Download Genetic Engineering of Plants for Resistance to Viruses

Richard Mundembe
Focus of the presentation
The talk will focus on methods of plant transformation that have
been used on non-model crops.
- Cowpea, cassava, sweet potato and banana will be used as the
main examples.
- Crop and traits of interest
- Method of transformation, efficiency and safety implications,
- Other methods of plant transformation and other non-model
plants will also be discussed in brief.
Methods of Plant
Transformation
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Agrobacterium-mediated transformation
Microprojectile bombardment/ biolistics
Direct protoplast transformation
Electroporation of cells and tissues
Electro-transformation
The pollen tube pathway method
Other methods such as infiltration,
microinjection, silicon carbide mediated
transformation and liposome mediated
transformation
Model plants
 Arabidopsis
 Nicotiana benthamiana, N. tabacum
 Tomato
 Rice
 Maize
Highly optimized methods
High transformation efficiencies
Non-model Plants
 Cowpea (Vigna unguiculata)
 Cassava (Manihot esculenta)
 Sweet potato (Ipomoea batatas)
 Banana (Musa spp)
 Many monocotyledonous cereal crops e.g. sorghum (Sorghum
bicolor; grain; sweet stem), pearl millet (Pennisetum glaucum,
mhunga), finger millet (Eleusine coracana, zviyo).
… and more
Recalcitrant to transformation and regeneration
(efficient, reliable and reproducible)
Cowpea transformation
Importance of cowpea
 source of dietary protein in traditional diets
 partially replenishes the soil nitrogen
 is used as fodder
 Currency of trade/barter
 Grown mainly by women
Cowpea production constraints
 Low yields of traditional varieties
 Viral, bacterial and fungal diseases in the field
 Post-harvest storage diseases and pests
http://www.fao.org/fileadmin/
Key dates of cowpea genetic transformation
From Diouf, 2011
Cowpea transformation
Methods of cowpea transformation
 Electro-transformation
 Agrobacterium-mediated
 Biolistics – Ivo et al., (2008), TF 0.9%
Molecular approaches to virus resistance
 Coat protein-mediated resistance
 RNA-mediated resistance
Cowpea aphid-borne mosaic virus (CABMV)
Molecular approaches to contain post-harvest damage
 Bruchid resistance, trypsin inhibitors
Hind III
pBI 121
LB
RB
Hind III
pBI121-CPk
Hind III
CP-MR
Delayed symptom development
LB
RB
pBI121-CPantisense
Antisense
LB
RNA-MR
pBI121-CPstop
RNA-MR
Hind III
Hind III
RB
Hind III
Hind III
LB
pBI121-CPcore
Modified symptoms
Delayed symptom development
RB
Hind III
Delayed symptom development
Recovery
Hind III
LB
RB
KEY
Promoter
Terminator
Kozak
consensus
sequence
T-DNA
borders
Coding
sequence
s
Illustration of the binary plasmids used for tobacco transformation by
Agrobacterium-mediated transformation, and effectiveness of each approach in
conveying virus resistance
Electro-transformation
 DNA can also be delivered into cells, tissues and
organs by electrophoresis (Ahokas 1989; Griesbach
and Hammond, 1994; Songstad et al., 1995).
 This method is known as transformation by
electrophoresis or electro-transformation.
 The tissue to be transformed is placed between the
cathode and anode.
 The anode is placed in a pipette tip containing agarose
mixed with the DNA to be used for transformation.
The assembly is illustrated in the next slide.
Cowpea Transformation by Electrotransformation
Diagrammatic illustration of the electro-transformation equipment and experimental set-up.
Electro-transformation of Cowpea
A common feature of the GUS positive plants is that the manipulations
were carried out on plants that had
 straight stems,
 first true leaves open and
 cotyledons still attached to the seedling.
No pre-treatment other than maybe punching the meristem appear to be
necessary.
Both DC and AC are effective in delivering DNA to the plant cells.
