The topic covers in detail breading techniques to develop nutritional profiling in crops

1. BREEDING FOR QUALITY TRAITS
(PROTEIN, OIL)
Presented by
S.S. UDAYA KARTHIKA
II. M. Sc.(Agri)-Genetics and Plant Breeding

2. QUALITY TRAIT
MORPHOLOGICAL ORGANOLEPTIC NUTRITIONAL BIOLOGICAL
SIZE
COLOUR
PALATABILITY
TASTE
AROMA
SMELL
JUICINESS
PROTEIN
OIL
MINERAL
VITAMIN
PROTEIN EFFICIENCY
RATIO
BIOLOGICAL VALUE

3. PROTEIN EFFICIENCY RATIO
• Gain in gram in body weight of an animal on
consumption of 1 gram of protein
• In cereals, high lysine content is directly
proportional to high PER value
BIOLOGICAL VALUE
• Nitrogen retained in the body of animal
• Total protein nitrogen absorbed by it from diet

4. NUTRITIONAL QUALITY
❖ PROTEIN QUALITY (AMINO ACID BALANCE)
❖ PROTEIN CONTENT
❖ PROTEIN DIGESTIBILITY
❖ OIL CONTENT
❖ OIL QUALITY (FATTY ACID COMPOSITION)
❖ VITAMIN CONTENT
❖ MINERAL CONTENT
❖ ABSENCE OF ANTINUTRITIONAL FACTORS

5. PROTEIN
ORGANIC MACROMOLECULES
LONG CHAIN OF AMINOACIDS
LINKED BY PEPTIDE BONDS
JOINING THE CARBOYL GROUP (-COOH)
GROUP OF ONE AMINO ACID WITH AMINO
GROUP (-NH2) OF OTHER AMINO ACID

6. ➢ PROTEIN – 20 AMINOACIDS
➢ AMINOACID SEQUENCE IS DETERMINED BY
✓ BASE SEQUENCE OF GENE ENCODING THAT PROTEIN
✓ FUNCTIONAL PROPERTIES OF PROTEIN
➢ NUTRITIONAL PROPERTIES DETERMINED BY
✓ COMPOSITION OF AMINOACID
✓ NOT BY AMINOACID SEQUENCE

7. PROTEIN QUALITY
8 ESSENTIAL AMINOACIDS
ISOLEUCINE( Ile )
LEUCINE( Leu )
LYSINE( Lys )
METHIONINE( Met)
PHENYLALANINE( Phe )
THREONINE( Thr )
VALINE( Val )
TRYPTOPHAN (Trp)

8. WHEAT
ATLAS 66 – SOURCE OF PROTEIN CONTENT GENES
OBTAINED GENES FROM “FRONDOSA”
▪ LEAF RUST RESISTANCE
▪ PROTEIN CONTENT
LANCOTA – IMPROVING PROTEIN CONTENT
ABSORB MORE SOIL NITROGEN
▪ HIGHER NITRATE REDUCTASE ACTIVITY
▪ THAN CHECK VARIETY “LANCER”

9. High lysine
mutant
Hiproly
barley
High lysine
trait
P721
Opaque
mutant
Opaque 2
gene
Vivek
QPM9

10. CASE STUDY 1

11. MAIZE-3rd major cereal crop
Mostly consumed in Africa, Latin America, Asia
❖ Zein – most abundant protein
❖ Highly unbalanced in composition
❖ Deficient in lysine and tryptophan
❖ Cannot be synthesized in body

12. ➢ Poor net protein
utilization
➢ Low biological value
➢ Cause malnutrition and
leads to Kwashiorkar
➢ Insufficient essential
aminoacids leads to
under nutrition in
✓ Infants
✓ Lactating mother
✓ Pregnant mother

13. QPM is an improved variety
of maize which contains
higher amount of lysine and
tryptophan in endosperm
than normal maize

14. QPM DISCOVERY
1920-Spontaneous mutant of maize
1964-Homozygous recessive o2 allele
Soft and opaque grains – Opaque2 (o2)
Reduce zein fraction(50%)
Lysine and Tryptophan proportion is doubled

15. ZEIN
MAJOR GROUPS MINOR GROUPS
19 Kd & 22 Kd α zeins
β-15 kD, γ-16 kD &27 kD,
σ-10kD zeins

16. Opaque 2 gene
Short arm of
chromosome 7
Encodes leucine zipper(bZIP)
class transcriptional factor
Downregulate
Cause opaque
phenotype
22-kDa α zeins

