High-value pleiotropic genes for developing multiple stress-tolerant biofortified crops for 21st-century challenges.pptx

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. 1 st Year Dept. of GPBR Submitted to :- Dr. M. Shanthi Priya Professor & Head Dept. of GPBR ACHARYA N.G. RANGA AGRICULTURAL 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 V ertical 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 2020 Wheat MACS 4058 (durum) Rich in protein (14.7 %), iron (39.5 ppm) and zinc (37.8 ppm) 2020 2020 HD 3298 Rich in protein (12.1 %) and iron (43.1 ppm) 2020 2020 HI 1633 Rich in protein (12.4 %), iron (41.6 ppm) and zinc (41.1 ppm) 2020 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 2020 Pusa HQPM 7 Improved Rich in provitamin-A (7.10 ppm), lysine (4.19 % in protein) and tryptophan (0.93 % in protein) 2020 2020 IQMH 203 (LQMH 3) Rich in lysine (3.48 % in protein) and tryptophan (0.77 % in protein) 2020 2020 Pearl Millet HHB 311 Rich in iron (83.0 ppm) 2020 2020 Finger Millet VR 929 ( Vegavathi ) Rich in iron (131.8 ppm) 2020 2020 CFMV1 (Indravati) Rich in calcium (428 mg/100g), iron (58.0 ppm) and zinc (44.0 ppm) 2020 2020 CFMV 2 Rich in calcium (454 mg/100g), iron (39.0 ppm) and zinc (25.0 ppm) 2020 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 se lection 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 T issue 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 E ffect 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 T he 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 E ffects of OsSGL o n 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 A pplication of 93-11-OE lines in hybrid rice breeding T he morphological marker of curling flag leaves facilitated the selection of positive transgenic plants

26. 26 P ossible 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, s hoot 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 I ncrease 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 R oot-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 CO 2 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). CO 2 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) H 2 O 2 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. 2 A ) R oot 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. 2 B ). 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. 2 D ). 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 H 2 O 2 was lower in the shoots of the transgenic plants than in the WT plants (Fig. 3 B ). 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. 3 C , 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 g s and PN Responses of 14-d-old seedlings to the drought stress

47. 47 T he 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 ). D egradation 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 signi fi cantly 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 signi fi cantly (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 T ransgenic 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

61. 61 CASE STUDY - 5 Aim: T o develop transgenic rice plants capable of accumulating sakuranetin , to enhance the nutritional value and disease resistance in rice grains.

62. 62 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

63. 63 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

64. 64 T he 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. 2 A–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. 2 D). As shown in Fig. 2 E, 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.

65. 65 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

66. 66 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 T he 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

68. 68 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. 4 E–H). In summary, these results show that the nutrition and quality of pOsGluD-1:: OsNOMT seeds were not affected.

69. 69 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

70. 70 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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73. S.V. AGRICULTURAL COLLEGE, TIRUPATI 73 Thank you