Modern agriculture confronts multifaceted challenges, encompassing biotic and abiotic stresses alongside malnutrition. Biofortified crops emerge as a pivotal solution, augmenting nutritional quality during plant growth. By harnessing specific genes with pleiotropic effects for stress tolerance, these crops exhibit heightened yields, resilience against pests and diseases, and adaptability to environmental stressors. This innovation not only secures food safety and nutrition but also fosters the development of "high-value farms," ensuring sustainable escalation in global food productivity and stable food prices. Conclusion: Integrating diverse transgenes and gene editing with omics approaches enhances stress tolerance and nutritional content in biofortified crops. This holistic strategy enables precise modifications to crop genomes and comprehensive insights into stress responses and nutrient metabolism, ensuring sustainable food production and nutrition security.

1. Submitted by :-
P .TEJASREE
TAD/2023-010
Ph.D. 1st Year
Dept. of GPBR
Submitted to :-
Dr. M. Shanthi Priya
Professor & Head
Dept. of GPBR
ACHARYA N.G. RANGAAGRICULTURAL UNIVERSITY
S.V. AGRICULTURAL COLLEGE, TIRUPATI
Course No :- GPB-691
Course Title :- Doctoral Seminar-I
High-value pleiotropic genes for developing multiple stress-
tolerant biofortified crops for 21st-century challenges

2. 2
Polygenic inheritance type of non-Mendelian inheritance where a trait is
influenced by multiple genes
Example: kernel colour of wheat
corolla length in tobacco
Non Mendelian Inheritance - Pleiotropy

3. 3
Jordan et al. 2019
Vertical pleiotropy Horizontal pleiotropy LD-induced horizontal pleiotropy
A popular method of measuring pleiotropy is to use knock-out genotypes in a
homogenous background, knock-in genotypes to validate the function of genes.

4. 4
Devendra Kumar Yadava et al. 2020

5. 5
Crop Feature Year of release
Rice
CR Dhan 315 Rich in zinc (24.9 ppm) 2020
Wheat
MACS 4058 (durum) Rich in protein (14.7 %), iron (39.5 ppm) and zinc (37.8 ppm) 2020
HD 3298 Rich in protein (12.1 %) and iron (43.1 ppm) 2020
HI 1633 Rich in protein (12.4 %), iron (41.6 ppm) and zinc (41.1 ppm) 2020
Maize
Pusa HQPM 5
Improved
Rich in provitamin-A (6.77 ppm), lysine (4.25 % in protein) and tryptophan (0.94 % in protein) 2020
Pusa HQPM 7
Improved
Rich in provitamin-A (7.10 ppm), lysine (4.19 % in protein) and tryptophan (0.93 % in protein) 2020
IQMH 203 (LQMH 3) Rich in lysine (3.48 % in protein) and tryptophan (0.77 % in protein) 2020
Pearl Millet
HHB 311 Rich in iron (83.0 ppm) 2020
Finger Millet
VR 929 (Vegavathi) Rich in iron (131.8 ppm) 2020
CFMV1 (Indravati) Rich in calcium (428 mg/100g), iron (58.0 ppm) and zinc (44.0 ppm) 2020
CFMV 2 Rich in calcium (454 mg/100g), iron (39.0 ppm) and zinc (25.0 ppm) 2020
Devendra Kumar Yadava et al. 2020

6. 6
Crop Feature Year of release
Lentil
IPL 220 Rich in iron (73.0 ppm) and zinc (51.0 ppm) 2018
Groundnut
Girnar 4 Rich in oleic acid (78.5 % in oil) 2020
Girnar 5 Rich in oleic acid (78.4 % in oil) 2020
Linseed
TL 99 High in linoleic acid (58.9%) 2019
Mustard
Pusa Double Zero
Mustard 31
Low in erucic acid (0.76 % in oil) and glucosinolates (29.41 ppm in seed meal) 2016
Pusa Mustard 32 Low in erucic acid (1.32 % in oil) 2020
Soybean
NRC 127 Free from KTI (Kutniz Trypsin Inhibitor) 2018
NRC 132 Free from lipoxygenase-2 2020
NRC 147 Rich in oleic acid (42.0%) 2020
Little Millet
CLMV1 Rich in iron (59.0 ppm) and zinc (35.0 ppm) 2020

7. 7
Devendra Kumar Yadava et al. 2020

8. 8
Ahmad et al. 2021
Advances in genome-editing technology and their applications
in crop improvement to achieve zero hunger
Improved plant architecture; modifications in plant
architecture via the CRISPR-Cas system can bring a new
green revolution. For example, DELLA proteins limit plant
growth and development ; thus, editing DELLA proteins
generated vigorous and short-stature rice lines.

