Vitamin A deficiency affects hundreds of thousands of children yearly. Golden rice was engineered to produce beta-carotene in the endosperm by introducing phytoene synthase and other genes. New lines of Golden rice produce higher levels of beta-carotene, up to 31 micrograms per gram, through genes from different plants controlled by specific promoters. This helps combat vitamin A deficiency in areas reliant on rice.

2. Introduction
 Vitamins are the very basic building blocks required over a long
period of time (lifelong) to build a strong, healthy, disease free
body.
 Vitamin A essential for vision (also known as Retinol), cell
growth, reproductive functions and maintaining immune system.
 Dependance on rice as predominant food source-VAD(vit A
deficiency).because in rice – no b carotene(vitamin A).
 VAD- 250,000-500,000 chilldren blind every year.
 Biofortified crop like Golden rice – offer sustainable solution to
VAD.

3.  Golden rice prototype (1999) – accumulated around 1.6ug/g β
carotene in the grain.
 New line GR2 (by tissue specific promoter) – produced 31ug/g
β carotene .
 Vitamin A - first synthesized in 1947 by two Dutch chemists,
David Adriaan van Dorp and Jozef Ferdinand Arens.

4. Increase severeness of common childhood infection
Impaired
Epithelial
integrity
Impaired
Impaired
skeletal
vision
growth
VAD
Reduced
Impaired
Immune
haemopoises
response

5. Improving the nutritional value of Golden rice
through increased pro-vitamin A content
Jacqueline A Paine1, Catherine A Shipton1, Sunandha Chaggar1,
Rhian M Howells1, Mike J Kennedy1, Gareth Vernon1, Susan Y
Wright1, Edward Hinchliffe2, Jessica L Adams3, Aron L Silverstone3
& Rachel Drake1

6. Golden Rice
 Variety of rice engineered to produce β-carotene (pro-vitamin A)
to combat vitamin A deficiency.
 Carotenoids- plant pigment- precursor of vitamin A so known as
pro-vitamin A
 Produce Carotenoids in the endosperm of grain so, giving
characteristic “ yellow colour ”.
 Phytotene synthase – limiting regulatory steps for Carotenoid
biosynthesis.

7.  In case of Canola seed, crtB (gene encoding bac phytotene
synthase) expression alone – increased carotenoids
production.
 In wild type Rice endosperm, first barrier to Carotenoid
biosynthesis – phytotene synthase and carotene desaturase –
provided by daffodil psy and crt transgenes.

8. Expression of a psy transgene increases the
carotenoid content of maize callus
Gene cassettes in the two plasmid used to cotransform maize callus. Both
contain the maize polyubiquitin1 promoter (Ubi1) and the nos terminator
(nos). (i) The seven similar plasmids constructed with the phytoene
synthase-coding region (psy) from each of the species listed below. (ii) The
phosphino N-acetyl transferase (pat) selectablemarker and beta-
glucuronidase (gus) gene cassettes.
Paine et al.Nature biotechnology(2005)

9. Photograph showing individual maize calli cotransformed with the
plasmid containing the maize psy (right, Zm psy) and an empty vector
(EV) control (left).
Paine et al.Nature biotechnology(2005)

10. Histogram showing the total colored carotenoid content of maize calli transformed with a
given psy gene (from Arabidopsis thaliana (At), Daucus carota (Dc), Narcissus
pseudonarcissus (Np), Zea mays (Zm), Capsicum annuum (Ca), Oryza sativa (Os) or
Lycopersicon esculentum (Le)). Data shown represents the 75th percentile for each
population of transgenic calli expressed as a percentage of the median empty vector (EV)
control value. The second y-axis (diamonds) shows the percentage of calli from each
population with a carotenoid content more than fivefold that of the EV median.
Paine et al.Nature biotechnology(2005)

11.  High level of carotenoid accumulate by the
mechanism of carotenoid sequestration including
crystallization, oil deposition, membrane
proliferation or protein lipid sequestration.
 Starchy food matrix of the rice grain + fat content
→ facilitate intestinal β carotene uptake.

