cotton biotechnology keerti s. rathore - cba · cotton biotechnology congresso brasileiro do...

41
Cotton Biotechnology Congresso Brasileiro Do Algodao , 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore Institute for Plant Genomics and Biotechnology & Dept. of Soil & Crop Sciences Borlaug Center for Southern Crop Improvement Texas A&M University

Upload: duongkiet

Post on 21-Jan-2019

217 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

Cotton Biotechnology

Congresso Brasileiro Do Algodao, 2015

Brazil Cotton Congress, 2015

Keerti S. Rathore

Institute for Plant Genomics and Biotechnology

& Dept. of Soil & Crop Sciences

Borlaug Center for Southern Crop Improvement

Texas A&M University

Page 2: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

CaMV 35S Promoter Activity in Cotton

Seed-specific Promoter (Cotton a-globulin B Gene)

Oil Quality Improvement (Antisense of D-12- desaturase)

Drought Tolerance (Tobacco Osmotin Gene)

Disease Resistance (T. virens Endochitinase Gene)

Broad-spectrum Disease Resistance (At NPR1 Gene)

Ultra-low Gossypol Cottonseed through RNAi

Tissue-specific & Inducible Promoters

Nematode Resistance through RNAi

Transgenic Traits & Related Research in Cotton at Texas A&M Univ.

Page 3: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

CaMV 35S Promoter

Activity in Seed

and Fiber Tissues

in Cotton

Sunil Kumar …… Rathore

(Plant Molecular Biology 2002)

This Promoter was

used to Drive the Bt Gene

in original Bollgard Cotton

Page 4: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

a-Globulin Promoter: a Strictly Seed-specific

Promoter from Cotton

16 dpa 18 dpa16 dpa 20 dpa19 dpa

30 dpa25 dpa Dry40 dpa Control

Sunilkumar …… Rathore (2002)

Transgenic Research 11: 347-359

U.S. Patent #7,626,081

Page 5: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

0

50

100

150

200

250

300

350

400

Stem Root Pollen SeedLeaf Floral

bud

GU

S A

CT

IVIT

Y

(nm

ole

4-M

U /

mg p

rote

in/

min

)

Seed-specific activity of α-globulin promoter

in cotton

0

50

100

150

200

250

300

350

400

Control

AGP:gusA

Sunilkumar …… Rathore (2002)

Transgenic Research 11: 347-359

U.S. Patent #7,626,081

Page 6: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

ggtaccggtccggaattcccgggctctcttgtagtcacattgactcttggttggcctatgtact

tagccttcaacgtttcgggtcgatactatgatcgattagcttcccactataacccttacggccc

catttactccgaacgcgagaggctacaagtttacatctccgatgctggtatagttgcggtaat

ttatgtactttataagattgctgcaacaaaagggctggcttggcttttatgcacttatggggtac

ctctacttattgtgaatgccttccttgtgttgatcacctacttgcaacatactcactcggcattgc

gcattacgactcgtctgaatgggattggtttcgaggagcattgtcgacgattgatcgagatta

cggggtgttgaacaaagtgttccataacatcaccgatacgcatgtggctcatcacctcttctc

aacgatgccacattatcatgcaatggaggccactaaagcaatcaaaccgatactcggcaa

gtattatcctttcgacgggacaccgatttataaggcaatgtggagggaggcaaaagagtgc

cttacgtcgaggctgacgttggtggtggtggtagcaaggtgtttttgggtaatcgtaacaagt

tctaaagacagaccaactgcctgatagctnggccggcaaatcgancgtaaaacgtaactt

attaga

698 bp

Cotton D-12-desaturase

antisense construct

Improving Cottonseed Oil Quality

Sunil Kumar …… Rathore (2005)

Plant Biotechnology Journal, 3:319-330

Page 7: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

Nu

ll

Nu

ll

Hem

i

Hem

i

Ho

mo

Ho

mo

Oleic acid

Linoleic acid

WT

10

20

30

30

40

10

20

0

50

60

404 14 15 13 8 1

% v

alu

es

fo

r ta

rget

fatt

y a

cid

s

OA

WT

LA

0 0 30 33 33 21

28 27 13 11 0 0

(a)

