Transgenic plant with improved nutritional quality

Presented by:
Kirti
Ph.D. (MBB)

Millions could be saved, if only ...
GM technology could help tackle
both poverty and health problems
facing developing countries — if
only those who oppose GM crops
would relax their stance and weigh
up the technology's costs and
benefits.

Golden Rice is now within reach

Why Rice?
• Global staple food and is especially
important in asia.
• Cultivated for over 10,000 years.
• Rice provides as much as 80 percent or
more of the daily caloric intake of 3 billion
people, which is half the world’s
population.
• To provide pro-vitamin A to third world i.e,
developing countries where malnutrition
and vitamin A deficiency are common.

• It is generally consumed in its milled form with outer layers
removed (Ye et al., 2000; Beyer et al., 2002).
• The main reason for milling is to remove the oil-rich aleurone
layer, which turns rancid upon storage.
• As a result, the edible part of rice grains consists of the
endosperm, filled with starch granules and protein bodies, but it
lacks several essential nutrients such as carotenoids exhibiting
provitamin A-activity.
• Vitamin A deficiency is a serious health
problem in at least 26 countries in Asia,
Africa and Latin America (Beyer et al.,
2002).

Who Began the Golden Rice Project?
The scientific details of the rice were first published in Science
(287:303-5, 2000), the product of an eight-year project by Ingo
Potrykus of the Swiss Federal Institute of Technology and Peter
Beyer of the University of Freiburg.
The Golden Rice Humanitarian Board encourages further
research to determine how the technology may play a part in the
ongoing global effort to fight VAD in poor countries.

inspiring to scientists all over the world…….
Funded by the Rockefeller Foundation, the Swiss Federal
Institute of Technology.
Syngenta, a crop protection company.

Golden Rice, the real thing
• Golden rice is a variety of Oryza sativa rice produced through genetic
engineering to biosynthesize beta-carotene, a precursor of vitamin A, in the
edible parts (endosperm) of rice.
• Vitamin A deficiency causes blindness among children and may even
lead to death.
According to the WHO, dietary vitamin A deficiency (VAD) causes some
250,000 to 500,000 children to go blind each year as a result of
xerophthalmia and keratomalacia.
• The US Recommended Daily Allowance (RDA) for vitamin A is 400µg
retinol/day for children aged 1-3 years and 800µg for adults.

Golden Rice – A golden opportunity?
• Vitamin A deficiency often occurs where rice is the staple food since rice
grain does not contain provitamin A i.e., β-carotene.
•Rice produces β-carotene in the leaves but not in the grain, where the
biosynthetic pathway is turned off during plant development.
•The resulting transgenic rice ‘golden rice’ contains good quantities of β-
carotene, which gives the grain a golden colour.
indicator of β-carotene concentration

Happy farmers, hungrier planet?
It has been shown that between 23 and 34 percent of child
mortality could be prevented by having a universal source of
vitamin A
Mothers about 40 percent of maternal mortality could be
prevented

Genetic engineering was the only way to produce GR
(‘Breeding where possible
Genetic modification where necessary‘)
there is no rice germplasm capable of synthesizing carotenoids
in the germplasm available.
Transgenic approach has become feasible during recent years
due to two reasons:
1. The rapid progress in the development of rice transformation
technologies through biolistic methods as well as using
Agrobacterium.
2. The availability of almost complete molecular elucidation of the
carotenoid biosynthetic pathway in numerous bacteria and plants
which provides ample choice of bacterial genes and plant cDNA
to select from.

Growers can reuse their seed as they please
Golden Rice offers people in developing countries a valuable and
affordable choice in the fight against the scourge of malnutrition.
A 2009 study of boiled golden rice fed to volunteers concluded that
golden rice is effectively converted into vitamin A in humans and a
2012 study of golden rice that fed 68 children ages 6 to 8, and
concluded that the golden rice was as good as vitamin A
supplements and better than the natural beta-carotene in spinach
(Tang et al., 2009).