The leaves of GUS positive plants had a sectored appearance;
 Kanamycin resistance was not an effective assay against germinating
cowpea seedlings The mechanism of DNA integration is probably nonhomologous recombination into sites on the genome that are
undergoing repair or replication
 Has potential for marker-free transformation
 Efficiency less than 0.3% (4 in 1200)
Agrobacterium-mediated
transformation
 In crown gall disease of dicotyledonous plants, caused
by Agrobacterium tumefaciens and hairy root disease
caused by Agrobacterium rhizogenes, the bacterium
transfers part of the DNA of its Ti or Ri plasmid DNA
respectively into the host plant where it becomes
integrated into the host genome (Herrera-Estrella et
al., 1983).
 The natural host range of the bacterium expanded
 Harnessed for use in Plant Biotechnology for in vitro
plant transformation, using various modified versions
of the Ti plasmid
Agrobacterium-mediated transformation of
Cowpea
T.J. Higgins method (Popelka et al., 2006)
Co-cultivation
 Agrobacterium strain containing pBSF16, in liquid medium
(MS/MES/vits/BAP/GA3/acetosyringone/DTT/cys)
 Cotyledonary nodes of 3 different cultivars
 Co-cultivation of explants for 6 days
Shoot initiation
 Shoots were initiated on MS/NTS/timentin.
 12 days, no selection for multiple shoots to appear.
Selection
 Selective medium (MS + 5 mg/l PPT),
 refreshed every 2 wk; remove dead tissue, 4 – 6 transfers
Agrobacterium-mediated transformation of
Cowpea
Shoot elongation
 green shoots were transferred to shoot elongation medium
(MS/GA3/Asp/IAA/timentin/5 mg/l PPT)
 sub-cultured every 2 wk until shoots were more than 1 cm long.
(14 weeks!)
Rooting
 Transfer from large culture jars for growth under selection.
 Some rooted, others needed to be grafted directly onto 10-dayold seedlings with the aid of a silicon ring (G, 17 weeks).
 Transfer to soil, high humidity chamber
 then to greenhouse.
 Transformation efficiency:
Cassava Transformation
Importance of Cassava
Important source of dietary carbohydrate, food security
Yields relatively well even under low rainfall and in poor soils,
but there is need for improvement
Has potential as an industrial crop – biofuels and starch
industries
Cassava production constraints
Viral (e.g. CMD), bacterial and fungal diseases in the field
Does not store well once removed from the soil
Methods of cassava transformation
Agrobacterium-mediated transformation
Microprojectile-mediated transformation / biolistics
RNA interference (RNAi)
 This is the process that depends on small RNAs (sRNAs) to
regulate the expression of the eukaryotic genome,
including maintenance of genome integrity, development,
metabolism, abiotic stress responses and immunity to
pathogens.
 micro RNAs (miRNAs) and small interfering RNAs
(siRNAs).
 siRNAs are derived from perfectly paired double stranded
RNA (dsRNA) precursors, that are derived either from
antisense or are a result of RNA-dependent RNA
polymerase (RDR) transcription.
 Hairpin RNA is more effective at inducing RNAi
History of Cassava Transformation
Li et al., 1996 - Agrobacterium-mediated transformation of
somatic cotyledons to then regenerate transgenic shoots by
organogenesis.
Schöpke et al. 1996,- microparticle bombardment of
embryogenic suspension-derived tissues and then regenerated
transgenic plantlets by embryo maturation. (= FEC suspension
cultures).
Gonzalez et al., (1998), Zhang et al., (2000) and Shrueder et al.,
(2001), Agrobacterium-mediated transformation of FEC
Optimisation by Bull et al, (2009) – SE induction, FEC
production, co-cultivation and selection.
Agrobacterium-Mediated Transformation of
Cassava Friable Embryonic Callus (FEC)
FEC are a specialized totipotent cell clusters
Methods for induction of FEC and Agrobacterium-mediated
transformation were optimized by Bull et al. (2009).
1. Somatic embryo production
2. Production of FEC
3. Co-cultivation of FEC with Agrobacterium Bull et al., 2009
4. Maturation and development of transformed FEC
5. Selection and regeneration of transgenic plants
Agrobacterium-Mediated Transformation of
Cassava Friable Embryonic Callus (FEC)
We have successfully used this method to transform
 TMS60444 (IITA model cultivar) and
 T200 (a commercial grown SA landrace)
using pCambia-based construct designed to convey resistance to
various CMD causing viruses by
 hp RNAi,
 replicase and
 antisense strategies.