PCR+/KanR

PCR- /KanS

Myristic

10

20

30

0

% v

alu

es

fo

r n

on

-ta

rget

fatt

y a

cid

s

acid acid acidPalmitic Stearic

(b)

0

10

20

30

Homo

Null

Hemi

Higher Oleic Acid and Lower Linoleic Acid Cottonseed Oil

Sunil Kumar …… Rathore (2005)

Plant Biotechnology Journal, 3:319-330

Page 8: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

Tobacco Osmotin-mediated Drought Tolerance in Cotton

WT 61-63 61-53Parkhi et al. (2009) Mol. Breed.

Page 9: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

Multiple drought treatments under greenhouse conditions:

Three cycles of drought at various stages of plant development

Wa

ter

Co

ns

um

pti

on

in

Co

tto

n

12346810 days 6 days

3 days water

withholding

0

5

10

15

20

25

30

35

40

45

0 10 20 30 40

Squar

e 60

1st B

loom 80 90 10

011

0

1st B

oll o

pen

130

140

150

160

Progression of Cotton Crop (Days)

Ra

te o

f W

ate

r U

se

(Source: Denise McWilliams (2003) Drought Strategies for Cotton. Cooperative Extension Service, Circular 582,

College of Agriculture and Home Economics, New Mexico State University, Las Cruces, New Mexico, USA)

Page 10: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

10 days of drought-stress

61-63 WT

Parkhi et al. (2009) Mol. Breed.

Page 11: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

0

20

40

60

80

100

120

140

160

WT 61-63 WT 61-63

Well-watered Drought-stressed

Yie

ld/p

lan

t (g

)

Seed Lint

Growth and yield measurements following multiple drought

treatments under greenhouse conditions: Yield (Seed and Lint)

**

**

**P<0.001; n=10

Seed/lint ratio

WT: 1.54; 61-63: 1.11

Parkhi et al. (2009) Mol. Breed.

Page 12: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

H1

21

B

WT

(Test tube assay) (Soil Based Assay)

Kumar et al 2009. Planta

Resistance to Rhizoctonia solani in transgenic cotton

expressing an endochitinase gene from Trichoderma virens

Page 13: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

Control

135-4

Resistance to Rhizoctonia solani in transgenic cotton expressing an

endochitinase gene from Trichoderma virens

High Disease

Pressure

Emani et al 2003

Plant Biotech J.

Page 14: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

Broad-spectrum Disease resistance in

transgenic cotton plants expressing

Arabidopsis-NPR1 gene

Page 15: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

Resistance to fungal pathogens in transgenic cotton lines

expressing AtNPR1.

Fungal pathogens

• Rhizoctonia solani

• Thielaviopsis basicola

• Alternaria alternata

• Fusarium oxysporum f.sp. vasinfectum isolate Fov11

• Verticillium dahliae

- non-defoliating pathotypes, TS-2 and EZ-2

Page 16: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

0

10

20

30

40

50

60

WT 68L-19 68L-20

Dis

ea

se

in

de

xNPR1-mediated Resistance to Rhizoctonia solani

Parkhi et al 2010.

Transgenic Research

Page 17: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

NPR1-mediated Resistance to

Thielaviopsis basicola (Black Root Rot)

Parkhi et al 2010.

Transgenic Research

Page 18: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

0

20000

40000

60000

80000

WT 68L-

19

68L-

20

Re

lative infe

cte

d a

rea/leaf

NPR1-mediated Resistance to Alternaria alternata

Parkhi et al 2010.

Transgenic Research

Page 19: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

0

2

4

6

8

10

WT DP444 68L-19 WT DP444 68L-19

Uninfected Infected

Sh

oo

t w

eig

ht

(g)

0

30

60

90

WT DP444 68L-19

Le

af

dis

ea

se

in

de

x

68L-19 WT DP444

NPR1-mediated Resistance to Fusarium oxysporum (Fov11)

Fusarium wilt-resistant,

commercial cotton variety

Parkhi et al 2010.

