Transcript of Richard Mundembe. Focus of the presentation The talk will focus on methods of plant transformation...
- Slide 1
- Richard Mundembe
- Slide 2
- 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.
- Slide 3
- Methods of Plant Transformation 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
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- Model plants Arabidopsis Nicotiana benthamiana, N. tabacum
Tomato Rice Maize Highly optimized methods High transformation
efficiencies
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- 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)
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- 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/
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- Key dates of cowpea genetic transformation From Diouf,
2011
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- 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
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- Illustration of the binary plasmids used for tobacco
transformation by Agrobacterium -mediated transformation, and
effectiveness of each approach in conveying virus resistance CP-MR
Delayed symptom development RNA-MR Delayed symptom development
Recovery Anti- sense RNA-MR Modified symptoms Delayed symptom
development
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- 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.
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- Diagrammatic illustration of the electro-transformation
equipment and experimental set-up. Cowpea Transformation by
Electro- transformation
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- 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 non- homologous 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) Electro-transformation of Cowpea
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- 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
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- 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
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- 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-day- old seedlings with the aid of a silicon ring (G, 17 weeks).
Transfer to soil, high humidity chamber then to greenhouse.
Transformation efficiency: Agrobacterium-mediated transformation of
Cowpea
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- 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
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- 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
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- History of Cassava Transformation Li et al., 1996 -
Agrobacterium-mediated transformation of somatic cotyledons to then
regenerate transgenic shoots by organogenesis. Schpke 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.
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- 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 4. Maturation and development of transformed FEC
5. Selection and regeneration of transgenic plants Bull et al.,
2009
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- 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
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- Microprojectile Bombardment/ Biolistics A gene transfer method
developed to transform crops that remained recalcitrant to
Agrobacterium - mediated 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.
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- 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 (non- homologous
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
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- 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.
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- Minimum cassette technology When only the required gene
expression cassettes (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.
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- 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
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- 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 -
- Slide 27
- 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
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- 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 -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)
- Slide 29
- 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
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- 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, Ramirez- Villalobos and de
Garcia, 2008.
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- 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)
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- 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.
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- Other methods
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- 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.
- Slide 35
- Acknowledgements Prof M.E.C. Rey Cassava transformation Prof I.
Sithole-Niang Cowpea transformation Sweet potato transformation -
Banana transformation - MCB Plant Biotechnology Group
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- Thank you