Nano-priming - An Emerging technology in Medicinal and Aromatic Plants.pptx

Nano-priming stands out as a transformative technique that significantly enhances seed germination and seedling growth by integrating nanotechnology with traditional seed priming methods. By treating seeds with nanoparticles, this technique induces specific physiological states that boost tolerance to various biotic and abiotic stresses, thereby improving overall plant growth, yield, and resilience. This is particularly beneficial for medicinal and aromatic plants, ensuring consistent germination rates and active compound concentrations. Nanoparticles such as silver, gold, zinc oxide and carbon-based compounds interact at the molecular level with seeds, enhancing their biochemical properties. This interaction leads to improved germination rates, stress resistance and seedling development. Additionally, nanoparticles can act as carriers for nutrients and growth regulators, providing controlled release and reducing the need for chemical fertilizers and pesticides, thus promoting sustainability.
Historical advancements in seed priming have paved the way for modern nano-priming techniques. From early observations of seed soaking to sophisticated nanotechnological applications, seed priming has continually evolved, improving agricultural productivity and sustainability. The integration of nanotechnology has brought about significant advancements in enhancing seed germination and crop productivity under stressful conditions. The use of nanoparticles in seed priming offers substantial benefits, including increased stress resilience, improved photosynthetic efficiency, enhanced starch and lipid metabolism and elevated secondary metabolite production. These advantages make nano-priming a promising approach for sustainable agriculture, helping farmers achieve higher yields while minimizing environmental impact. As research and development continue, optimizing the physico-chemical properties of nanomaterials will be crucial for maximizing the effectiveness of nano-priming, ensuring improved agricultural outcomes and contributing to global food security.

1. WELCOME

2. UNIVERSITY OF HORTICULTURAL SCIENCES, BAGALKOT COLLEGE OF HORTICULTURE, BENGALURU-560065 Speaker : Shivanand D Ainapur UHS21PGD422, III Ph.D. Department of PSM&AC Major Advisor: Dr. Maruthi Prasad B. N. Associate Professor, Department of PSM&AC Seminar-IV Nano-priming: An emerging technology in medicinal and aromatic plants 2 July 2024 Department of PSM&AC 2/48

3. CONTENTS 2 July 2024 3 Department of PSM&AC 3/48

4. The induction of a particular physiological state in plants by treating the seeds prior to sowing with natural or synthetic compounds to promote crop performance by enhancing tolerance to biotic and abiotic stress. 02-07-2024 Dept. of FSC Principle : To minimize the period of emergence and to protect the seeds from stresses during the critical phase of seedling establishment. Heydecker et al. , 1973 Introduction 4/48

5. Timeline of seed priming development towards nano-priming Seed priming Gaius Secundus (23 A.D) Theophrastus (371 B.C) Charles Darwin (1855) Soaked cucumber seeds in water Dried the soaked seeds before sowing Cucumber seed priming with honey Ells (1963) Seed priming with seawater treatment Seed priming with nutrition Heydeeker (1973 ) Khodakovskaya et al., (2009) Seeds treated with nanocarbon Seed priming with PEG Oliver de Serres (1539) Seed priming with salt solution Koehler (1967) Berric & Drennam (1971) Studied the effect of seed priming time Seed Nano-priming Technology 5/48 Nile et al ., 2022

6. Types of seed priming techniques 6/48 Nile et al ., 2022

7. Nanoparticles used in Nano seed priming Metal-based Nanoparticles : Silver nanoparticles, zinc oxide nanoparticles and copper nanoparticles Carbon-based Nanoparticles: Carbon nanotubes and graphene nanoparticles have unique physical and chemical properties Metal-Organic Frameworks (MOFs): MOFs are crystalline nanoporous materials that can serve as carriers for nutrients, water or bioactive compounds, providing sustained release and targeted delivery to seeds 7/48 Drummer et al ., 2021

8. Synthesis of nano-particles 8/48 Drummer et al ., 2021

9. Figure 1. Physico -chemical properties for nanomaterial for seed priming Particle size – ranging from 1-100 nm Particle shape – spherical, rod-shaped, tubular and plate like Zeta potential – indicative of surface charge Composition – ZnO , Fe 2 O 3, SiO 2 & Ag-NPs Surface corona – layer of molecules that adsorb on the Surface of NPs Aggregation - NPs clump together Drummer et al ., 2021 9/48