Transgenic Research

Page 20: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

Non defoliating isolate Defoliating isolate

Response of NPR1- cotton transformants to Verticillium dahliae isolates

Longitudinal section

of the 5th internode

Parkhi et al 2010.

Transgenic Research

Page 21: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

0

20

40

60

80

100

120

WT 68L-19 68L-20

Ren

ifo

rm n

em

ato

des/g

so

il

**

*

0

0.5

1

1.5

2

2.5

WT 68L-

19

68L-

20

WT 68L-

19

68L-

20

Uninfected Infected

Nu

mb

er

of

bo

lls/p

lan

t

**

0

5

10

15

20

25

30

WT 68L-

19

68L-

20

WT 68L-

19

68L-

20

Uninfected Infected B

oll

weig

ht/

pla

nt

(g;

FW

)

**

NPR1-mediated resistance to reniform nematodes

0

4

8

12

16

WT 68L-19 68L-20

No

. of bolls

at giv

en n

odes

Node 5-7 Node 8-10

Parkhi et al 2010.

Transgenic Research

Page 22: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

Gossypol Elimination from Cottonseed

Page 23: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

Presence of toxic gossypol prevents the use of cottonseed as food &

also limits its use as feed

Global Cotton Lint Production in 2011 = 26.1 MMTGlobal Cottonseed Production in 2011 = 48.8 MMT (~11 MMT Protein)

Can meet the protein requirements of 600 million people/year (50 g/day)

Page 24: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

• Protein content of the seed is about 22.5%; good quality protein.

• Its use for human consumption or animal feed is limited by the presence of gossypol in the seed glands.

• Gossypol is toxic to non-ruminant animals, including pigs, chicken and humans. It damages heart and liver.

• Meal used as feed for the cattle – gossypol binds to lysine thus reducing the nutritional value of the meal.

• Gossypol typically removed during the processing of cottonseed to obtain edible oil – gossypol contamination in oil known to cause sterility in men.

Gossypol

Page 25: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

• 3.66 kg of crude protein is converted to 0.83 kg of

milk protein per day

• Feed conversion ratio for beef cattle is 5.8

Cows are Inefficient and Wasteful

Page 26: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

Animal FCR Reference

Beef 5.8a Klopfenstein et al., 1991

Pig 3.3a Losinger, 1998; Cromwell, 1991

Duck 2.705b Shalev and Pasternak, 1989

Turkey 2.102b Shalev and Pasternak, 1989

Chicken 2.047b Shalev and Pasternak, 1989

Shrimp 2.0c Tacon, 2002

Tilapia 1.5d McGinty and Rakocy, 1989; Boyd et al., 2005

Salmon 1.1d Vill amar, 2002

Channel Catfish 1.0d Lovell, 1990

1

Feed Conversion Ratio (FCR)

for Various Animals

aFeed grain/weight gain ratio

bTotal feed utilization of live weight for seven week-old broilers

cDry aquafeed/weight gain ratio

dFeed/weight gain ratio

1.5

5.8 3.3

2.7

2.1

2.05

2.0

1.1 1.0

Page 27: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

There is tremendous potential for utilizing cottonseed for human

nutrition, either directly as food or indirectly as feed for non-

ruminants, if gossypol can be eliminated from the seed.

Page 28: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

• McMichael discovered a GLANDLESS mutant of cotton

in 1954

• Seed from glandless cotton was gossypol-free

• National and International breeding programs launched to transfer the trait to commercial varieties

• Animal nutrition studies showed glandless cottonseed to be a relatively good source of feed for pigs, chicken, and shrimp

• Glandless cottonseed was even considered fit for human consumption

Attempts Using Plant Breeding

Texas A&M University tested and promoted glandless

cottonseed as a snack food - TAMUNUTS

Page 29: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

Traditional Insect Pests that Cause Greater Damage to Glandless Cotton

Cotton Bollworm (Helicoverpa zea)

Tobacco Budworm (Heliothis virescens)

Beet Armyworm (Spodoptera exigua)

Non-traditional Insects that Damage Glandless Cotton

Black Fleahopper (Spanogonicus albofasciatus)

Pillbug (Porcellia sp.)

