Pelagia Research Library - iMedPub · 2016-09-21 · Mukesh Kumar et al Asian J. Plant Sci. Res., 2011, 1 (1):5-21 _____ 7 Pelagia Research Library for 48 h in a sealed desiccator

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Asian Journal of Plant Science and Research, 2011, 1 (1): 5-21

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Analysis of Biochemical and physiological changes during ultra-dessication in Radish (Raphanus Stivus)

Mukesh Kumar *, Anjali Kak , Satyapal Singh*

*Departement of Biotechnology, Singhania University, Jhunjhunu, Rajasthan, India

Germ plasm Conservation Division, NBPGR, New Delhi, India ______________________________________________________________________________ ABSTRACT This research aimed to determine whether ultra-dry storage improves the longevity of radish. Currently seed germplasm is dried to 3–7% moisture content (mc) before storage at subzero temperatures. In the present study, seeds were dried to different low moisture contents and stored under various conditions, to identify any increase in seed longevity. Seeds were dried to 2–3.4% in radish(Raphanus sativus).Seed conditioned to various mc’s were sealed hermetically and stored at temperatures of -20°C, 10°C and ambient for 14years. Seed germination and vigour was assessed at yearly intervals.Moisture content of radish(Raphanus sativus) seeds was dried to 10%, 7.7%, 6.3%,4.2% and 3.4% (w.b.) in a desiccating container with silica gel, and stored at 45°C, 25°C and 15°C for 24 months. The data from 24 months showed that the optimum moisture content for storage varies with temperature. After ultra drying some physiological indices were tested. The results Indicated that Dehydrogenase, lipid peroxidation , electrical conductivity higher than those of the control seeds,. The results indicate that moisture content of seed was a key index for storage at ambient temperature (25°C) and 3.81% seem to be the best moisture content for ultra-dry seeds in our research. RAPD markers were also used to evaluate the genetic fidelity of seeds, all RAPD profiles from ultra-dry seeds were monomorphic and similar to non-ultra-dry seeds, we conclude that variation is almost absent in ultra-dry storage. From these results, we suggest that seed moisture content less than 5% enhances longevity and ultra-dry could be an economical way for conservation of the plant genetic resource. Keywords: Moisture content; Physiological indices; RAPD; Seed storage; Ultra-dry; Radish. ______________________________________________________________________________

Mukesh Kumar et al Asian J. Plant Sci. Res., 2011, 1 (1):5-21

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INTRODUCTION The radish (Raphanus sativus) is an edible root vegetable of the Brassicaceae family that was domesticated in Europe in pre-Roman times. They are grown and consumed throughout the world. Radishes have numerous varieties, varying in size, color and duration of required cultivation time. Broadly speaking, radishes can be categorized into four main types (summer, fall, winter, and spring) and a variety of shapes, colours, and sizes, such as black or multi-coloured radishes, with round or elongated roots that can grow longer than a parsnip. Summer radishes mature rapidly, with many varieties germinating in 3-7 days and reaching maturity in three to four weeks. A common garden crop in the U.S., the fast harvest cycle makes them a popular choice for children's gardens. Harvesting periods can be extended through repeated plantings, spaced a week or two apart. Radishes grow best in full sun and light, sandy loams with pH 6.5 - 7.0. They are in season from April to June and from October to January in most parts of North America; in Europe and Japan they are available year-round due to the plurality of varieties grown. As with other root crops, tilling the soil helps the roots grow. Most soil types will work, though sandy loams are particularly good for winter and spring crops, while soils that form a hard crust can impair growth .The depth at which seeds are planted affects the size of the root, from 1 cm deep recommended for small radishes to 4 cm for large radishes. However, reports on the protection germplasm resource and biochemical basis of seed storage in radish and sunflower are few. Hence, our aim was to investigate seed germination ability and viability in these seeds after ultra-drying and to explore the physiology mechanism of ultra-dry storage.