12. Carotenoid enhancement of the rice endosperm by
transformation with psy orthologues and crtI.
T-DNAs used to generate transgenic rice plants. The T-DNA comprised
the rice glutelin promoter (Glu) and the first intron of the catalase gene
from castor bean (I), E. uredovora crtI functionally fused to the pea
RUBISCO chloroplast transit peptide (SSUcrtI) and a phytoene synthase
from each of five plant species (psy), with a nos terminator, as well as a
selectable marker cassette comprising the maize polyubiquitin (Ubi1)
promoter with intron, hygromycin resistance (hpt) and nos terminator.
Paine et al 2005 ,Nature biotechnology

13. Photograph of polished wild-type and transgenic rice grains
containing the T-DNA with the daffodil psy (Np) or maize psy
(Zm) showing altered color due to carotenoid accumulation.
Paine et al.Nature biotechnology(2005)

14. Histogram showing the total carotenoid content of T1 rice seed
containing a T-DNA (as above) with the psy gene from either rice,
maize, pepper, tomato or daffodil from the five events with the
highest carotenoid content for each T-DNA.
Paine et al.Nature biotechnology(2005)

15.  Analysis of T2 seed showed -carotenogenic
ability was stable and heritable for all psy
cDNAs, and high levels of carotenoids were
again observed in seed from homozygous
progeny containing the maize psy/crtI
transgenes (over 16 mg/g).

17.  Why daffodil psy show low expression?
 daffodil psy – itself barrier to even higher level of
carotenoid accumulation in Golden rice.
 Although there was no shortage of precursor geranyl
geranyl diphosphate and no problem with product
sequestration.
 Overcome by providing psy of other species.
 Reason for differing efficacy of psy1 – difference in
transgene transcription under the control of same promotor.

18.  The increase in total carotenoid content brought about by the
more highly effective psy genes is largely due to a preferential
increase in b-carotene rather than a proportional increase in all
carotenoids.
 increases in the amount of b-carotene in transgenic tomato were
associated with a reduced total carotenoid content possibly
because of feedback inhibition at the level of phytoene synthase
activity.

19.  Explanation for high b-carotene level:-
 Downstream processing of carotene to xanthophylls does
not keep pace with the rate of flux through the pathway
when an efficacious PSY is expressed. so, b-carotene
accumulate.
e. Pathway endpoint may be influenced by sequestration,
rendering b-carotene inaccessible to downstream
hydroxylases.

20. Golden rice 2
Schematic diagram of the T-DNA in pSYN12424 used to create Golden Rice 2. The T-
DNA components with a selectable marker cassette comprising the maize polyubiquitin
(Ubi1) promoter with intron, phosphomannose isomerase gene (pmi) and nos
terminator. The use of an intron was abandoned because it was shown to have no effect
on carotenoid accumulation.
The golden rice reported here has up to 37 µg/g carotenoid of which 31 µg/g is b-
carotene (23 fold increase).

21. Engineering the b-carotene biosynthetic pathway into rice endosperm
Heldt-Plant biochemistry

22. Engineering the provitamin A biosynthetic pathway into rice endosperm
Xudong Ye,1*† Salim Al-Babili,2* Andreas Klo¨ ti,1‡ Jing Zhang,1
Paola Lucca,1 Peter Beyer,2§ Ingo Potrykus1§
 Immature rice endosperm – synthesize geranylgeranyl diphosphate –
uncoloured carotene.
 β carotene synthesis require –
 Phytotene synthase (psy)
 Phytotene desaturase (crt)
 ζ carotene desaturase Introduce 2 double bond
 Lycopene β cyclase
 Agrobacterium mediated transformation – to introduce the entire β carotene
biosynthetic pathway in rice endosperm.

23. Structure of T-DNA region and representative Southern blots of pB19hpc
used in single transformation
LB, left border;
RB, right border;
“!”, polyadenylation
signals;
p, promoters; psy,
phytoene synthase;
crtI, bacterial
phytoene desaturase;
tp, transit peptide.

24. Structure of T-DNA region and representative Southern blots of
pZPsC and pZLcyH used in co-transformation

25. Phenotypes of transgenic rice seeds
Panel 1, untransformed control; panels 2 pZPsC/pZLcyH co-transformants lines z5
through 4, pB19hpc single transformants (panel 1), z11b (panel 2), z4a (panel 3),
lines h11a (panel 2), h15b (panel 3), h6 z18 (panel 4).
(panel 4).

26. Analysis of carotenoid from transgenic lines
Line h2b
Control seed (Single transformants)
Line z!!b Line z4b
(Co-transformats) (Co-transformats)