BIOL4411

Plant & Food Biotechnology

Professor M. L. Chye
Email: mlchye@hku.hk
1
Review

2
Description Approx. No. of hours
Introduction 4
Content and assessment
Importance of plants
Introduction to genetic engineering in plants

Tools for plant gene transfer 4

Promoters, reporter genes, selectable markers, vectors
Agrobacterium vectors
Techniques in plant gene transfer

Genetic engineering of biosynthetic pathways in transgenic plants 4

Gene silencing in plants
Discovery
Applications
Reduction of lignin, ethylene, polyphenol oxidase
Improvement of protein composition in transgenic plants
Improvement of vitamin content
Other examples of biofortified food

Development of pest and disease resistant plants ~5

Engineering plants resistant to microbial pathogens (bacteria)
Engineering virus-resistant plants (viral coat protein, RNAi)
Engineering insect-resistant plants
Production of herbicide-tolerant transgenic plants

Plants as bioreactors ~3
Biofuels
Plant made pharmaceuticals, edible vaccines
Chloroplast transformation

Genetically-modified crops: regulations, testing and labelling, public perceptions 2 3

L7-8: Tools for plant gene transfer

promoters

marker genes - reporter genes

- selectable markers
vectors
Agrobacterium vectors
Techniques in plant gene transfer
Tools for plant gene transfer
Promoters
Marker genes
Vectors
A. Viral vectors #
B. Agrobacterium vectors *

Techniques in plant gene transfer

i) Agrobacterium-mediated transformation *
ii) protoplasts
iii) microprojectile bombardment
iv) microinjection
v) plant viruses

i-iv: stable transformation, integration into plant genome

v : no integration #
 Plant Mol Biol Labfax Ed. RRD Croy

i)
***********

ii)

iii)

iv)
Plant Mol Biol Labfax Ed. RRD Croy
 Plant Mol Biol Labfax Ed. RRD Croy

Western blot analysis detects accumulation of foreign protein

*
In nature…

Agrobacterium
tumefaciens
contains
Ti
(Tumour-inducing)
plasmid;
causes
crown gall
Agrobacterium
rhizogenes
contains
Ri
(Root-inducing)
plasmid;
causes
hairy root
 Tools for plant gene transfer Agrobacterium

• Agrobacterium tumefaciens
• A broad host range plant pathogen forming
tumors on many dicots
• Contains Ti (Tumor-inducing) plasmid that
causes crown gall disease

https://apps.extension.umn.edu/garden/diagnose/plant/images/butternut/burl3_200px.jpg 11
https://i1.wp.com/bio-caretechnology.com/wp-content/uploads/2018/03/Crown-gall-on-peach-ncsu-photo.jpg?w=395
https://upload.wikimedia.org/wikipedia/commons/thumb/4/43/Growth_on_tree_trunk_-_geograph.org.uk_-_724687.jpg/200px-Growth_on_tree_trunk_-_geograph.org.uk_-_724687.jpg
 In nature, A. tumefaciens enters through wound sites
In the lab, A. tumefaciens infects through cut edges (wound sites) of tissues

The formation of “crown galls” on dicotyledonous plants after infection by the soil
bacterium A. tumefaciens
12
E.D. Gardner, Simmons, Snustad, Principles of genetics.
Transfer of T-DNA into plant genome
Ti plasmid contain T-DNA (Transferred-DNA)
which can be integrated into the plant genome

(1 circular &
1 linear)

Transformation of plant cells by Agrobacterium tumefaciens harboring a wildtype Ti plasmid. Cells in the plant tumor
contain the T-DNA segment of the Ti plasmid integrated into the DNA of plant chromosomes. The integration site
random (After M.-D. Chilton, 1983 “A Vector for Introducing New
appears to be selected at random or nearly at random.
Genes into Plants,”Sci. Amer. 248(6): 51-59)
13
E.D. Gardner, Simmons, Snustad, Principles of genetics.
Ability for gene transfer
- development of A. tumefaciens as vector

DNA cloned in T-DNA between borders

transferred

Elimination of T-DNA genes for tumorous

growth

14
 What gets transferred?
T-DNA (flanked by 23 bp repeats) 23 kb
• transferred into plant cells
• integrated into nuclear genome
• genes expressed nitrogen source for
Agrobacterium
• genes for auxin, cytokinin and opine production
are dispensable
• DNA cloned between borders (LB-RB) in T-DNA
are transferable