MATERIALS AND METHODS

Freshly harvested seeds of radish (Raphanus sativusL. cv. Pusa Chetki), were obtained in 1996. Seeds were adjusted to different moisture levels by drying over regularly regenerated silica gel in a desiccator or humidifying at 25°C for varying periods of time. Moisture contents were determined by the high low constant temperature oven methods and are expressed on a dry weight basis. The hermetically sealed packets were kept at -20°C, 10°C, and ambient conditions of our laboratory in Sinhania University,Jhunjhunu (Rajasthan) (25–45° C). Seed ultra-drying treatment and pre-humidification Seeds were packed in plastic net bags; the ratio of the seeds to silica gel was 1:5 (w/w). Seed bags were buried into silica gel in a desiccator at normal atmospheric temperature (25°C) for 15 d to reduce the moisture content of seeds to 10%, 7.7%, 6.3%, 4.2% and 3.4%. The ultra dried seeds were kept in sealed aluminium foil packages for experiment. The rapid uptake of water by dry seeds can result in imbibitions injury (Powell and Matthews, 1978). Imbibitions injury can be avoided by conditioning(humidifying) the seeds in a moist atmosphere (close to100% RH) in order to raise seed moisture contents before the seeds are set to germinate in contact with liquid water(Ellis et al.,1985). In our research to avoid the imbibitions injury, the ultra-dried seeds were hydrated for 48 h in a sealed desiccators containing saturated CaCl2 solutions (RH is 35%) and then they were hydrated


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for 48 h in a sealed desiccator containing saturated NH4Cl solutions (RH is 75%) at normal atmospheric temperature (25-30°C) (Huang et al., 2002) before the germination assessment and the following experiment. Seed moisture content (%): It was determined gravimetrically by the low constant temperature oven method where the seeds were dried at 103±20C for 17 hrs. (ISTA, 1993 rules). The moisture content was calculated using the formula: M2 – M3 MC = X100 M2 – M3 M1 = Weight of empty bottle with its lid. M2 = Weight of bottle with lid and sample before drying. M3 = Weight of bottle with lid and sample after drying. Seed Germination Test: The primary objective of the germination test is to determine the potential of a seed to produce normal, healthy seedlings. Seeds were placed on the top of substrata or germination Paper. The plated seeds were maintained at 32° C temperature. Four replications of 25 seeds each were sown. The emergence of both radical and plumule were recorded after the 7th day of sowing. Germination percentages were recorded every alternate day, accounting the percent of seeds with protrusion of radical as indication. (ISTA, 1993). The length of roots and shoots were recorded in cm and calculated for total number of seed plated. Seed and Seedling Vigour (Abdul-Baki, 1969); The seedling vigour index was calculated using formulae Vigour index = [Root length + Shoot length (in cm)] x Germination (%) Speed of germination calculated using formula.

∑ nd Speed of germination =

∑ n n = number of seeds which germinate on day “d” d = number of days counted from the beginning of germination test. Electrical Conductivity of leachates: It was measured by weighing two replicates of 10 seeds each and soaking them in 10 ml of deionised water at 250C for 18hours and conductivity was measured of resultant leachate water every 2 hrs using (Control Dynamics, India) conductivity meter identifying period for achieving optimum level of conductivity, which then can be uniformly used for all conductivity experiments in radish seeds.


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Lipid peroxidation : It was measured by Heath and Parker method (1968) using TBA- TCA reagent (0.5% Thiobarbituric acid in 20% trichloroacetic acid). A sample of 0.25 gm of seed material was homogenized in 2 ml of distilled water and 4 ml of TBA-TCA reagent. These samples were incubated at 90°C for 30 min. After incubation, the samples were cooled in ice bath and centrifuged at 10,000 g for 10 min. Supernatants were collected and the (OD) was measured at 532 and 600 nm. After subtracting the absorbance of nonspecific (600 nm) from specific (532 nm) the net absorbance was expressed in terms of absorbance at 532 n mol /mg d.wt, which indicated the level of malondialdehyde (MDA) produced as a result of lipid per oxidation. Enzyme estimations Dehydrogenase For estimation of dehydrogenase enzyme activity, embryos axes of 15 seeds of each treatment were soaked with 1% triphenyl tetrazolium chloride (TTC) solution in test tubes and incubated for 18 hours in dark at 270C. After that thoroughly washed with distilled water and surface drying, the red colored formazan produced. After, incubation was extracted with 5ml of 2-methoxy ethanol and absorbance value of the solution were recorded at 470 nm. These test tubes were kept on shakers for 4-6 hours for effective extraction. Dehydrogenase activity was determined by triphenyl tetrazolium chloride (TTC) method (Kun and Abood, 1949). Reaction: Tetrazolizum chloride Dehydrogenase Formazan (red coloured) Protein extraction Radish seeds (25-30 seeds) were crushed with mortar and pestle to fine powder. This powdered material was transferred to test tubes to which 7 ml of defatting solvent mixture (Chloroform: Methnol : Acetone ) was added, mixed well and covered with aluminium foil. After 4 hrs the solvent mixture was decanted and again added 7 ml of defatting solvent mixture. This process was repeated several times until the oil was removed completely. The defatted seed powder was air-dried. The fine powdered pellet of the seed material was transferred to the eppendorf tubes for protein extraction. To 0.03 g of air-dried seed powder 200 µl of Tris: glycine buffer (pH 8.3) was added and was left overnight in the refrigerator. The sample was then centrifuged at 10,000 rpm for 10 minutes and supernatant was collected in separate eppendorf tubes. Gel Preparation and Casting: Separating gel (30%) : Tris: HCl buffer (pH 8.8),Distilled water,30% running gel acryl amide, 5 % APS, 10 % SDS,TEMED Stacking gel (30%):Tris: HCl buffer (pH 6.8) ,Distilled water, 30% stacking gel acryl amide,5 % APS ,10 % SDS,TEMED The separating gel were mixed well, (TEMED is added just before pouring) and gel was poured between the plates filling 3/4th of the cassette. The gel was allowed to polymerize. Following polymerization stacking gel mixture was added into the cassette carefully and a comb was inserted. The stacking gel was allowed to polymerize for 3 - 4 minutes. After the polymerization the comb was removed carefully and the wells were washed with distilled water.


