Plant biotechnology with methods for standard function

About sterlization and other types used in plants biotechnology and plant biology laboratory

1. Direct DNA TransferDirect DNA Transfer

Introduce naked DNA into cells (plant or Introduce naked DNA into cells (plant or
animal)animal)

Can assay expression of the gene immediately, Can assay expression of the gene immediately,
or select cells that are permanently or select cells that are permanently
transformedtransformed

DNA introduction methods:DNA introduction methods:
1.1.ChemicalChemical
2.2.ElectroporationElectroporation
3.3.MicroinjectionMicroinjection
4.4.Particle bombardment (Biolistics)Particle bombardment (Biolistics)

2. Chemically-induced transformationChemically-induced transformation

Usually use on cells without wallsUsually use on cells without walls

Multiple protocols:Multiple protocols:
1.1.put DNA inside artificial membranes put DNA inside artificial membranes
(liposomes), they will fuse with plasma (liposomes), they will fuse with plasma
membranemembrane
2.2.Bind DNA with polycations to neutralize Bind DNA with polycations to neutralize
charge, some cells endocytose the complexcharge, some cells endocytose the complex
3.3.Combine (2) and (1)Combine (2) and (1)

3. Fuse with plasma membrane

4. Endocytosis

7. A
PM
B a b

8. Targeted DNA encapsulated in a
spherical lipid bilayer termed a liposome
In the presence of PEG, endocytosis
occurs.
After endocytosis, the DNA is free to
recombine and integrate with the host
genome
Liposome

9. Intensive electrical field leads to pores
on plasma membrane, allowing DNA to
enter

Use on cells without walls (plant Use on cells without walls (plant
protoplasts or animal cells )protoplasts or animal cells )

High-voltage pulses cause pores to form High-voltage pulses cause pores to form
transiently in cell membrane; DNA pulled transiently in cell membrane; DNA pulled
in by electrophoresis or diffusion (?)in by electrophoresis or diffusion (?)

DrawbackDrawback - its more cumbersome to - its more cumbersome to
regenerate plants from single regenerate plants from single
protoplasts than from the tissue protoplasts than from the tissue
transformations with transformations with AgrobacteriumAgrobacterium
ELECTROPORATION

10. ElectroporationElectroporation

12. Uses fine glass needles to inject the
foreign DNA directly into the host cell
Developed to inject DNA into
protoplasts, cultured embryonic cell
suspensions and multicellular structures
Time consuming
MICROINJECTION

13. Microinjection of
DNA into the
pronucleus of a
newly fertilized
egg
From Primrose, Molec. Biotechnology
needle
~ 1-2
picoliter
vol is
injected
~5-40% of
animals
will
contain
transgene

15. [Photo of a Microinjection apparatus(courtesy of A. Yanagi]
Making Transgenic Plants
Microinjcetion

16. Particle Bombardment (Biolistics)Particle Bombardment (Biolistics)

Less limitations than electroporationLess limitations than electroporation

Can use on cells with walls, or essentially Can use on cells with walls, or essentially
any tissueany tissue

Can transform organellesCan transform organelles

Method: Method:
1.1.Precipitate DNA onto small (micron) tungsten or gold Precipitate DNA onto small (micron) tungsten or gold
particlesparticles
2.2.Accelerate particles to high speeds to penetrate cells Accelerate particles to high speeds to penetrate cells
and tissuesand tissues
3.3.Perform selective growth and regeneration of Perform selective growth and regeneration of
transgenic plants as described for Agro-mediated transgenic plants as described for Agro-mediated
transformationtransformation

17. Biolistics involves the direct delivery of DNA
coated metal particles into the cell
Gold or tungsten particles a few microns in
size are coated with recombinant DNA or
RNA and the particles are literally shot into
the cell
Once inside the cell, the DNA soaks off the
particle and becomes integrated into the
cell genome

20. The Helium Gas Gun – Circa 2000

22. Starting conditions for several types of
biological systems

23. The Hand-Held Gas Gun
Purpose:
Introduce DNA into cells
that are below the top
surface layer of tissues
(penetrate into lower
layers of a tissue)

26. Agrobacterium mediated Agrobacterium mediated
plant transformationplant transformation
•Plant tissue to be transformedPlant tissue to be transformed
•Vector selectionVector selection
•Vector constructionVector construction
•Agrobacterium strain selectionAgrobacterium strain selection
•Transfer the Vector in to Transfer the Vector in to
AgrobactreriumAgrobactrerium

