Protein and fats are important for our dietary requirement. The nutritionally improved fat and protein necessary for healthy food.

1. Breeding, Genomics and Transgenics for
special compounds - proteins and fats.
Presented by Presented to
RAJU RAM CHOUDHARY Dr. Mukesh kumar
Adm. No. 2019A48D
Assignment on

2. What are the various approaches?
 Biofortification through fertilizer application
 Improvement through conventional breeding
 The identification of quality protein and quality oil varieties
and the use of marker assisted selection to introgress such
traits into widely cultivated, adapted germplasm
 Mutagenesis has also been used to produce crops with higher
nutrient levels, including the lysine-enriched maize opaque-2
mutant
 Many transgenic strategies are also available to enhance the
nutritional value of crops; these strategies offer a rapid way to
introduce desirable traits into elite varieties
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3. Crop-wise improvements
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4.  Proteins are an essential component of the diet.
 Protein are organic macromolecules consisting of a long chain
of amino acids linked with each other by peptide bonds formed
by carboxyl(-COOH) group of one amino acid with amino
group(-NH2) of other amino acid
 The nutritional properties of proteins are determined by their
amino acid composition
 There are 21 amino acids which are important in human
nutrition. These can be classified into two groups, viz.
1. essential amino acids &
2. non essential amino acids.
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Protein content & Quality

5. 5
 EAA can’t be synthesized in human body & their requirement has to
be met through dietary intake
 There are ten EAA (methionine, isoleucine, leucine, lysine,
threonine, tryptophan, valine, phenylalanine, histidine, & arginine).
 Out of these arginine & histidine are considered non essential for the
adult.
 The non-EAA can be synthesized in human body & they need not be
supplied through diet.
 The quality of protein is determined by the content of essential
amino acids.

6. Crop Limiting amino acids
Cereals lysine, threonine and
sometimes tryptophan
Pulses Methionine
Nuts and Oilseeds Lysine
Green leafy vegetables Methionine
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EAA deficient in some crop plants

7. 1. GENETIC APPROACHES
 Maize seeds was very low in Lys, a major effort initiated at the
mid 20th century to identify high-Lys corn varieties by genetic
approaches
 These efforts resulted in the discovery of the high-Lys opaque2
mutant, which contains low levels of the Lys poor seed storage
proteins (called zeins) and a compensatory increase in Lys,
Trp, and and non-zein seed proteins as well as free Lys and
Trp, compared to normal maize.
 The QPM genotypes are generally associated with an
increased level of the 27-kD gamma-zein storage protein,
which somehow compensates for the reduced level of the Lys-
poor alpha and beta-zein storage proteins
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8. Pathway of synthesis
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.
Abbreviations:
AK, aspartate
kinase; ASAD,
aspartic
semialdehyde
dehydrogenase;
DHDPS,
dihydrodipicolinat
e synthetase;
HSD, homoserine
dehydrogenase;
HSK, homoserine
kinase; TS,
threonine
synthetase

9. 9
Manipulation of amino acid synthesis pathways in plants
 Two key enzymes are aspartate kinase (AK), which functions early in
the pathway and is inhibited by both lysine and threonine.
 Dihydrodipicolinate synthase (DHDPS), 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 DHDPS transgene or
by knocking out the lysine catabolism pathway (bifunctional
LKR/SDH enzyme) , resulting in 12-fold or five fold gains in lysine,
respectively.
 Where both the transgene and knockout were combined in the same
Arabidopsis line, increases of 80-fold over wild-type levels were
achieved (Zhu X and Galili G, 2003).

10. • Genetic mutations in the tobacco (Nicotiana tabacum) DHDPS
gene, rendering the enzyme Lys insensitive, or constitutive
expression of a bacterial Lys-insensitive DHDPS in transgenic
tobacco or Arabidopsis (Arabidopsis thaliana) plants caused Lys
overproduction in all plant organs, including the seeds
• However, high levels of Lys in all plant tissues can cause
abnormal vegetative growth and flower development that, in turn,
reduce seed yield
• Targeting the expression of bacterial Lys-insensitive DHDPS to
seeds of tobacco, using a seed-specific promoter, resulted in
plants with normal growth characteristics that also accumulate
higher amounts of Lys in their seeds, but not yet nutritionally
desirable levels
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11. • The expression of a bacterial feedback-insensitive DHDPS
under the control of an embryo specific promoter caused a
100-fold increase in the accumulation of seed free Lys level
• High Lys levels were also obtained when the expression level
of LKR/SDH was suppressed. Suppression of these genes in
the embryo led to an accumulation of 0.2 mg/g DW of soluble
Lys in maize seeds, whereas the suppression in the endosperm
resulted in an accumulation of 0.9 mg/g DW
• Monsanto/Renessen generated a high-Lys maize genotype
(LY038) expressing a bacterial feedback-insensitive DHDPS
in an embryo-specific manner.
• First GM crop with high nutritional value to be approved for
commercial use in a number of countries

