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Advance breeding for salt stress in flower crops seminar 2 converted
- 2. Division of Floriculture and Landscaping Seminar FLA691 ICAR- IARI (Indian Agricultural Research Institute) New Delhi 110 012 By: Mohammad Musa Daudzai 11373 Ph. D 1st year Trimester (III) 2018-19Chairman: Dr. Prabhat Kumar.
- 4. 4 IndianAgriculturalResearchInstitute,NewDelhi Salinity is defined as the presence of excessive amounts of soluble salts that affect the normal functions of plant growth. In India 6.73 million hectares are salt-affected Soil salinity is a worldwide issue threatening land productivity. 30% loss of arable land 2025 50% of all arable land will become impacted by salinity by 2050 Salinity
- 5. Global scenario of soil problem Regions Total area Saline soils Sodic soils Mha Mha % Mha % Africa 1,899 39 2.0 34 1.8 Asia, the Pacific andAustralia 3,107 195 6.3 249 8.0 Europe 2,011 7 0.3 73 3.6 LatinAmerica 2,039 61 3.0 51 2.5 Near East 1,802 92 5.1 14 0.8 NorthAmerica 1,924 5 0.2 15 0.8 Total 12,781 397 3.1% 434 3.4% IndianAgriculturalResearchInstitute,NewDelhi Singh, 2018
- 6. Extent of salt affected soils in India (ha). 6 State saline sodic Total Andhra Pradesh 77598 196609 274207 Andaman & Nicobar Island 77000 0 77000 Bihar 47301 105852 153153 Gujarat 1680570 541430 2222000 Haryana 49157 183399 232556 Karnataka 1893 148136 150029 Kerala 20000 0 20000 Madhya Pradesh 0 139720 139720 Maharashtra 184089 422670 606759 Orissa 147138 0 147138 Punjab 0 151717 151717 Rajasthan 195571 179371 374942 Tamil Nadu 13231 354784 368015 Uttar Pradesh 21989 1346971 1368960 West Bengal 441272 0 441272 Total 2956809 3770659 6727468 IndianAgriculturalResearchInstitute,NewDelhi Extent of salt affected soils in India (ha).
- 7. 7 Species Dry wt (DW) reduction with 14.2 dS. m-1 EC as compared with control (4.0 dS.m-1) Most tolerant Snapdragon and petunia DW reduced ≥50% and < 70% Slightly tolerant Begonia, Fuchsia, Coleus, Marigold, Vinca, Verbena and geranium DW reduced ≥70% and < 90% Moderately sensitive Impatiens, euphorbia and Salvina DW reduced by ≥90% Extremely sensitive Zinnia and pansy 100% mortality at 14.2 dS. m-1 EC IndianAgriculturalResearchInstitute,NewDelhi Villarino, 2011
- 8. 8 Country Total land Area cropped Mha Area irrigated Area of irrigated land that is salt-affected Mha % Mha % China 97 45 46 6.7 15 India 169 42 25 7.0 17 Russia 233 21 9 3.7 18 United States 190 18 10 4.2 23 Pakistan 21 16 78 4.2 26 Iran 15 6 39 1.7 30 Thailand 20 4 20 0.4 10 Egypt 3 3 100 0.9 33 Australia 47 2 4 0.2 9 Argentina 36 2 5 0.6 34 South Africa 13 1 9 0.1 9 Subtotal 843 159 19 29.6 20 World 1,474 227 15 45.4 20 FAO, (2007) IndianAgriculturalResearchInstitute,NewDelhi
- 9. 9 Breeding for salinity resistance Conventional Introduction Selection Hybridization Mutation IndianAgriculturalResearchInstitute,NewDelhi
- 10. 10 Genetic engineering The change in genetic make up of living cells by inserting desired gene through a vector in called genetic engineering . Transgenes: The plants obtained through genetic engineering contain a gene or genes usually from unrelated organism. The plants containing them are know as transgenic plants. . Gene transfer methods Agrobacterium- mediated Direct Chemical methods , Electroporation Particle gun delivery, Lipofection Microinjection, Macro injection Pollen transformation, Laser-induced Fiber mediated gene Trensfers Singh, 2018 IndianAgriculturalResearchInstitute,NewDelhi
- 11. 11
- 12. 12 Floriculture Genetic Engineering Flower colour modification Long life Flowers Morphological Modification Morphological of floral scent Abiotic stress TanaKa et al., 2005 Biotic stress
