1. SUCCESS STORIES OF
V ITAMINE & AMINO ACIDS
ENHANCEMENT IN
TRANSGENIC CROPS

2. What is a Transgenic Crop?
Transgenic indicates that a transfer of
genes has occurred using recombinant
DNA technology.
Generally a transgenic crop contains one or
more genes that have been inserted
artificially either from an unrelated plant
or from different species altogether.

3. How are Transgenic Crops
Made?
• In order to make a transgenic crop, there are
five main steps:-
1. Extracting DNA.
2. Cloning a gene of interest.
3. Designing the gene for plant infiltration.
4. Transformation.
5. Breeding.

4. STEPS INVOVED IN TRANSGENICS:-

5. VITAMINES
VITAMINE
S

6. VITAMIN E
•Vitam in E is a group of eight hyd rophobic com pound s
(known as vitam ers), the m ost potent of which is a-
tocopherol.
Vitam in E is obtained m ainly from seed s.
•Function:- prevent the oxidation and polymerization of
unsaturated fatty acids in body.
•Deficiency:-
•general wasting
•kid ney d egeneration
•infertility
•The levels of vitam in E activity can be increased either by
increasing the total am ount of vitam in E or by shifting the
m etabolic flux toward s a-tocopherol

7. In plants, tocopherol synthesis requires input from two
metabolic pathways :-
1.T shikimae pahw ygener t homogentisic acid, w f ms t aomaic r oft
he t t a aes hich or he r t ing ocopher ,
ol
2.T side cha is der ed fom phytyldiphosphate, apr oft met l yhr olphosphae(M pahw y
he in iv r oduct he hyer t it t EP) t a.
•These precursors are joined together by homogentisic acid
prenyltransferase(HPT) to form the intermediate 2-methyl-6-
phytylbenzoquinol(MPBQ) .
•M Qis t substae f t o enzy t
PB he r t or w mes, ocopher cy a a M Qmet lr nsf a
ol cl se nd PB hyta er se.
Tocopherol cyclase form d -tocopherol
MPBQ methyltransferase forms 2,3-dimethyl-5-phytylbenzoquinol.
T a ion oft
he ct ocopher cy a on 2,3 hy-5 phyybenzoquinol
ol cl se -dimet l t l
produces g-t
ocopher . B h g-t ol nd ocopher ae substaes f g-tocopherol
ol ot ocopher a d-t ol r r t or
methyltransferase pr oducing a- a b-t
nd ocopher , r iv y
ol espect el .

8. shikimate pathway MEP pathway
Homoginistic acid Phytyldiphospate
HPT
2 methyle-6-phytyle
bezoquinol {MPBQ} MPBQ
Tocopherol
Methyle
Cyclase
transferase
2,3 dimethyle 5-
d-tocopherol
phytylebenzoquinol
Tocopherol
TMT Cyclase
TMT
a & b-tocopherol g-tocopgerol
Tocopherol synthesis

9. •Shintani and Della-Penna expressed the Arabid opsis
genes encod ing g-tocopherol m ethyltransferase (g-TM T)
in Arabid opsis seed s, resulting in a fund am ental shift of
g/d-tocopherol a/b-tocopherol
this showed that nutritional enhancem ent in plants was
possible without altering total vitam in E levels.
•The expression of Arabid opsis hom ogentisic acid
prenyltransferase (H PT) prod uced twice the level of
vitam in E found in norm al seed s.

10. In case of soybean
• The Arabid opsis genes encod ing 2-methyl-6-
phytylbenzoquinol (MPBQ)
methyltransferase and g -TMT were used .
• Transgenic soybeans showed a significant
elevation in the total am ount of vitam in E
activity (fivefold greater than that of wild -type
plants), which was attributable m ainly from its
norm al 1 0% of total vitam in E to over 95% .