18. PLEIOTROPIC EFFECTS OF THIS OPAQUE (O2) GENE
Soft endosperm
Damaged kernles
Susceptibility to pest and diseases
Reduced yield
Inferior food processing

19. Due to its
pleotropic effect
,opaque
appearance and
unpleasant taste
which results in low
grain yield

20. 3 Genetic systems involved in QPM breeding
Recessive mutant allele of opaque -2 gene
Endosperm hardness modifier gene
Amino acid modifiers/gene

21. In 1976, Bjarnason et al, found set of QTLs in
opaque-2(chromosome 7) to improve hard &
vitreous kernels
In 1980, Vasal et al, combined opaque-2 allele with QTLs
to produce elite germplasm with hard kernel & increase
in lysine & tryptophan content

22. Recently, In 2016, Liu et al.2016, found a 15.26 kb duplication segment
(qy27) at 27 kDa γ zeins locus is reported to be a major modifier QTL to
enhance the expression of protein & lead to endosperm hardness
Multiple genes are involved in the control of aminoacid.
Three loci are implicated in control of lysine level

23. CONVENTIONAL BREEDING
(PHENOTYPIC SCREENING)
MARKER ASSISTED SELECTION
(GENOTYPIC SCREENING)
Both genes o2 and endosperm modifier
genes are selected
DNA fingerprinting to help in matching
physical properties of QPM.
Light based screening 3 SSR markers –Foreground selection
marker for o2 gene
Seeds placed on Plexiglass surface
above a light and screened endosperm
DNA Extraction – identifying QPM
plants early
Human error or mis-classification may
occur
PCR based Co-dominant markers-
robust, significant and reproducible
Confirmation by molecular markers or
amino acid content profiling
CML 145*CML 212
Vivek QPM 9
BREEDING APPROACHES

24. MOLECULAR APPROACHES
TECHNIC PLANT MATERIAL AIM REFERENCE
SSR QPM & non-QPM Marker assisted introgression
of o2 trait
Manna, R;Okello, D.K.
ISSR Primers and variety
diagnostic markers
QPM & non-QPM Molecular breeding program
development
Nkongolo,K.K;Daniel,G.
SDS-PAGE QPM grain Protein identification Zhang,Z;Song,L.
Proteomic analysis QPM inbred lines Pfunde,C.N.
ISSR and RAPD markers QPM & non-QPM Assess the level of genetic
variation
Mbuya,K.
SNP markers QPM lines Genetic diversity studies Pfunde,C.N.
SNP-based genetic
distance
QPM inbred lines Heterotic grouping Badu-Apraku,B.

25. COUNTRY VARIETY
DESIGNATION
VARIETY TYPE AREA OF ADAPTION YEAR OF
RELEASE
India Shakthi OPV Tropical rainfed 1971
India Rattan OPV Tropical rainfed 1971
India Protina OPV Tropical rainfed 1971
India Shaktiman 1 Hybrid Tropical rainfed 2001
India HQPM1 Hybrid Tropical rainfed 2006
India HQPM7 Hybrid Tropical rainfed 2008
India Vivek QPM9 Hybrid Mid-
Altitude/Highland,
rainfed
2008
List of QPM varieties released till 2015

26. OILS AND FATS
Esters of glycerol & aliphatic straight chain fatty acids
CH2OH R1-COOH CH2OOC-R1
| |
CHOH + R2-COOH CHOOC-R2 + 3H20
| |
CH2OH R3-COOH CH2OOC-R3
(Glycerol) (Fatty acids) Triglyceride(natural oil/fats)
Esterification

27. Types and proportion of fatty acids present in the triglycerides
Saturated fatty
acid crops
Oil palm
Coconut
Cocoa
Oleic-Linoleic Acid
Crops
Maize
Groundnut
Soybean
Safflower
Sesame
Cotton
Erucic Acid Crops
Rapeseed
Turnip rape,
Toria
Mustard

28. Breeding Approaches
• Domestication
• Selection
• Mutation
• Hybridization
• Interspecific Hybridization
Biotechnological
Approaches
• Micropropagation
• Somo clonal Variation
• Anther culture
• Somatic hybridization
• Genetic engineering

29. MUTATION BREEDING IN
OILSEED CROPS
Oilseed crops – economically important crops
Major crops- Soybean, rapeseed, Groundnut, Cottonseed
Minor crops- Linseed, Fenugreek