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Gel Electrophoresis and Staining procedure:- The electrophoresis tank was filled with tank buffer and the buffer circulation unit was kept on for the continuous exchange of tank buffer between the upper and lower chambers. The gel cassette was inserted into the buffer chamber. Wells were loaded with 60 µl of protein sample (30µl of protein extract & 30µl of working sample buffer) boiled for 10 min before loading and run at constant current 2.0 mA per well in stacking gel and raised to 2.5 mA per well in separating gel and allowed to run until dye reaches 2 cm above the edge of the cassette. Firstly gel was fixed with fixing solution for about 30-45 min. Then the gel was stained with staining solution for about 10-12 hrs, washed and destained with distilled water several times until the band becomes visible. RAPD ANALYSIS DNA extraction 1. Grind up to 100 mg plant sample under liquid nitrogen to a fine powder and quickly transfer to a sterile 1.5 –ml or 2- ml eppendrof tube. 2. Add 400 µl PX1 Buffer and 4 µl R Nase A stock solution (100 mg/ml ) to the tissue powder and vortex vigorously and Incubate the mixture at 65°C for 10 minute. 3.Meanwhile, preheat dd H₂0 (pH 9.0), 10 Mm Tris-HCL (pH 9.0), or TE buffer (500µl /prep) at 65°C for DNA elution. 4. Add 130 µl PX2 buffer to the lysate, and vortex the mixture. Incubate it on ice for 5 minutes. 5. Place a shearing tube onto a collection tube. Apply lysate (or lysate supernatant) to the Shearing tube and centrifuge for 2 minute. Transfer the flow – though lysate from the collection tube to a new sterile tube. 6. Determine the volume of flow –through lysate obtained. Add 0.5 volume of PX3 Buffer and mix by pipetting . Add 1 volume of 98-100% ethanol to the mixture and mix by pipetting. 7. Place a DNA easy plant Mini column onto a Collection tube. Apply 650 µl of the ethanol added sample (including any precipate ) from at 8,000 x g (10,000 rpm) for 1 minute. Discard the flow-through. 8. Repeat step 6 for the rest of the sample. 9.Wash the colume twice with 0.7 ml WS Buffer by centrifuging for 30-60 seconds. Discard the flow-through. Centrifuge for another 2 minutes to remove any ethanol residue Purification of DNA Added RNAse (0.2 ml) to the extracted DNA solution and incubated at 37 °C for 1 hour. Then added equal volume of phenol : chloroform (1 : 1), mixed properly for 2 min and spin for 5 min. DNA supernatant was pipette out .Precipitated the DNA by adding 1/10 volume of 3M NaOAc and 2.5 times of the total volume chilled ethanol. Mixed and spool out the DNA. The extra salts were removed by two washings with 70 % ethanol. DNA was dried under vaccum and dissolved in minimum volume of TE (10:1) at room temperature and store frozen at –20 °C. Quantification and Dilution of DNA samples Quantification of DNA was done using DNA Quant flourimeter. Flourimetric estimation measures the fluorescence emitted by double stranded DNA-Hoechst 33258 dye complex, which is directly proportional to the amount of DNA in the sample. Prior to actual quantification, the flourimeter was calibrated with 2 µl (1 mg/ml) of standard calf-Thymus DNA in 2 ml of assay solution. For quantification, 2 µl of unknown DNA sample was added to 2 ml of assay solution (blank). The two were mixed thoroughly and measurement recorded.