11. VITAMIN A
Vitam in A d eficiency is prevalent in the d eveloping world
and is probably responsible for the d eaths of two m illion
child ren annually.
• Deficiency :- blind ness
•H um ans can synthesize vitam in A if provid ed with the
precursor m olecule b -carotene (provitam in A), a pigm ent
found in m any plants but not in cereal grains.
•Therefore, a strategy was d evised to introd uce the correct
m etabolic steps into rice end osperm to facilitate b -
carotene synthesis.

12. GOLDEN RICE
•Professor Ingo Potrykus, Dr. Peter
Beyer & other European scientists in
august 1999.
•At Swiss Federal Institute of
Technology & University of Freiburg
in Germany.
•Produced by combining genetic
material from:-
•daffodils,
•peas, and
•Japonica rice.

13. Donor DNA
Plasmid vector
Selectable
antibiotic
resistance
marker Donor DNA cut
with EcoRI
Donor DNA fragments
Vector cut
with EcoRI
Add DNA ligase Plasmids
Tetracycline-resistant
Bacterial colony from
transformed cell
Introduce into
E. coli
Recombinant DNA
Transformed cell

14. PLANT GENE TRANSFER
VIA AGROBACTERIUM
The bacterium that
causes crown gall
disease in plants has
a natural vector for
transformation of
desirable traits from
one plant to another.
T-DNA

15. Agrobacterium tumefaciens
A specific gene is chromosomal
“cut out” of the DNA
Plasmid DNA is donor DNA using
cut open with the same enzyme.
an enzyme.
plasmid DNA
New gene is When the plant cell
inserted into divides, each daughter
the plasmid. cell receives the new
Plasmid is transformed gene, giving the whole
into Agrobacterium. plant a new trait.
When mixed with plant The new gene is transferred
cells, Agrobacterium into the chromosomal DNA
duplicates the plasmid. of the plant cell.

16. Cloned Gene in Vector DNA Molecule
Biolistic bombardment
Transformation of
(gene gun)
Agrobacterium
Protoplast transformation Agrobacterium-mediated
followed by cell wall transformation of plant
regeneration cell
Migration and integration of
gene into nucleus
Plant cells Regeneration of
grown in genetically
tissue culture modified plant
from tissue
culture

17. BIOSYNTHESIS OF b-CAROTENE
• Joining of two geranylgeranyl
diphosphate(GGDP) molecules to
form the precursor phytoene.
•The conversion of phytoene into
b-carotene requires three
additional enzyme activities:
•phytoene desaturase
•b-carotene desaturase
•lycopene b-cyclase.
•Cereal grains, such as rice,
accumulate GGDP but lack the
subsequent enzymes in the
pathway, so the genes for all three
enzymes are required to form b-
carotene.

18. •This has led to sim ilar progress in other crops,
includ ing, m ost recently, “yellow potato’, ‘orange
cauliflower’, carrots with enhanced b-carotene in
the taproot and tom atoes with the b-carotene
m etabolic pathway transferred to the plastid s.
•A recently d eveloped potato variety containing the
phytoene synthase, phytoene d esaturase and
lycopene b-cyclase from E rwinia herbicola
contained 1 1 4 mg carotenoid s per gram of d ry
weight and 47 mg b-carotene per gram of d ry
weight.

19. FOLATES
• Folate is a B-group vitamin critical for normal
cellular function and division.
• Deficiency:-
• megaloblastic anaemia
• cardiovascular disease
• cancers and
• cognitive decline
• spina bifida and anencephaly

20. •Folate is produced from multistep process from:-
• pteridine synthesized in cytosol
•glutamate moieties
•p-aminobenzoate(PABA) synthesized in plastid
•These moieties are then transported to the mitochondria,
where they condense to form dihydropteroate and are
conjugated to glutamate.
•Rice plants transgenic for wheat 6-hydroxymethyl-7,8-
dihydropterin pyrophosphokinase/7,8-dihydropteroate
synthase(HPPK/DHPS) which operates at a central point in the
production pathway, gives elevated folate levels.