10. Figure 2. Mechanism of nano-priming in seed germination 5 4a 4b 3 6 2 7 9 8 10/48 Nile et al ., 2022 Induced expressions of cell division ( CycB ), Cell wall extension (NtLRX1 gene) & enhanced cell respiration enzyme (dehydrogenase) Improved seed germination rate, SVI, induced Rubisco activity, altered PSI & PSII and Improved photosynthesis Facilitate water uptake, ROS diffusion Membrane damage NPs H 2 O 2 /ROS production (Oxidative stress) Water molecules Aquaporins (PIP1 & PIP2) Induced radical growth Starch Soluble sugars

11. Table 1 . Influence of nano-priming plant growth and metabolism Effect on plant physiology Changes in chemical constituents Molecular changes in plant Growth & biomass, Photosynthesis [Chlorophyll] Photosynthetic quantum efficiency, Gas exchange traits, Carotenoids, Cellular electron exchange, Soluble proteins, Relative water content, Antioxidant enzymes [ POX, SOD, CAT & APX ] Gibberellic acid IAA ABA:GA α-amylase Soluble sugar Aquaporins Lipids DNA content DNA repair Cell wall extension ( NtLRX1 ) Cell division ( CycB ) Aquaporin ( PIP1, PIP2, NIP1, TIP3 & TIP4 ) Phynylalanine ammonia lysase ( PAL1 ) Anthocyanin synthase 1 ( ANS1 ) and Anthocyanin pigment 1 ( PAP1 ) 11/48 Nile et al ., 2022

12. Figure 3. Influence of nano-priming plant metabolism 12/48 Nile et al ., 2022

13. Figure 4. Effect of nanopriming agents to improve seed germination in abiotic and biotic stress conditions Nile et al ., 2022 13/48

14. Effect of nanoparticles on seed g ermination and Seedling g rowth of Boswellia Ovalifoliolata – An endemic and e ndangered m edicinal t ree Objectives : To investigate the effect of biologically synthesized SNPs on seed germination and seedling growth Savitramma et al ., 2012 Nano Vision – NAAS 7.11 14/56 Department of Botany, Sri Venkateswara University, Tirupati, Andhra Pradesh, INDIA .

15. Material and methods SNPs prepared biologenically using the bark extract of Boswellia ovalifoliolata The seed germination experiment was carried out with four sets, each set with five test tubes containing MS basal medium without growth regulators Five seeds were placed in each test tube and observed for germination Savitramma et al ., 2012 15/56 Treatment set Details First set – Control MS basal medium Second set MS basal medium + 1 ml of SNPs (10 μ g/ml) Third set MS basal medium + 2 ml of SNPs (20 μ g/ml) Forth set MS basal medium + 3 ml of SNPs (30 μ g/ml)

16. Figure 5: (a) The colour change of bark extracts, ( b ) Absorption at 430nm in UV-Vis Spectroscopy (c) SEM image of synthesized silver nanoparticles of stem barks of Boswellia ovalifoliolata Savitramma et al ., 2012 16/56 a. b. c.

17. Table 2. Effect of Silver nanoparticles on in-vitro seed germination and seedling growth of Boswellia ovailifoliolata values are an average of five replications ± SE. S. No. Concentration Germination Percentage Germination period (Days) Seedling growth (cm) 1. Control 70 ± 2.5 17 ± 3 3.0 ± 0.5 2. 10 m g /ml 91 ± 3.2 8.0 ± 1 5.5 ± 1.0 3. 20 m g / ml 92 ± 2.0 8.0 ± 2 6.3±1.7 4. 30 m g /ml 95 ± 3.1 9.0 ± 2 10.6 ± 0.3 Figure 6. Effect of biologically synthesized silver nanoparticles on seed germination and seedling growth of Boswellia ovaliofoliolata a) Control, b) SNPs 10 m g/ml, c) 20 m g/ml and d) 30 m g/ml Savitramma et al ., 2012 17/48 Inference

18. Stimulating effect of biogenic nanoparticles on the germination of basil ( Ocimum basilicum L.) seeds Objectives : To investigate the effects of various concentrations of biosynthesized NPs on the germination and germination index of basil seeds Sencan et al ., 2024 Sci. Rep . – NAAS 10.60 18/48 Department of Chemical Engineering, Biology and Bioengineering, Suleyman Demirel University, 32260 Isparta , Turkey.