Spotted Cucumber Beetle (Diabrotica undecimpunctata)

Grape Colaspis (Maecolaspis flavida)

Green Dock Beetle (Gastrophysa cyanea)

Blister Beetle (Epicauta vittata)

Currently, there is no large-scale cultivation of glandless cotton

Page 30: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

Desired Trait

Seeds:

Gossypol < 450 ppm

Leaf & other Organs:

Full Compliment of Gossypol

and Related Terpenoids

for Defense

Page 31: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

O

HO

HO

HO

HO

CHO

OH

CHO

HO

HO

O

O

CHO

HO

HO

O

O

+

Desoxyhemigossypol

(dHG)

Heliocide H2

Heliocide H3

Hemigossypol

(HG)

+

CHO

HO

HO

O

O

Hemigossypolone

(HGQ)

CHO

HO

HO

O

O

Heliocide H1

CHO

HO

HO

O

O

HeliocideH4

Myrcene

-Ocimene

HO

HO

CHO

OH

OH

OH

HCO

OH

Gossypol

(G)

OH

8-Hydroxy-(+)-

-cadinene

(+)--CadineneOPP

Farnesyldiphosphate

(FDP)

dCS

XO

HO

HO

HO

HO

CHO

OH

CHO

HO

HO

O

O

CHO

HO

HO

O

O

+

Desoxyhemigossypol

(dHG)

Heliocide H2

Heliocide H3

Hemigossypol

(HG)

+

CHO

HO

HO

O

O

Hemigossypolone

(HGQ)

CHO

HO

HO

O

O

Heliocide H1

CHO

HO

HO

O

O

HeliocideH4

Myrcene

-Ocimene

HO

HO

CHO

OH

OH

OH

HCO

OH

Gossypol

(G)

OH

8-Hydroxy-(+)-

-cadinene

(+)--CadineneOPP

Farnesyldiphosphate

(FDP)

dCS

X

O

HO

HO

HO

HO

CHO

OH

CHO

HO

HO

O

O

CHO

HO

HO

O

O

+

Desoxyhemigossypol

(dHG)

Heliocide H2

Heliocide H3

Hemigossypol

(HG)

+

CHO

HO

HO

O

O

Hemigossypolone

(HGQ)

CHO

HO

HO

O

O

Heliocide H1

CHO

HO

HO

O

O

HeliocideH4

Myrcene

-Ocimene

HO

HO

CHO

OH

OH

OH

HCO

OH

Gossypol

(G)

OH

8-Hydroxy-(+)-

-cadinene

(+)--CadineneOPP

Farnesyldiphosphate

(FDP)

dCS

XO

HO

HO

HO

HO

CHO

OH

CHO

HO

HO

O

O

CHO

HO

HO

O

O

+

Desoxyhemigossypol

(dHG)

Heliocide H2

Heliocide H3

Hemigossypol

(HG)

+

CHO

HO

HO

O

O

Hemigossypolone

(HGQ)

CHO

HO

HO

O

O

Heliocide H1

CHO

HO

HO

O

O

HeliocideH4

Myrcene

-Ocimene

HO

HO

CHO

OH

OH

OH

HCO

OH

Gossypol

(G)

OH

8-Hydroxy-(+)-

-cadinene

(+)--CadineneOPP

Farnesyldiphosphate

(FDP)

dCS

X

O

HO

HO

HO

HO

CHO

OH

CHO

HO

HO

O

O

CHO

HO

HO

O

O

+

Desoxyhemigossypol

(dHG)

Heliocide H2

Heliocide H3

Hemigossypol

(HG)

+

CHO

HO

HO

O

O

Hemigossypolone

(HGQ)

