Physiological, biochemical and molecular changes due to seed priming in Cucumber (Cucumis sativus L.)

Transcription

1 Physiological, biochemical and molecular changes due to seed priming in Cucumber (Cucumis sativus L.) 6/26/2013 Dr. SOWMYA, K.J University of Agricultural Sciences, National Seed Project(Crops) GKVK, Bangalore, India. 1

2 Introduction Seed priming is a pre-sowing treatment that involves controlled hydration of seeds, sufficient to allow pre-germinative metabolic events to take place and to restrict radicle protrusion through the seed coat (Heydecker et al., 1973). 2

3 Origin and Taxonomy Common name: Cucumber Scientific name: Cucumis sativus Family : Cucurbitaceae Origin: India Ploidy level: 2n = 14 Mode of pollination : Cross pollinated Period of crop growth : Annual 3

4 Objectives To know the physiological, biochemical and molecular changes due to seed invigoration To know the protein marker (s) associated with invigoration 4

5 Material and Methods Vigour levels (V): V 1 - Fresh seeds - high vigour (>90%) V 2 - low vigour (<60%) Treatments (T): T 1 - Control T 2 - Water T ppm T 4 - KNO 1% T 5 - PEG 6000, -1.5 MPa T 6 - Cow dung slurry (1:1 w/v) T 7 - PSB Total treatment combinations: 2 x 7 = 14 Replications: Three Design: Factorial CRD Treatment details 5

6 Observations : Physiological changes Seed moisture content (%) [ISTA (2007)] First count germination (%) [ISTA (2007)] Final count germination (%) [ISTA (2007)] Mean germination time (in days) [Ellis and Roberts (1981)] Time to 50 % germination (T 50 ) [ Coolbear et al. (1990). ] Speed of germination (BRI) [(Bartlett, 1973)] Germination energy (%) [Wang (2008) ] Coefficient of velocity of germination (%) [Nichols and Heydecker (1968)] Mean seedling length (cm) Mean seedling dry weight (mg) Seedling vigour index -I Seedling vigour index -II [Abdul-Baki and Anderson (1973) ] 6

7 Observations : Biochemical changes Electrical conductivity (dsm -1 ) [ISTA (2007)] Total soluble seed protein (%) [Lowry et al. (1951)] Total soluble sugars (%) [Dubois et al. (1951)] Total dehydrogenase activity [Perl et al. (1978)] Amylase activity [Bernfeld (1955) ] Peroxidase activity [Sadasivam and Manickam (2008)] Catalase activity [Goth (1991)] DNA content (µg/g of seed) [Doyle and Doyle (1990) ] 7

8 Molecular changes Electrophoretic characterization of total soluble seed protein through NATIVE- PAGE Electrophoretic characterization of total soluble seed protein through SDS- PAGE [Laemmeli (l970)] Elecrophoretic analysis of esterase (EST) and [Glaszman et al. (1988) ] Elecrophoretic analysis of Peroxidase isozyme (POX) [Glaszman et al. (1988) ] 8

9 Experimental Results Physiological Biochemical & Molecular aspects 9

10 Table 1. First and final count germination as influenced by vigour levels and priming treatments in cucumber Treatments First count germination (%) Final count germination (%) HV LV Mean HV LV Mean T T T T T T T Mean SEm± CD (P=0.01) CV (%) SEm± CD (P=0.01) V CV (%) T V x T Treatments (T): T 1 - Control; T 2 - Water ; T ppm; T 4 - 1%; T 5 - PEG 6000, -1.5 MPa; T 6 - Cow dung slurry (1:1 w/v); T 7 - PSB 10

11 Table 2. Mean germination time, coefficient of velocity of germination and Seedling Vigour Index (SVI) based on seedling dry weight as influenced by vigour levels and priming treatments in cucumber Mean germination time(days) CV of germination (%) SVI Treatments HV LV Mean HV LV Mean HV LV Mean T T T T T T T Mean SEm± CD (P=0.01) CV (%) SEm± CD (P=0.01) CV (%) SEm± CD (P=0.01) V CV (%) T V x T NS Treatments (T): T 1 - Control; T 2 - Water ; T ppm; T 4 - 1%; T 5 - PEG 6000, -1.5 MPa; T 6 - Cow dung slurry (1:1 w/v); T 7 - PSB 11