12. Modifying biosynthetic and catabolic fluxes of Lys
• A chimeric gene encoding a bacterial feedback-insensitive
dihydrodipicolinate synthase (DHDPS) enzyme of Lys synthesis
was expressed under the control of a seed-specific promoter in an
Arabidopsis knockout mutant lacking a bifunctional Lys-
ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH)
enzyme of Lys catabolism
• This genotype (termed the KD genotype) led to a nearly 64-fold
increase in the level of seed free Lys. Yet, seed germination of the
KD genotype was significantly retarded
• The boosting of Lys synthesis and blocking its catabolism in the KD
genotype had a major influence on the levels of several TCA cycle
metabolites, indicating that the suboptimal germination of this
mutant is due to a negative influence on the cellular energy status
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13. Increase the protein sink for Lys
 This can be done by transforming plants with genes encoding
stable proteins that are rich in the desired amino acid(s), and
can accumulate these proteins to high levels
1. Natural genes encoding Lys-rich proteins derived from
different plant or non-plant sources;
2. Natural genes that have been mutated to increase the number
of Lys codons and make proteins richer in Lys; and
3. Synthetic genes encoding Lys-rich proteins
 The most significant increases in seed Lys levels were
obtained by expressing a genetically engineered gene encoding
HORDOTHIONINE12 or the BARLEY HIGH LYSINE8
(BHL8) protein, which contain 28% and 24% Lys, respectively
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14. • These proteins accumulated in transgenic maize seeds to 3% to
6% of total grain protein, when introduced together with a
bacterial DHDPS resulted in an elevation of total Lys to over
0.7% of seed dry weight, compared to around 0.2% in wild-
type maize
• BHL8 is a recombinant protein derived from a barley
CHYMOTRYPSIN INHIBITOR-2, which was genetically
engineered to substantially increase the number of Lys codons
and those of other essential amino acids, based on a three-
dimensional structure analyses
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15. Methionine pathway
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16. Enhancing the level of free Met in plants
 Traditional plant breeding methods have failed to increase the
level of Met in crop plants
 metabolic engineering approach leading to higher Met content
with minimal perturbation to the plant phenotype was the
manipulation of the first committed enzyme of the Met
biosynthesis pathway, Cystathionine Gamma Synthase (CGS)
 Overexpression of Arabidopsis CGS (AtCGS) in transgenic
Arabidopsis led to 6.2-fold elevation of soluble Met content in
leaves
 The transcript level of Arabidopsis CGS (AtCGS) is negatively
regulated by the Met downstream product, S-adenosyl-Met
(SAM), via a post-transcriptional mechanism
 Thus, it was suggested that the level of Met could not increase
behind a certain threshold
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17. • Met Catabolic product, S-adenosyl-Met, is a precursor for the
hormone ethylene, for polyamines, and it is also the primary methyl-
group donor for multiple biological processes, such as DNA
replication, cell wall development, and secondary metabolite
production.
 A compound posttranscriptional control mechanism involving
interactions with a highly regulatory multicomponent domain located
in the N terminus of the mature CGS polypeptide
 Interestingly, mutations in this region, or its deletion, result in
overproduction of Met, which is likely independent of a reduction of
SAM synthesis
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18. • Utilization of CGS enzymes with either deleted or mutated N-
terminal domains currently appears as a potentially promising
approach to increase the production of free Met with minimal
negative effects.
• Another approach to increase the Met content in plants
involved the expression of sulfur-rich proteins
• The Brazil nut and sunflower 2S albumins have been
transgenically expressed in seeds of a number of plant species,
including tobacco, canola and soybean.
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19. Fat Quality Improvement
• Vegetable oils account for ~85% of the world’s edible fat and oil
production
• Oil palm, soybeans, rapeseed and sunflower, which together
account for ≈ 79% of the total production.
• The degree of desaturation of plant oils can profoundly affect
their physical properties and health benefits
• Many health studies have linked high dietary saturated fat to
obesity and cardiovascular disease and so oils with lower SFA