19. Material and methods Biosynthesis of NPs (Ag-, ZnO - & Fe 3 O 4 -NPs) using lavender ( Lavandula officinalis L.) flowers and thyme leaves ( Origanum minutiflorum O. Schwarz & P.H. Davis) Treatments – primed for 24 h Relative seed germination (%), Relative root growth (%), Germination Index (GI), Germination vigour index (GVI) & Seed water content (SWC) Sencan et al ., 2024 Nanoparticles Ag-NPs & ZnO -NPs Magnetite (Fe 3 O 4 )-NPs Concentration (mg L -1 ) 0 (Control), 25, 50, 100, 200 0 (Control), 50, 100, 200 & 400 19/48

20. Figure 7. Response of NPs on germination index . Bars indicate standard errors of the means ― SE ( n = 20); different letters over identical bars indicate significant differences (Duncan post-hoc test; P ≤ 0.05). Sencan et al ., 2024 20/48 Germination Index (%)=   GP – 85%, Root length – 19.4 mm & GI – 139.15% GP – 8-43%, Root length – 16.6 mm & GI – 119.68% GP – 88%, Root length – 13.2 mm & GI – 90%

21. Figure 8. Response of NPs on germination vigor index . Bars indicate standard errors of the means ― SE ( n = 20); different letters over identical bars indicate significant differences (Duncan post-hoc test; P ≤ 0.05). Sencan et al ., 2024 21/48 Germination vigour index (%) =   GVI by 21%, Seedling length – 44.6mm GVI by 12%

22. Figure 9. Effects of NPs in seed water content . Bars indicate standard errors of the means ― SE (n = 20); different letters over identical bars indicate significant differences (Duncan post-hoc test; P ≤ 0.05). Sencan et al ., 2024 22/48 SWC (%) = x 100  

23. Figure 10. The water-holding capacity of basil seeds germinated at different concentrations of NPs on the third day of germination: a. control; b. Fe3O4- NP (50 mg/L); c. ZnO-NP (25 mg/L); d. Ag-NP (400 mg/L). Sencan et al ., 2024 23/48 Inference

24. The impact of priming with Al 2 O 3 nanoparticles on growth, pigments, osmolytes and antioxidant e nzymes of Egyptian Roselle ( Hibiscus sabdariffa L.) cultivar Objectives : To investigate the effi cacy of various doses of Al 2 O 3 tested on Egyptian roselle cultivars to unravel their potential for tolerance Latef et al ., 2020 Agronomy – NAAS 9.70 24/48 Botany and Microbiology Department, South Valley University, Qena 83523, Egypt .

25. Material and methods Synthesis of Al 2 O 3 Nanoparticles Seed treatment – 5 groups Growth traits, photosynthetic pigments, organic solutes, Malondialdehyde and antioxidant enzyme assays Set Treatment 1 st Set – Control Priming with distilled water for 12 h 2 nd Set Primed with 0.01% Al 2 O 3 NPs for 12 h 3 rd Set Primed with 0.05% Al 2 O 3 NPs for 12 h 4 th Set Primed with 0.1% Al 2 O 3 NPs for 12 h 5 th Set Primed with 0.5% Al 2 O 3 NPs for 12 h Latef et al ., 2020 25/48

26. Figure 11. Effects of seed-priming with aluminum nanoparticles (Al 2 O 3 NPs) on (A) chlorophyll a, (B) chlorophyll b and (C) carotenoids content in roselle plants Latef et al ., 2020 26/48

27. Figure 12. E ff ects of seed-priming with aluminum nanoparticles (Al 2 O 3 NPs) on ( A) fresh weight, (B) dry weight, (C) root length and shoot length and (D) leaf area in roselle plants Latef et al ., 2020 27/48