12 Control Water PEG KNO3 Ethrel Cowdung Slurry PSB control (dry) water MPa 100ppm KNO Cowdung slurry Influence of various priming treatments on germination in cucumber 6/26/2013 PSB UAS, Bangalore, India 12

13 Table 3. Conductivity of seed leachate, total dehydrogenase activity, as influenced by vigour levels and priming treatments in cucumber Treatments Conductivity of seed leachate (ds/m) Total dehydrogenase activity (A 480 ) HV LV Mean HV LV Mean T T T T T T T Mean SEm± CD (P=0.01) CV (%) SEm± CD (P=0.01) V CV (%) T V x T Treatments (T): T 1 - Control; T 2 - Water ; T ppm; T 4 - 1%; T 5 - PEG 6000, -1.5 MPa; T 6 - Cow dung slurry (1:1 w/v); T 7 - PSB 13

14 Table 4. Peroxidase activity, catalase activity and DNA content as influenced by vigour levels and priming treatments in cucumber Treatments Peroxidase activity ( A 436/min/g of seed) Catalase activity (mmol H 2 O 2 degraded/min) DNA content (µg/g of seed) HV LV Mean HV LV Mean HV LV Mean T T T T T T T Mean SEm± CD (P=0.01) CV (%) SEm± CD (P=0.01) CV (%) SEm± CD (P=0.01) CV (%) V T V x T Treatments (T): T 1 - Control; T 2 - Water ; T ppm; T 4 - 1%; T 5 - PEG 6000, -1.5 MPa; T 6 - Cow dung slurry (1:1 w/v); T 7 - PSB 14

15 Influence of seed priming on native protein profile in cucumber V 1 T 1 V 1 T 2 V 1 T 3 V 1 T 4 V 1 T 5 V 1 T 6 V 1 T 7 V 2 T 1 V 2 T 2 V 2 T 3 V 2 T 4 V 2 T 5 V 2 T 6 V 2 T 7 Vigour Levels: V 1 - Fresh seeds - high vigour (>90%); V 2 - low vigour (<60%); Treatments: T 1 - Control; T 2 - Water;nT ppm; T 4 - KNO 1%; T 5 - PEG 6000, -1.5 Mpa; T 6 - Cow dung slurry and T 7 - PSB 26 Polymorphic bands (Rm value: to 0.846).

16 0.000 V 1 T 1 V 1 T 2 V 1 T 3 V 1 T 4 V 1 T 5 V 1 T 6 V 1 T 7 V 2 T 1 V 2 T 2 V 2 T 3 V 2 T 4 V 2 T 5 V 2 T 6 V 2 T Zymogram of native protein due to seed priming in cucumber

17 Influence of seed priming on total soluble seed protein (SDS-PAGE) profile in cucumber M V 1 T 1 V 1 T 2 V 1 T 3 V 1 T 4 V 1 T 5 V 1 T 6 V 1 T 7 V 2 T 1 V 2 T 2 V 2 T 3 V 2 T 4 V 2 T 5 V 2 T 6 V 2 T kDa 68kDa 43kDa 29kDa 20kDa 14.3kDa 27 bands (Rm value: to 0.966) polymorphic regions : B, C, E, F, G and H. 6.5kDa 3kDa Vigour Levels: V 1 - Fresh seeds - high vigour (>90%); V 2 - low vigour (<60%); Treatments: T 1 - Control; T 2 - Water;nT ppm; T 4 - KNO 1%; T 5 - PEG 6000, -1.5 Mpa; T 6 - Cow dung slurry and T 7 - PSB 6/26/