9. 9
Shahzad et al. et al. (2021)
Various approaches for biofortification
Foliar application nutrients are applied in liquid form in
aerial parts of plants and got absorbed through stomata
and epidermis. And readily enters in to food chain.
Mineral fertilization through soil application available
for uptake and as a result their accumulation in eatable
parts of plants is increased. rhizobium bacteria,
mycorrhizae fungi, etc., help plants in nutrient
acquisition through mutualism. Conventional breeding
by crossing two parents possessing contrasting
phenotypes and selection in subsequent segregation
generations based on trait of interest.Knocking out of
genes involved in biosynthesis of anti-nutrient
compounds. lectins, phytic acid, saponins, lathyrogens,
protease inhibitor, a-amylase inhibitors, and tannins
restrict bioavailability of essential micronutrients. Genes
involved in biosynthesis of anti-nutrients could be
repressed through RNAi for reduced accumulation of
these compounds. Overexpression of gene responsible
for micronutrient accumulation in plants leads toward
micronutrient biofortification. Different genes involved
in biosynthesis of pro-vitamin A (CrtB), iron
homeostasis (Fer1-A), and flavonoids production (C1)
has been transferred across species for biofortification

10. 10
Amjad M. Husaini 2022
An overview of the 21st-century challenges and the high-value
genes for breeding nutrient-dense weather-resilient crops

11. 11
There is a well-known correlation between stress tolerance and activities of the major antioxidative enzymes viz. superoxide dismutase
(SOD), catalase (CAT), ascorbate peroxidise (APX), guaicol peroxidase, glutathione synthase and glutathione reductase
MAJOR-EFFECT MULTI-ROLE GENES FOR CHALLENGING SITUATIONS Transgenes encoding ROS scavenger proteins
Amjad
M.
Husaini
2022

12. 12
Transgenes encoding transcription factors
In order to impart tolerance against multiple stresses, a good strategy is to overexpress the transcription
factor encoding genes that control stress-responsive multiple genes of various pathways.
Amjad M. Husaini 2022

13. 13
Amjad M. Husaini 2022

14. 14
Transgenes encoding protein kinases
Perception and signaling pathways are vital components of an adaptive response for plants’ survival under stress conditions. Mitogen-
Activated Protein Kinases (MAPKs) are serine/threonine protein kinases, perform a vital role in signal transduction pathways
Amjad
M.
Husaini
2022

15. 15
Osmotin is a cysteine-rich PR-5c protein. It was discovered as a thaumatin-like stress-responsive protein synthesized and accumulated by cells
under salt and desiccation stress. It plays a major role in protecting plant plasma membranes under low plant water potential
Osmotin
Amjad M. Husaini 2022

16. 16
GENES FOR MINERAL (IRON, ZINC, COPPER) BIOFORTIFICATION
Application of mineral micro- and macro- nutrients coupled with breeding varieties with enhanced uptake of mineral
elements, is a good strategy for biofortification of edible crops
overexpression of YSL and NAS may increase metal uptake and translocation, especially iron, zinc, manganese and copper in transgenic plants.
Amjad M. Husaini 2022

17. 17
Plant genetic modification by insertion of genes
involved in stress response pathways is one approach to
increase stress-tolerance in crops.
CASE STUDIES

18. 18
CASE STUDY - 1
Aim to understand the molecular mechanisms underlying the
stress tolerance and grain length regulation mediated by OsSGL

19. 19
Materials and Methods
Plant material : Seeds of rice cultivar PA64S (O. sativa L. ssp. indica) - heat stress (45 °C, 2 h, under light), cold
stress (4 °C, 16 h, without light) treatments and moderate drought resistance protocols with 20% (M/V) PEG6000 .
Vector construction and plant transformation: For OsSGL overexpression vector construction
(pCaMV35S::OsSGL::NOS), the cDNA fragment with the whole open reading frame of OsSGL with hpII selection
marker followed by agrobacterium mediated transformation. RNA was extracted for microarray and qRT-PCR
analyses.
Phenotypic measurements: grain length, grain weight and grain number per panicle
Histological analysis and microscopy observation: measurements of vascular elements were performed using the
Leica Qwin software.
Subcellular localization of the OsSGL protein : pCaMV35S::OsSGL::GFP was ligated into the pCAMBIA1300
vector. GFP fluorescence was observed with a Leica MZ16FA fluorescent stereomicroscope.

