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1 1 1 Welcome seminar series 2011-12

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GENETIC ANALYSIS FOR SALT TOLERANCE IN RICE (Oryza sativa L.) Major Guide:- Dr. P.B. Patel, Assistant research scientist, CSSRS-Danti,NAU,Navsari. SPEAKER : YASHLOK SINGH Co- Guide:- Dr.M.K.Mahatma, Asst. professor Dept.of Biotechnology 2

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Introduction Salt tolerance mechanism Breeding for salt tolerance Saltol QTL Review of research work Conclusion Content 3

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5 Rice (Oryza sativa L., 2n= 24) is originated from Southeast Asia. Rice is short day, C3 plant and hydrophilic in nature. Rice is the staple food of more than 50% of word population including Asia, Latin America and South Africa etc. Rice rank second to wheat in terms of area harvested but in the terms of importance as a food crop rice provide more calories per hectare than any other cereals. Asia cultivates 137 m ha of rice of which India’s share is 45 m ha (Anon., 2010). The salinity and sodicity are the important factors adversely affecting the soil health and crop production. The total salt affected area in India approximately 8.1 m ha, out of which 3.2 m ha in coastal saline (Anon., 2009). Of course, this is dynamic problem. Total salt affected area in Gujarat is 1.2 lakh ha. In South Gujarat alone around 70,000 ha area (59% of total salt affected area of Gujarat) is salt affected, which cover coastal parts of Bharuch, Surat, Navsari and Valsad districts. In coastal areas of south and middle Gujarat, paddy is a major crop during kharif season. In coastal salt affected soil, the gap between achievable and existing productivity in paddy indicates that there is a good scope to raise the production and productivity through crop improvement. INTRODUCTION

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EXTENT OF SALT-AFFECTED SOILS Asia, Pacific and Australia (M ha) 195 249 Source : FAO database (2009) Total : 444 M ha 6

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What are the salt-affected soils ? Saline soils: Soluble salts – Cl- & SO4-2 of Na+ - salinity could vary in season Sodic Soils: CO3-2 & HCO3- of Na+ and Na+ on colloidal surface of clay micelle 7

Extent and distribution of salt - affected soil in India. (‘000 ha.) : 

Extent and distribution of salt - affected soil in India. (‘000 ha.) Source –CSSRI, Karnal (2009) 8

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Distribution of salt affected soils in Gujarat state (ha) Source –Khar Land Development Authourity ,Gujarat ( 2008) 9

Different rice breeding /testing centres for sodicity and salinity stresses in India. : 

Different rice breeding /testing centres for sodicity and salinity stresses in India. CSSRI,Karnal Gautam et al.(2008) 10

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Collaborating Countries / Institute in Asia Iran Pakistan CRRI, India BRRI, Bangladesh BINA CSSRI, NDUAT India Vietnam KAU, TNAU, India Indonesia Philippines Myanmar Thailand Sri Lanka 11

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Selectivity of ions Intracellular compartmentation Osmoregulation Organic solutes Inorganic solutes Antioxidants and ROS- scavenging LEA-type protein synthesis 12

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Salt Resistance Primary Stress Avoidance Tolerance Salt Exclusion Salt Extrusion Salt Dilution Avoidance of ion balance strain Tolerance of ion balance strain Low salt permeability Salt extrusion pump Water absorption Mineral binding with organic substances Mechanism of Salt resistance under primary stress condition 13

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Secondary stress Osmotic stress Nutrient stress Avoidance (Osmotic adjustment) Tolerance Deficiency Toxicity Accumulation of organic solutes in cytoplasm and salts in vacuoles Increase in wall extensibility Increase in leaf thickness Avoidance Tolerance Selectivity of ion uptake Replacement of K by Na Avoidance Tolerance Salt glands and bladders Control of transport to shoot Succulence Mechanism of Salt resistance under secondary stress condition Ion compartmentation 14

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Sucrose Trehalose Sorbitol Inositol Mannitol Glycerol Arabinitol Pinitol Other polyols Proline Betaine Glutamate Aspartate Glycine Choline Polyamines Oxalate Malate Carbohydrate and polyols Nitrogenous compound Organic acid Important osmolytes that accumulates in plants during salinity 15

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Generation and scavenging of superoxide radical and hydrogen peroxide, and hydroxyl radical-induced lipid peroxidation and glutathione peroxidase-mediated lipid (fatty acid) stabilization 17

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SOS signaling pathway for ion homeostasis under salt stress in rice. 18

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Traditional (conventional ) approach Somaclonal Variation Anther Culture Mutation Breeding Marker assisted selection(MAS) Transgenic approach 19 Method of breeding for salinity tolerance

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Modified standard evaluation score (SES) of visual salt injury at seedling stage in rice. Source –IRRI, Philippines(2009) 20

