Recent Advances in Breeding Golden Rice in Bangladesh

Partha S. Biswas, PhD

Project Leader, Golden Rice Project, BRRI, Bangladesh

Donald J. MacKenzie, PhDRegulatory Affairs & Stewardship Leader, Golden Rice Project,

IRRI, Philippines

Violeta Villegas, PhDCoordinator, Golden Rice Project, IRRI Philippines

Death becoming blind and suffering from other VAD related diseases

Consequences of VAD

Measles

Complex anemia

Blindness

9/15/20162

Approaches for combating VAD

Dietary diversification

Food fortification

Biofortification: a sustainable complementary approach

9/15/2016 3

Vitamin A deficiency in Bangladesh

0

5

10

15

20

25

Preschool agechildren

School agechildren

NPNL

1997

2012

BRRISource: ICDDRB, 2013

Animal source Vitamin A intake by food security status in preschool age children

BRRISource: ICDDRB, 2013

Richard J. Deckelbaum, 2005

Why biofortify rice for Vitamin A ?

Thus even a small increase in the nutritive valueof rice means healthier people

9/15/2016 6

Golden Rice

• A new type of rice that contains beta-carotene, giving its a unique yellow-orange color

• Beta-carotene is converted into vitamin A as required by the body

• Golden Rice 2 was developed in Japonica rice cultivar Kaybonnet through transformation –introducing maize phytoene synthase gene (Zmpsy1), bacterial phytoene desaturase (crtI), and selectable marker (pmi) from E. coli

GR2E BRRI dhan29

GR2E BRRI dhan29 Golden Rice was developed and selected at IRRI from cross between GR2E Kaybonnet Golden Rice and BRRI dhan29

Import Permit No.

IP-12.11.0000.012.39.026.14-8422 (2 March 2015)

Contained trial, 2015

Dr Robert Zeigler, DG, IRRI visited GR Screenhouse on 07 September 2015

Dr. VioletaVillegas

visited GR Screenhouse

on 03 September

2015

Selection criteria No. of entries

Very similar to BRRI dhan29 in grain type and growth

duration and plant height

13

Very similar to BRRI dhan29 in grain type and plant

height but 2-8 days late in maturity

30

Medium bold type grain and 2-5 days late in maturity 23

Grain finer than BRRI dhan29 and 2-5 late in maturity 8

Similar to BRRI dhan29 in grain type and growth

duration and plant height but BLB infestation was

noticed

8

Non uniform 88

Total 170

Summary results of homozygous GR2E BRRI dhan29 Golden

rice tested in contained trial at BRRI, April-September 2015

Genotype Statistic GD (days) PN PH (cm) PL (cm) FG/

plant

SSt (%) TC

(ug/g)

GY (g)

GR2E

BRRI

dhan29

(BC5F3)

Mean 132.7 9.5 128.8 26.6 71.2 58.9 11.0 11.5

SD 2.6 1.1 4.9 1.9 17.6 8.4 3.6 3.5

CV 2.0 11.3 3.8 7.1 24.7 14.3 32.3 30.5

Range (129 -138) (1.1-11.3) (116.7 -

139.3)

(21 -

30.3)

(32.2 -

178.2)

(27.2 -

80.5)

(3.5 -

26.3)

(6.4 -

27.5)

Control

BRRI

dhan29

Mean 128.5 10.25 132 29.15 82.7 57.25 - 15.6

SD 0.71 0.07 5.23 1.20 36.91 10.82 - 2.26

CV 0.55 0.69 3.96 4.12 44.63 18.90 - 14.50

Range (128 - 129) (10.3 -

10.3)

(135.7 -

128.3)

(30 -

28.3)

(108.8 -

56.6)

(49.6 -

64.9)

- (17.2 -

14.0)

Mean difference

(%)