CHO

HO

HO

O

O

Heliocide H1

CHO

HO

HO

O

O

HeliocideH4

Myrcene

-Ocimene

HO

HO

CHO

OH

OH

OH

HCO

OH

Gossypol

(G)

OH

8-Hydroxy-(+)-

-cadinene

(+)--CadineneOPP

Farnesyldiphosphate

(FDP)

dCS

X

O

HO

HO

HO

HO

CHO

OH

CHO

HO

HO

O

O

CHO

HO

HO

O

O

+

Desoxyhemigossypol

(dHG)

Heliocide H2

Heliocide H3

Hemigossypol

(HG)

+

CHO

HO

HO

O

O

Hemigossypolone

(HGQ)

CHO

HO

HO

O

O

Heliocide H1

CHO

HO

HO

O

O

HeliocideH4

Myrcene

-Ocimene

HO

HO

CHO

OH

OH

OH

HCO

OH

Gossypol

(G)

OH

8-Hydroxy-(+)-

-cadinene

(+)--CadineneOPP

Farnesyldiphosphate

(FDP)

dCS

X

O

HO

HO

HO

HO

CHO

OH

CHO

HO

HO

O

O

CHO

HO

HO

O

O

+

Desoxyhemigossypol

(dHG)

Heliocide H2

Heliocide H3

Hemigossypol

(HG)

+

CHO

HO

HO

O

O

Hemigossypolone

(HGQ)

CHO

HO

HO

O

O

Heliocide H1

CHO

HO

HO

O

O

HeliocideH4

Myrcene

-Ocimene

HO

HO

CHO

OH

OH

OH

HCO

OH

Gossypol

(G)

OH

8-Hydroxy-(+)-

-cadinene

(+)--CadineneOPP

Farnesyldiphosphate

(FDP)

dCS

XO

HO

HO

HO

HO

CHO

OH

CHO

HO

HO

O

O

CHO

HO

HO

O

O

+

Desoxyhemigossypol

(dHG)

Heliocide H2

Heliocide H3

Hemigossypol

(HG)

+

CHO

HO

HO

O

O

Hemigossypolone

(HGQ)

CHO

HO

HO

O

O

Heliocide H1

CHO

HO

HO

O

O

HeliocideH4

Myrcene

-Ocimene

HO

HO

CHO

OH

OH

OH

HCO

OH

Gossypol

(G)

OH

8-Hydroxy-(+)-

-cadinene

(+)--CadineneOPP

Farnesyldiphosphate

(FDP)

dCS

X

O

HO

HO

HO

HO

CHO

OH

CHO

HO

HO

O

O

CHO

HO

HO

O

O

+

Desoxyhemigossypol

(dHG)

Heliocide H2

Heliocide H3

Hemigossypol

(HG)

+

CHO

HO

HO

O

O

Hemigossypolone

(HGQ)

CHO

HO

HO

O

O

Heliocide H1

CHO

HO

HO

O

O

HeliocideH4

Myrcene

-Ocimene

HO

HO

CHO

OH

OH

OH

HCO

OH

Gossypol

(G)

OH

8-Hydroxy-(+)-

-cadinene

(+)--CadineneOPP

Farnesyldiphosphate

(FDP)

dCS

X

X8-Hydroxy-(+)-

(HGQ)

dCS

(+)--Cadinene-Cadinene

Hemigossypolone

(dHG)Desoxyhemigossypol

(HG)Hemigossypol

(G)

Gossypol

-OcimeneMyrcene

Farnesyldiphosphate

+ +

(FDP)

Heliocide H2 Heliocide H3 Heliocide H1 Heliocide H4

O

HO

HO

HO

HO

CHO

OH

CHO

HO

HO

O

O

CHO

HO

HO

O

O

+

Desoxyhemigossypol

(dHG)

Heliocide H2

Heliocide H3

Hemigossypol

(HG)

+

CHO

HO

HO

O

O

Hemigossypolone

(HGQ)