The Science of Golden Rice
• Biosynthetic pathway of provitamin A is a continuation of
lycopene pathway.
• Immature rice endosperm is capable of synthesizing GGDP
(geranyl geranyl diphosphate) but subsequent stages of
pathway are not expressed in this tissue.
• The exogenous lyc gene has a transit peptide sequence
attached so it is targeted to the plastid, where geranyl geranyl
diphosphate formation occurs.

How does it work?
• The addition of 3 genes in the rice genome will complete the
biosynthetic pathway.
Phytoene synthase (psy) gene –derived from daffodils (Narcissus
pseudonarcissus)
fused to rice endosperm-specific glutelin (Gt1) promoter
• (Phytoene synthase is a transferase enzyme involved in the
biosynthes of carotenoids. It catalyzes the conversion of geranyl
geranyl pyrophosphate to phytoene).
Three steps required to convert : phtoene to β-carotene
Phytoene desaturase (pds) and ζ-carotene desaturase
to introduce double bonds to form lycopene.

Lycopene cyclase – from soil bacteria Erwinia uredovora
form rings in the beta-carotene (biosynthesis of carotenoids in the
endosperm).
Bacterial carotene desaturase capable of introducing all four
double bonds can be substituted for the Phytoene desaturase and
ζ-carotene desaturase.
Manipulation of Golden rice would require the introduction of 3
genes :
Phtoene synthase, Carotene desaturase, Lycopene beta-cyclase.
The daffodil psy gene rice glutelin promoter construct was inserted
into the vector pZPsC, along with the bacterial carotene desaturase
gene, (crt1) controlled by the 35S promoter.

Both enzymes were targeted to the plastid (the site of GGDP
synthesis): psy gene by its own transit peptide and the crt1 gene by
fusion to a pea ribulose-1,5-bisphosphate carboxlase/oxygenase
small subunit (rbcs) transit peptide sequence.
The lycopene β-cyclase gene with a functional transit peptide was
inserted into vector pZLcyH under the rice endosperm-specific
glutelin promoter along with hygromycin resistance marker gene.
(a) pZPsc
(b) pZLcyH

(c) pB19hpc
The three vectors constructed. pB19hpc is used in single transformation whereas
pZPsC and pZLcyH are used in co-transformation. LB, left borders; RB, right
borders; p, promoter; Gt1 glutelin; psy, phytoene synthase; crtl, bacterial carotene
desaturase; lcy, lycopene β-cyclase; aphIV, hygromycin resistant gene. These
three vectors were then spliced into a T-DNA vector for transformation
experiments. (Beyer et al., 2002).
Co-transformation
500 precultured immature embryos were inoculated with an
Agrobacterium mixture of LBA4404/pZCycH and LBA4404/pZLcyH
(Ye et al., 2000; Beyer et al., 2002).
The co-transformed plants were analyzed by Southern
hybridization (Ye et al., 2000; Beyer 2002).

• The presence of pZPsC was analyzed by restriction digestion (Ye
et al., 2000).
• To determine the formation of β-carotene, mature seeds from the
transform lines and control plants were air dried, de-husked and
polished with emery paper (Ye et al., 2000; Beyer et al., 2002).
The colour of the transformed endosperms was observed (Ye et
al., 2000; Beyer et al., 2002).
Single Transformation
• 800 rice immature embryos were inoculated with Agrobacterium
LBA 4404/pB19hpc with the presence of hygromycin (Ye et al.,
2000; Beyer et al., 2002).

• The hygromycin-resistance plants were analyzed by Southern
hybridization for the presence of psy and crtl genes (Ye et al.,
2000; Beyer et al., 2002).
• The endosperm of these plants' seeds were isolated and
appeared yellow, indicating carotenoid production (Ye et al.,
2000).
• High Performance Liquid Chromatography (HPLC) analysis
revealed the presence of β-carotene in transgenic endosperm
(Beyer et al., 2002).

