Presented by: Kirti Ph.D. (MBB)

Transgenic plants with improved nutritional quality

A good start is a food start!

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

NormalVitamin 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 APathway

is completeand functional

GoldenRice

• 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 generationGolden 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 increasingZn 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 EngineeringFriday, 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 acidsCereals lysine, threonine, sometimes

tryptophan

Pulses Methionine, tryptophan

Nuts & oilseeds Lysine

Green leafy vegetableMethionine

Leaves & 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.

Thank you !!!!!!