Richard M undembe Focus of the presentation The talk will focus on methods of plant transformation that have been used on nonmodel crops Cowpea cassava sweet potato and banana will be used as the main examples ID: 933514
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Slide1
Transformation of Non-Model Plants on the Sub-Saharan African continent
Richard
M
undembe
Slide2Focus 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.
Slide3Methods 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
Slide4Model plants
Arabidopsis
Nicotiana
benthamiana
,
N.
tabacum
Tomato
Rice
Maize
Highly optimized methods
High transformation efficiencies
Slide5Non-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)
Slide6Cowpea 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/
Slide7Key dates of cowpea genetic transformation
From
Diouf
, 2011
Slide8Cowpea 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 resistanceRNA-mediated resistanceCowpea aphid-borne mosaic virus (CABMV)Molecular approaches to contain post-harvest damageBruchid resistance, trypsin inhibitors
Slide9Illustration 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
Slide10Electro-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.
Slide11Diagrammatic illustration of the electro-transformation equipment and experimental set-up.
Cowpea Transformation by Electro-transformation
Slide12A 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 replicationHas potential for marker-free transformationEfficiency less than 0.3% (4 in 1200)
Electro-transformation of Cowpea
Slide13Agrobacterium-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 expandedHarnessed for use in Plant Biotechnology for in vitro plant transformation, using various modified versions of the Ti plasmid
Slide14T.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 cultivarsCo-cultivation of explants for 6 daysShoot initiationShoots were initiated on MS/NTS/timentin. 12 days, no selection for multiple shoots to appear.SelectionSelective medium (MS + 5 mg/l PPT),
refreshed every 2 wk; remove dead tissue, 4 – 6 transfers
Agrobacterium-mediated transformation of Cowpea
Slide15Shoot elongationgreen 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
Slide16Cassava 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
Slide17RNA 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
Slide18History 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 FECOptimisation by Bull et al
, (2009) – SE induction, FEC production, co-cultivation and selection.
Slide19Agrobacterium-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 Agrobacterium4. Maturation and development of transformed FEC5. Selection and regeneration of transgenic plants
Bull et al., 2009
Slide20Agrobacterium-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
h
p
RNAi
,
replicase
and antisense strategies.Transformation efficiencies are relatively high (…)Evaluation of levels of resistance is ongoingp
Cambia map
Slide21Microprojectile 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.
Slide22Microprojectile 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
Slide23Microprojectile Bombardment
R
esult 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.
Slide24Minimum 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.
Slide25Microprojectile 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, InsertSouthern analysis - pending
Slide26Sweet 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 fieldLow yields from recycled disease-infested planting material, and poor farming practicesNematodes - Stem nematode (Ditylenchus
destructor)Insect damage – in the field and in storage - weevils (Cylas
formicarius), Water stress -
Slide27Sweet 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
Slide28Sweet 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)
Slide29Banana 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 identifiedPratylenchus
Helicotylenchus
Slide30Banana 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 traitsMolecular pharming
Banana ECS, Ramirez-Villalobos and de Garcia, 2008.
Slide31Banana Transformation
Methods
of
Banana
transformation
Protoplast
electroporation
Agrobacterium
-mediated
transformation
Microprojectile
-mediated transformation /
biolistics
– GUS, HygVariable 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)
Slide32PEG-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
R
egeneration
of whole plants
is unreliable
for some important species
.
Slide33Other methods
Slide34Non-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 groundnutIndigenous vegetables such as Okra (
Corchorus tridens/olitorius;
derere, idelele), Spider flower (Cleome
gynandraruni
;
runi
/
nyeve
, elude)
Not all are candidates for transformation.
Slide35Acknowledgements
Prof M.E.C. Rey – Cassava transformation
Prof I.
Sithole-Niang
–
Cowpea transformation
Sweet potato transformation -
Banana transformation -
MCB Plant Biotechnology Group
Slide36Thank you