Transformation efficiencies are relatively high (…)
Evaluation of levels of resistance is ongoing
pCambia map
Microprojectile
Bombardment/ Biolistics
 A gene transfer method developed to transform
crops that remained recalcitrant to Agrobacteriummediated transformation
 The DNA construct, attached to a microprojectile
(gold or tungsten),
 is delivered at high speed across the various plant
cell barriers (cell wall, cell membrane, cytoplasm,
nuclear envelop, to enter the nucleoplasm)
 transient expression or integration (whole or
fragments) into the plant genome may occur.
Microprojectile Bombardment
 Delivery into the nucleus results in 45 x higher
likelihood of transient expression in cytosol, and 900 x
higher than in vacuole (Yamashita et al., 1991).
 The mechanism of integration is thought to be (nonhomologous integration)
 Efficiency of transformation is influenced by the stage
of the cell cycle, higher expression if close to the time
the nuclear membrane disappears at mitosis
Microprojectile Bombardment
 Result in transformants with higher copy numbers,
especially with amounts of bombarding
 Integration into the same or tightly linked loci, most
likely in relation to replication forks or integration hot
spots resulting from initial integration events
 Rearrangements (deletions, direct repetitions, inverted
repetitions, ligation, concatamerization) may occur
prior to, or during integration
 90% of integrations are into random sites within
transcriptionally active regions.
Minimum cassette technology
 When only the required gene expression cassettes
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(promoter, coding region of interest, terminator) is
bombarded into the plant cells
Sometimes co-transformed together with marker genes to
be removed before commercialization
Screening and selection might be more difficult, probably
depending on detection of the gene sequence or gene
product of interest,
But the approach is very attractive since absence of reporter
genes and selection markers results in address the biosafety
concerns of consumers and are safer for the environment
Marker genes also limit options for gene stacking in an
original transgenic line.
Microprojectile bombardment of Cassava
Results from our lab (Poster)
Optimisation of parameters for biolistic transformation of
cassava FEC
 Linear and circular constructs
 Gold particle size
 Helium pressure
 Minimum cassettes
GUS assay, Hyg re-rooting assay,
PCR – GUS, Hyg, Insert
Southern analysis - pending
Sweet Potato (Ipomoea batatas) Transformation
Importance of sweet potato
Important source of food crop – roots and foliage
Controversial alternative biofuel substrate
Can be stored in the soil until needed
Sweet potato production constraints
Viral (e.g. SPFMV), bacterial and fungal diseases in the field
Low yields from recycled disease-infested planting material, and
poor farming practices
Nematodes - Stem nematode (Ditylenchus destructor)
Insect damage – in the field and in storage - weevils (Cylas
formicarius),
Water stress -
Sweet Potato Transformation
Regeneration
– relatively easy, from protoplasts, via shoot organogenesis, from
leaves, roots and stem internodes. Somatic embryogenesis can
be induced from axillary bud shoot tips, apical and bud
meristems, and leaf, petiole, stem and root explants
Methods of sweet potato transformation
Electroporation of protoplasts (Nishiguchi et al., 1992)
Agrobacterium-mediated transformation of leaf and stem
explants.
Efficiency – can be higher than 2%
Biolistics
Sweet Potato Transformation
Viruses – SPFMV, CPMR
Nematodes – oryzacystatin-I gene, OC1 (Gao et al., 2011), ,
Insect resistance - weevil (Cylas formicarius) – Garcia et al., 2007,
field trials, cowpea trypsin inhibitor (CpTI), snowdrop lectin
(GNA)
Other traits: - granule-bound starch synthaseI (GBSSI),
- tobacco microsomal ω-3 fatty acid desaturase (NtFAD3),
- starch branching enzyme II (IbSBEII)
- bar gene
- Xerophyta viscosa peroxiredoxin 2, XvPrx2, gene conferring
drought stress tolerance (Kamwendo, P.M., 20xx)
Banana and Plantain (Musa spp) Transformation
Importance of Banana
Important source of dietary carbohydrate and income
Banana production constraints
Viral (e.g. Banana bunchy top virus),
bacterial (banana Xanthomonas wilt, BXW) and
fungal (Fusarium wilt by Fusarium oxysporum) diseases in the
field;
Nematodes – Radopholus similis – natural resistance identified
- Pratylenchus
- Helicotylenchus
Banana Transformation
Methods of Banana transformation
Target tissue is embryogenic cell suspension (ECS) –
establishment is not routine, because of low embryogenic
response, long time needed, somaclonal variation, and
contamination.