20. 20
Tissue specificity of OsSGL expression in the transgenic rice
expressions detected in
leaf (A), internode (B), coleoptile (C), hulls of young
spikelets (D), leaf sheath (E), stamen (F) root (G),
pistil of mature spikelets before flowering (H)
longitudinal section of rice root at seedling stage (I)
Transverse section of leaf blade (J)
The high levels of expression in these tissues suggest
that OsSGL may play an important role in regulating
rice vegetative and reproductive developments

21. 21
Biological Role of OsSGL
shoot apical meristem
transition stage from the vegetative
to the reproductive phase
primary branches formation stage secondary branches formation stage flower organs differentiation stage
93-11(WT)
93-11-OE
developmental processes of spikelets and panicles of 93-11 and 93-11-OE plants grown in parallel showed that the
rachis meristem and spikelets at both primary and secondary branch primordia formation and flower organ
differentiation stages were markedly larger in 93-11-OE than those observed in the wild-type 93-11

22. 22
Effect of overexpression of OsSGL on cell number and size
These results demonstrate that OsSGL positively
affects grain size by increasing both cell number
and cell size leading to the enhanced
longitudinal growth of the rice grains
Spikelets 6 days before heading
- longer
A cross section of the spikelets revealed that
the inner parenchyma cell layer of
palea/lemma in 93-11-OE contained 35.0–
60.5% more cells than in the 93-11 hull and
that its cells were 18.4– 29.6% larger (Fig.
3C–J).
longitudinal axis of the panicle
parenchyma
cell
numbers(C)
and
(D)
sizes
Cross-sections
of
florets
cut
horizontally
lemma palea
93-11 (WT)
93-11-OE lemma palea
Furthermore, inspection of longitudinal
palea and lemma sections showed that the
inner parenchyma cell layer of 93-11-OE
contained 42.7% more cells than 93-11,
which were on average 40.3% larger

23. 23
Panicles of 93-11 (left) and 93-11-OE (right)
1 cm 3 cm 10 cm 20 cm
Biological Role of OsSGL
phenotype of longer panicles in 93-11-OE appeared at the late stage of panicle development

24. 24
OsSGL might also play a role in dry matter accumulation during grain milk filling, thereby regulating grain weight
The FW and DW of 93-11-OE grains were 33.4% and 28.1% heavier than those of 93-11 grains, consistent with the longer ovaries and grains observed in 93-11-OE

25. 25
Effects of OsSGL on yield
22.2% increase in panicle length
25.7% in grain number per panicle
24.8% longer, 8.6% narrower
16.3% heavier
(ms) PA64S × C3–1(transgenic)
LYP9-OE
PA64S × 93-11 (WT)
LYP9
average increase of 12.1% in grain yield
Application of 93-11-OE lines in hybrid rice breeding
The morphological marker of curling flag leaves facilitated the
selection of positive transgenic plants

26. 26
Possible role of OsSGL in drought resistance - overexpression of OsSGL enhanced
drought tolerance of the transgenic lines and promoted plant growth
moderate drought stress with 20% (M/V) PEG6000 in hydroponics
normal growth conditions

27. 27
rice grain size four genes positively regulating GW2, GW5, GS5, GW8
cell cycle G1/S-phase transitions: elevated in the
OsSGL-overexpressing lines
cytokinin signalling
OsSGL May Function via Cytokinin Signal Transduction Pathway

28. 28
Conclusion
The study revealed that overexpression of the OsSGL gene in rice results in increased grain length, grain
weight, and grain number per panicle, leading to a significant increase in yield. Microscopical analysis
indicated that OsSGL overexpression promoted cell division and grain filling. Furthermore, gene expression
analysis suggested that OsSGL may regulate stress tolerance and cell growth by modulating the cytokinin
signalling pathway and influencing the expression of genes involved in stress response and cell cycle
regulation. Overall, this study enhances in understanding the molecular mechanisms underlying rice stress
tolerance and grain length regulation and provides insights into strategies for improving crop yield.