CHO

HO

HO

O

O

Heliocide H1

CHO

HO

HO

O

O

HeliocideH4

Myrcene

-Ocimene

HO

HO

CHO

OH

OH

OH

HCO

OH

Gossypol

(G)

OH

8-Hydroxy-(+)-

-cadinene

(+)--CadineneOPP

Farnesyldiphosphate

(FDP)

dCS

XO

HO

HO

HO

HO

CHO

OH

CHO

HO

HO

O

O

CHO

HO

HO

O

O

+

Desoxyhemigossypol

(dHG)

Heliocide H2

Heliocide H3

Hemigossypol

(HG)

+

CHO

HO

HO

O

O

Hemigossypolone

(HGQ)

CHO

HO

HO

O

O

Heliocide H1

CHO

HO

HO

O

O

HeliocideH4

Myrcene

-Ocimene

HO

HO

CHO

OH

OH

OH

HCO

OH

Gossypol

(G)

OH

8-Hydroxy-(+)-

-cadinene

(+)--CadineneOPP

Farnesyldiphosphate

(FDP)

dCS

X

O

HO

HO

HO

HO

CHO

OH

CHO

HO

HO

O

O

CHO

HO

HO

O

O

+

Desoxyhemigossypol

(dHG)

Heliocide H2

Heliocide H3

Hemigossypol

(HG)

+

CHO

HO

HO

O

O

Hemigossypolone

(HGQ)

CHO

HO

HO

O

O

Heliocide H1

CHO

HO

HO

O

O

HeliocideH4

Myrcene

-Ocimene

HO

HO

CHO

OH

OH

OH

HCO

OH

Gossypol

(G)

OH

8-Hydroxy-(+)-

-cadinene

(+)--CadineneOPP

Farnesyldiphosphate

(FDP)

dCS

X

dCH

O

HO

HO

HO

HO

CHO

OH

CHO

HO

HO

O

O

CHO

HO

HO

O

O

+

Desoxyhemigossypol

(dHG)

Heliocide H2

Heliocide H3

Hemigossypol

(HG)

+

CHO

HO

HO

O

O

Hemigossypolone

(HGQ)

CHO

HO

HO

O

O

Heliocide H1

CHO

HO

HO

O

O

HeliocideH4

Myrcene

-Ocimene

HO

HO

CHO

OH

OH

OH

HCO

OH

Gossypol

(G)

OH

8-Hydroxy-(+)-

-cadinene

(+)--CadineneOPP

Farnesyldiphosphate

(FDP)

dCS

X

Biosynthetic Pathway for Gossypol and Related Terpenoids

dCS = -cadinene synthase

dCH = -cadinene hydroxylase

(G)

Gossypol

O

HO

HO

HO

HO

CHO

OH

CHO

HO

HO

O

O

CHO

HO

HO

O

O

+

Desoxyhemigossypol

(dHG)

Heliocide H2

Heliocide H3

Hemigossypol

(HG)

+

CHO

HO

HO

O

O

Hemigossypolone

(HGQ)

CHO

HO

HO

O

O

Heliocide H1

CHO

HO

HO

O

O

HeliocideH4

Myrcene

-Ocimene

HO

HO

CHO

OH

OH

OH

HCO

OH

Gossypol

(G)

OH

8-Hydroxy-(+)-

-cadinene

(+)--CadineneOPP

Farnesyldiphosphate

(FDP)

dCS

X

(Cytosol)Mevalonate (MVA) pathway

IPP

DMAPP

Page 32: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

0

2

4

6

8

10

12

Wild-type

3 4 5 6 7 8 9 10

14

LCT66-2

mg

go

ss

yp

ol

mg

-1ti

ss

ue

LCT66-32

10.45 0.54 0.54 0.07* 0.46 0.13*

(n=10) (n=8) (n=8)

1 2 3 4 5 6 7 8 9 101 2 3 4 5 6 7 8 9 101 2

Gossypol Levels in Individual Wild-type and T1 Cottonseeds

Sunilkumar …… Rathore (2006)