28. Figure 13. Effects of seed-priming with aluminum nanoparticles (Al 2 O 3 NPs) on (A) soluble sugar, (B) soluble protein, (C) total free amino acid and (D) proline in roselle plants Latef et al ., 2020 28/48

29. Figure 14 . Effects of seed-priming with aluminum nanoparticles (Al 2 O 3 NPs) on the activities of (A) superoxide dismutase (SOD), (B) catalase (CAT), (C) peroxidase (POD) and (D) ascorbate peroxidase (APX) in leaves of roselle plants Latef et al ., 2020 28/48

30. Figure 15 . Effects of seed-priming with aluminum nanoparticles NPs) on malondialdehyde content (MDA) in roselle plants Latef et al ., 2020 30/48

31. Figure 16 . ( A ). Principal component analysis (PCA) to understand the variability relationships of parameters and treatments in roselle plants. ( B ) The treatments’ rectangles in different colors included (control) set was primed with distilled water for 12 h. The 2nd set was primed with 0.01% Al 2 O 3 NPs for 12 h. The 3rd set was primed with 0.05% Al 2 O 3 NPs for 12 h. The 4 th set was primed with 0.1% Al 2 O 3 NPs for 12 h. The 5th set was primed with 0.5% Al 2 O 3 NPs for 12 h Latef et al ., 2020 31/48 Inference

32. Application of chitosan nano-priming on plant growth and secondary metabolites of Pancratium maritimum L. Objectives : To estimate the potential of primed seed with chitosan nanoparticles on seed growth and yield by inducing plant secondary metabolism Allam et al ., 2024 BMC Plant Biol. – NASS 9.90 32/48 Department of Botany and Microbiology, Alexandria University, Alexandria, Egypt .

33. Material and Methods Preparation of chitosan nanoparticles S eeds (Coated and uncoated) were imbibed in each concentration of chitosan nanoparticles ( CsNPs ) (0.1, 0.5, 1 mg/ ml) for 4, 8 and 12 h Germination bioassay – Petri dish assay Growth experiment – In plastic pots Growth Parameters – Germination percentage, Gemination velocity, Speed of germination, Germination energy, Germination index, Mean germination time and Seedling vigour index Determination of plant biomass and water content Determination of phytochemicals [Alkaloid] – GC-MS Antioxidant activity Allam et al ., 2024 33/48

34. Figure 17 . Effect of different concentrations of nano-priming usi n g chit o san nanoparticles (0.1, 0 5 and 1 mg/ml) on germination of coated and uncoated seeds under vari o us seed priming trea t ments fo r the three different soaking durations. Coated Seeds Uncoated seeds 34/48 Allam et al ., 2024

35. F igure 18. Variations of a: Germination Percentage (GP%), b: Germination Velocity ( GVe ) c: Speed of Germination (SG) and d: Germination energy (GE) in coated and uncoated seeds of Pancratium maritimum L. in pot experiment in response to different concentration of nano priming under different soaking durations Allam et al ., 2024 35/48 G Ve =   GP (%) =   SG =   GE = x 100  

36. Figure 19. Variations of e : Germination index (GI), f: Mean germination time (MGT), g: shoot root ratio, h: seedling vigor index (SVI) in coated and uncoated seeds of Pancratium maritimum L. in pot experiment in response to different concentration of nanopriming under different soaking durations Allam et al ., 2024 36/48 GI =   MGT =   SVI = Seedling length * GP%

37. Figure 20 . Variations of i : Biomass in coated and uncoated seeds of Pancratium maritimum L. in pot experiment in response to different concentration of nanopriming under different soaking durations Allam et al ., 2024 37/48