29. 29
CASE STUDY - 2
Plant Physiology® , July 2018, Vol. 177, pp. 1078–1095
The aim of this study was to enhance root size and architecture in barley
plants by manipulating the levels of the plant hormone cytokinin

30. 30
Materials and Methods
Transgenic Barley Generation: Transgenic barley plants were created by introducing a gene encoding
CYTOKININ OXIDASE/DEHYDROGENASE (CKX), an enzyme responsible for cytokinin degradation,
under the control of a root-specific promoter and Western blot analysis to confirm CKX overexpression.
Gene expression analysis of CKX and other genes involved in cytokinin signalling and root development using
quantitative real-time PCR (qRT-PCR)
Phenotypic Analysis: The root size and architecture parameters such as root length, branching, biomass
allocation, shoot growth and seed yield were measured.
Nutrient Analysis: Concentrations of macro elements and microelements in the leaves - using inductively
coupled plasma mass spectrometry (ICP-MS).
Drought Stress Response: Transgenic lines were subjected to long-term drought conditions - drought stress
responses such as stomatal conductance, photosynthetic rate and osmotic adjustment.

31. 31
RT-qPCR analysis showing root-specific expression of the rice genes
Rice UBQ5 and eEF-1α were used as reference genes
RETROTRANSPOSON PROTEIN EXPRESSED PROTEIN PEROXIDASE PROTEIN
Identification and Validation of Root-Specific Promoters for Root Engineering in Barley

32. 32
Expression of root-specific promoters of rice in transgenic Arabidopsis plants
Expression of pEPP:GUS in transgenic Arabidopsis plants
Expression of the reporter gene was mostly confined to roots
Root-specific expression was strongest in the vasculature but hardly visible in
primary and lateral root meristems
Reporter gene expression was absent in rosette leaves of five-weeks-old plants and
reproductive organs
Expression of pPER:GUS in transgenic Arabidopsis plants
Root-specific expression was mainly confined to the vasculature but absent in
primary and lateral root meristems
Expression of the reporter gene was mostly confined to roots
These results indicated that the EPP and PER promoters mediate root-
specific expression in monocotyledonous and dicotyledonous species,
thus being suitable to drive CKX gene expression

33. 33
Generation of Transgenic Barley Plants with Increased CKX Activity in
Roots
Expression of CKX2 under the control of the EPP promoter
in roots at different developmental stages, no shoot
CK concentrations in roots.

34. 34
Root-specific expression of CK oxidases enhances root system size
Root phenotypes of 2 week-old transgenic
lines grown in hydroponic culture
Total root length and surface area were
calculated using the WinRHIZO software
Increase of the total root length by 24%
to 70% and of the total root surface area
by 12% to 50% in transgenic plants
compared with the wild type (Fig. 2, B
and C). Root biomass of transgenic
plants was increased by up to 47% in
comparison with wild-type roots (Fig.
2D). In contrast, the shoot biomass of the
transgenic lines was comparable to that
of the wild type, except for line
pEPP:CKX1-109, which showed a 15%
increase in shoot biomass (Fig. 2E).

35. 35
Root-Specific Expression of CKX Does Not Cause a Yield
Penalty
root-specific expression of CKX genes caused
root enhancement but did not significantly
affect shoot growth or seed yield in the
transgenic lines.

36. 36
Root-specific expression of CKX enhances mineral element accumulation in leaves
In leaves from 8-week-old soil-grown transgenic
plants, concentrations of numerous mineral
elements were higher in lines expressing CKX2

37. 37
concentrations of most of the elements
were similar in all lines. However, the
concentrations of Ca, Cu, and Zn were
increased consistently in seeds of
transgenic plants.
Element concentration in seeds of
transgenic barley

38. 38
Transgenic plants withstand long-term drought better than the wild type
transgenic plants withstood prolonged water
deficit better than wild-type plants, evident
from the higher CO2 assimilation rate in the
transgenic plants
In transgenic plants, stomatal conductance
was reduced to 25% to 29% and
transpiration rate was reduced to 30% to
32% of control conditions (Fig. 6, A and
B). CO2 assimilation rate 36% to 45% in
the transgenic lines
Together, these results indicated that
transgenic plants withstood prolonged
water deficit better than wild-type
plants. The accumulation of sugars is
important for osmotic adjustment
under drought stress