30. Three step process of mutation breeding
a)Inducing mutations
b)Screening for putative mutant candidates
c)Mutant testing and official release
Mutation based plant breeding aimed to upgrade well adapted
varieties by altering one or two major traits
Mutation breeding-most viable method.
Induced mutation / molecular mutation breeding-improving food
security

32. VARIETIES YEAR CROP IMPROVED CHARACTER
TG1(Vikram) 1973 Groundnut Oil content and TMV
resistance
RLM 198 1975 Mustard Oil content, Early
maturity
RLM 514 1980 Oriental mustard Oil content, Less erucic
acid
RL 1359 1987 Chinese mustard Oil content, Short
duration
TGS 1(Somnath) 1989 Groundnut Oil content , Sequential
flowering
UMA 1990 Sesame Early and uniform
maturity, Oil content
OIL CROP VARIETIES IN INDIA

33. CASE STUDY 2
The use of invitro mutagenesis and directional OP based selection to
develop peanut varieties with increased oil content in kernels

34. Materials and Methods
1.Peanut tissue culture:
a)Basal medium
b)Embryo induction medium
c)Mutagenesis medium-mutagen(pingyangmycin)
d)Embryo germination & screening medium
*6-benzyl amino purine(BAP)
*Hydroxyproline(HYP)-OP regulator
All the media are adjusted to pH 5.8 and autoclaved at
120 C for 20 mins.

35. 2.Determination of optimal conc of the mutagen
Cotyledons are removed
Embryos are surface disinfected and soaked in sterile water for 10-12
hour.
Explants dissected from embryos and cultured in culture bottle (36
explants per bottle) containing 45mL of mutagenesis medium supplemented
with PYM at 0,1,2,3,4 or 5 mg/L. 4 weeks later somatic embryo induction rates
were calculated by formula:
Somatic embryo no of explants forming somatic embryos *100
Induction (%)= No of explants
The embryo induction rate was appx 50% with no PYM, so we selected 4mg/L
PYM as the optimal conc for mutagenesis

36. 3.Mutagenesis, selection, grafting, and transplantation of
regenerated plants with HYP tolerance
After regeneration of plantlets from somatic embroys, they
are subcultured.
1.5 cm height regenerated plant are grafted to the
rootstock from Huayu 20
After 3 days sterile culture, they are transferred to pots
containing mixture of soil: vermiculite :turfy soil(1:1:1)
Three weeks later, they are transplanted in the field and
seeds of M1 generation is harvested.

37. 4.Selection of mutants from progeny of HYP Tolerant plants
Individual plants are selected from M2 to M5 generations. Seeds
from the generations and parents are planted in the field.
Selection was performed based on plant type, branch number,
pod shape, and pod number per plant.
5.Measurement of oil content
Mature dry seeds were sent to the Quality Inspection and Test
Centre for Oilseeds and measured using residue method.

38. 6.Yield comparison test
The offspring of M6 generation with higher yield and oil
content greater than 55% was chosen for the yield comparison
test.M7 and M8 generations accessed in separate experiment
using RCBD with 3 replications .Huayu 20 was used as a control
7.Variety regional test for variety registration
Tests were performed using RCBD with 3 replications at
6 locations and Baisha 1016 used as control. At maturity pods
are harvested and weighed.
8.Statistical analysis

39. ➢ Here, we used HYP to increase the osmotic pressure from 77.0 to 85.5
mmol kg–1 in the embryo germination and screening media.
➢ Water generally flows from low OP to high OP regions.
➢ Because of the increased OP in the media, we assumed that the somatic
embryos or regenerated plantlets with normal oil content would die due to
water loss, and only those with dehydration tolerance would be able to
survive.
➢ When the somatic embryos that survived in vitro mutagenesis were
incubated and screened on somatic embryo germination medium
supplemented with 6 mmol L–1 HYP (OP=85.5 mmol kg–1), only about 0.5%
survived, and 15 regenerated plantlets were obtained.
RESULTS

41. Based on the results for yield and oil content presented here, three lines were
submitted for regional testing. Line 4-11-1-1 was tested in Anhui in 2015; lines 1-3-6-3
and 2-1-9-1 were tested in Liaoning in 2016 and 2017, respectively. In each of these
provinces, tests were conducted at six locations with Baisha 1016 being included as a
control. The average yield was 16.6, 13.9 and 14.4% higher for lines 4-11-1-1, 1-3-6-3
and 2-1-9-1, respectively, than for the control variety.