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DNA samples were diluted with sterilized distilled water in accordance with the quantified measurement to yield a working concentration of 10 mg/µl. These standard DNA solutions were further used for the PCR amplification. Selection of primers Initially 12 primers of OP (Operon Technologies, USA) were screened with the 4 randomly selected samples. Out of these 12 primers screened 8 primers were selected for the further study. Selection criterion involves presence of polymorphic bands, satisfactory amplification and band resolution. Primer sequences are as follows P-01, P-21, P-22, and P-23. PCR Amplification Pipette out accurately into sterile 0.2 ml micro tubes the reagents in the following order and quantity as mentioned below: H2O:12.8 µl., Buffer : 2.5 µl.,MgCl2 : 2.5 µl, DNTP :0.5 µl, Taq : 0.2 µl, DNA: 4 µl, Primer:2.5 µl. The appropriate amount of the mix was aliquot to each tube and DNA template was added separately in each tube. Setting PCR reactions for RAPD Thawed PCR reagents - Mgcl2, primers, buffers, d NTPs, taq DNA polymerase. Then PCR master mix was prepared by adding components, vortexed vigorously and dispensed aliquots of master mix into PCR tubes. Template DNA was added and mixed gently by spinning for 1 min. The reaction tubes were placed on the thermo cycler plate. At the end of the run the tubes were taken out and 2.5 µl of 10 X loading dye was added. The mixture was spin for 2-5 sec and stored at 4 °C till electrophoresis. Thermo cycler program is ISSR. Gel electrophoresis 0.8 % agarose gel was prepared in 1 X TAE buffer. The gel was poured into the gel tray with comb placed and let it to polymerize for at least 30 min. The comb was removed and the gel was placed into the electrophoresis tank filled with 1 X TAE buffer. 25 µl of the amplified DNA was loaded into the wells. The gel was allowed to run at 90 V until the dye runs 5-6 cm from the wells. The gel was placed in staining tray and allowed to stain with ethidium bromide stain. The gel was illuminated with UV light and photographed under trans-illuminator. RAPD marker DNA of seeds derived from radical and the method described by Hanania et al. (2004).For PCR amplification; eight arbitrary 10-base primers were selected for PCR amplification. Amplification reactions were performed with 25 dm3 of 10×assay buffer, 2.0 of 1.25 mM each of dNTP’s, 15 ng of the primer, 1×Taq polymerase buffer, 0.5 units of Taq DNA polymerase (TaKaRa), 2.5 mM MgCl2, and 30 ng of genomic DNA. DNA amplification was performed in a Perkin Elmer Cetus 480 DNA Thermal Cycler programmed for 45 cycles as follows: 1st cycle of 3.5 min at 92°C, 1 min at 35°C, 2 min at 72°C; followed by 44 cycles each of 1 min at 92°C, 1 min at 35°C, 2 min at 72°C followed by one final extension cycle of 7 min at 72°C. The amplification products were separated by electrophoresis in 1.2% (w/v) agarose gels with 0.5×TBE buffer, stained with 0.2 mg dm-3 ethidium bromide. A 1 kb DNA ladder was used as molecular standards and the bands were visualized and analyzed by JD-801 Gel Electrophoresis Image analytic system (Jiangsu, China). All the reactions were repeated at least twice.


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Data analysis Each band was treated as one of the marker. Scoring of bands was done from photographs. Homology of bands was based on distance of migration in the gel. Presence of band was scored as “1”, absence of a band as “0” and missing data was denoted by “9”.