39. 39
ABA homeostasis and Proline concentrations in pEPP:CKX transgenic lines
Under control conditions, the steady-state levels
of ABA and its catabolites were low and similar
or slightly lower in transgenic as compared with
wild-type plants (Fig. 7A).
Drought caused an 11-fold increase in the ABA
level of the wild type and a 4- to 5-fold increase in
transgenic plants (Fig. 7A). The accumulation of
PA and DPA in response to drought was lower in
the transgenic lines than in wild type
Gene expression analysis showed that transcript
levels of gene involved in ABA synthesis
(HvNECD2; E), a gene involved in ABA
degradation (HvABA-8’-OH; F), and the Pro
synthesis gene (HvP5CS1; G) at the eight to nine
tiller stage as determined by RT-qPCR.
Under drought conditions, their concentrations
increased less strongly in CKX-transgenic
barley, indicating, similar to the behavior of
ABA, reduced drought sensitivity

40. 40
The study successfully demonstrated that enhancing root size and architecture in barley through cytokinin
modulation can lead to several beneficial outcomes. The transgenic barley plants with enlarged root systems
showed improved nutrient efficiency, as evidenced by increased concentrations of essential nutrients in leaves
and seeds. Additionally, these plants exhibited dampened stress responses to long-term drought conditions,
indicating enhanced drought tolerance. Importantly, the root engineering approach did not penalize shoot growth
or seed yield, suggesting that the transgenic plants were not limited in their resource allocation. Overall, this
work highlights the potential of root engineering as a promising strategy to improve nutrient efficiency,
biofortification, and drought tolerance in cereal crops.
Conclusion

41. 41
CASE STUDY - 3
Aim: overexpression of ApKUP3 gene affects on K+ accumulation, growth
performance and physiological response to drought stress in transgenic rice plants.

42. 42
Transgenic Rice Development: The CaMV35S :: ApKUP3 construct to overexpress the ApKUP3 gene in rice,
leading to enhanced tolerance to K deficiency and drought . (high-affinity potassium transporter from Alternanthera
philoxeroide)
Experimental Conditions: Seedlings were subjected to different treatments including potassium deficiency,
control and excess potassium concentrations as well as drought stress induced by PEG 6000 supplementation.
Physiological Analyses: Various parameters such as net photosynthetic rate, stomatal conductance, proline
content, antioxidant enzyme activities (SOD, POD, CAT, APX) H2O2 content and potassium content were
measured.
Molecular Analysis: The behaviour of the transgene and putative stress-responsive antioxidation genes was
analysed using Northern blot and real-time quantitative polymerase chain reaction (RT-qPCR)
Materials and Methods

43. 43
plasmid construct with ApKUP3 open reading frame driven by the CaMV 35S
promoter
The Northern blot analysis shows that ApKUP3 was constitutively expressed
in both shoots and roots of all the three T1 generation rice lines

44. 44
The responses of 14-d-old
seedlings of WT and transgenic
plants to various external K+
concentrations
ApKUP3 overexpression affect on overall plant growth and
development
The total fresh masses of the ApKUP3
overexpressing transgenic plants were ~34 % (K+
deficiency), ~37 % (control), and ~30 % (K+
excess) higher than those of the WT plants (Fig.
2A)
Root biomass of the transgenic lines was obviously
increased together with an enhanced total root
length under the K+ deficiency compared to that in
the WT plants (Fig. 2B).
The tissue K+ content was also increased in the
transgenic lines especially under the K+ deficiency
(with a ~67 % increase in shoots and ~40 % in
roots) (Fig. 2D).
ApKUP3 overexpression improved plant
performance and a K+ accumulation, especially
under unfavorable K+ nutrient conditions

45. 45
ApKUP3 overexpression affect on plant response to
drought stress
The water loss and content of H2O2
was lower in the shoots of the
transgenic plants than in the WT
plants (Fig. 3B).
Correspondingly, significantly
higher activities of SOD, POD, and
APX were observed in the leaves
of the transgenic plants than in the
WT plants from day 15 to day 21
(Fig. 3C,D,E). However, no
difference in CAT activity was
found between the WT and
transgenic plants (Fig. 3F).