21. The folate production pathway. PABA is synthesized from
chorismate in the chloroplast, pterin is synthesized in the cytoplasm.
These are transported into the mitochondria where the two are
condensed and the product glutamated.

22. PROCEDURE
•Wheat HPPK/DHPS cDNA was isolated.
•It was cloned into Sma1-Sac1 digested bombardment vector
pUbi.gfp.nos. so as to replace the green fluorescent protein(gfp)
fragment with HPPK/DHPS.
•Introduced the gene under the control of the maize ubiquitin
promoter into the Australian rice variety Jarrah of Oryza Sativa
via particle bombardment using the Biolistic PDC-
1000/He system.
•Transgenic plants were selected for by growth on hygromycin
media, and further subject to PCR for the presence of the HPPK/
DHPS gene.

23. AMINO ACIDS

24. Introduction
The nutritional quality of cereals and legumes has been improved by
using biotechnological methods. Two genetic engineering
approaches have been used to improve the seed protein quality.
• First case:- A transgene (e.g. gene for protein containing sulphur
rich amino acids) was introduced into pea plant (which is deficient
in methionine and cysteine, but rich in lysine) under the control of
seed-specific promoter.
Second case:- The endogenous genes are modified so as to increase
the essential amino acids like lysine in the seed proteins of cereals.
These transgenic routes have helped to improve the essential amino
acids contents in the seed storage proteins of a number of crop
plants. E.g. overproduction of lysine by de-regulation.

25. Enhancement of Mithionine & cysteine in pea
• It is based on INTODUCTION OF TRANSGENE APPROACH.
•a new gene encoding for storage protein rich in
deficient amino acid is intoduced into crop to correct
its amino acid deficiency.
•vicilin seed storage protein of pea(Pisum sativum) have 7%
lysine is deficient in the sulfur-containing amino acids
methionine and cysteine.
•Sunflower seeds protein, sunflower albumin 8(SFA8) has
23% mithionine+cysteine content.

26. •Gene cod ing for SFA8 is isolated and fused with vicilin gene
prom otor.
•Transferred the viciline gene prom otor-SFA gene construct into pea.
Viciline promotor-SFA8
gene construct
•This has enhanced the level of sulphur containing
am ino acid s upto 40%

27. Enhancement of Lysin in corn
•Corn has become the most productive major crop.
•Deficiency:- lysine
Strategies for lysine genetic engineering in corn:-
Supressing α-zein production:-
•Natural maize opaque mutants have nutritionally poor corn protein
known as α-zein.
•RNAi mechanism has been used to specifically suppress α-zein
production in transgenic corn, resulting in a doubling of the lysine
content of corn grain from 2400 ppm to 4800 ppm2.
•α-zeins comprise roughly 40% of the total kernel protein, but contain
almost no lysine. By reducing α-zeins, other lysine-containing kernel
proteins were comparatively increased, raising the lysine content in
corn protein from 2.8% to 5.4%.
•

28. RNAi mechanism to supress the α-zein production

29. genetically modify Lysine metabolic pathway
•lysine, along with methionine, threonine, and isoleucine, is
derived from aspartate.
•dihydrodipicolinate synthase (DHDPS) catalyzes the first
committed step of lysine biosynthesis.
•A bifunctional enzyme, lysine-ketoglutarate
reductase/saccharopine dehydrogenase (LKR/SDH), is responsible
for lysine catabolism.
•The free lysine level in plant cells is thought to be regulated by:-
• lysine feedback inhibition of DHDPS and
• feed-forward activation of LKR/SDH.

30. To Enhance the levels of lysine:-
•Activated the expression of a lysine feedback-
insensitive DHDPS from Corynebacterium
glutamicum, CordapA.
•suppressed the enzyme LKR/SDH
•To further enhance the accumulation of free lysine in
corn, we recently developed transgenic corn lines that
combine CordapA expression and LKR/SDH
suppression7, by using a novel bifunctional transgene
cassette.