RESULTS

Seed Germination The initial seed germination of fresh seeds when the experiment was started in 1996 was 96%. After 14 years of storage at different temperature and moisture conditions the seed viability declined .Table 1.0 depicts the effect of different moisture levels at different temperatures on the seed viability. The seeds which were equilibrated to low level of moisture 3.4% and 4.2 % showed higher percentages of survival at all the temperatures including ambient temperature. At high moisture 6.3%, 7.7% and 10% and ambient temperature the seeds deteriorated with storage time. Although deterioration occurred in all the treatments at all the temperatures but the extent of deterioration was faster and maximum at ambient temperature where the seeds lost viability significantly after 14 years of storage. Seeds stored at 10% at ambient temperature and with 10% at all the temperatures completely lost their viability as all the seeds were dead after 14 years. The seed viability declined with storage time which was highly significant in the seeds maintained at ambient temperature compared to other temperatures of 10°C and 20°C. In case of ultra dry seeds the reduction in viability at all the temperatures was insignificant compared to the control. This clearly shows that ultra dry condition of seeds at all the temperatures performed well and viability was very close to the control. Seedling length and vigour index The initial value of shoot length was7.1 and root length was 9.5 cm in fresh control seedlings. The root and shoot length used as a measure of vigour showed a steady decline. Both shoot and root length decreased significantly when seeds were stored at higher mc. The trends in changes of root and shoot length were similar to those for germination. Changes in shoot length were more pronounced under high mc at all the three temperatures. Amongst the treatments 3.4%, 4.2% and 6.3% mc at all the three temperature were found to be the best with a maximum root length of 18.7cm, 18.3cm and 17.5cm respectively. A significant reduction in the vigour index was observed with storage period in all the conditions but the extent was high at ambient temperature where the reduction was highly significant. The mean vigour index of 1608 gradually decreased with increase in storage period in all the treatments. The change in vigour index is closely followed by changes in viability. Storage under high mc (7.7%) and ambient temperature resulted in a significant fall in the vigour index (176) after 14 years of storage. Dehydrogenase activity Activity of enzyme dehydrogenase declined with seed deterioration .In fresh seeds the activity of enzyme dehydrogenase (∆ OD/g fresh wt) was found to be 0.8549. Similar activity has been observed in the seeds maintained at 3.4%, 4.2% MC at all the three temperatures after 14 years of storage. However, at ambient temperature the activity was significantly lowered when seeds were stored with 7.7% and 10% of MC. However the decline in activity was less pronounced at


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4.2% MC at all the temperatures. Moisture content seems to have more deleterious effect on dehydrogenase activity compared to temperature .Further appraisal revealed that in spite of having same germination percentage of various treatments there was variation in their dehydrogenase enzyme activity. This could probably because of the internal state or the vigour status of the seed which has not been reflected by the germination percentage of the seeds. Lipid peroxidation Radish being an oil rich seed the extent of lipid peroxidation is of greater importance in the context of its storability pattern. Result of the data on lipid peroxidation showed that the values were least in healthy and 98 % viable seeds and showed and increased trend with seed deterioration. The values of lipid per oxidation were found to be significantly different under different treatments which increased with seed deterioration. The low value of 0.0954 malionaldehyde content (n mol /mg dwt.) in fresh control increased twice. The SDS –PAGE protein profile The SDS –PAGE protein profile did not show significant changes except that some of the high mobility low molecular weight bands disappeared during deterioration. The sharp bands disappeared or their intensity decreased as the viability decreased. The alteration in the banding pattern might be due to the loss of low molecular weight proteins under deterioration at ambient temperature and high moisture. In ultra dry seeds there were only minor changes of one or two bands in the protein banding pattern. Conversely ,the protein banding profile showed some new bands and disappearance of some existing bands in deteriorated seed samples .Similar reports were found in fresh and aged seeds in cotton (Varier and Dadlani 1992)and wheat seeds (Yogeesha et al.,1998).However ,the number of protein fractions decreased due to biochemical changes associated with seed ageing in tomato (Chakrabarti et al.1994) .Protein bands with mol. wt 250,130, 95, 72, and 55 kda were altogether absent in all the treatments .Some band are present between 55 and 36 Kda. From 28 to 11 Kda the entire band are present in all the treatments. Monitoring of genetic fidelity by RAPD In order to confirm genetic fidelity (at molecular level) of seeds after ultra-dry treatment, the seeds were screened with the 8 random RAPD primers, one primer that produced distinct amplification profiles. The representative profile of the ultra-dry seeds and the control (non-ultra dry) with primer is shown in Figure . It was obvious that the ultra-dry seeds showed identical RAPD profiles (i.e. nopolymorphism was observed). These results confirmed the genetic fidelity of the ultra-dry seeds. Several studies on the effect of seed deterioration at DNA level pointed out that chromosomal aberrations , point mutations and decrease in the activity of DNA repair enzymes are some of the major events , occurring during the process of ageing in seeds( Larson, 1997 and McDonald, 1999).While RAPD markers would scan the entire genome at random irrespective of being expressed or non -expressed, it is possible that most of the polymorphism observed in the amplified fragments are interspersed in the regions which are not highly conserved. Hence these sectors are likely to be fragmented or altered more at random than the regions which are highly conserved. In the present study there was not much difference in the RAPD profile of the aged


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and control seeds .The RAPD clustering showed the possible accumulation of aberrations in non-coding part, the junk DNA.

Table Effect of Moisture and Temperature on Germination of Radish seeds.

Figure 1. Figure 2.