46. 46
transgenic plants showed a higher total fresh mass and non-chlorotic leaves accompanied by significantly
higher amounts of total chlorophyll and proline, enhanced gs and PN
Responses of 14-d-old seedlings to the drought stress

47. 47
The molecular mechanisms underlying the relation between antioxidant
enzyme activities and drought tolerance
The genes encoding SOD, POD, and APX had
a higher expression in the transgenic plants
than in the WT plants with different dynamics
under the PEG treatment
No difference in the transcription of three
OsCAT genes, was found between the WT
and transgenic plants

48. 48
The overexpression of ApKUP3 in rice plants resulted in enhanced potassium nutrition
and improved tolerance to drought stress. Transgenic plants exhibited increased root
formation, higher potassium content, reduced H2O2 levels, and elevated activities of
antioxidant enzymes compared to wild-type plants. These findings suggest that
ApKUP3 plays a crucial role in plant response to abiotic stresses and may serve as a
valuable target for enhancing crop resilience and productivity in challenging
environmental conditions
Conclusion

49. 49
CASE STUDY - 4

50. 50
Materials and Methods
Transgenic Plant Development: Transgenic rice plants were created by introducing the OsSRDP gene,
controlled by a stress-inducible promoter (AtRd29A) into the background of cv. Pusa Sugandh 2 (PS2).
Molecular Analysis: The integration and copy number of the transgene were confirmed qRT-PCR and
microarray analysis identify differentially expressed genes and pathways associated with stress tolerance
Experimental Stress Conditions: The transgenic plants were subjected to various abiotic stresses such as
drought, salinity, cold, and heat to evaluate their resilience compared to non-transformed PS2 plants.
Physiological Assessments: Several physiological parameters were measured, including relative water
content (RWC), photosynthetic pigments, proline accumulation, and accumulation of reactive oxygen
species (ROS). Cell membrane injury under cold stress and resistance to rice blast fungus were assessed.

51. 51
This plant transformation construct, pCAMBIA1300- pAtRd29A-OsSRDP-NosT (pC1300::SRDP),
was used for rice Agrobacterium genetic transformation in to PS2 (drought susceptible) cultivar
Construction of recombinant plasmid (pC1300::SRDP) and rice transformation

52. 52
Phenotypic and physio-biochemical trait analyses of
the AtRd29A::OsSRDP transgenic rice plants and WT
in response to water-deficit stress. (A) Phenotypic
appearance of WT and AtRd29A::OsSRDP transgenic
rice plants at the active tillering stage under well
water condition, before imposing drought stress, (B,
C) WT and AtRd29A::OsSRDP transgenic plants
subjected to drought stress for 7 and 14 days,
respectively, and (D) recovery of plants after 10 days
of re-watering.
Analysis of OsSRDP gene expression under drought stress
transgenic lines remained healthy and were
able to retain turgidity without any stress
symptoms during this short stress period
(Figure 2B). Transgenic plants remained green,
though they did show leaf rolling and wilting
(Figure 2C). recovered more vigorously,
whereas just one or a few leaves of WT plants
recovered greenness (Figure 2D).

53. 53
stress-inducible OsSRDP confers drought tolerance in rice
RWC declined to 58%–70% RWC in the transgenic
plants and 40% in the WT plants after 14 days of
drought stress and Ten days after re-watering, RWC
increased up to 67%–75% in all the transgenic plants as
compared to WT plants (49%), whose leaves had
almost dried out. (Figure 2E).
Degradation of photosynthetic pigments in
AtRd29A::OsSRDP transgenic plants ranged from
17% to 34%, while it was 45% in WT plants (Figures
2G, H). After 10 days of re-watering,
AtRd29A::OsSRDP transgenic plants exhibited a
higher quantum of photosynthetic pigments (8%–
27%) compared to WT plants (10%).
AtRd29A::OsSRDP transgenic rice plants showed 18,
14, and 20-folds more accumulation of proline in the
DUF-1, DUF-2, and DUF-3 lines, respectively, after
14 days of water-deficit stress (Figure 2F). They also
showed a lesser reduction of proline content (1.4-1.6
fold) than WT plants (2.6 fold), after 10 days of re-
watering.
AtRd29A::OsSRDP transgenic plants showed
enhanced drought tolerance as demonstrated from
their RWC, proline content, photosynthetic
pigments and recovery after drought stress

54. 54
RSA was studied in the AtRd29A:: OsSRDP transgenic
lines and WT plants under well-watered conditions as
well as in response to drought stress. Interestingly, no
noticeable differences could be observed between WT
and transgenic plants in the root phenotype or RSA
parameters, namely, total root length, diameter,
surface area, and volume of root under either well-
watered or moisture-deficit conditions. stress-induced
expression of OsSRDP does not have any significant
impact on enhancing the root system architecture in
transgenic rice plants, even under drought stress
Analysis of root system architecture transgenic plants under drought stress