- 13. Stress Gene Origin Transgenic plant References Salt (iron) Fro2 Arabidopsis thaliana Rhododendron Dunemann et al., 2002 Drought P5CS D1-pyrroline-5 carboxylate Synthetase (proline) Arabidopsis thaliana Petunia Mika Yamada et al., 2005 Heat AtDREB1A Arabidopsis Chrysanthemum Bo Hong et al., 2009 Salt and drought AtNHX1 Arabidopsis Petunia Ping Hong et al., 2009 Drought RhNAC2 and RhEXPA4 Rose Rose Fanwei Dai et al., 2012 Salt, cold and drought ICE1 Chrysanthemum dichrum Chrysanthemum grandiflorum Lin Chen et al., 2012 Salt NnCIPK (calcineurin B-like protein kinase ) Lotus Lotus Ruijie Liua et al., 2014 Salt, drought and temperature P5CS1 and P5CS2 Chrysanthemum lavandulifolium Chrysanthemum × morifolium Mi Zhang et at., 2014
- 14. 14 Tissue Culture Anther Culture Embryo Rescue Zhu et al, 2013 Intergeneric hybrid between Chrysanthemum × morifolium & Artemisia japonica achieved via embryo rescue shows salt tolerance IndianAgriculturalResearchInstitute,NewDelhi
- 15. Case study 1 Objectives ❖To isolate CBF homolog gene named ClCBF1 from Chrysanthemum lavendulifolium ❖To study the overexpression of ClCBF1 in C. morifolium “White Snow”
- 16. Materials and methods Isolation of ClCBF1 gene Genetic transformation of Chrysanthemum morifolium “White Snow” Confirmation of transgenic plants Assessment of salinity and drought tolerance C.lavandulifolium 8–10 expanded leaf stage, leaves were harvested to extract RNA. Agrobacterium tumefacien strain used for transformation of C. morifolium ‘White Snow’
- 17. Results Quantitative real-time PCR analysis of expression patterns of CICBF1 in different tissues in response to salt and drought treatments in Chrysanthemum lavandulifolium. (A)Expression patterns of CICBF1 in roots, stems and leaves, under non-stress (B) NaCl-treated (C) Drought treated conditions.
- 18. Salinity tolerance of WT and transgenic plants (A) Phenotypes under salt - treated conditions. (B) MDA
- 19. 19 Salinity tolerance of WT and transgenic plants
- 20. Conclusion • A salt and drought-inducible CBF transcription factor (ClCBF1) was isolated from Chrysanthemum lavandulifolium. • Overexpression of ClCBF1 in C. morifolium ‘White Snow’ resulted in enhanced tolerance to salt and drought stresses. • These results indicated that ClCBF1 might directly or indirectly regulate stress-related genes to increase salt and drought tolerance.
- 21. 21 Case study 2 Objective ❑Isolation and characterization of P5CS gene from Lilium regale ❑To study the gene expression in Arabidopsis thaliana IndianAgriculturalResearchInstitute,NewDelhi
- 22. 22 Materials and Methods: Cloning P5CS cDNA and Sequence analysis Plant material Real-time quantitative PCR Arabidopsis Transformation and Stress Treatments Proline Determination Seeds of L. regale, 2-week stratification @ 4°C for germination, transferred into potting trays (540 mm × 280 mm × 40 mm) filled with (peat: vermiculite: perlite = 3:1:1,v/v/v). Seedlings in greenhouse 16 h light/8 h dark, 25°C day/18°C night thermoperiod & high ventilation,fertilized with Hoagland solution every 2 d. Plants of 2 weeks old used ( drought & salt treatment. salt stress experiment, seedlings were watered with Hoagland nutrient solution containing 250 mM NaCl gradually. Leaf & root samples collected @ 0, 2, 4, 6, 9, 12 h during the treatment. 20 plants taken @ each time point & frozen in liquid nitrogen rapidly for further total mRNA extraction & proline content determination. Wei et al., 2016 IndianAgriculturalResearchInstitute,NewDelhi
- 23. 23 Wei et al., 2016 Proline metabolism of Lilium. regale under drought and salt stress Expression levels of three LrP5CS genes by quantitative RT-PCR analysis in leaf (A, B) IndianAgriculturalResearchInstitute,NewDelhi
- 24. 24 Roots Wei et al., 2016 Proline metabolism of Lilium. regale under drought and salt stress IndianAgriculturalResearchInstitute,NewDelhi
- 25. 25 Wei et al., 2016 IndianAgriculturalResearchInstitute,NewDelhi (A) Representative seedlings after subjected to different conditions including control (non-stress), drought and salt treatments. The growth of LrP5CS transgenic Arabidopsis in soil
- 26. 26 Survival rates of seedlings after the treatments above.