(3.4 LTS, MTS.AMB.) (4.2 LTS, MTS, AMB)

Moisture in %

No of seeds germinates. LTS MTS Ambient

3.4

R1 25 23 24 R2 24 23 21 R3 25 24 23 R4 24 23 23

% 98 93 91 4.2

R1 23 21 23

R2 25 25 21 R3 24 24 22 R4 25 20 23

% 97 90 89 6.3

R1 24 23 18

R2 25 21 21 R3 23 22 22 R4 24 23 23

% 96 89 84 7.7

R1 18 19 _ R2 17 15 _ R3 20 18 _ R4 19 14 _

% 74 66 - 10

R1 - - _ R2 - - _ R3 - - _ R4 - - _

% - - -


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Figure 3. Figure4.

(6.3 LTS,MTS,AMB) (7.7 LTS,MTS,AMB)

Figure 5

(10 LTS,MTS,AMB)

Table 2.1 Effect of Moisture and Temperature on seedling length

Table 2.2 Effect of Moisture and Temperature on Vigour Index

Moisture in %

Seedling length in cm.

LTS MTS Ambient 3.4

R1 19.5 17.6 21.4 R2 13.4 23.2 14.3 Mean 16.4 20.4 17.8

4.2

R1 16.7 16.6 24 R2 20.3 17.8 21.3 Mean 18.5 17.2 22.6

6.3

R1 17.2 23.2 2.2

R2 22.4 17.8 2.0 Mean 19.8 20.5 2.1

7.7

R1 19.6 23.6 4.2 R2 22.1 20.1 4.3

Mean 20.8 21.8 4.2

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R1 11.6 20.9 0 R2 10.8 22.9 0

Mean 11.2 21.9 0

Moisture in %

Vigour index. LTS MTS Ambient

3.4 1607 1897 1619 4.2 1794 1548 2011 6.3 1900 1824 176 7.7 1539 1438 0 10 0 0 0


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Table: 3 Effects of Moisture and Temperature on Electric Conductivity.

Table 4.0 Effect of Moisture and Temperature on Enzyme Dehydrogenase

Table 5.0 Effect of Moisture and Temperature on the Lipid Peroxidation

Moisture in %

E C 0f per gram of sample in ms/cm. LTS MTS Ambient

3.4

R1 5.93 0.68 0.71 R2 4.25 0.46 0.56

MEAN 5.09 0.57 0.63 4.2

R1 4.26 0.70 0.52 R2 5.3 0.64 0.46

MEAN 4.78 0.67 0.49 6.3

R1 5.29 0.50 2.66 R2 0.79 0.59 3.08

MEAN 3.04 0.54 2.87 7.7

R1 0.59 0.58 3.19 R2 0.76 0.77 2.96

MEAN 0.67 0.67 3.07 10

R1 0.59 1.36 2.84 R2 0.65 0.74 2.8

MEAN 0.62 1.05 2.82

Moisture in %

Optical Density at 470 nm.

LTS MTS Ambient 3.4

R1 0.0995 0.0684 0.0626 R2 0.0714 0.0897 0.1725

Mean 0.0854 0.0790 0.1175

4.2

R1 0.1449 0.0766 0.0638 R2 0.0814 0.0444 0.0895 Mean 0.1131 0.0605 0.0766

6.3

R1 0.0640 0.0130 0.1770 R2 0.1043 0.1105 0.1782 Mean 0.0841 0.2065 0.1776

7.7

R1 0.0741 0.2125 0.0375 R2 0.1026 0.1036 0.0802 Mean 0.0883 0.1580 0.0588

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R1 0.1736 0.1712 0.0837 R2 0.0710 0.0581 0.0639 Mean 0.1223 0.1146 0.0738

Moisture in %

Difference of 600-532 nm wave length. LTS MTS Ambient

3.4

R1 0.0953 0.1086 0.1019 R2 0.1026 0.0927 0.1327

MEAN 0.0989 0.10065 0.1173 4.2

R1 0.0968 0.1042 0.1123 R2 0.0980 0.1367 0.1251


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Table 6.0 Effect of Moisture and Temperature on SDS-PAGE

Band no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14

1 250 + - - - - - - - - - - - - - 2 130 + - - - - - - - - - - - - - 3 95 + - - - - - - - - - - - - - 4 72 + - - - - - - - - - - - - - 5 55 + - - - - - - - - - - - - - 6 - - + + + + + - - - + + + + 7 36 + - - - - - - - - - - - - - 8 28 + + + + + + + + + + + + + - 9 - + + + + + + + + + + + + + 10 17 + + + + + + + + + + + + + + 11 - + + + + + + + + + + + + + 12 - + + + + + + + + + + + + + 13 11 + + + + + + + + + + + + + -