55. 55
transgenic plants showed less ROS
accumulation in response to drought stress
WT and the AtRd29A:: OsSRDP transgenic rice
lines following 2 weeks of drought stress
revealed much stronger dark blue NBT staining
in WT than that of the three AtRd29A::OsSRDP
transgenic lines (Figure 4A). Likewise, WT
plants showed more reddish brown DAB
staining compared to AtRd29A::OsSRDP
transgenic lines during water stress. results
revealed that WT plants had a significantly
higher accumulation of ROS
nitrobluetetrazolium (NBT) and diaminobenzidine (DAB)

56. 56
imposition of salt stress with 150 mMNaCl
for 7 days, most of the WT plant’s leaves
were severely withered, while
AtRd29A::OsSRDP transgenic seedlings
survived moderately without serious rolling
and wilting of leaves (Figure 5B). half of the
transgenic seedlings could recover by the
sixth day while almost 85% of WT seedlings
became pallid and died (Figure 5C),
transgenic lines maintained less decay
(8%– 9%) of photosynthetic pigments
than WT plants (Figures 5D, E).
transgenic seedlings showed
significantly less reduction of fresh
weight (45.5%–51.7%) and dry weight
(40%–47.4%) as compared to the
corresponding WT (54.4 and 52.3%)
under salt stress (Figures 5G, H).
Transgenic plants also showed
significantly (2.3-fold) higher levels of
proline accumulation compared to WT
plants
Stress- induced expression of OsSRDP in rice results in improved salinity tolerance

57. 57
Stress- induced expression of OsSRDP in rice results in improved cold tolerance
12 days of cold stress, WT plants showed severe yellowish
and wrinkled leaves, unlike transgenic lines (Figure 6B).
transgenic seedlings showed moderate wilting, retaining their
greenness, and showing new younger leaves upon recovery
(Figure 6C), with an average survival rate of 47%–62%,
significantly higher than that of the WT plants (21%) (Figure
6F). after 12 days of cold stress, we found >40% electrolyte
leakage in WT plants, while it was<30% in the transgenic
lines (Figure 6E). Likewise, the MDA - malondialdehyde
(ROS) contents of three different AtRd29A::OsSRDP
transgenic lines were significantly lesser (0.6-1 fold) when
compared with that of WT plants (Figure 6D).

58. 58
Transgenic plants showed resistance to rice blast fungus M. oryzae
The disease symptoms were recorded
in the form of chlorotic lesions after
72 hpi. In the case of AtRd29A::
OsSRDP transgenic plants, no
lesions were observed on the leaves
(Figure 8), whereas WT and
AtRd29A::OsCHI2 transgenic plants
showed lesions of size ranging from
1 mm to 4 mm diameter. These
results clearly indicated that
AtRd29A::OsSRDP transgenic plants
were resistant to rice blast disease

59. 59
Upregulation of ROS scavenging genes in the transgenic lines under multiple abiotic stresses
expression level of OsSOD (superoxide dismutase) and OsPOD (peroxidase) was significantly higher in the transgenic plants, 8-13 and 2.7-6 folds, respectively, as compared to WT plants
under water-deficit stress (Figures 7A, B). Similarly, the expression level of the OsSOD gene increased more than 4.6-6.7 and 5.2-8.6 folds in AtRd29A::OsSRDP transgenic lines in
comparison to the WT plants under salt and cold stresses, respectively (Figures 7C, E). The transcript level of the OsPOD gene was significantly higher by 1.9-3.3 and 2.8-5 folds under salt
and cold stresses, in the transgenic rice lines (Figures 7D, F). Thus, the upregulation of ROS scavenging genes was found to be associated with the tolerance of AtRd29A::OsSRDP transgenic
plants under multiple abiotic stresses.

60. 60
The study concludes that the stress-inducible expression of the OsSRDP gene
significantly enhances tolerance to multiple abiotic stresses (drought, salinity, cold) and
a biotic stress (rice blast fungus). Bioinformatics analysis identified potential interaction
partners for the gene, suggesting its involvement in complex stress response pathways.
Overall, the findings suggest that OsSRDP could be a valuable candidate for improving
stress resilience in rice through genetic engineering approaches.
Conclusion

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CASE STUDY - 5
Aim: To develop transgenic rice plants capable of accumulating sakuranetin,
to enhance the nutritional value and disease resistance in rice grains.