38. Table 3. Mean values of a. alkaloids and b. Pancratistatin content (%) (mean ± S.D., n = 3) of Pancratium maritimum L. in response to different concentrations of nano priming under different soaking durations a. Alkaloids 4h 8h 12h F P Control 13.54 bc ± 1.74 13.54 a ± 1.74 13.54 b ± 1.74 - - Nano 0.1 mg/ml 9.45 deB ± 0.42 12.47 abA ± 0.48 13.46 bA ± 0.54 55.936 * <0.001 * Nano 0.5 mg/ml 16.34 aA ± 0.53 9.87 cdB ± 0.86 10.47 deB ± 0.39 98.518 * <0.001 * Nano 1 mg/ml 8.69 eB ± 0.66 9.87 abA ± 0.73 9.65 deA ± 0.33 3.318 0.107 F 28.091 * 24.978 * 42.253 * P <0.001 * <0.001 * <0.001 * b. Pancratistatin 4h 8h 12h F P Control 2.13 a ± 0.22 2.13 a ± 0.22 2.13 a ± 0.22 - - Nano 0.1 mg/ml 2.14 aA ± 0.05 2.15 aA ± 0.05 1.98 abA ± 0.27 1.029 0.413 Nano 0.5 mg/ml 2.19 aA ± 0.06 1.9 abA ± 0.19 1.87 abA ± 0.17 3.671 0.091 Nano 1 mg/ml 2.10 aA ± 0.10 1.84 abA ± 0.00 2.16 abA ± 0.21 4.717 0.059 F 1.908 4.116 * 3.596 * P 0.121 0.006 * 0.011 * Allam et al ., 2024 38/48

39. Table 4. Mean values of a. Lycorin content and b. Antioxidant content (%) (mean ± S.D., n = 3) of Pancratium maritimum L. in response to different concentration of nanopriming under different soaking durations a. Lycorine 4h 8h 12h F P Control 29.45 e ± 1.22 29.45 f ± 1.22 29.45 e ± 1.22 - - Nano 0.1 mg/ml 29.64 deB ± 0.37 29.47 fB ± 0.39 46.74 abA ± 0.50 1669.25 * <0.001 * Nano 0.5 mg/ml 38.46 bA ± 0.52 36.95 dB ± 0.48 36.45 dB ± 0.18 18.595 * 0.003* Nano 1 mg/ml 41.36 aC ± 0.36 54.36 aA ± 0.18 48.21 aB ± 0.32 1458.31 * <0.001* F 120.911 * 745.604 * 42.253 * P <0.001 * <0.001 * <0.001 * b. Antioxidant 4h 8h 12h F P Control 9.12 f ± 0.27 9.12 e ± 0.27 9.12 d ± 0.27 - - Nano 0.1 mg/ml 12.14 bcB ± 0.32 12.64 bcAB ± 0.44 13.24 bA ± 0.19 8.356* 0.018* Nano 0.5 mg/ml 12.89 bA ± 0.35 11.67 dB ± 0.22 11.58 cB ± 0.32 17.549* 0.003* Nano 1 mg/ml 14.31 aC ± 0.10 16.98 aA ± 0.53 15.24 aB ± 0.22 40.550* <0.001* F 75.785* 150.746 * 130.155 * P <0.001* <0.001 * 0.011 * Allam et al ., 2024 39/48 Inference

40. Seed nano‑priming using silica nanoparticles: Effects in seed germination and physiological properties of Stevia r ebaudiana Bertoni Objectives : T o identify and select the most effective nanoparticles in terms of germination parameters and physiological properties to enhance stevia seed germination Hasanaklou et al ., 2023 Chem. Biol. Technol. Agric. – NAAS 12.77 40/48 Department of Nanotechnology and Plant Molecular Physiology, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran .

41. Material and methods Synthesis of nanoparticles ( nSiO 2 (I) and nSiO 2 (II) showed the silica NPs synthesized at 85 °C and 25 °C, respectively) Seed treatment with different concentrations (0, 1, 5, 10, 25, 50, and 100 ppm) of nSiO 2 (I), nSiO 2 (II), and commercial bulk SiO 2 (bSiO 2 ) Germination percentage, germination rate & seed vigour Starch and sucrose determination Antioxidant enzymes activity [CAT & POX] Hasanaklou et al ., 2023 41/48

42. Figure 21. Impacts of different priming agents ( nSiO 2 (I), nSiO 2 (II) and bSiO 2 at concentrations of 1, 5, 10, 25, 50 and 100) on A. seed germination percentage, B. germination rate, C. seed vigor index D. root dry weight, E. shoot dry weight and F. seedling dry weight Hasanaklou et al ., 2023 42/48