PNAS 103:18054-18059

U.S. Patent #7999148

Page 33: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

mg

te

rpe

no

ids

mg

-1ti

ss

ue

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Bract

0.72

0.60

0.48

0.36

0.24

0.12

0.00

Floral Bud

1.8

1.5

1.2

0.9

0.6

0.3

0.0

Boll

mg

te

rpe

no

ids

mg

-1ti

ss

ue

G HGQ H

Terminal bud

Root

5

4

3

2

1

0

G

Petal

8

6

4

2

0

G MG DMG

2.5

2.0

1.5

1.0

0.5

0.0

G HGQ H G HGQ H

G HGQ H dHG dMHG

0.10

0.08

0.06

0.04

0.02

0.00HG MHG

0.6

0.5

0.4

0.3

0.2

0.1

0.0

Levels of Gossypol and Related Terpenoids in

Terminal Bud, Floral Bud, Bract, Petal, Boll, and Root Tissues

Wild-type Control

LCT66-2

LCT66-32

ULGCS trait is

completely tissue

specific

Sunilkumar …… Rathore (2006)

PNAS 103:18054-18059

U.S. Patent #7999148

Page 34: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

Ultra-low Gossypol Cottonseed (ULGCS) – Field Trials 2009, 2010 & 2011

• ULGCS Stability

• ULGCS Specificity

• Fiber/seed Yield & Quality

Page 35: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

6,466,35

7,48

0,15 0,14 0,140,31 0,21 0,38

0,45

0

1

2

3

4

5

6

7

8

2009 2010 2011

ug

goss

ypo

l/m

g se

ed

-ke

rne

l tis

sue

***

Coker 312 66-49B 66-81 66-250

Coker 312

66-49B

66-81

66-250

ULGCS Trait is stable under field conditions

Page 36: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

0

50

100

150

200

250

2009 2010 2011

Yie

ld (

g/p

lan

t)

Seed

** **

0

20

40

60

80

100

120

2009 2010 2011Y

ield

(g/

pla

nt)

Fiber

**

**

**

**

**

Coker 312

66-49B

66-81

66-250

Coker 312

66-49B

66-81

66-250

No adverse effect on yield

Page 37: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

No adverse effect on

fiber quality

Page 38: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

ULGCS trait is stable under field conditions

Foliage and Floral tissue have normal terpenoid levels

No adverse effect on yield or quality of fiber/seeds

Page 39: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

Regular CottonSeed Gossypol: 1%

Insect Protected Foliage

Glandless CottonSeed Gossypol: 0.001%

No insect protection

Engineered Cotton(ULGCS)

Seed Gossypol: 0.01%

Insect Protected Foliage

Seed

ker

ne

lLe

afM

agn

ifie

d L

eaf

Page 40: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

Other possible projects for collaboration

Tissue-specific & Inducible Promoters

Nematode Resistance through RNAi

Resistance to old world bollworm and boll weevil

through Bt or RNAi????

Genome editing system for cotton????

Page 41: Cotton Biotechnology Keerti S. Rathore - CBA · Cotton Biotechnology Congresso Brasileiro Do Algodao, 2015 Brazil Cotton Congress, 2015 Keerti S. Rathore ... C h icke n 2.047 b S

USDA-ARS

Robert Stipanovic

Lorraine Puckhaber

Alois Bell

IPGB, TAMU

G. Sunilkumar

LeAnne Campbell

Chandrakanth Emani

Sabarinath Sundaram

Sylvain Marcel

Sreenath Reddy Palle

Devendra Pandeya

Lauren Tollack

Vilas Parkhi

Vinod Kumar

Sameer Joshi

Shanna Sherwood

Madhusudhana Janga

Teressa McIntyre Sam Reddy

Robert A. Creelman

J. Liu

J. Shah

Cotton Inc., TxCOT, TFFC, THECB & TALR

C. Wayne Smith

Steve Hague

Charles Kenerley

Jim Starr

Clint Magill