-Carotene Pathway Problem in Plants
IPP
Geranylgeranyl diphosphate
Phytoene
Lycopene
 -carotene
(vitamin A precursor)
Phytoene synthase
Phytoene desaturase
Lycopene-beta-cyclase
ξ-carotene desaturase
Problem:
Rice lacks
these enzymes
Normal
Vitamin A
“Deficient”
Rice

The Golden Rice Solution
IPP
Geranylgeranyl diphosphate
Phytoene
Lycopene
 -carotene
(vitamin A precursor)
Phytoene synthase
Phytoene desaturase
Lycopene-beta-cyclase
ξ-carotene desaturase
Daffodil gene
Single bacterial gene crtI;
performs both functions
Daffodil gene
-Carotene Pathway Genes Added
Vitamin A
Pathway
is complete
and functional
Golden
Rice

• In one transgenic line, β-carotene content was as high as 85%
of the total carotenoids present in the grain.
• One explanation is that enzymes downstream along the
pathway, such as lycopene cyclases (lyc) and alpha- and beta-
carotene hydroxylases (hyd) are still being produced in non-
transformed rice endosperm, while psy and phytoene
desaturase (pds) and ζ-carotene desaturase (zds) are not.
• Synthesis of lycopene by psy and crt1 in transgenic plants
provides the substrate for these downstream enzymes.

The fact that a psy transgene alone led to phytoene accumulation but
not to desaturated products (Burkhardt et al., 1997) is evidence for
the absence of at least one active desaturase, namely pds.
Similarly, the expression of crtI alone did not produce any colour in
rice endosperm, because of the lack of psy activity.

Why do you think that Potrykus and his
co-workers initially used the less
effective biolistic transformation
method?
Rice is a monocot, and till then, the A. tumefaciens
method was restricted for use with dicots.

Improvements made to Golden rice
• The pmi (phosphomannose isomerase or mannose 6-phosphate
isomerase) mannose-selection gene was substituted to avoid
antibiotic selection using the hygromycin-resistance gene.
• The golden Rice trait was genetically engineered into indica rice
cultivars. Indica rice is consumed by 90% of the Asian
population, whereas the original Golden Rice was produced
using the japonica variety Taipei 309.
• Subsequent research indicated that the lycopene beta-cyclase
transgene was not required to produce beta carotene in the
endosperm.

A new Golden Rice generation
Golden Rice 2
Further work by Syngenta to optimize beta-carotene production showed
that the daffodil phytoene synthase was rate limiting and psy gene from
maize was much more effective (resulting in the greatest accumulation of
total carotenoids and -carotene)
After trying with psy genes from different sources it turned out that the
maize and rice genes gave the best results (Paine et al., 2005).
In the process, Golden Rice lines were obtained that accumulated up
to 37 μg/g carotenoids, of which 31 µg/g was β-carotene (as compared
to the first generation Golden Rice (original golden rice was called GR1)
where only 1.6 μg/g were obtained.

• Transformation of rice with the construct pSYN12424 resulted in
a 23 fold increase in carotenoids compared with the original
Golden Rice and has been named Golden Rice 2.
• To construct Golden Rice 2, the phytoene synthase gene (psy)
from maize and the carotene desaturase gene (crtI) from
Erwinia uredovora were inserted into rice.
Gt1p, crtI, nos, Zea mays phytoene synthase (psy), Zea mays
polyubiquitin Ubi–1 promoter with intron, E. Coli phospho-mannose
isomerase (pmi) selectable marker.

In 2006, Stein, for India, finds that the newer GR would reduce the
burden of VAD in India by 5-54%, depending upon assumptions
about adoption and who consumes it.
Although some beta-carotene is destroyed during cooking and not
all of it is absorbed into the body, the level of beta-carotene in
Golden Rice 2 is comfortably enough to prevent VAD in people
eating ordinary amounts of rice.

GR2 GR1
Wild-Type
Tiny grain with a giant footprint
The image clearly shows the progress made since the proof-of-concept stage of
Golden Rice. The new generation, also known as GR2 contains β-carotene levels
that will allow to provide an adequate amount of provitamin A in for children's diets in
SE Asia.