43. Figure 22. Impacts of different priming agents ( nSiO 2 (I), nSiO 2 (II) & bSiO 2 at concentrations of 1, 5, 10, 25, 50, and 100) on A. α-amylase, B. cotyledon starch, C. root sucrose and D. shoot sucrose content Hasanaklou et al ., 2023 43/48

44. Table 5. CAT and POX activity and H 2 O 2 concentration in the treated seed with SiO 2 NPs Treatments Concentration (ppm) CAT activity (U mg -1 Protein ) POX activity (U mg -1 Protein ) H 2 O 2 concentration ( umol g -1 FW) Control - 24.77 ― 0.29 0.044 ― 0.0003 4.22 ― 0.09 bSiO 2 1 27.74 ― 0.30 0.045 ― 0.0004 4.36 ― 0.04 5 29.82 ― 1.58 0.046 ― 0.0010 4.36 ― 0.07 10 30.38 ― 0.37 0.049 ― 0.0004 4.24 ― 0.05 25 31.61 ― 0.18 0.050 ― 0.0007 4.42 ― 0.03 50 31.20 ― 0.79 0.050 ― 0.0009 4.63 ― 0.01 100 30.24 ― 0.24 0.052 ― 0.0005 4.70 ― 0.02 nSiO 2 (I) 1 29.88 ― 0.15 0.047 ― 0.0006 3.1 ― 0.04 5 31.45 ― 0.49 0.056 ― 0.0039 3.46 ― 0.04 10 32.41 ― 0.54 0.058 ― 0.0007 3.68 ― 0.10 25 33.09 ― 0.25 0.057 ― 0.0030 3.87 ― 0.18 50 32.81 ― 0.46 0.056 ― 0.0005 4.53 ― 0.06 100 31.72 ― 0.39 0.055 ― 0.0004 4.69 ― 0.02 nSiO 2 (II) 1 29.29 ― 0.37 0.052 ― 0.0012 3.50 ― 0.05 5 29.93 ― 0.37 0.055 ― 0.0004 3.62 ― 0.06 10 36.15 ― 0.29 0.057 ― 0.0003 3.67 ― 0.10 25 34.66 ― 0.21 0.057 ― 0.0003 4.48 ― 0.04 50 33.25 ― 0.23 0.056 ― 0.0003 4.55 ― 0.04 100 32.90 ― 0.65 0.051 ― 0.0005 4.65 ― 0.04 Hasanaklou et al ., 2023 44/48

45. Figure 23. Pearson correlation coefficients among 14 quantitative traits on stevia seedlings under the influence of SiO 2 NPs. The correlation coefficients with absolute values higher than 0.20 and 0.26 were significant at the statistical probability level of 5 and 1 per cent, respectively. Hasanaklou et al ., 2023 45/48 Inference

46. Available nanoparticles for agricultural use Cost of NPs ZnO -NPs – 20-100$ kg -1 Ag-NPs – 100-300$ g -1 Copper NPs – 50-100$ g -1 TiO 2 -NP – 20-50$ g -1 Au-NPs – 80-150$ g -1 Chitosan NPs – 300-500$ 100g -1 Lipid NPs – 200-500$ g -1 Magnetite NPs – 380-2255 $ kg -1 MgO 2 -NPs – 20-50$ g -1 ( Source: Cognitive Market Research , Fortune Business Insights , Fact.MR & IMARC ) 46/48

47. Limitations of nanotechnology in agriculture sector 47a/48 Saritha et al ., 2023

48. Future prospects of nano-priming Optimization of size, shape, concentration and treatment duration in various plant species Development of new nano-enabled materials based on bio-polymers viz., chitosan, sodium aginate , xanthane gum, lignin and gum arabic etc. Genome-wide transcriptomic study in different nano-priming conditions will be useful in understanding the commonly controlled networks responding to NPs Utilization of various aquaporin family gene mutants is probably useful to dissect additional transcription co-factors correlated with the expression of aquaporin genes in primed seeds Kandhol et al ., 2022 & Nile et al ., 2022 47b/48

49. Conclusion 48/48