content are desirable to address this health issue
• Trans fatty acids are generated during partial hydrogenation of
polyunsaturated vegetable oils (particularly soybean and
cottonseed oils)
• Trans fatty acids have a strong association with heart diseases
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20. Main objectives
Increase content of ‘‘healthy’’ fatty acids and
reduce unhealthy’’ fatty acids
Improve oil stability to expand applications
and reduce the need for hydrogenation.
 Expand the repertoire of fatty acids through
exploitation of genetic diversity and enzyme
engineering.
Reduction in the anti-nutritional facts
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21. Codex Standard for Vegetable Oils
Lauric acid (C 12:0) Not present in discernible amounts
Myristic acid (C 14:0) < 0.1
Palmitic acid (C 16:0) 7.5 - 20.0
Palmitoleic acid (C 16:1) 0.3 - 3.5
Heptadecanoic acid (C 17:0) < 0.5
Heptadecenoic acid (C 17:1) < 0.6
Stearic acid (C 18:0) 0.5 - 5.0
Oleic acid (C 18:1) 55.0 - 83.0
Linoleic acid (C 18:2) 3.5 - 21.0
Linolenic acid (C 18:3) < 1.5
Arachidic acid (C 20:0) < 0.8
Behenic acid (C 22:0) < 0.3
Erucic acid (C 22:1) Not present in discernible amounts
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22. Pathways
• The levels and types of fatty acid desaturation can be changed
by modulating the expression of several different classes of
lipid biosynthetic genes
• The initial steps of plant lipid biosynthesis occur in the plastid
via the fatty acid synthase complex where the growing fatty
acid chain is attached to an acyl carrier protein (ACP).
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25. Condensation
Condensation reactions to add two carbon units to the growing acyl
chain (supplied by malonyl-ACP) are performed by ketoacyl
synthase (KAS) enzymes.
 Ketoacyl synthase enzyme Complex
• KAS I performs intermediary elongations
• KAS II specializes in converting palmitoyl-ACP to stearoyl-ACP
• KAS III performs the initial condensation reactions
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26. Desaturation in Plastid
• Initial desaturation of stearoyl-ACP occurs via stearoyl-ACP
desaturase (SAD) to form oleoyl-ACP.
• Synthesis reactions are terminated by cleavage of the fatty acid
from ACP by fatty acyl-ACP thioesterases (FATs) that have
varying specificities.
• In Arabidopsis, FATA has preference for oleoyl-ACP and FATB
forpalmitoyl-ACP
• The newly synthesized fatty acids are incorporated into
plastidial lipids in the highly specialized thylakoid membranes
or are exported to the cytosol.
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27. • Further fatty acyl desaturations are performed with acyl
chains attached to phosphatidylcholine (PC)
• FAD2 is a Δ12-desaturase and converts oleic acid (18:1) to
linoleic acid (18:2) and
• FAD3 is a Δ15-desaturase and converts linoleic acid (18:2) to
linolenic acid (18:3)
• These two enzymes are instrumental in affecting the degree of
unsaturation in seed oils and have opened routes to develop
oils highly enriched in oleic or linolenic acids in oilseed crops.
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28. Manipulating Plastidial Fatty Acid Biosynthesis Enzymes
• The fatty acyl-ACP thioesterases and KAS II
enzymes are the primary targets for modifying
desaturation
• Additionally, the plastidic SAD can be
downregulated to increase the amount of SFAs.
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29. Fatty Acyl-ACP Thioesterases
 The acyl-ACP-thioesterase FATB has a preference for palmitic
acid (16:0) as a substrate.
 Loss of FATB function in several plant species results in lower
levels of 16:0 in seed oils.
 Downregulation of FATB in Arabidopsis using
a. A microRNA with a napin seed-specific promoter (50% reduction in SFA)
b. Antisense construct with a constitutive viral promoter (45% reduction in
SFA)
c. T-DNA insertional mutant(50% reduction in SFA)
 High yields of medium-chain fatty acid lauric acid (12:0) were
achieved in canola by expressing the California bay laurel
(Umbellularia californica) 12:0-ACP thioesterase. The oil was
approved for commercialization in North America as
Laurical™ canola for a potential cocoa butter alternative
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30. KAS II Engineering
• Seed-specific RNAi downregulation of KAS II in
cottonseed increases 16:0 content by twofold to 53%
• Introduction of soybean KAS II transgenes decreases
16:0 in canola and maize.
• The ZF protein was designed to bind to a sequence
upstream of promoters of two endogenous canola KAS
II genes.
• Expression of the artificial ZF-enhancer gene driven by