18 M V 1 T 1 V 1 T 2 V 1 T 3 V 1 T 4 V 1 T 5 V 1 T 6 V 1 T 7 V 2 T 1 V 2 T 2 V 2 T 3 V 2 T 4 V 2 T 5 V 2 T 6 V 2 T 7 Region A Region B Region C Region D Region E Region F Region G Zymogram of SDS-PAGE protein due to seed priming in cumber 18

19 Influence of seed priming on Esterase (EST) isozyme in cucumber V 1 T 1 V 1 T 2 V 1 T 3 V 1 T 4 V 1 T 5 V 1 T 6 V 1 T 7 V 2 T 1 V 2 T 2 V 2 T 3 V 2 T 4 V 2 T 5 V 2 T 6 V 2 T 7 Vigour Levels: V 1 - Fresh seeds - high vigour (>90%); V 2 - low vigour (<60%); Treatments: T 1 - Control; T 2 - Water;nT ppm; T 4 - KNO 1%; T 5 - PEG 6000, -1.5 Mpa; T 6 - Cow dung slurry and T 7 - PSB Polymorphic bands with Rm values: 6 (0.242), 7 (0.257), 8 (0.300), 16 (0.635), 17 (0.678), 19 (0.721), 20 (0.771), 21 (0.785), 22 (0.800), 23 (0.864) and 24 (0.892)

20 0.000 V 1 T 1 V 1 T 2 V 1 T 3 V 1 T 4 V 1 T 5 V 1 T 6 V 1 T 7 V 2 T 1 V 2 T 2 V 2 T 3 V 2 T 4 V 2 T 5 V 2 T 6 V 2 T Zymogram of esterase (EST) isozyme due to seed priming in cucumber 20

21 Influence of seed priming on Peroxidase (POX) isozyme in cucumber V 1 T 1 V 1 T 2 V 1 T 3 V 1 T 4 V 1 T 5 V 1 T 6 V 1 T 7 V 2 T 1 V 2 T 2 V 2 T 3 V 2 T 4 V 2 T 5 V 2 T 6 V 2 T 7 Vigour Levels: V 1 - Fresh seeds - high vigour (>90%); V 2 - low vigour (<60%); Treatments: T 1 - Control; T 2 - Water;nT ppm; T 4 - KNO 1%; T 5 - PEG 6000, -1.5 Mpa; T 6 - Cow dung slurry and T 7 - PSB Monomorphic bands (Total of six bands having Rm value of to 0.565) 21

22 0.000 V 1 T 1 V 1 T 2 V 1 T 3 V 1 T 4 V 1 T 5 V 1 T 6 V 1 T 7 V 2 T 1 V 2 T 2 V 2 T 3 V 2 T 4 V 2 T 5 V 2 T 6 V 2 T Zymogram of peroxidase (POX) isozyme due to seed priming in cucumber

23 CONCLUSION The first count & CV of germination Significantly higher in HV seeds compared to LV seeds KNO 3 primed seeds Higher first count & CV of germination compared to unprimed seeds in both HV & LV seeds HV seeds had higher TDH activity, TSP and DNA content compared to LV seeds Activities also higher in KNO 3 primed seeds in contrast with unprimed seeds 23

24 CONCLUSION Continued. TSS of seed leachate Significantly lower in HV seeds than LV seeds Lower in KNO 3 primed seeds compared to unprimed seeds The amylase, catalase & peroxidase activities Significantly higher in KNO 3 primed seeds compared to unprimed HV seeds showed increased activity of these enzymes compared to LV Conversely, TSP, esterase & peroxidise enzyme profiles revealed polymorphism with respect to expression of peptides due to priming induced proteins. The enhanced biochemical activities, particularly in low vigour seeds might have resulted in the enhanced seed quality attributes following priming in cucumber. 24

25 acknowledgement DST, Ministry of Science and technology, New Delhi, India Department of Seed Science and Technology, UAS, Bangalore, India Indian Institute of Horticultural Research, Hessaraghatta, Bangalore, India THANK YOU for your attention 25