15
 Regeneration after DNA transfer
Ti plasmid
chromosome
DNA encoding desired gene
A. tumefaciens

Callus

Untransformed control plant 4 Transformed plant

3 regenerated

Transformation in the Laboratory

1 Pieces of tomato leaves are cultured together with Agrobacterium

2 The leaf pieces are moved to a medium that kills the bacteria and untransformed cells
3 The pieces are moved to a medium that induces the formation of shoots
16
Chrispeels, M.J. and D.E. Sadava. Plants, genes, and agriculture.
 Agrobacterium vector – general
features
• T-DNA (transferred DNA)
• Flanked by RB: right border; LB: left border
• Binary vector/ Shuttle vector
• Able to replicate in E. coli & A. tumefaciens
• Allows rec DNA manipulations in E. coli before transfer to
Agrobacterium
• Consists
• Origin of replication (ori) in E. coli
• Ori in Agrobacterium
• Selectable marker in bacteria e.g. tetracycline (in pGA482),
spectinomycin, kanamycin
• Selectable marker in plant e.g. kanamycin
• Restriction cloning sites 17
 Selection in
Right Border
plant (Kmr)

Cloning sites
Replication in
A. tumefaciens
T-DNA

Replication in
Selection in E. coli
bacteria (Tcr)

pRK2 origin Left

of transfer Border
An G. 1986 Plant Physiol 81, 86-91 18
 Binary vector system
• Binary: pair of plasmids, both replicate in Agrobacterium
• D-Ti plasmid (provides vir functions in trans)
• Disarmed = Lacks phytohormone biosynthesis genes (no tumours)
• vir genes are required for transfer and integration
• Vir proteins cut at LB & RB and facilitate integration
• Binary/Shuttle vector with gene of interest targeted for
integration

• Enable mobilization from E. coli to Agrobacterium and

replication in both

• Contains plant selectable marker and gene of interest

between LB & RB
19
 Tri-parental mating
• Binary vectors:
for mobilization from E. coli into Agrobacterium

• 3 bacteria involved
• Agrobacterium: recipient with D-Ti plasmid

• E. coli helper strain:

provides transfer and mobilization (mob) functions
(mobilizes Plant Transformation Vector to Agrobacterium)

• E. coli with binary (plant transformation) vector

20
 With 1 With
binary vector 3 D-Ti

2
E. coli mob
helper

Plant Transformation Vector

Tri-parental

Able to replicate in E. coli

Able to replicate in Agrobacterium
Spectinomycin resistance in bacteria
Kanamycin resistance in plant
Gene flanked by LB-RB
Integrate into
plant chromosome

21
Gasser & Fraley 1989 Sci 244:1293
 Binary vector used in
Agrobacterium transformation
T-DNA region of pBI141
ATG TGA
RB LB
NOS-Pro NPT II (Kmr) NOS-ter Actin Promoter β-Glucuronidase (GUS) NOS-ter

BamH I
Hind III
Sph I
Pst I

SnaB I

EcoR I
Sst I
NOS: nopaline synthase
NPT II: Selectable marker
Gus: Reporter marker
RB: Right Border LB: Left Border

The one you use in the practical is pBI121 with CaMV 35S-GUS
22
 Regeneration after transformation

Callus Further
Transformed developed callus
cells
Root & shoot
development

Whole plant in soil Regenerated

plant 23
 Agrobacterium Transformation
Advantages Disadvantages
• Natural means of transfer, • Several elite monocot crops,
minimal equipment and facility
required and less expensive; legumes, and woody plants
• Capable of infecting intact plant are recalcitrant to A.
cells, tissues, and organs;
• Transformed plants can be
tumefaciens-mediated
regenerated rapidly; transformation
• The transfer of relatively large
segments of DNA without
rearrangements;
• Integration of T-DNA in genome is
a relatively precise* process;
• Stability of gene transfer is
excellent

* without loss of bases from the T-DNA side

Agrobacterium transformation using leaf discs

1’45”

Full version at https://www.youtube.com/watch?v=NXNFR4cj68U

25
 Floral-dip transformation

10’21”

https://www.jove.com/video/1952/floral-dip-transformation-arabidopsis-thaliana-to-examine-ptso2
Mara, C., Grigorova, B., Liu, Z. Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::β-glucuronidase Reporter Gene Expression. J. Vis. Exp.
(40), e1952, doi:10.3791/1952 (2010). 26
10’
 Agroinfiltration
a suspension of A. tumefaciens is introduced into leaf by direct infiltration
to induce transient expression of foreign genes