1 2 3 4 5 6 7 8 9 10 11 12 13 Fig: - Photograph of SDS-PAGE gel. Lane 1-13:- 1-7.7%MTS, 2-7.7%LTS, 3-6.3%Ambient, 4- 6.3%MTS, 5-6.3%LTS, 6- 4.2%Ambient, 7- 4.2% MTS, 8- 4.2%LTS, 9- 3.4%Ambient, 10-3.4%MTS, 11-3.4%LTS, 12-control, 13-ladder.

MEAN 0.0974 O.1204 0.1187 6.3

R1 0.0954 0.1187 0.0917 R2 0.0984 0.0977 0.1070

MEAN 0.0969 0.1082 0.0993 7.7

R1 0.0524 0.1070 0.0840 R2 0.0808 0.0913 0.1135

MEAN 0.0702 0.0991 0.0987 10

R1 0.0969 0.0949 0.1659 R2 0.0961 0.1044 0.0994

MEAN 0.0965 0.0996 0.1326


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DISCUSSION Temperature and moisture content has been indicated to be important factors, which influences the seed longevity during processing and storage of seeds. Therefore, the present investigations on Radish has been undertaken to understand the effect of different temperatures and mcs on the seed storability. The present study showed that germinability of radish seeds can be maintained for a prolonged duration if the seed internal moisture content is maintained at lower level of 3.4% and 4.2% and 6.3%. Analysis of data further revealed that seed internal moisture content has a more pronounced effect on seed germinability as compared to temperature. At low mc of 3.4% and 4.2% and 6.3% viability decreased by only 6% and 8% and 12% respectfully. Whereas at high mc 7.7% and 10% mc the viability was reduced to 0 at the end of 14 years at ambient temperature. Deteriorative reactions frequently proceed in the seed more readily if the moisture content is higher, and consequently the moisture condition would constitute a threat to longevity of seed survival.


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Vigour and related parameters Loss of viability always precedes loss of vigour. The changes in seedling vigour index also closely followed the changes in the viability since vigour index is directly dependent upon shoot length, root length and germination percent. Loss of vigour is associated with seed deterioration and is influenced by composition of seed, particularly the food reserves or the efficiency of mobilization of nutrients. In the present study, the highest vigour was observed in the control seeds, which decreased with seed storage under different conditions of temperature and humidity. The present studies showed a decrease in shoot length which was found to be directly proportional to the seed viability and it also confirms with the earlier reports in a number of crops (Harrington, 1972; Dey and Basu, 1982; Yadav et al., 1987; Dharamlingam and Basu, 1990). Shanmugavel (1993) observed a close association between the loss in seed germination and reduction in seedling vigour in soybean during the course of ageing at 70% RH. In the present investigation, it has been observed that seedling length and vigour index declined steadily over the storage period and were more pronounced at higher moisture contents and ambient temperature. Seed deterioration is generally initiated in the meristematic tissue. Meristematic areas and particularly the radical meristem appear most prone to deterioration. At least two reasons are forwarded to explain this observation. Generally, the radical of most seeds is located at the funicular end of the seed, an area permitting rapid entry and penetration of water during imbibition (McDonald, 1999) along with likely intake of oxygen as well. In addition, meristematic regions are energy intensive and possess high numbers of mitochondria compared to other cellular tissues. Lipid peroxidation occurring within mitochondria of the radical tip would explain the reduced seedling growth characteristics of deteriorated seeds. Electrical conductivity of seed leachates Membrane is the most important site of a seed which appears to be effected by seed deterioration/ ageing (Ching and Schoolcraft, 1968). Degradative changes in cellular membranes are (is one of) the early events of seed ageing (Heydecker, 1972). Enhanced solute leakage from imbibed seed is associated with the loss in seed vigour and viability (Matthews and Bradnock, 1968; Dadlani and Agrawal, 1985). In the present study also, increase in electrolyte leakage was noted before the reduction in germination. The decrease in membrane integrity and occurrence of membrane lesions might play a significant role in the deterioration of seeds has been supported by the work on solute leaching accompanying a fall in germ inability and viability Nutile, 1964., Powell and Matthews , 1977 ., Halder et.al., 1982) . The increase in the amount of electrolytes is found to be proportional to the seed deterioration, attaining maximum values when seeds lost viability completely during storage at high humidity (Table 3.0). At these levels the seeds showed very low germination. Therefore seed deterioration primarily results in loss of solutes thereby resulting in increase in the electrical conductivity which could be interpreted in terms of irreversible changes in membrane structure and loss of unique chemical structure of membranes essential for viability. In present study radish seeds showed reduced level of leaching under low mc and low temperature in comparison to seeds stored at high mc and ambient temperature. The leaching of total electrolytes was always less in seeds stored under desiccation and in this case below 3.4% moisture content. The increase in the amount of electrolytes is found to be proportional to the