3.3 -7.3 -2.4 -8.7 -14.0 2.9 NA -26.0

p-Value 0.215 0.051 0.287 0.032 0.038 0.2 NA 0.048

Agronomic performance of GR2E BRRI dhan29 Golden rice tested in

contained trial at BRRI, April-September 2015

0

50

100

150

200

250

300

350

400

450

5000

.7

2.9

5.0

7.1

9.3

11

.4

13

.5

15

.7

17

.8

19

.9

22

.1

24

.2

26

.3

28

.5

30

.6

32

.7

34

.9

37

.0

39

.1

41

.2

43

.4

45

.5

47

.6

49

.8

51

.9

54

.0

56

.2

58

.3

60

.4

62

.6

64

.7

66

.8

69

.0

71

.1

73

.2

Fre

qu

ency

of

pla

nts

Grain yield/plant (g)

Control BRRI dhan29

0

10

20

30

40

50

60

3.9 5.1 6.2 7.4 8.6 9.8 11.0 12.1 13.3 14.5 15.7 16.8 18.0 19.2 20.4 21.5 22.7 23.9 25.1 More

Freq

uen

cy o

f p

lan

ts

Total carotenoids (ppm)

Confined Field Trial, Boro 2016

Raising of seedlings at the transgenic screenhouse, Boro 2015-16.

(Dated on 23 January 2016)

Dr. Jiban Krishna Biswas, DG BRRI, Dr. Md

Shahjahan Kabir, Director (Administration

and Common Service), Dr. Md Anser Ali,

Director (Research) and Dr. Tamal Lata

Aditya, Head, Plant Breeding inaugurated

the transplanting activity planting the first

row by themselves and BRRI-IBC members

and Sonia Sultana, DD, DOE-Gazipur

District and Dr. Dilafroza Khanom, Head,

Biotechnology Division, BARI observed the

transplanting activities. (Dated on 14

February 2016)

A view of crop of pro-vitamin A enriched GR2E BRRI dhan29 showing

very uniform crop under confined field trial, Boro 2015-16

Gel image of GR2E specific STS marker analysis of the entries tested in CFT, Boro 2016.

Lane 1-23, 27-49 and 53 – 81 represent GR2E BRRI dhan29 introgression lines, lane 24,

50, 82 for null segregant, lane 25, 51, 81 for wild type BRRI dhan29 and lane 26, 51 and 83

for no DNA control

A view of grains of some pro-vitamin A enriched GR2E BRRI dhan29 showing very similar shape and size of grain to BRRI dhan29 under confined field trial, Boro 2015-16.

BRRI dhan29

A view of panicle of a pro-vitamin A enriched GR2E BRRI dhan29 line showing very similar architecture and size of panicle to BRRI dhan29 under confined field trial, Boro 2015-16

GR2-E Golden Rice

BRRI dhan29

Cooked rice of GR2E BRRI dhan29 Golden Rice at 2 months of

storage after harvest in room temperature

GR2-E BRRI dhan29 Golden Rice BRRI dhan29

Days to Maturity

Plant Height (cm)

Panicle Number

Panicle Length

(cm)

Filled Grains per

Plant

Percent Sterility

(%)

Event GR2E (n=69)

MeanRange

146.7(143–152)

106.8(101–115)

13.59(10–16.2)

26.6(22.6–28.6)

158(125–217)

17.68(10.4–27.1)

BRRI dhan29 (n=3)

MeanRange

147.7(147–149)

107.9(102–111)

12.27(10.6–13.6)

27.42(26–29.2)

155.5(145–163)

20.15(18.5–21.4)

Null Segregants (n=3)

MeanRange

146.3(145–148)

108.9(105–114)

13.53(12.2–14.2)

26.2(25–27.2)

152.9(128–167)

15.5(11.3–21.1)

p-Values

GR2E vs. Control 0.4470 0.2684 0.0625 0.1800 0.8038 0.3324

GR2E vs. Null 0.7650 0.0408 0.9399 0.5044 0.6207 0.3979Null vs. Control 0.3511 0.5897 0.0628 0.2901 0.7533 0.2680

Comparison of agronomic and phenotypic parameters between event GR2E, control BRRI dhan29, and null-segregants

Grain Length (mm)

Grain Width (mm)

