Cisgenic barley for animal feed

1
Cisgenic barley for animal feed  Holme IB,
Dionisio G, Brinch-Pedersen H, Vincze E, Borg S,
Lange M, Holm PB University of Aarhus, Faculty of
Agricultural Sciences, Research Centre
Flakkebjerg, Department of Genetics and
Biotechnology, DK-4200 Slagelse Schørring J,
Kichey T, Jahn T, Sandøe P, Lassen J, Miele H,
Gylling M University of Copenhagen, Faculty of
Life Sciences, Thorvaldsensvej 40, DK-1871
Frederiksberg C Hauge Madsen K, Haastrup M The
Danish Agricultural Advisory Service, Udkærsvej
15, DK-8200 Århus N Eriksen L and Hjortsholm K
Sejet Plant Breeding, Nørremarksvej 67, DK-8700
Horsens
2
Cisgenesis instead of transgenesis

• Cisgenesis is based on the use of the gene pool
  from the plant itself or close relatives.
• Marginal difference between plants produced by
  conven-tional and GM breeding
• Allows augmentation of particular traits
  (insertion of additional gene copies)
• Might be more acceptable by the public

3
Main principles of transgenesis and cisgenesis
Transgenesis
Cisgenesis
Backbone in 50 of transformants
Discard integrations with backbone
Antibiotic resistance gene
Elimination of antibiotic resistance gene
Promoter and terminator from other species
Genomic clones from the plants own genome
including promoter and terminator regions
cDNA clones from any species
4
(No Transcript)
5
Advantages of cisgenesis as compared to
traditional breeding

• Transfer of gene of interest only
• Eliminates many generations of backcrossing
• Eliminates linkage drag

6
Backcross breeding
Cisgenesis
Receptor
Donor
Receptor
R-gene from crossable plant in Agrobacterium

x
F1
Receptor
x
r
BCn
After Jacobsen and Schouten, 2007
7
Objectives for the project Cisgenic barley for
animal feed

• Evaluate the concept of cisgenesis with regard to
  
• Development of cultivars with improved
  properties
• Ethic and social aspects
• Economy and downstream handling

8
Cisgenic barley for animal feed
Development of transformation vectors
Transformation of barley
Down- stream handling Economics Breeding
Phytase potential Nitrogen use efficiency
Ethics and public perception
Characterization in the greenhouse
Field trials
Preben Bach Holm
9
Nitrogen use efficiency Thomas Kichey, Thomas
Jahn and Jan K. Schjørring, Plant and Soil
Science Laboratory, Department of Agricultural
Science, KU-LIFE
Insertion of extra copies of genes
for Glutamine synthetase (GS1)
N remobilization Phosphoenol Pyruvate
Carboxylase (PEPc) Supply of C-skeletons Citrate
Synthase (CS) Tonoplast Intrinsic Proteins
(TIP1 and TIP2) Improve NH4 handling
capacity
10
Phytase potential University of Aarhus,
Department of Genetics and Biotechnology
Phytases are enzymes that degrade phytic acid
releasing bio- available phosphate
Phytic acid
Myo-Inositol-(1,2,3,4,5)-pentakisphosphate
70-80 of the phosphate in seeds are bound as
phytic acid. Phytic acid is largely indigestible
by non-ruminant animals.
11
Phosphate utilization in pigs
Non-ruminants fed with cereal based diets will
suffer from phosphate deficiency and the feed
have to be supplemented with extra phosphate.
Poulsen, 1996.
12
Increase the endogenous phytase level of the
barley seed
The phytases Purple acid phosphatase (PAP) and
Multiple inositol polyphosphate phosphatase
(MINPP) together account for the phytase activity
expressed in barley during grain filling and seed
germination.
PAP isoforms PhyIa, PhyIb, PhyIb2 MINPP
isoforms PhyIIa1, PhyIIa2, PhyIIb a isoforms
are mainly expressed during grain filling
b-isoforms are mainly expressed during seed
germination
13

Isolation of genomic clones for
cisgenesis (present status)
Isolation and characterizations of genomic clones
incl. promotor and terminator for the of TIP2,
GS1a, PAP1a, MINPPIIa2 Genomic sequence of TIP2
available in DNA DataBase Barley genomic lambda
library screened with cDNA for PAP, MINPP and GS1
14
Purple acid phosphatase Hv_PAP_PhyIa
Promoter-region
Coding-region
Terminator-region
2388
597
2310
0
2310
5295
4698
BbsI
PsiI
15
pGreen/pSoup (Hellens et al., 2000, Thole et al.,
2007) In Agrobacterium, pSoup provides
replication in trans for pGreen Advantages
pGreen small and larger fragments can be
inserted Some pGreen have two left
borders (pClean) pSoup
available with T-DNA (pClean)
Present status gTIP2, gGS1a, gPAP1a
16
Vector used for transgenesis (control)
CAMV35S-cDNA
Present status cDNA of TIP2 cDNA of GS1a cDNA of
PAPIa cDNA of MINPPIIa2
17
Transformation targets
Immature embryos Ovules (isolated just
after fertilization) Allows transformation
without selection markers
18
Acknowledgements Directorate for Food, Fisheries
and Agri Business Thank you for your
attention