Status of Cassava Genetic Transformation at CIAT.

1. Status of Cassava Genetic Transformation at CIAT.
www.ciat.cgiar.orgSince 1967 / Science to cultivate change
July 17th / 2014
Paul Chavarriaga Aguirre, PhD.

2. What are Genetically Modified Plants?
Plants whose genetic constitution has been
changed using non-conventional breeding
methods.
Agrobacterium tumefaciens is the
natural and most favored vector to
transfer genes to plants.

3. GM in the World: > 17 years & >180 M ha

4. GM Adoption in Colombia

5. How to Transfer Genes to Plants?
• Combining Biotech Tools : Cloning, Bacteria,
Cell Culture, Marker genes, etc.
Friable Embryogenic Callus
FEC

6. Summary of GMs at CIAT
(doesn’t include rice, sugar cane nor lulo)
Year Species Traits
1993 to 1998 M. esculenta-Mper 183 Herbicide Tolerance (HT; bar)
1994 S. guianensis (Aubl.) Sw. CIAT 184 HT
2002 Phaseolus sp Visual Marker (VM)
2002 M. esculenta 60444, CM3306-4 & SM1219-9 Bt genes
2002 Heliconia stricta VM
2003 M. esculenta 60444 Starch (antisense)
2005 & 2009 M. esculenta 60444 VM
2007 M. esculenta 60444 Flowering (inducible FT gene)
2009 to 2014 M. esculenta 60444 Cassava Bacterial Blight
2009 S. lycopersicon var. UNAPAL-Arreboles Bt genes
2010 M. esculenta 60444 Promoters
2010-2012 M. esculenta 60444 Pro-Vitamina A
2013-2014 M. esculenta 60444 & SM1219-9 Haploids (HT)
2013-2014 M. esculenta 60444 PPD (SOD overexpression)
2014 M. esculenta 60444 African Virus (tasiRNAs)
1983: First Transgenic Plants in the World (tobacco & petunia)
(Annual Miami Winter Symposium of Mol. Biol. of Plants and Animals, Miami, January /1983)

7. First GM-Cassava Plant not HT at High ppt (0,2 to 1,5 g/l).
(Sarria et al 1998; Echeverry et al 2003)
Tabaco 53-52 108 137-31 207 Mper 183 Mper 183 pGV1040
600 pb
pGV1040
217 pb

8. Southern for T0 Transgenic Cassava Plants with Bt gene (2002)
(TMS60444 = 2 to 4; CM3306-4 = 5 to 16; SM1219-9 = 17; Control =18)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

9. Improving Genetic Transformation of Cassava in 2005
60444 SM1219-9
Protocolo T1

10. In vitro plants of cassava
flower after induction of
an heterologous FT gene
from Arabidopsis.
(unpublished)

11. 0
5
10
15
20
25
Root P Stem Root C Leaf
GUSActivity(pmol4MU/min/μgprotein)
CP2::GUSPlus
NT
Promoter and Gene Expression Analysis.
Beltrán et al 2010

12. crtB
crtI
crtY
2X35S
LLBB RRBB
hpt II CP1 TP-CrtB-NOS
HindIII
ScaI
LB RB
EcoRIScaI
35S PatII TP-CrtBPatI PatII TP-Crt ITP-CrtYnpt II
Genetic Constructs for Carotenoids

13. GM Cassava with Higher Carotenoid Content
(>7-8 mg/g FW)
pCasPhyt-12
1221-55
Wt-60444
Welsch et al 2012; Univ. Of Freiburg, Germany.

14. Current Biosafety Cassava Field
Water, chemicals &
tools deposit

15. 0.47 0.70
1.52
2.53
2.95
5.89
7.38
8.84
11.30
20.97
0.00
5.00
10.00
15.00
20.00
25.00
60444
1257-14
1260-14
P12
1221-92
1257-15
1221-88
1252-1
1221-7
1252-10
1721-5
1257-2
1721-16
1260-6
1221-4
1252-8
1721-21
1221-75
1252-2
promedio
1252-11
1721-18
1221-84
1257-10
1221-6
GM905-52
1257-13
1221-63
GM905-21
1721-13
1221-30
1260-1
1221-22
1221-74
1221-12
1257-7
1221-55
GM905-60
GM905-57
TCCinmg/gFW
Effect of crtB gene in segregating, yellow-rooted cassavas,
heterozygous for PSY2(y2/y1) gene. (Chavarriaga 2012)