Thousand Seed Weight

(g)

Yield(t/ha)

Amylose(% of

Starch)

Event GR2E (n=69)

MeanRange

8.43(7.86–8.83)

1.804(1.69–1.92)

20.81(18.9–23.8)

6.96(5.15–8.16)

23.45(18.1–29.4)

BRRI dhan29 (n=3)

MeanRange

8.58(8.45–8.77)

1.807(1.75–1.86)

21.4(20.8–22.4)

7.02(6.56–7.37)

28(27.8–28.2)

Null Segregants (n=3)MeanRange 8.57

(8.37–8.69)1.821

(1.79–1.86)21.58

(21.1–22)7.30

(7.08–7.64)23.87

(21.5–28.3)

p-Values

GR2E vs. Control 0.1345 0.8803 0.2897 0.8325 0.0011

GR2E vs. Null 0.1531 0.4748 0.1659 0.2631 0.7653Null vs. Control 0.9555 0.6737 0.7788 0.3504 0.2033

Comparison of agronomic and phenotypic parameters between event GR2E, control BRRI dhan29, and null-segregants (Contd)

0

5

10

15

20

25

30

35

40

45

8.4

9.1

9.4

9.8

10

.1

10

.4

10

.8

11

.1

11

.4

11

.8

12

.1

12

.4

12

.8

13

.1

13

.4

13

.8

14

.4

Average total carotenoids content (ug/g) at two months after harvest in the backcross introgression lines of GR2-E BRRI dhan29 Golden Rice grown under confined field trial condition, Boro 2015-16

Average : 11.2SD: 1.12

Sl . Designation DM PH GL GB TGW YLD Amy TC GA

1 IR 112062 GR 2-E:14-40-7-8 147 112 8.4 1.8 21.8 7.2 25.3 10.9 Tr2 IR 112062 GR 2-E:14-40-7-16 151 105 8.5 1.7 21.1 7.1 25.9 12.5 Tr3 IR 112060 GR 2-E:2-9-89-16 144 108 8.4 1.8 20.4 7.5 25.9 11.2 Tr4 IR 112060 GR 2-E:2-17-36-10 146 105 8.3 1.8 20.9 7.0 27.9 10.1 Tr5 IR 112062 GR 2-E:14-40-7-21 145 112 8.4 1.8 20.2 7.8 24.5 10.7 Tr6 IR 112060 GR 2-E:2-7-63-1 147 108 8.6 1.8 20.9 7.3 24.6 10.9 Tr7 IR 112062 GR 2-E:14-40-7-23 143 103 8.5 1.8 20.8 7.4 25.8 9.8 Tr8 IR 112060 GR 2-E:2-7-63-2 145 105 8.4 1.8 21.3 7.8 24.7 8.5 Tr

9 IR112064 GR2-E:3-6-73-2(Null segregant)

146 109 8.4 1.8 21.6 7.3 23.8 - Tr

10 BRRI dhan29 (Ck) 148 108 8.8 1.8 21.4 7.0 28 - TrLSD (0.05) 3.1 2.8 0.24 0.24 1.44 0.7 - -

List of selected backcross introgression lines of provitamin A

enriched GR2E BRRI dhan29

Note: DM = Days to maturity, PH = Plant height (cm), GL = Grain length (mm), GB = Grain breadth (mm), TWG = Thousand grain weight (g), YLD = Yield (t/ha), Amy = Amylose (%), TC = Total carotenoids (ug/g) content at two months after harvest, Tr = Translucent

Reaction of provitamin A enriched GR2E BRRI dhan29 Golden Rice to different diseases under confined field trial conditions

•None of the major diseases like Blast, Sheath blight, Bacterial blight

and Tungro were observed in BRRI dhan29 or the transgenic GR2E

BRRI dhan29 Golden Rice.

Reaction of provitamin A enriched GR2E BRRI dhan29 Golden Rice introgressed lines to different insect pests under confined field trial condition

• Only grasshoppers, leaf folder and stem borer infestation was

observed, however, infestation was below the economic threshold

level (ETL) both in vegetative and reproductive stage of the test

entries including non-transgenic BRRI dhan29.

