred, multiplied and expressed in plant cells. They spread systemically
within the host plant where the new genetic material is expressed.
• Various viruses used for gene transfer are: Gemini virus, Cauliflower mossaic
virus(CaMV), RNA plant virus, Complementary DNA, etc.

9. Direct or Vectorless DNA transfer
• The term direct or vector less transfer of DNA is used when the
foreign DNA is directly introduced into the plant genome. Direct DNA
transfer methods rely on the delivery of naked DNA into the plant
cells.
Limitations of direct DNA transfer:
• The major disadvantage of direct gene transfer is that the frequency
of transgene rearrangements is high. This results in higher transgene
copy number, and high frequencies of gene silencing.

10. Production of Transgenic plants
using Direct or Vectorless Gene
Transfer

12. Applications of Gene Transfer in plants
• Marker genes may render plants resistant to antibiotics that are not
used medically (e.g., agromycin, canamycin) or tolerant to certain
herbicides.
• Several dicotyledonous plants (dicots) are affected by crown gall
disease e.g. grapes, roses, stone-fruit trees.
• Certain sugars (e.g. glucose, galactose, xylose) that induce virulence
genes are used for production of virulence proteins.
• Hairy roots can be cultured in vitro, and thus are important in plant
biotechnology. Hairy root systems are useful for the production of
secondary metabolites, particularly pharmaceutical proteins.

13. • Members of Cruciferae, Datura are genetically transformed using
Cauliflower mosaic virus(CaMV).
• A wide range of crop plants (monocotyledons and dicotyledons)
which attract plant biotechnologists to employ Gemini viruses for
gene transfer.
• Gemini virus vectors can be used to deliver, amplify and express
foreign genes in several plants/ explants (protoplasts, cultured cells).
• A gene sequence encoding chloramphenicol resistance (enzyme-
chloramphenicol acetyltransferase) has been inserted into brome
mosaic virus genome(cDNA Transfer). This gene expression, however,
has been confined to protoplasts.
• In case of vegetatively propagated plants, transmission of desired
traits can be done using viral vectors e.g. potatoes.

14. • Electroporation and Bombardment has been successfully used for the
production of transgenic plants of many cereals e.g. rice, wheat,
maize(first commercial genetically modified (CM) crops such as maize
containing Bt-toxin gene were developed by this approach).

15. What are Plant Secondary Metabolites?
• Plant secondary metabolites can be defined as compounds that have
no recognized role in the maintenance of fundamental life processes
in plants, but they do have an important role in the interaction of the
plant with its environment.
• They mostly have an ecological role as attractants of pollinating
insects or in defense mechanisms against predators.

16. Application of PTC has 3 major aspects
1. breeding and genetics:
• micropropagation – using meristem and shoot culture to produce
large numbers of identical individuals;
• selection – screening of cells, rather than plants, for
advantageous characters;
• crossing distantly related species by protoplast fusion and
regeneration of the novel hybrid;

17. • production of dihaploid plants from haploid cultures to
achieve homozygous lines more rapidly in breeding programs;
• transformation, followed by either short-term testing of genetic constructs
or regeneration of transgenic plants;
• removal of viruses by propagation from meristematic tissues;
2. model system for study of plant cell genetics, physiology, biochemistry,
and pathology;
3. production of secondary metabolites – growth in liquid culture as
source of products.

18. Applications of Plant Tissue Culture for
production of secondary metabolites
• Shikonin by Lithospermum erythrorhizon was accumulated in higher
levels in cultured cells than in the intact plants.
• Rosmarinic acid by Coleus bluemei was obtained in higher
concentration using Plant Tissue Culture.
• Higher quantities of berberine have been obtained from growing cells
of Coptis japonica.
• Metabolites can be produced in Root Culture such as a form of root
culture has been developed using A.rhizogenes(the cause of hairy
root culture).

19. Some Secondary metabolites produced in Cell
and Root Culture
• L-DOPA: important neurotransmitter used in Parkinson's Disease
• Shikonin: antibacterial and antiulcer agent
• Anthraquinone: used for dyes and medicinal purposes
• Opiate alkaloids: particularly codeine and morphine
• Berberine: an alkaloid for cholera and bacterial dysentry
• Valprotriates: as sedative
• Gingsenosides: for medicinal purposes
• Quinine: for malaria
• Cardenolides: for heart disease

21. Other applications of Plant tissue culture
• In food additives
• In development of aromas and fragrances
• Development of pigments
• Production of pharmaceuticals.

22. References
• http://www.biologydiscussion.com/genetics/en
gineering/methods-of-gene-transfer-in-plants-
2-methods/10824
• https://www.researchgate.net/publication/309
392143_Gene_transfer_methods_and_applicati
ons_in_plants
• http://www.academia.edu/3617256/Gene_Tran
sfer_Technologies_in_plants_Roles_in_improvin
g_Crops
• https://in.images.search.yahoo.com/
• https://www.researchgate.net/publication/525
8172_Production_of_Secondary_Metabolites_U
sing_Plant_Cell_Cultures
• https://www.slideshare.net/shivam_hayabusa/
production-of-secondary-metabolites

23. Thankyou