Status of Cassava Genetic Transformation at CIAT.

www.ciat.cgiar.orgSince 1967 / Science to cultivate change

July 17th / 2014Paul Chavarriaga Aguirre, PhD.

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.

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

GM Adoption in Colombia

How to Transfer Genes to Plants?• Combining Biotech Tools : Cloning, Bacteria,

Cell Culture, Marker genes, etc.

Friable Embryogenic CallusFEC

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 HT2002 Phaseolus sp Visual Marker (VM)2002 M. esculenta 60444, CM3306-4 & SM1219-9 Bt genes2002 Heliconia stricta VM2003 M. esculenta 60444 Starch (antisense)2005 & 2009 M. esculenta 60444 VM2007 M. esculenta 60444 Flowering (inducible FT gene)2009 to 2014 M. esculenta 60444 Cassava Bacterial Blight2009 S. lycopersicon var. UNAPAL-Arreboles Bt genes2010 M. esculenta 60444 Promoters2010-2012 M. esculenta 60444 Pro-Vitamina A2013-2014 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)

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

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

Improving Genetic Transformation of Cassava in 2005

60444 SM1219-9Protocolo T1

In vitro plants of cassava flower after induction of an heterologous FT gene from Arabidopsis.(unpublished)

0

5

10

15

20

25

Root P Stem Root C Leaf

GU

S A

ctiv

ity (p

mol

4M

U/m

in/μ

g pr

otei

n)

CP2::GUSPlusNT

Promoter and Gene Expression Analysis. Beltrán et al 2010

crtB

crtI

crtY

2X35S LLBB RRBB

hpt II CP1 TP-CrtB-NOS

HindIII

ScaI

LB RB

EcoRI ScaI

35S PatII TP-CrtB PatI PatII TP-Crt I TP-CrtY npt II

Genetic Constructs for Carotenoids

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.

Current Biosafety Cassava Field

Water, chemicals & tools deposit

60444

1257-14

1260-14P12

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

promed

io

1252-11

1721-18

1221-84

1257-10

1221-6

GM 905-52

1257-13

1221-63

GM 905-21

1721-13

1221-30

1260-1

1221-22

1221-74

1221-12

1257-7

1221-55

GM 905-60

GM 905-570.00

5.00

10.00

15.00

20.00

25.00

0.47 0.701.52

2.53 2.95

5.89

7.38

8.84

11.30

20.97

TCC

in m

g/g

FWEffect of crtB gene in segregating, yellow-rooted cassavas, heterozygous for PSY2(y2/y1) gene. (Chavarriaga 2012)

NSF-BREAD/2010Fast Breeding for Slow Crops: Doubled Haploids in Cassava and Banana.

UC-Davis, IITA, CIAT.

Bill y Melinda Gates Foundation, Seattle, WA, 2011

Come on, give us the money!

Mmmmh…I don’t know…!

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

Human

CENP-A

= Pl

ant C

ENH3

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

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 38hyg

38 32 15 9 8

SM1219-9 2 2hyg

1 1 1 0 0

TOTAL 273 263 128 10748 28 31

(45%) (26%) (29%)

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)

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

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

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 )

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.

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 fromIICI (DDPSC).

• 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

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.

Perspectives:1. Selection Markers: HT vs Antibiotics vs Embryogenesis Genes2. 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.

Funding:1. DfID-UK2. Dutch-Government3. Harvest Plus4. Bill & Melinda Gates Foundation-USA5. NSF-USA6. Colciencias-Colombia7. MADR-Colombia

The

Team

: A P

ricel

ess L

oad!

Thank you…