• Vertebrate pests were not observed in experimental plot since the

confined trial plot was protected by active barrier system (ABS) and

chicken net.

Study on weediness, aggressiveness and dormancy period of provitamin A enriched GR2E BRRI dhan29 Golden Rice

• No unusual difference in morphological traits was

observed between transgenic GR2E BRRI dhan29

Golden Rice and non-transgenic BRRI dhan29 plants.

• No unusual difference in grain shattering was observed

between transgenic and non-transgenic plots.

• No unusual difference in the survivability of volunteers

or ratoons was observed between the transgenic and

non-transgenic plots.

• Also, no significant difference in dormancy period was

observed between transgenic and non-transgenic

materials.

SAFETY MEASURE ADOPTED FOR RISK MANAGEMENT DURING CONDUCTING TRIAL

Uprooting of seedlings from the seedbed in the contained screenhouse (Plate A), labelled properly with

tag (Plate B) and packed in poly vinyl plastic box (Plate C). Biosafety Officer Dr. Md Abdul Kader is

observing the packaging activities (Plate D).

DC

A B

A. Transportation of seedlings from screen house to CFT site and transplanting

A

C D

B

Transportation of seedlings to CFT site from transgenic screenhouse of BRRI. Biosafety Dr. Md Abdul Kader (left hand

side in plate A) observed and stewarded the seedlings transportation, Principal Investigator Dr. Partha S. Biswas (Right

hand side in Plate A) and Scientific Officer (Golden Rice Project) Md. Maksudul Haque(Plate B and D) were on board

in the car during seedling transportation.

Receipt of seedlings at the CFT site and distribution to the plots. Boxes were opened in presence of the

Biosafety Officers (Plate A), Md Maksudul Haque, Scientific Officer is seen to supervise seedling distribution to

the corresponding plots (Plate B- C). Primary containers (plastic bags) were destroyed by burning after

transplanting (Plate D).

AB

C D

Established crop of pro-vitamin A enriched BRRI dhan29 Golden Rice event GR2E in the confined filed trial. Three rows non-transgenic BRRI dhan29 were grown around each block and also 5 rows of corn plants were grown around the trial plots

B. Raising of guard rows and pollen barrier

3 rows BRRI dhan29

C. Isolation distance

D. Post planting field monitoring for unusual weed and abnormal plantsdevelopment

Regular monitoring of transgenic plots and premises of the experimental area for the existence of abnormal plants and weedy relatives. Ploughing of fallow land around experimental area for incorporating weeds into soil at interval of 15 days

E. Rat invasion management

Installation of net over the crop of pro-vitamin A enriched BRRI dhan29 event GR2E in the confined field trial to keep away the stray birds

F. Installation of net over the trial plots

G. Crop harvest and disposal of crop residues and straw:

H. Transfer of seeds to greenhouse

Seeds were collected in brown paper bags and transported to transgenic greenhouse in securely locked PVS box for additional data recording

H. Post harvest field monitoring

Post harvest monitoring of perpetuation of volunteer plants or ratoons at the experimental areas in the CFT site. A. Experimental plots were flooded immediate after harvest for 5-6 days to allow any dropped seeds to germinate, B. Demarked area for field monitoring of prohibited plants with red flags after puddling of the soil of the experimental area and premises with power tiller, C. Searching for rice seedlings during field monitoring, D. Field monitoring team (DOE personnel, Agronomist assigned for safety issues and BSO) with Principal Investigator at CFT site during a field monitoring

Marker assisted introgression of GR2E locus from GR2E BRRI dhan29 Golden Rice into BRRI dhan28, BRRI dhan49 and BRRI dhan62

Further Work with GR2E

Future Outlook

Multi-location testing of GR2-E BRRI dhan29 Golden Rice under Confined Field Condition

Materials : 8-10 entries of BC5F6 generationNo. of Sites : 5 Sites across the countryTest season: Boro 2017 and Boro 2018