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Reasons of Limited Success 21 Salt stress seldom happen in isolation Highly variable environment, large G/E Lack of efficient / precise screening procedure Lack of mechanism understanding Low priority and less number of researchers involved

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Salt Stresses and Associated Complexities 22

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Mostly quantitative trait Breeding for Grain Yield Salt-Tolerance 23

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1. Restricting the entry of toxic ions at root level - Exclusion 2. Transporting the toxic ions to stem, leaf sheath or older leaves – plant level compartmentation 4. Sequestration of the toxic ions to vacuole or cell wall – cell level compartmentation 3. Excretion of salt through salt glands, salt-hairs or bladders – in most halophytes Predominant salt-tolerance mechanisms operating in plant Na+ Cl- 24

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Physiology: traits associated with salinity tolerance

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Morphological Symptoms v     White leaf tip followed by tip burning (salinity)     Leaf browning & death (sodicity)   Stunted plant growth     Low tillering     Spikelet sterility     Low harvest index     Less florets per panicle     Less 1000 grain weight     Low grain yield     Change in flowering duration     Leaf rolling     White leaf blotches     Poor root growth     Patchy growth in field Manifestation of Salt Stress 26

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First symptom “Leaf tip burning” “Leaf tip burning extends toward base through Lamina” “Ultimate death of leaf – always from oldest to youngest” Salinity symptoms at the vegetative stage 27

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Effect of salinity at Reproductive stage – Spikelet Sterility 28

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Effect of salinity at reproductive stage – papery sterile spikelets 29

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SALINITY Genetics of Salt Tolerance Inheritance Pattern 30

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Genetics of Salt Tolerance P1 X P2 F1 X F1 X P1 31

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Performance of FL478 (tolerant line) and IR29 (susceptible variety) rice seedlings under saline solution . (Source: IRRI (2010) 32

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Physiological & Biochemical v     High Na+ transport to shoot v     Preferential accumulation of Na in older leaves v     High Cl- uptake v     Lower K+ uptake v     Lower fresh and dry weight of shoot and roots v     Low P and Zn uptake v     Increase of toxic organic compatible solutes v     Increase in Polyamine levels Causes of Salt Stress in rice 33

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Breeding Strategy Identification of the genotypes based on the inherent physiological mechanism responsible for salinity tolerance Inter-mating of the genotypes with high degree of expression of the contrasting salinity tolerance mechanism Identifying / screening of the recombinants for pooling/ pyramiding of the mechanisms 34

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Identify the donors for predominant physiological mechanisms responsible for salt tolerance Na+ exclusion, Tissue Tolerance K+ uptake, Preferential accumulation of Na+ in stem, leaf sheath, older leaves etc. Early vigour However, none of the rice variety posses all the possible positive mechanism conferring salinity tolerance. Breeding Strategy 35

Grouping of the rice varieties on the basis of Na accumulation per day : 

Grouping of the rice varieties on the basis of Na accumulation per day

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Grouping of the rice varieties on the basis of K accumulation per day 37

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Rice variety A Good excluder + poor tissue tolerance Rice variety B Poor control at root level + High tissue tolerance Dustbin Garbage Na+ Rice variety C Good excluder + High tissue tolerance K+ 38

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An Ideal High Yielding Salinity Tolerant Variety of Rice Highly tissue tolerant Good Excluder- Minimum per day uptake of Na+ High uptake of K+ per day Low Cl- uptake Low Na+/ K+ ratio Good initial vigour Agronomically superior with high yield potential (plant type + grain quality) 39

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C52903S C1733S R2374B C52903S C1733S R2374B RM283 R844 S2139 RM23 RM140 RM113 S1715 S13994 RM9 RM5 C1456 RM237 RM246 0.0 27.4 28.4 40.0 75.3 77.2 91.9 98.2 99.1 103.1 119.5 123.5 129.9 RM283 R844 S2139 RM23 RM140 RM113 S1715 S13994 RM9 RM5 C1456 RM237 RM246 0.0 27.4 28.4 40.0 75.3 77.2 91.9 98.2 99.1 103.1 119.5 123.5 129.9 0.0 1.0 1.8 1.9 1.2 1.3 Short arm of chromosome 1 Progress of Saltol locus Saturated map of the Chromosome 1 (Saltol segment) is developed Closely linked markers linked to the saltol locus identified. 60.6 40

Mapping Salinity Tolerance Genes at Reproductive Stage : 

Mapping Salinity Tolerance Genes at Reproductive Stage QTLs for salinity tolerance genes at seedling stage are different from reproductive stage Seedling stage tolerance in chrom- 1. Reproductive stage tolerance in chrom- 3, 4, and 7. 41

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Early Seedling Stage Boot stage Vegetative / tillering stage Rice Growth Stages vs. Salt Tolerance Germination Stage Ripening

Donors : 