Cons behind ‘magic rice ’
Greenpeace and associated GMO opponents regard “golden
rice” as a “Trojan horse” that may open the route for other GMO
applications.
Health
 By promoting GE rice you encourage a diet based on one staple
rather than an increase in access to the many vitamin-rich food
plants. These plants would address a wide variety of
micronutrient deficiencies, not just VAD.
 GE crops (including GE rice) have the potential to cause allergic
reactions.
 Supply does not provide a substantial quantity as the
recommended daily intake.

• Environment
 Loss of Biodiversity. May become a gregarious weed and
endanger the existence of natural rice plants.
 Genetic contamination of natural, global staple foods.
• Culture
 Some people prefer to cultivate and eat only white rice based
on traditional values and spiritual beliefs.
• Financial interest
 The majority of patents for genetically engineered plants are held
by a few large multinational companies. So it's in their financial
interest – and not ours, the public – to get us hooked on their
seed

Improve level of IRON AND ZINC in Rice grains
Iron deficiency is the most widespread micronutrient deficiency
world-wide.
Affecting an estimated one-third of the world’s population and
causing 0.8 million deaths annually worldwide.
Anemia caused by iron deficiency triggers serious disorders such
as abortion, brain damage in infants, increase susceptibility to
infection.

Rice is a poor source of most of many essential micronutrients,
especially iron (Fe) and zinc (Zn), for human nutrition
(Zimmermann and Hurrell, 2002).
According to the World Health Organization (2010),
approximately two billion people suffer from iron deficiency.
The polished rice contains an average of only 2 mg kg-1 Fe and
12 mg kg-1 Zn (IRRI, 2006), whereas the recommended dietary
intake of Fe and Zn for humans is 10-15 and 12-15 mg per day,
respectively (Welch and Graham, 2004).

Rice actually has a lot of iron, but only in the seed coat.
Because unpeeled rice quickly becomes rancid in tropical and
subtropical climates, the seed is removed for storage.
A major cause is the poor absorption of iron from cereal and
legume-based diets high in phytic acid.
Besides having inherently low levels of Zn, wheat grain is also
rich in substances limiting utilization (bioavailability) of Zn in the
human digestive tract, such as polyphenols and phytic acid
(Welch and Graham, 2004).

Phytic acid is the major storage compound of phosphorus in grain.
By binding Zn, phytic acid reduces solubility of Zn in food and
restricts its utilization and retention in human body.
Most of the seed-Zn is located in the embryo and aleurone layer,
whereas the endosperm is very low in Zn concentration (Ozturk
et.al., 2006).
According to a Zn-staining study in wheat seed (Fig. 3), Zn
concentrations were found to be around 150 mg kg−1 in the
embryo and aleurone layer and only 15 mg kg−1 in the
endosperm (Ozturk et.al., 2006).

Embryo Aleuron Endosperm
Diphenyl thiocarbazone (DTZ) staining a wheat seed. When reacting with
Zn, DTZ forms a red DTZ-complex which indicates localization of Zn
(Ozturk et.al., 2006)
DTZ staining at increasing
Zn concentrations, mg kg-1

Global distribution of Zn deficiency-affected areas (Alloway
2004)

Over one third of the world's soils are considered Fe deficient.
In order to deal with the limiting amounts of Fe, plants have
evolved several strategies to obtain Fe from the soil.
The Strategy I mechanism includes proton extrusion to
solubilize Fe(III) in the soil, reduction of the solubilized
Fe(III) by a membrane-bound Fe(III) chelate reductase and
subsequent transport of the resulting Fe(II) into the plant root
cell by the Fe(II) transporter IRT1.

Strategy II is a chelation-based strategy involving release of
Fe(III)-specific phytosiderophores (PS) and subsequent uptake of
the Fe(III)-phytosiderophore complexes via a specific transport
system.