57”

https://www.youtube.com/watch?v=GHc7PU_jG2M
27 57”
 Example Qs
• Explain how binary vector can be used in plant
genetic engineering

• Discuss T-DNA

• Describe the required bacterial strains,

plasmids and essential features involved in
“triparental mating”

28
 Tools for GE:
Markers &
transformation
Transient expression

Agrobacterium: Recipient with D-Ti plasmid

E. coli helper strain: provides transfer and
mobilization (mob) functions (mobilizes Plant
Transformation Vector to Agrobacterium)
E. coli with binary vector: for plant transformation
 Tools for plant gene transfer
Protoplasts
• Isolated plant cells with no cell wall
Maize Lc (controls anthocyanin pigmentation)
visualize transformed maize cells after bombardment with gold particles coated with
plasmid CaMV35S-Lc

Transformed protoplasts containing CaMV 35S-Lc with anthocyanin

Embryogenic-derived protoplast Mesophyll-derived protoplast

30
Lusardi et al. 1994. Plant Journal 5: 571-582
METHOD

• Ideal for gene transfer

• DNA uptake promoted by

- polyethylene glycol (MW4000-6000, 15-20%)
- electroporation which stimulates
permeability of cell membranes
- Agrobacterium

• Eg rice transformation
 Advantages
1. Plasmalemma freely accessible
• DNA enters every protoplast
• Concentration controlled experimentally
2. Enzymatic or mechanical isolation induce wound
response
• Potentially competent cells become competent
• Increases regeneration and transformation
3. DNA reaches every competent cell
• Increases recovery from a given population
• Transgenic plants regenerated from ONE protoplast are
genotypically identical (uniform genetic make up)

32
 Advantages
4. DNA uptake is a physical process
• Applicable to any plant
• Not limited by host range
• No biological vector (in comparison to Agrobacterium)
5. Efficient integrative event: stable transformant
6. Efficient co-transformation (for non-selectable genes)
7. No barrier for gene transfer
• Virtually every protoplast is transformable
(different efficiencies)

33
 Disadvantages
1. Severe problems related to plant regeneration
(delicate!)
2. Conditions for such regeneration available for few
plant species than for multicellular explants
3. Regeneration depends upon parameters not under
experimental control
• For example: species- and genotype-dependent
competence for wound response

34
 Example: Production of herbicide-
resistant rice plants from protoplasts

• Use of bar (herbicide Basta) in making herbicide-

resistant rice (Oryza sativa)
• Use protoplasts isolated from suspension cultures
of immature embryo callus
• Transformed using PEG-mediated DNA uptake
• Transformed calli selected in medium containing
PPT for 2-4 weeks

Basta = PhosPhinoThricin tripeptide

35
Rathore, Chowdhury and Hodges. 1993. Plant Mol. Biol. 21: 871-884
 bar-containing vector
Bam HI
Hind III Sma I Bgl II Eco RI
nos
CaMV 35S promoter bar poly A pGEM-3
810 bp 550 bp 260 bp

GATCTACCATGAGC
For optimal translation in eukaryotes

Plasmid pG35barB (4.35 kb)

50 µg/ml protoplast suspension

36
 Use of bar as selectable marker
Growth of protoplast-derived microcalli

Untransformed Untransformed Transformed

No PTT with pG35barB
With 4 mg/L PPT

Rathore, Chowdhury and Hodges. 1993. Plant Mol. Biol. 21: 871-884 37
 Use of bar as selectable marker
Leaves from transgenic plants

Treated with
Untreated T1 T2 T3 T4 T5
500 mg/l PPT
Putative transgenic plants
Control Treated with 500 mg/l PPT
Rathore, Chowdhury and Hodges. 1993. Plant Mol. Biol. 21: 871-884 38
 Can co-transform: use >1 plasmid
Bam HI
Hind III Sma I Bgl II Eco RI
1 nos
CaMV 35S promoter bar poly A pGEM-3
810 bp 550 bp 260 bp
pG35barB
Kpn I Bam HI
Hind III Sma I Sst I Xba I
2 GUS nos
Rice actin promoter intron uidA poly A pBluescript II-KS
2100 bp 1890 bp 260 bp

Plasmid pACT 1-D (7.2 kb)