16. NSF-BREAD/2010
Fast Breeding for Slow Crops: Doubled Haploids in Cassava and Banana.
UC-Davis, IITA, CIAT.
Bill y Melinda Gates Foundation, Seattle, WA, 2011

17. Come on, give us
the money!
Mmmmh…
I don’t know…!

18. Schueler & Sullivan. Annu. Rev. Genomics Hum. Genet. 2006. 7:301–13

19. Maruthachalam & Chan (2010) Haploid plants produced by centromere-
mediated genome elimination. Nature (464):615-619

20. Summary of Cassava Transgenic Events for Haploids.
Construct
cv
(genotype)
# of plants
received
for analysis
Number
of plants
analyzed
Marker
gene
PCR (+)
Total
events
Copy number by Southern
blot
1 2 >3
GFP-
TailSwap
TMS60444 60 60 bar 15 15 2 4 9
SM1219-9 49 49 bar 4 0 0 0 0
TailSwap TMS60444 124 114* hyg 70 59 30 15 14
GFP-
CENH3
TMS60444 38 38
hyg
38 32 15 9 8
SM1219-9 2 2
hyg
1 1 1 0 0
TOTAL 273 263 128 107
48 28 31
(45%) (26%) (29%)

21. A sample of 107 Southern-proved transgenic lines transformed
with genes to induce haploidy in Cassava and transferred to
Greenhouse (BREAD Project ; UC-Davis and IITA)

22. CBB.
Paula Díaz & Camilo López, UNAL-Bogotá, Col.
T-DNA pCAMBIA1305.2
T hptII RXam2 GUSPlus35SP P TT
Overexpression
RXam2: QTL explaining ≈62%
resistance against XamCIO151

23. CBB.
• Seven constructs introduced into cassava
• First four produced >180 plant lines
• Second three will produce >>500 lines

24. tasiRNAs to Combat Multiple African Viruses.
(Vincent Fondong, Univ. of Delaware)
“This method exploits a special 22-nucleotide miRNA of Arabidopsis thaliana, miR173,
which can trigger production of another class of small RNAs called trans-acting small
interfering RNAs (tasiRNAs). We show that fusion of gene fragments to an upstream
miR173 target site is sufficient for effective silencing of the corresponding endogenous
gene. MIGS can be reliably used for the knockdown of a single gene or of multiple
unrelated genes.”
(Felippes et al 2012, The Plant Journal )

25. tasiRNAs to Combat Multiple African Viruses.
(Vincent Fondong, Univ. of Delaware)
• Six constructs already introduced in cassava.
• Hundreds of lines isolated.
• Plants produced in vitro and sent to Delaware.
• NO testing at CIAT in GH nor Field.

26. From Few to Countless Transgenic Lines:
What Changed?
• FEC Purity, Optical Density, Bacteria Aggressiveness, Co-culture
Conditions, Dedication and Concentration, etc.*
• * We knowledge the wise advise provided by Deepika Chauhan and Nigel Taylor from
IICI (DDPSC).

27. • Current efficiency per 100 mg FEC:
– Average 4,85 transgenic lines… and rising!
– Range 0,4 to 10,7
– Genotype-dependant
34 mg of FEC

28. Conclusion:
After 16 years of hard work, lots of pressure,
criticism (good and bad), and lots of self-
confidence, the pipeline to produce transgenic
cassava is going thru one of its best moments,
though not the best yet. There is still a lot of
room for improvement, which may lead us to
produce CIAT’s first GM cassavas useful for
breeders, producers and/or consumers. Hope
and confidence remain high.

29. Perspectives:
1. Selection Markers: HT vs Antibiotics vs Embryogenesis Genes
2. TALENs & CRISPERs for Haploids and Carotenoids.
3. Genes for Embryogenesis (Next Seminar).
4. Synthetic Seeds (Next Seminar).
5. Increase Yield (Stomata Opening).
6. High-Carotene FEC Lines & Virus Tolerance.

30. Funding:
1. DfID-UK
2. Dutch-Government
3. Harvest Plus
4. Bill & Melinda Gates Foundation-USA
5. NSF-USA
6. Colciencias-Colombia
7. MADR-Colombia

31. TheTeam:APricelessLoad!

32. Thank you…