• Several groups have initiated efforts to increase iron by
expressing the ferritin gene from soyabean (Goto et al., 1999;
Drakakaki et al., 2000) and bean (Lucca et al., 2000) in rice
seed.
• Malnutrition of Fe and Zn which may weaken immune function
and impair growth and development of human (Welch, 2002)
afflict more than 50% of the world’s population (Tucker, 2003;
Welch, 2005).
• Zn and Fe deficiencies ranked 5th and 6th among the 10 most
important factors in developing countries.

Approaches for increasing the amount of iron
absorbed from rice-based meals
1. Introduced a ferritin gene from Phaseolus vulgaris
into rice grains, increasing their iron content up to two-
fold.
2. To increase iron bioavailability, introduced a
thermotolerant phytase from Aspergillus fumigatus into
the rice endosperm.
3. As cysteine peptides are considered a major
enhancer of iron absorption, overexpressing the
endogenous cysteine-rich metallothionein-like protein.

Plant Genes Help to Mobilize and Store Iron
One gene encodes nicotianamine synthase, the enzyme that
produces nicotianamine.
Nicotianamine chelates (metal ion) iron temporarily and facilitates its
transport in the plant.
Nicotianamine synthase is expressed under a constitutive promoter.
The second gene encodes the protein ferritin (consists of 24
subunits), which functions as a storage depot for up to four thousand
iron atoms per protein molecule in both plants and humans.
Iron Biofortification of Rice Targeted Genetic Engineering
Friday, January 22, 2010
By Christof Sautter and Wilhelm Gruissem
• .

Since the ferritin gene is under the control of an endosperm-
specific promoter, ferritin comprises a sink for iron in the center of
the endosperm.
The synergistic action of these two genes allows the rice plant to
absorb more iron from the soil, transport it in the plant, and store it
in the rice kernel.
A third gene encoding phytase was also engineered into this rice
line.
Phytase degrades phytate, a compound that stores phosphate
and binds divalent cations like iron and thus inhibits their
absorption in the intestine.

The genetically engineered lines expressing nicotianamine
synthase, ferritin, and phytase (NFP-line) contain up to a 6.3-fold
increase of iron in the endosperm of polished kernels as compared
to wild type.
It is significantly more than the lines that contain only single genes,
i.e., nicotianamine synthase (NAS) or ferritin (FER).
Maintenance of Iron Homeostasis
One obstacle to iron biofortification of plants is the toxicity of iron
when it accumulates to higher concentrations in cytoplasm.

Plants regulate the uptake and concentration of iron in their cells by
altering nicotianamine concentration through the activity of
-nicotianamine synthase (NAS)
-or a degrading enzyme, nicotianamine amino transferase (NAAT),
in response to an iron-dependant signal.
Constitutive expression of nicotianamine synthase in combination
with ferritin in the endosperm increases iron in sink tissue, but does
not change iron homeostasis in leaves, despite higher levels of
nicotianamine.
Expression of the gene for nicotianamine-degrading NAAT is
stimulated, by higher levels of nicotianamine in leaves of NFP-
plants.

Iron biofortification in rice by the introduction of
multiple genes involved in iron nutrition
(Masuda et.al., 2012; Science Reports)
• Goto et.al., 1999 generated transgenic rice plants that
expressed the soybean ferritin gene, SoyferH1, in the
endosperm using the endosperm-specific 1.3-kb GluB1 rice
promoter; the transformants showed higher Fe accumulation
in brown rice seeds.
• Qu et.al., 2005 expressed SoyferH1 under the control of both
the OsGlb1 promoter and 1.3-kb GluB1 promoter to further
increase seed Fe concentration.
• But enhancement of ferritin expression did not produce further
increases in seed Fe content.

Therefore, in addition to increased Fe storage in seeds,
enhanced Fe uptake from the soil and enhanced translocation
within the plant body were thought to be required to further
improve Fe biofortification in seeds.
Takahashi et.al., 2003 produced NA-deficient transgenic tobacco
plants that showed young leaves with serious chlorosis, and Fe
and Zn concentrations in the leaves and flowers decreased as a
result of disrupted internal metal transport.
Enhancement of Fe flux into the endosperm occurs by
expression of the Fe(II)-NA transporter gene OsYSL2.
Koike et.al., 2004 identified the rice NA-Fe(II) transporter
gene OsYSL2, which is preferentially expressed in leaf phloem
cells, the vascular bundles of flowers, and developing seeds,
suggesting a role in internal Fe transport.