31. •An inverted repeat sequence corresponding to partial
LKR/SDH cDNA was inserted into the intron of an
expression cassette containing CordapA as the coding
region.
•Principle:- the expression of this transgene should
generate an intron-derived, double-stranded RNA
against LKR/SDH and an mRNA encoding CordapA.

33. Lysin enhancement in sorghum
•Sorghum is one of the m ain staples of the world ’s poorest an m ost food -
insecure people.
•It have low nutritional quality because of low lysine content.
•Genetically enhancing the nutritional quality of grain sorghum by the
introd uction of genes encod ing:-
•Methionine-rich maize beta-zein
•L ysine-rich barley chymotrypsin inhibitor C I-2 proteins.
TRA NSG E NIC STRA TE GIE S
1 . Transgenic sorghum plants were prod uced via A grobacterium-
mediated transformation using im m ature zygotic em bryos as
explant.
2. by particle bombardment O f im m ature inflorescences and shoot

34. •Dihydropicolinate synthase, the first enzyme of the lysine-
specific pathway
•A functional gene which codes for a feedback insensitive
dihydropicolinate synthase, was introduced into the genome of
sorghum with the goal of producing transgenic sorghum plants
with increased lysine content
RE QIURE ME NTS:-
•Two transformable sorghum genotypes
•five A frican sorghum genotypes which are highly
regenerable and transform able.
•The plant expression vectors containing:-
1 . The reporter gene uid A (GU S),
2. The selectable m arker genes bar or hpt II,
3. The lysine-rich C I-2 gene und er control of the gam m a-zein
prom oter.

35. •Four constructs were prepared for particle
bombardment-mediated transformation of grain
sorghum:-
•One construct containing the wild type CI-2 gene
driven by the maize gamma-zein (γ-zein) promoter
•Three constructs were prepared containing the
genetically engineered CI-2 gene, with additional
lysine substitutions in a reactive loop or hairpin
region, driven by the maize gamma-zein (γ-zein)
promoter.
•Two constructs were prepared containing the
methionine-rich beta-zein gene or fusion protein gene
driven by gamma-zein promoter, respectively

36. Vector is introd uced into the sorghum genom e via A grobacterium-
mediated transformation of selected sorghum genotypes.

37. Enhancing protein quality in amaranthus albumin
potatoes
‘Increased nutritive value of transgenic potato by expressing
a nonallergenic seed albumin gene from Amaranthus
hypochondriacus’
• Potato is the fourth most abundant global crop and used for food,
animal feed and production of starch and alcohol
• Limited in lysine, tyrosine, methionine and cysteine
•Transformed potato with seed albumin from Amaranthus
hypochondriacus .
• Expression in tuber 5-10 fold higher with GBSS promoter than with 35S
promoter
•Total protein content also increased (35-45%)

38. • A gene that encod es a seed -specific protein, am aranth
seed album in (Am A1 ) from Amaranthus
hypochond riacus
• The Am A1 protein has great potential as a d onor protein
for the following reasons:-
(i) It is a well-balanced protein in term s of am ino acid
com position and even better than the values
recom m end ed by the World H ealthO rganization for
a nutritionally rich protein;
(ii) It is a nonallergenic protein in its purified form
(iii) It is encod ed by a single gene and thus would
facilitate gene transfer into target plants with less
d ifficulty.

39. • The expression plasm id pSB8 was constructed by
using AmA1 cod ing sequence along with 1 02 bp of 39
AmA1 untranslated region under the control of CaMV
35S promoter in pBI121and pSB8G, wherein 35S
prom oter was replaced by GBSS prom oter
2 alternative pSB8
constructs. p35S CaMV AmA1 Nos 3’
Promoters
constitutive or
tuber-specific
pGBSS AmA1 Nos 3’ pSB8G