42. These three lines were named varieties: Yuhua 4 (line 4-11-1-1), Yuhua 9
(line 1-3-6-3) and Yuhua 14 (line 2-1-9-1). The oil content was 57.9, 61.1 and
59.3% for Yuhua 4, Yuhua 9 and Yuhua 14, respectively. Among all peanut
varieties currently planted worldwide, Yuhua 9 has the highest oil content.
Conclusion
This study demonstrated that the combination of PYM as a mutagen
and in vitro screening with HYP is a promising way to create new peanut
mutant and obtain varieties with high oil content. It provides an effective
approach for peanut breeding and will be useful for breeding peanut
varieties with higher oil content.

43. Photographs of representative somatic embryos that
formed from embryonic leaflets on the induction
medium without (A) or with (B) pingyangmycin (PYM)
at 4 mg L–1.
Photographs of representative somatic embryos and
plantlets that survived pingyangmycin (PYM)
mutagenesis and subsequently survived on a medium
supplemented with 6 mmol L–1 hydroxyproline (HYP)
after 3 wk (A), 5 wk (B) and 8 wk (C)

44. Photographs of representative grafted plants with hydroxyproline (HYP) tolerance
and their appearance when transplanted in the field and at pod harvest.
A and B, grafted plants with HYP tolerance.
C, No. 14 regenerated plant had purple stems and branches.
D, an HYP-tolerant plant and its pods (the arrow points to the graft union)

45. Photographs showing representative trait variation in the M2 generation.
A, Huayu 20.
B, progeny of the No. 14 plant.
C, progeny of the No. 8 plant.
D, pods from the No. 7 plant.

46. Perspectives of Breeding for
altering Sunflower Oil quality to
obtain novel oils
Sunflower competes Soybean, Groundnut, Rapeseed
Good quality of oil – PUFA – reduce cardiac risk
Oil content – 35-45%

47. Concept of Oil quality is changed by
➢ Saturated fatty acid composition
➢ Triacylglycerol profile
➢ Tocopherol content & composition
OIL QUALITY
“Palmitic acid” – undesirable- detrimental atherogenic effect- rises serum cholesterol

48. High oleic acid %
High linoleic acid
Low linoleic acid
Linoleic acid-
essential for
human
High oleic hybrid
–above 70%
1.Seed yield/unit area
2.Oil content
3.Oil quality
BREEDING OBJECTIVE

49. Effect of Environmental factors
Instability of genotypes across environment may be
❖ Planting location
❖ Planting date result in different during maturation stage
❖ High or low rainfall in growing season
❖ Presence of modifier genes
Environment variability in oil content
Genotypic factors variability in fatty acid composition

50. Early sowing - Oleic acid Linoleic acid
At seedling stage,
High night temperature Oleic acid Linoleic acid
Low rainfall
Accumulation of oil in seed requires
a)below 25 C mean daily temperature
b)sufficient soil moisture
c)absence of disease
Use of wild species:
Sunflower lines with best oil concentration and quality improvement
Helianthus deserticola (desert sunflower)
Helianthus anomalus (sand sunflower)

51. Effect of mutation
1st artificial mutation by Soldatov (1976)
2.5% Dimethyl sulphate
VNIMK Pervenets High oleic variety
X-rays
Sunflower seeds 30% palmitoleic acid
Sodium azide
Sunflower seeds 35% stearic acid

52. Genetic basis of sunflower
❖ P1, P2, P3 – 3 genes in control of high palmitic acid
❖ Es1, Es2, Es3 – genes in control of stearic acid
❖ O11 – principal gene – control of high oleic acid
content
❖ Tph1, Tph2 – control altered tocopherol composition
❖ Two potential QTLs for OA & LA content identified
with markers ORS 762 & HO-Fsp-b explained more
than 57.66-66.6% of phenotypic variation.

53. These markers /QTLs useful in MAS breeding programe to improve oil quality
Molecular markers for some of the traits have been developed for
High stearic & oleic acid content
High beta & gamma tocopherol contents
Polymorphic markers applied for selection in backcross b/w high oleic donor &
2 low oleic recurrent parents.

54. CONCLUSION
➢ Quality protein maize has nutritional quality can alleviate
protein malnutrition in maize dependent countries.
➢ Though conventional methods of breeding are still in use
,marker assisted breeding with markers for foreground
selection can expedite good breeding process in all crops.
➢ This saves time, money and labour resources with high genetic
gain.