OsYSL2 knockdown mutant plants exhibit a 30% decrease in Fe
concentration in the endosperm.
Simple overexpression of OsYSL2 by the 35S promoter did not
increase Fe concentration in seeds.
In contrast, enhancement of OsYSL2 expression under the control
of the rice sucrose transporter promoter OsSUT1, which drives high
expression in immature seeds during the seed maturation stage.
increased Fe concentration in polished rice seeds by up to
threefold.

Introduction of mugineic acid synthase gene was reported as
another approach to increase Fe concentration in seeds.
In graminaceous plants, NA is the precursor of mugineic acid
family phytosiderophores (MAs), which are natural Fe(III) chelators
used in Fe acquisition from the rhizosphere.
Graminaceous plants synthesize and secrete MAs into the
rhizosphere by TOM1 transporter.
They form Fe(III)–MAs complexes and are taken up into the root via
YS1 and YSL transporters.
Rice biosynthesizes 2′-deoxymugineic acid (DMA), which facilitates
Fe uptake and internal transport.

Rice lacks IDS3 gene (MA synthase gene) and does not produce
MA.
Fe concentration in polished rice seed increased up to 1.25 to 1.4
times by introduction of barley IDS3 genome fragment.
Each of these approaches could increase Fe concentration in
polished rice seeds.
But a higher Fe concentration in seeds was required to reduce the
human Fe deficiency anemia health problem.
A combination of these transgenic approaches would further
increase the Fe concentration in seeds.
New transgenic rice lines with enhanced Fe accumulation in seeds
using the Soybean ferritin gene under the control of two
endosperm-specific promoters, the OsGlb1 and GluB1.

These seeds exhibited enhanced Fe transportation within the plant
body due to over expression of NAS and enhanced Fe translocation
to seeds due to OsYSL2 expression under the control of
the OsSUT1 promoter and OsGlb1 promoter.
Gene insertion, ferritin accumulation in seeds, and higher
expression of OsYSL2 and HvNAS1 were confirmed.
Abundant NA facilitates formation of Fe(II)–NA, which is stable
under higher pH conditions, such as in phloem sap (pH 8.0).
Consequently, Fe(II) transport in the plant body, including the
phloem, is improved by NAS overexpression.
Increasing the NA concentration by enhancing NAS expression may
improve the bioavailable mineral content of rice grains.

Fe is well absorbed by the human gastrointestinal tract from
soybean ferritin (Lonnerdal B, 2009).
Increased NA in rice will likely reduce the rates of high-blood
pressure disease (Usuda K. et.al., 2009).
Zn concentration also increased in Fer-NAS-YSL2 lines.
Some reports show that higher NA production increases the Zn
concentration in seeds of rice plants (Masuda H. et.al., 2009).
Endosperm-specific ferritin expression also contributes to the
increased Zn concentration in rice seeds (Vasconcelos M. et.al.,
2003).

Feed Crops with Improved Proteins and
Amino Acids

Seeds of higher plants contain large quantities of storage proteins.
These proteins have been classified on the basis of their solubility
in various solvents.
Albumins (soluble in water)
Globulins (soluble in salt solution)
Prolamins (alcohol soluble)
Glutelins (soluble in acidic or
basic solution)
Wheat, barley, maize, sorghum accumulate major storage
proteins which are low in lysine.
Storage proteins of legumes are insufficient in sulfur-
containing amino acids.
found in dicot plants
found in monocot plants

Barley, rice, wheat, sorghum are also low in threonine and maize in
tryptophan.
Food Limiting amino acids
Cereals lysine, threonine, sometimes
tryptophan
Pulses Methionine, tryptophan
Nuts & oilseeds Lysine
Green leafy vegetable
MethionineLeaves & grasses
EAA DEFICIENT IN SOME VEGETARIAN FOODS