Use of GUS as a non-selectable marker to test the principle

39
 Gus assay of PPT-resistant plants

a1-6: Untransformed controls

b1-6 & c1-4: 10 PPT-resistant plants developed from individual callus
Only b4, c2 & c4 are PPT-resistant & express GUS activity
40
Rathore, Chowdhury and Hodges. 1993. Plant Mol. Biol. 21: 871-884
 Herbicide-resistant rice
• Calli resistant to PPT are capable of regeneration
• Phosphinothricin acetyltransferase (PAT) assays
confirmed expression of bar
• Co-transformation efficiency (using gus) was 30%
• Leaves resistant to Basta application
• T0 plants were self-fertile
• bar and gus genes were transmitted to T1 and T2
progenies (confirmed by Southern blot)
• Leaves resistant to Basta application
41
Tools for plant gene transfer
Microprojectile bombardment

• Particle gun or biolistics

• DNA-coated metal microparticles
• Tungsten (toxic oxide; 0.5-10 µm)
• Gold (inert; 1-3 µm)
• High-velocity acceleration of microparticles into
living cells
• Penetrate cell wall & membrane
• DNA to every type of cell/tissue

42
 Gene gun or biolistic method

3’13”

43
Modified from https://www.youtube.com/watch?v=VqklR_8YRfA
 Gene gun or biolistic method

A gene gun us used for bombarding cells with

genetic information, it is also known as biolistic
particle delivery system.

44
https://upload.wikimedia.org/wikipedia/commons/thumb/d/d3/Genegun.jpg/220px-Genegun.jpg
https://upload.wikimedia.org/wikipedia/commons/thumb/5/53/Gene_Gun.svg/220px-Gene_Gun.svg.png
Both methods deliver DNA into LIVING cells

Ti-plasmid
DNA Bombardment can
be used for
species that are
difficult to
transform by
Agrobacterium-
mediated
transformation

45
Chrispeels and Sadava. Plants, genes, and agriculture.
 Macrotargeter

Expression of GUS in all cell types in rice leaf tissue after

bombardment. CaMV 35S-GUS was used.
46
Bhattacharyya-Pakrasi et al. 1993 Plant J 4:71-79
 Macrotargeter
Involves large population
of totipotent cells
Difficult to control
which cells to
‘transform’
Inefficient for a
population of cells
that are mainly
difficult to
regenerate

47
http://i.imgur.com/DoXu3tn.jpg
 Microtargeter

http://grade12biotransgenicplants.blogspot.hk/2013/06/the-gene-gun.html
https://www.turbosquid.com/3d-models/3d-gene-gun-model/804835 48
http://www.bio-rad.com/webroot/web/images/lsr/products/gene_transfer_rnai/product_detail/global/lsr_gene_gun.jpg
 Microtargeter

Restriction

https://d1o50x50snmhul.cloudfront.net/wp-content/uploads/2006/11/25773001.jpg
Potrykus 1992 Nature 355: 568-569 49
 Microtargeter
Meristem cells are
unspecialized, undifferentiated
and give rise to various plant
organs . . .
. . . .
. . . .
.. . . . . .
. .
. . . .
.. . .
. . .
. .
. .

Shoot meristem of a cereal seedling and particle distribution under the line-crossing,
which is used for aiming purposes. The ring characterizes the targeting area of about 150 µm.
The second cell layer (basis for sexual offspring) is indicated by drawn-in nuclei.

50
https://media1.britannica.com/eb-media/99/5599-004-D6C19960.jpg Potrykus 1992 Nature 355: 568-569
 Microtargeter

100 µm 10 µm
10 µm

Cells expressing GUS White arrows: particles

Gene expression in vegetative

shoot apical meristems of
wheat
Evaluation was conducted 3 d
200 µm
10 µm after bombardment
Cells expressing anthocyanin m, tip of meristem
51
Bilang et al. 1993 Plant Journal 4:735-744
 Advantages
1. Easy to handle
2. One shot gives multiple hits (many cells)
3. Cells survive intrusion of one particle
4. Genes on particle retain biological activity
5. Broad utility (effective among all plant species
tested)
6. Different target cells - pollen, cell culture cells,
meristems and cells in differentiated tissues
Embryos and meristems to optimize regeneration
7. Target at the surface or in deeper layers of organs
8. Method dependent mainly on physical parameters 52
 Disadvantages
1. Inefficient in yielding stable integrative events esp.
those not using embryogenic suspensions
• Lower yield than protoplasts
• Large numbers necessary to hit a rare competent cells
• Frequency of gene transfer for transient assay (10-4-10-3)
• Only 2-5% of transient transformants become stably
transformed
2. Costly instrumentation
3. Plants regenerated from tissues are usually chimeric
• Due to random bombardment of small no. of cells in multiple
system
• However transformants can be sorted and stabilized when
selectable markers are used
53
Transgenic cereal by
particle bombardment
of meristems

a) GUS expression

b) Embryogenesis after
bombardment

c) Regeneration

d) Transgenic cereal
plants

Barcelo P et al.
Plant Journal 5: 583-592
Example
Production of transgenic wheat
by particle bombardment of
calli derived from immature embryos