- 27. 27 Wei et al., 2016 IndianAgriculturalResearchInstitute,NewDelhi The growth of LrP5CS transgenic Arabidopsis in agar plates
- 28. 28 Conclusion •The three P5CS genes in Lilium regale play distinct roles respectively in proline accumulation and abiotic stress tolerance. •Duplication of LrP5CS provides new evidence for the evolution of P5CS in monocots. Wei et al., 2016 IndianAgriculturalResearchInstitute,NewDelhi
- 29. 29 Case study 3 Objective This study investigated the importance of DgWRKY2 as a transcription regulator under salt stress. IndianAgriculturalResearchInstitute,NewDelhi
- 30. 30 Materials and Methods: Plant material Cloning of DgWRKY2 & Sequence Analysis Generation of Transgenic Chrysanthemum Expression of DgWRKY2 under Salt Treatment Salt Treatment of Transgenic Chrysanthemum 20-day-old seedlings planted in basins filled with 1:1 mixture of peat & perlite, incubated for 3 days & watered once daily (70% of field capacity). Seedlings at the six- leaf stage harvested, frozen in liquid nitrogen immediately, & stored at −80 C0 for RNA extraction. Plant Materials: wild-type Dendranthema grandiforum— ‘Jinba’. Chrysanthemum seedlings grew on MS culture medium for 20 days. He et al., 2018 IndianAgriculturalResearchInstitute,NewDelhi
- 31. 31 Salt stress WT OE-17 OE-21 0d 5d 10d 15d WT OE-3 OE-11 OE-17 OE-21 OE-24 Relativeexpressionlevel 01234 Salt-Tolerance Analysis of DgWRKY2 Transgenic Chrysanthemum He et al., 2018 IndianAgriculturalResearchInstitute,NewDelhi
- 32. Od 5d 10d 15d Chlorophyllcontent(mg/gFW) 0.00.40.81.21.62.02.42.8 32 WT OE-17 OE-21 Survivalrate(%) 020406080100 Analysis of chlorophyll content and under salt stress He et al., 2018 IndianAgriculturalResearchInstitute,NewDelhi
- 33. 33 15d10d5d0d WT 0E-17 OE-21 15d10d5d0d WT 0E-17 OE-21 He et al., 2018 Accumulation of H2O2, O2 −, and MDA in DgWRKY2 Transgenic Chrysanthemum under Salt Stress (A) Diaminobenzidine (DAB) staining of chrysanthemum leaves during salt stress treatment (B) Nitroblue tetrazolium (NBT) staining of chrysanthemum leaves during salt stress IndianAgriculturalResearchInstitute,NewDelhi
- 34. 34Table: Primers and their sequence in experiment He et al., 2018 Cloning of DgWRKY2 and Sequence Analysis IndianAgriculturalResearchInstitute,NewDelhi
- 35. 35 CAT 0.00.81.62.43.24.0 Od 5d 10d 15d P5CS 0246810 Od 5d 10d 15d He et al., 2018 Expression of Abiotic Stress-Related Genes in DgWRKY2 Transformed Chrysanthemum IndianAgriculturalResearchInstitute,NewDelhi
- 36. 36 APX 12345 Od 5d 10d 15d DREB1A 0123456 APX 012345 Od 5d 10d 15d He et al., 2018 Expression of Abiotic Stress-Related Genes in DgWRKY2 Transformed Chrysanthemum IndianAgriculturalResearchInstitute,NewDelhi
- 37. 37 He et al., 2018 Expression of Abiotic Stress-Related Genes in DgWRKY2 Transformed Chrysanthemum IndianAgriculturalResearchInstitute,NewDelhi
- 38. 38 Conclusion •DgWRKY2 confers salt tolerance to transgenic chrysanthemum by enhancing antioxidant and osmotic adjustment. •This study suggests that DgWRKY2 could be used as a reserve gene for salt-tolerant plant breeding Key. He et al., 2018 IndianAgriculturalResearchInstitute,NewDelhi