Three molecular approaches are being used in
altering amino acid sequence
1. Identification of naturally occuring seed storage plant with high
levels of desired amino acids, followed by cloning the
corresponding gene and expressing it at high levels in the
species distinctly differ from the sources of genes.
2. Modification by recombinant DNA technologies so that they
encode proteins similar to wild type proteins but possess
higher levels of desired amino acids.
3. Modification in the pool size of the desired amino acids for the
synthesis of seed storage proteins by an alternative metabolic
pathway.
57

Examples of expression of recombinant storage proteins with
desirable amino acid profiles:
Expression of pea (Pisum sativum) legumin, which has a high
lysine content, in rice and wheat grains (Stoger et.al., 2001).
The expression of sunflower seed albumin, which is rich in
methionine, in the laboratory model lupin (Molvig et.al., 1997).

In India, a genetically modified potato has been developed by a
coalition of charities, scientists, government institutes and industry
as part of a 15-year plan to combat malnutrition amongst India's
poorest children.
The 'protato', contains a gene AmA1 from the South American
amaranth plant, resulting in an increased protein content of 2.5 per
cent.
AmA1 gene from the Prince’s feather (Amaranthus
hypochondriacus), which encodes seed albumin, was expressed in
potato and was shown to double the protein content and increase the
levels of several essential amino acids (Chakraborty et.al., 2000).
The protato has high levels of essential amino acids, lysine and
methionine.
Protein-rich potato
• .

GM maize with increased lysine (LY038) was developed by
inserting a cordapA gene from a common soil
bacteria Corynobacterium glutamicum.
Enhanced production and accumulation of free lysine (Lys) in
the GM corn kernel made body weight gain, feed conversion and
carcass yields of experimental poultry and swine comparable
with animals fed with Lys supplemented diets, and higher than
those fed with conventional maize diets (Lucas DM et.al., 2007).
Lys-enriched maize with the gene sourced from potato, was also
found to be safe as conventional maize (He XY et.al., 2009).
LY038 has been commercialized and incorporated in feed meals
since 2006.

In all higher plants, lysine, threonine and methionine are
synthesized from aspartic acid via a pathway that is highly
branched and under complex feedback control.

Two key enzymes are aspartate kinase (AK), which functions
early in the pathway and is inhibited by both lysine and
threonine.
Dihydrodipicolinate synthase (DHPS), which functions in the
lysine-specific branch and is inhibited by lysine alone.
Feedback- insensitive versions of the bacterial enzymes have
been expressed in plants with promising results:
the free lysine content of Arabidopsis seeds was increased either
by expressing a bacterial, feedback-insensitive DHPS
transgene
or by knocking out the lysine catabolism pathway,
resulting in 12-fold or fivefold gains in lysine, respectively.

Where both the transgene and knockout were combined in the
same Arabidopsis line, increases of 80-fold overwild-type
levelswere achieved (Zhu X and Galili G, 2003).
 Protein-enriched soybean event M703 was found to contain more
digestible amino acids lysine, methionine, threonine, and valine,
and had a higher level of metabolizable energy (Edwards HM et.al.,
2000).
 A maize γ-zein gene encoding a sulphur amino acid rich protein
was used to transform alfalfa and trefoil (Lotus corniculatus) under
CaMV 35S promoter and RUBISCO small subunit promoter.
Expression level was rather low to the extent of 0.05% of alcohol
soluble protein.

To increase methionine level, a new methionine-rich zein,
normally expressed at low levels was expressed at a high level
using the 27 kDa zein promoter.
This protein called the high sulphur zein (HS 7) was 21 kDa and
contained 37% Met.
Biotechnology offers great potential for the production of novel
design crops, which are the sole solution to safeguard the supply of
sufficient quantities of safe & healthy food tomorrow.

Transgenic plant with improved nutritional quality

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