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Materials and Methods
Transgenic Plant Development: Transgenic rice plants were developed by introducing the NOMT (naringenin 7-
O-methyltransferase) gene under the control of the OsGluD-1 endosperm-specific promoter into rice cells.
Validation of Sakuranetin Accumulation: Liquid chromatography tandem mass spectrometry (LC-MS/MS) was
used to quantify sakuranetin levels in the seeds of transgenic rice plants at different stages of development.
Evaluation of Disease Resistance: The panicle blast resistance of transgenic rice plants was assessed and
compared to wild-type rice plants.
Assessment of Nutritional and Quality Indicators: soluble sugars, total amino acids, total flavonoids, amylose,
total protein, and free amino acid content, were analyzed. The phenotypes traits such as grain width, grain
length, and 1000-grain weight were also evaluated.
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) Imaging: MALDI-MS imaging
to detect the content and spatial distribution of sakuranetin and other nutritional metabolites

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The Accumulation Pattern of Sakuranetin in Rice:
naringenin was high in the shoots of rice seedlings,
gradually increased in roots with growth and
development, and was rarely present in seeds at the
filling and mature stages

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The accumulation pattern of the sakuranetin in rice seeds
The GUS staining patterns revealed that
OsNOMT was highly expressed in the leaves and
leaf sheaths of rice seedlings, and was slightly
expressed in the ridges and embryos of seeds at
the filling stage, with no signals in roots, husks,
and endosperm (Fig. 2A–C). The quantitative
real-time PCR (qRT-PCR) results also showed
that OsNOMT was highly expressed in the shoots
of rice seedlings. The expression levels decreased
gradually with the growth time, while it was
almost not expressed in roots and seeds (Fig.
2D). As shown in Fig. 2E, in general agreement
with the OsNOMT expression pattern and
naringenin content, sakuranetin content was high
in the shoots of rice seedlings and decreasing
with growth and development time, whereas it
was not detected in roots.
These results indicate that
sakuranetin is absent or present in
rice seeds at very low abundance.

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Engineering the Biosynthesis of Sakuranetin in the Rice Endosperm
A) Western blot analysis
the protein levels of
OsNOMT-GFP in 7-
day-old shoots of
p35S::OsNOMT-GFP
B) qRT-PCR analysis
of the expression levels
of OsNOMT in 7-day-
old shoots of
p35S::OsNOMT-GFP.
C) LC-MS/MS
analysis of the
sakuranetin content
in 7-day-old shoots
D) LC-MS/MS analysis of
the sakuranetin content in
15 DAF seeds of
p35S::OsNOMT-GFP

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No change
specific expression of OsNOMT in endosperm resulted in the accumulation
of sakuranetin in rice seeds
15 DAF
25 DAF
content of sakuranetin in rice seeds at the filling stage were found to be notably higher than wild type in three transgenic lines

67. 67
The panicle of pGluD- 1::OsNOMT had more seeds than the
wild type. Further detection of the relative fungal growth by
DNA-based qPCR revealed that the M. oryzae biomass of
transgenic panicles was much less than wild type.
endosperm-specific expression of OsNOMT successfully increased the rice blast resistance

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The Nutrition and Quality of pOsGluD-1::OsNOMT Seeds Were not Affected
The contents of total amino acid
content, total soluble sugars,
total flavonoid, amylose, total
protein and free fatty acid in the
mature seeds were detected, and
there was no significant
difference between pOsGluD-
1::OsNOMT plants and wild
type (Fig. 4E–H). In summary,
these results show that the
nutrition and quality of
pOsGluD-1::OsNOMT seeds
were not affected.

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The Growth and Development of pOsGluD-1::OsNOMT Plants Were not Affected
based on our observations in the phytotron and the field, we also found the vegetative and reproductive phenotypes of p35S::OsNOMT-GFP were not significantly different from the WT
at all stages of growth and development. This suggested that the accumulation of sakuranetin in various tissues of rice does not influence its growth and development
14-day-old reproductive stage maturation stage
Mature grains
Husked grains

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The study successfully developed a biofortified rice plant with enriched sakuranetin
content in the endosperm, demonstrating enhanced nutritional quality and potential
health benefits. The findings suggest that the overexpression of OsNOMT in rice can
lead to significant improvements in metabolite accumulation and phenotypic traits,
highlighting the potential of biofortified rice in addressing nutritional deficiencies and
enhancing crop resilience.
Conclusion

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Thank you