Marker genes bar gene encoding PPT-resistance

and GUS, each expressed from the maize ubiquitin
promoter
 Time frame for transgenic Bobwhite wheat
Plant Seeds
~55 d Transfer to root media
Anthesis 14 d Acclimation

~15 d Transfer to soil

Embryo excision 14 d

5 d Callus initiation Move to greenhouse

~35 d
Bombardment
Selection Anthesis T0 plants
~100 d ~30 d
Shoot formation
Mature seeds

Excision to anthesis: ~168 d 56

anthesis = open flowers Weeks et al 1993 Plant Physiol 102:1077-1084 6 months
Immature wheat Proliferation of 30 d callus tissue, Gus Bialaphos resistant
embryos excised 15 d callus tissue assayed 2 d after tissue, shoot
post anthesis bombardment regeneration

Basta resistant Regeneration of root Potted transgenic plants Mature T0

tissue, 70 d after Wildtype; Transgenic + Basta; transgenic plant
bombardment Wildtype + Basta

T1 plants + Basta
Selection of Left: wildtype ;
Basta resistant T1 Right: transgenic
progeny
57
Weeks et al 1993 Plant Physiol 102:1077-1084
Tools for plant gene transfer
Microinjection
Microinjection of Nicotiana tabacum SR1 protoplast

Courtesy of D.H.H. Steinbiss, Max-Planck-Institut für Züchtungsforschung, Köln, FRG

58
 Microinjection
• Precise delivery of macromolecules into specific
intracellular compartments of living cells
• Uses micromanipulator (microcapillaries and
microscopic devices) to deliver DNA into defined
cells
that the injected cell survives and proliferates
• Cells used:
protoplasts, zygotic- & microspore- derived
proembryos (chimera results)
• Like biolistics, it delivers DNA into walled plant cells

59
 Micromanipulators

http://media.americanlaboratory.com/m/20/article/184955-fig1.jpg 60
http://www.leica-microsystems.com/products/light-microscopes/accessories/details/product/leica-micromanipulator/
 Microinjection

1’19”

https://www.youtube.com/watch?v=h-Bfc1GPWpE :murine pronucleus 61

 Advantages
1. Quantity of DNA delivered can be optimized
2. Can decides which cell to deliver DNA into
3. Delivery is precise, even into the nucleus, and is under
visual control
4. Cells of small structures (e.g. microspores and few-celled
proembryos, which are not available in the large quantities
for biolistic bombardment) can be precisely targeted
5. Defined microinjected cells can then be microcultured
6. If protocols exist for the culture of zygotic proembryos,
microinjection can be used to transform every species 62
 Disadvantages
1. Only one cell receives DNA per injection
2. Requires handling skills
3. Costly instrumentation

63
What marker
gene was used
here?
Anthocyanin regulatory gene
Maize Lc gene,
encoding a key regulatory protein that
controls anthocyanin pigmentation

a) embryogenic-derived protoplast
b) mesophyll-derived protoplast
Arrows in a and b, Lc-expressing cell

c) Somatic cells for microinjection

d) Arrows, Lc expression after injection
e) Shoot apical meristem for injection
f) Lc expression after microinjection
g) and h) Red sectors in leaf

Lusardi et al. 1994. Plant Journal 5: 571-582

Plant Physiol.156: 474–478 (2011)

66
Methods to introduce recombinant gene: PEG-mediated transformation of
protoplasts
5 min

PEG-mediated Transformation
https://www.jove.com/video/2560/efficient-polyethylene-glycol-peg-mediated-transformation-moss

A simple and efficient method to transform moss, Physcomitrella pantens protoplasts

 Example Qs
Write short notes on:
a) microprojectile bombardment in plant gene transfer;
b) use of protoplasts in plant gene transfer.

Discuss advantages and limitations in use of the following:

a) Microprojectile bombardment
b) Protoplasts

68