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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