 Agronomic traits
 Quality traits
 Molecular pharming
Banana ECS, RamirezVillalobos and de Garcia, 2008.
Banana Transformation
Methods of Banana transformation
 Protoplast electroporation
 Agrobacterium-mediated transformation
 Microprojectile-mediated transformation / biolistics – GUS, Hyg
Variable transformation frequencies, depending on cultivar.
Agro. better than biolistics in a wider range of cultivars.
 Maize cystatin and synthetic repellent genes (plantain,
Tripathi et al., 2011)
 Bacterial – over-expression of sweet pepper plant like
ferredoxin protein, Pflp, and hypersensitive response
assisting protein, Hrap. (Abubaker et al, 2011)
 Fungal resistance – pathogenesis-related protein genes
as candidates for GE (FW) … van der Berg et al., 2011)
PEG-mediated transformation of protoplasts
 Plant cell walls are removed by enzymatic degradation to produce
protoplasts.
 Polyethylene glycol (PEG) causes permeabilization of the plasma
membrane, allowing the passage of macromolecules into the cell.
Electroporation of Protoplasts
 Aelectric pulse permeabilizes the plasma membrane of the protoplasts.
 The cell wall and whole plants can be regenerated, if procedures exist.
The transgenic plants generated have characteristics similar to those of
plants derived from direct transformation methods.
 Carrier DNA (usually ~500 bp fragments of calf thymus DNA)
included in the transformation mixture increases transformation
efficiency, but increases prevalence of transgene rearrangements and
integration of superfluous sequences.
Protoplast cultures are not easy to establish and maintain
Regeneration of whole plants is unreliable for some important species.
Summary of other plant transformation methods
Transformation
Method
Electroporation of
cells and tissues
Short
Description
High voltage
discharge is
used to open
pores on the cell
membrane and
carry DNA into
the cell
Pros
Cons
Higher
regeneration
success than
with protoplasts
Protocol for
regeneration
required
Microinjection
DNA delivered
through a needle
into cells
immobilized by
microtools
Can potentially
be used for the
introduction of
whole
chromosomes
Practical only for
protoplasts.
Tobacco,
Petunia, rape
and barley
Silicon carbide
mediated
transformation
Silicon carbide
whiskers coated
with DNA pierce
and enter the
cells
The method is
widely
adaptable, and
requires little
DNA
Tobacco, maize,
rice, other
grasses.
The pollen tube
pathway
DNA delivered to
ovule via cut end
of pollen tube
Apparently
widely
applicable.
Low
transformation
efficiencies.
Silicon carbide
whiskers are a
health risk to
experimenter.
Apparently widely
applicable, but
particular
protocols need to
be developed
Liposome
mediated
transformation
Liposomes
loaded with DNA
are made to fuse
with protoplast
membrane
Uptake depends
on the natural
process of
endocytosis
Effective only for
protoplasts
Success for
tobacco and
wheat
Infiltration
A suspension of
Agrobacterium
cells habouring
the DNA
construct of
interest is
vacuuminfiltrated into
inflorescences
Simple
procedure
Not generally
applicable to
most species
Very efficient for
Arabidopsis
Other methods
Main Results
Achieved
Maize, rice,
tobacco, wheat
Successful for
rice, wheat,
soybean, water
melon and
Petunia hybrida
Non-model Plants (cont.)
 Many monocotyledonous cereal crops e.g. sorghum (Sorghum
bicolor; grain; sweet stem), pearl millet (Pennisetum glaucum,
mhunga), finger millet (Eleusine coracana, zviyo).
 Ginger, Zingiber officinale Roscoe (Zingiberaceae),
 Bambara groundnut
 Indigenous vegetables such as Okra (Corchorus
tridens/olitorius; derere, idelele), Spider flower (Cleome
gynandraruni; runi/nyeve, elude)
Not all are candidates for transformation.
Acknowledgements
 Prof M.E.C. Rey – Cassava transformation
 Prof I. Sithole-Niang – Cowpea transformation
 Sweet potato transformation -
 Banana transformation  MCB Plant Biotechnology Group
Thank you