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seed deterioration, attaining maximum values when seeds lost viability to 27% or lower. Below these levels the seeds did not germinate. Protein profile The SDS –PAGE protein profile did not show significant changes except that some of the high mobility bands disappeared during deterioration. The sharp bands disappeared or their intensity decreased as the viability decreased. The alteration in the banding pattern might be due to the loss of low molecular weight proteins under deterioration at ambient temperature and high RH. Conversely, the protein banding profile did not vary between fresh and aged seeds in cotton (Varier and Dadlani 1992) and wheat seeds (Yogeesha et al., 1998). However, the number of protein fractions decreased due to biochemical changes associated with seed ageing in tomato (Chakrabarti et al.1994) Dehydrogenase Activity The data as periodic profile of absorbance (A) of formazon revealed that high intensity of formazon was retained at low mc. Ray and Gupta (1980) also noted reduced dehydrogenase activity in terms of formazon formation in redish seeds undergoing deterioration. Lipid peroxidation and Radical scavenging enzymes An increase in the lipid peroxidation was reported in seeds/embryonic axes of several recalcitrant seed species during desiccation (Hendry et, al., 1992., Finch-Savage 1992a, Chandel et.al., 1995). Under the present studies there is a progressive increase in the lipid peroxidation during storage in sunflower seeds (Table5.0). These studies indicated a good correlation of increased electrolyte leakage with increased lipid peroxidation content, which indicates the destruction of membranes caused by peroxidation of lipids during storage. A decrease in the enzymic protection was probably associated with the lipid peroxidation in redish seeds. The increase in malondialdehyde throughout the levels of ageing in seeds has been reported by several workers (Harman and Mattick, 1976., Stewart and Bewley, 1980, Sung and Jeng, 1994). Changes in the membrane integrity results in the efflux of electrolytes in seed steep water (increase in EC values). Lipid peroxidation in stored seeds is the possible reason for the loss in seed viability during storage at high humidities. Hence it can be inferred that stress due to high temperature and humidity adds to the deleterious lipid peroxidation reactions. Perhaps the changes in seed MDA content during ageing support the hypothesis that loss of seed viability is associated with lipid peroxidation. Molecular analysis of seed ageing Several studies on the effect of seed deterioration at DNA level pointed out that chromosomal aberrations , point mutations and decrease in the activity of DNA repair enzymes are some of the major events , occurring during the process of ageing in seeds( Larson, 1997 and McDonald, 1999).While RAPD markers would scan the entire genome at random irrespective of being expressed or non –expressed, it is possible that most of the polymorphism observed in the amplified fragments are interspersed in the regions which are not highly conserved. Hence these sectors are likely to be fragmented or altered more at random than the regions which are highly conserved. In the present study there was not much difference in the RAPD profile of the aged


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and control seeds .The RAPD clustering showed the possible accumulation of aberrations in non-coding part, the junk DNA. From the above set of experiments it can be concluded that ultra-dessication can be used as a method for safe conservation of radish seeds both by the scientists as well as by the farmers. At ambient storage, most species showed maximum germination when seeds were stored at moistures less than 4% with the corresponding moisture level dependent on the crop species. This value (at which germination is maximum) is within the range of the FAO/IPGRI recommendation of 5–3% moisture (FAO/IPGRI, 1994) Based on this data; we conclude that drying below the currently recommended seed moisture level prolongs the life spans of seeds in radish crops, at least under ambient storage.

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[28] Shen, D. and X. Qi. 1998. Seed Sci. Res. 8(Suppl 1): 47-53. [29] Meng, S.C., H.Y. Zhang, P.Y. Liu, and X.H. Kong. 2003. Acta Agric. Sin. 18: 26-23. [30] Kun, E. and L. Abood. 1949. Science 109: 144-146.

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Pelagia Research Library - iMedPub · 2016-09-21 · Mukesh Kumar et al Asian J. Plant Sci. Res., 2011, 1 (1):5-21 _____ 7 Pelagia Research Library for 48 h in a sealed desiccator