Donors Traditional Donors Pokkali Cheriviruppu Nonabokra Bhirpala SR26B Hasawi Improved Donors FL478 PSBRc 88 / CSR23 CSR28 IR4630-22-2-5-1-3 CSR10 BR47 Rice : Salt - sensitive crop : Enormous variability 43

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Salt tolerant rice varieties released for commercial cultivation in different countries 44

Review of Research work : 

Review of Research work

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Screening 46

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Table -1: Survival scoring of varieties under saline condition SD- Survival day, RL- Root length, SL- Shoot length, RW- root weight, SW- shoot weight, HT- Highly tolerant, T-Tolerant, MT- Moderately tolerant, S- Susceptible, HS- Highly susceptible Vietnam Lang et al. (2001) 47

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Table 2: Salinity score , Na+, K+ content and Na+/K+ ratio in shoot of rice genotypes grown under salinized solution. Pakistan Ali et al. (2004) 48

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Table 3: Grain yield and yield components under saline and normal soil field conditions. Pakistan Ali et al. (2004) 49

Table 4: Salt tolerant rice genotypes and their salient traits. : 

Table 4: Salt tolerant rice genotypes and their salient traits. Karnal Gautam et al.(2008) 50

Table 5: Distribution of 11 genotypes under salt stress at seedling stage : 

Table 5: Distribution of 11 genotypes under salt stress at seedling stage Bangladesh Bhowmik et al. (2009) 51

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Table 6 - Chlorophyll a, Chlorophyll b and total Chlorophyll, Proline content and Na/K ratio of seedling in two rice genotype KKU,Thailand Pajuabmon et al. (2009) 52

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Gene Action 53

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Table 7: Estimation of general combining ability (GCA) effects for various characters of rice in a diallel cross Pakistan Mahmood et al.(2002) 54 Significant at 5 % level

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Table 8: Estimation of SCA effect for various character of rice varieties in a diallel cross Pakistan Mahmood et al.(2002) 55

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Bangladesh Saharay and Islam (2008) Table 9: Genetic parameter and x2 values of joint scaling test in an additive dominance model for salinity tolerance scores in six crosses of rice 56 ** Significant @ 1 %

Table 10 : Gene effects for salinity tolerance scores under six parameter model in rice : 

Table 10 : Gene effects for salinity tolerance scores under six parameter model in rice Bangladesh Saharay and islam (2008) 57 ** Significant @ 1 % & * significant @ 5 %

Table 11- Promising hybrids for grain yield per plant (g) with heterobeltiosis, relative heterosis and component traits based on pooled over environment in rice (L X T) : 

Table 11- Promising hybrids for grain yield per plant (g) with heterobeltiosis, relative heterosis and component traits based on pooled over environment in rice (L X T) PL= Panicle length GPP= Grains per panicle PTP=Productive tillers per plant TW = 1000- Grains weight HI= Harvest index PC= Protein content NAU, Navsari Sharma (2008) 58 ** Significant @ 1 %

Table 12: Performance of different varieties under normal and saline conditions {16 (dS m-1)} : 

Table 12: Performance of different varieties under normal and saline conditions {16 (dS m-1)} NAU, Navsari Patel et al. (2010) 59

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Molecular Approaches 60

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Khan Kaen , Thailand Kanlaya et al.(2005) Figure1 : The SDS-PAGE profiles of polypeptides extracted from controlled (C) and salt-treated (S) roots (A), leaf sheaths (B), leaf laminae (C) of rice cv. Leuang Anan 61

QTLs for salinity tolerance : 

QTLs for salinity tolerance 1 2 3 4 5 6 7 8 9 10 11 IRRI Bonilla et al.(2002) 62 Figure-2

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Saltol 1 2 3 4 5 6 7 8 9 10 11 12 Sub1 Evaluation of salinity + submergence tolerant plants by submerging in saline water IRRI Bonilla et al.(2002) 63 Figure-3:

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64 The rice productivity under salt affected soil can be increased through selection of tolerant variety. Screening for salinity should be major areas for large scale testing of salt tolerance. Breeding approaches will help to enhance the genetic and physiological basis of resistance against salinity. Under Salt tolerant mechanisms, the level of tolerance can be enhanced by High tissue tolerant, Good excluder, Minimum per day uptake of Na+, High uptake of K+ per day, High K/Na ratio, Good initial vigour and Agronomically superior with high yield potential (plant type + grain quality). Good donors (Pokkali, Nonabokra, CSR-10, KDML-105), and reliable phenotyping is the key to success. Multiple abiotic stress tolerance is possible and should be addressed because salt stress invariably occurs with other abiotic stresses or mineral toxicity/ deficiency. Transfer of Saltol QTL in different varieties will validate the effectiveness. Use of salt-tolerant varieties is a viable proposition to reclaim the salt-affected areas and to enhance the productivity of rice under salinity condition. Conclusion

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Thank you … 65

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