a constitutive promoter results in increased KAS II
transcript levels in leaves and seeds and a decrease in
total seed SFAs from 7.5 to 5.2% in one of the events
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31. Modulation of Desaturation by Extra-Plastidial Desaturases
 In soybean, conventional mutations in FAD2 genes provide
higher 18:1 and lower PUFA levels
 use of seed specific antisense RNA expression targeting of
FAD2-1 genes that has resulted in high-oleic soybeans (with
lower SFA levels), commercially marketed by DuPont Pioneer
under the trade name Plenish®
 Using TALENs, two FAD2 gene homologs were modified via
NHEJ imperfect repair to increase 18:1 and decrease PUFA
content
 PUFAs decreased in these seeds (from 63 to 8%) with a
concomitant increase in 18:1 (from 20 to 80%)
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32. Combined Approaches
• Monsanto’s Vistive Gold® soybeans expression of both
plastidial FATB and extra-plastidial FAD2 genes is suppressed
via RNAi
• These alterations result in seed oil with 72% 18:1 and 6% SFAs
(compared to 22% and 15%, respectively, in conventional
soybeans), with 16:0 lowered from 11 to 2.5%
• Cottonseed oil is rich in 16:0 (25%) and also has high PUFA
content (58% 18:2) contributing to lower oxidative stability in
high-heat applications
• Two seed-specific RNAi constructs were designed to lower SAD
and FAD2 activities. Transgenic seed expressing a FAD2
hpRNA decreased 18:2 from 58.5 to 3.7% with a concomitant
increase in 18:1 to 78.2%
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33. Increasing Oil Accumulation in Plants
• Total oil can be increased by modifying enzymes directly
involved in TAG synthesis
• This pathway (the Kennedy pathway) involves three sequential
acylations of glycerol via three acyltransferases (Fig. 1). The
final acyltransferase DGAT acylates sn-1-2-diacylglycerol
(DAG) using acyl-CoA as a substrate and is considered to be
the rate-limiting step
• There are two classes of DGATs in plants: typical DGAT1
proteins are about 20 kDa larger than DGAT2 and are
constitutively expressed, whereas DGAT2 has a less defined
physiological function and is highly expressed in the seeds.
Overexpression of DGAT1 in Arabidopsis and canola
increases seed oil content
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34. • Expression of DGAT2A from Umbelopsis ramanniana (a
soil fungus) in soybean leads to an increase of 1.5% in seed
oil on a dry seed matter basis across multi-year field trials
• The increase in oil content is accompanied by decreases in
soluble sugars and a slight increase in protein amount. Thus,
expression of the modified DGAT1 maintains the total
protein level and shifts carbon from sugars to oil
• Other approaches to increase total seed oil involve
overexpression of WRI1 and LEC2 lipid biosynthesis
transcription factors and altering starch metabolism to direct
carbon partitioning to lipid synthesis
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35. GENETIC ENHANCEMENT FOR DOUBLE LOW
CHARACTERISTICS IN INDIAN RAPESEED MUSTARD
• The rapeseed mustard cultivars being grown in India have high
amounts of erucic acid (40-50%) in the seed oil and high
glucosinolate (80-160 μmoles/g) in the oil free meal
• The popularly grown high yielding B.juncea var. Varuna was
crossed with low glucosinolate B.juncea line BJ-1058 for transfer of
low glucosinolate. Simultaneously, a unique three way
cross; B.juncea (var. Varuna x Zem-1) x BJ-1058 was made for
transfer of double low characteristics
• The zero erucic acid strains of B.napus were developed at TERI
from the advanced generation transgressive segregants of
intergeneric crosses of Brassica.
• The newly developed double low strains of B.napus i.e
TERI(OO)R985 and TERI(OO)R986 having zero erucic acid in the
seed oil and low glucosinolate (12-15 μmoles /g) in the oil
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36. Variety Pusa Double Zero Mustard-1 (PDZ-1)
• First Double Zero (erucic acid less than 2% and glucosinolates
less than 30 PPM) Indian mustard variety developed by IARI
• Crossing Pusa Mustard-21 (LES-1-27) x NUDHYJ-3 and
subsequently following the pedigree method of selection
• Pusa Mustard-21 is low erucic acid popular variety of Indian
mustard developed by Division of Genetics, ICAR-IARI, New
Delhi and NUDHYJ-3 is a double low germplasm accession.
• Single plants of medium duration, profuse branching with low
erucic acid in oil and low glucosinolates in defatted seed meal
were selected using pedigree selection method in the
segregating generations during generation advancement
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37. SYMBOL OF TRUST