27. Agrobacterium mediated Agrobacterium mediated
transformationtransformation

Identify a suitable explantIdentify a suitable explant

Co-cultivate with the agrobacteriumCo-cultivate with the agrobacterium

Kill the agrobacterium with suitable Kill the agrobacterium with suitable
antibiotic (which does not harm plant antibiotic (which does not harm plant
tissue- tissue- carbinecillin, cefotaxime, timitin))

Select transformed plant cellsSelect transformed plant cells

Regenerate whole plantsRegenerate whole plants

28. Explant

29. Components of transformation
vector  

The plasmid must be able to replicate both in The plasmid must be able to replicate both in
E. coliE. coli and and AgrobacteriumAgrobacterium

Additional selectable marker need to be Additional selectable marker need to be
included for identificationincluded for identification

Border sequences need to be incorporated Border sequences need to be incorporated
for for Agrobacterium Agrobacterium mediated transformation mediated transformation

Genes to be integrated that originates from Genes to be integrated that originates from
prokaryote or non plant eukaryote - may prokaryote or non plant eukaryote - may
need to be plant like by using appropriate need to be plant like by using appropriate
promoters and terminatorspromoters and terminators

30. A selectable marker gene
Remarkably few
Kanamycin resistance, nptII

NOS PROMOTER --- NPT II --- NOS 3'
POLYADENYLATION SEQUENCE
Hygromycin resistance, hpt
Bar gene (for resistance to herbicide
phosphinothricin)
DHFR gene (for resistance to methotrexate)
ESPS gene (for resistance to Round-up
herbicide)

31. A scorable marker gene
(optional, but very useful)
NPT II activity
Opine production
beta-Glucuronidase activity (GUS)
Green fluorescent protein (GFP)
Luciferase activity

32. Origins of replication for replication
of the plasmid in bacteria

33. Gene of interest
Can come from any source
Usually chimeric (made up from parts
of different genes)
Promoter: CaMV 35S, others
Terminator: Nos, CaMV 35S, others
Other elements

34. How is Plant Transformation with How is Plant Transformation with
Agrobacterium Agrobacterium Done?Done?
Co-cultivate the engineered A. tumefaciens
strain containing the gene of interest with an
explant from which regenerated plants can
be obtained.
Culture the explant on regeneration medium
in the presence of a selective agent (such as
kanamycin if the T-DNA contains NPT II) and
an antobiotic to kill the Agrobacterium or slow
its growth (carbinecillin, cefotaxime, timitin).

35. How is Plant Transformation with How is Plant Transformation with
Agrobacterium Agrobacterium Done?Done?
After some period of time, select
regenerated shoots (or embryos) that
are resistant.
Screen the regenerated shoots for
expression of the scorable marker.
Root shoots and produce plants

36. How is Plant Transformation with
Agrobacterium Done?
Test the transgenic plants for the
presence and expression of the
introduced genes.
Produce progeny of the transgenic
plant and determine whether the
introduced gene is heritable.

37. In planta transformation of
Arabidopsis

38. Vacuum infiltration
Vacuum infiltration was done by placing
flowering plants upside down in a beaker with
a solution containing Agrobacterium
tumefaciens and 5% sucrose. Plants were
placed in such a way that only inflorescences
were submerged.
This beaker and plants were placed in vacuum
chamber and the vacuum (0.05 bar) was held
for several minutes.

39. Floral dip
Plants were placed in a similar way as
vacuum infiltration but no vacuum was
applied.
Plants were kept in the solution for
several minutes before removal.

40. Floral spray
Floral spray was done by spraying
Arabidopsis flowers with Agrobacterium
solutions.
It is much less harmful to the plants and
can be applied many times.

41. Root elongation zone is the most highly
susceptible area to Agrobacterium-
mediated transformation.
For floral dip/spray methods, visible
immature floral buds are the most
susceptible to transformation.

42. Use silicon carbide fibers to punch
holes through cultured plant cells
Silicon carbide fibers and cultured
plant cells are added to a tube and
vortexed vigorously
The mechanical force generated by
the vortex drives the fibers into the
cell
Silicon Carbide Fibers