Seed fortification with chicory (Cichorium intybus L.) leaf extract to enhance seed vigour, viability and productivity in blackgram (Vigna mungo L.

Transcription

1 Legume Research, 38 (6) 2015 : Print ISSN: / Online ISSN: AGRICULTURAL RESEARCH COMMUNICATION CENTRE Seed fortification with chicory (Cichorium intybus L.) leaf extract to enhance seed vigour, viability and productivity in blackgram (Vigna mungo L.) C. Vanitha* and M. Kathiravan 1 Agricultural College and Research Institute, Madurai , India. Received: Accepted: DOI: /lr.v38i ABSTRACT Study was carried out to evaluate the effect of herbal fortification of seeds with chicory leaf extract on seed physiological properties related to vigour, viability and yield potential in blackgram cv. ADT 3. The treatment was effected through soaking of one month old seeds with fresh chicory (Cichorium intybus L) leaf extract at 10, 20 and 25 per cent concentrations followed by dehydration in a drying chamber at 30 C for three days to reach original moisture content (10 ± 0.25% on wet weight basis). Part of the treated seeds was subjected to artificial and natural ageing process. Treated and aged seeds when evaluated through germination per cent, speed of germination, dry matter production and biochemical properties, significant differences were evident with 20 per cent concentration. The magnitude of reduction after ageing in respect of the aforesaid parameters was more in control and the least in herbal fortified seeds. Treated seeds (20% extract) exhibited higher activity of free radical scavenging enzyme of catalase (2122 units), lower amounts of leachate amino acids (40.15 µg) and lipid peroxidation value (0.144) and also an enhancement in dry weight accumulation (20.23 g), leaf area index (5.35) and net assimilation rate (0.41 mg) leading to higher yield per plant (6.15 g) under field conditions. Key words: Blackgram, Chicory leaf extract, Fortification, Productivity, Seed vigour, Viability. INTRODUCTION Black gram (Vigna mungo L), one of the principal pulse crop in India, is next to chickpea and redgram in area and production. The increase in yield achieved in these crops is far from satisfactory in spite of research attempts made by way of genetic improvement in terms of higher harvest index and response to management practices. Of the several reasons attributed to this poor performance, seed quality is considered to be very important. Hydration-dehydration of fresh seeds would result in reduced storability and especially in legumes, this treatment would result in soaking injury. Such a disadvantage reported in pulses could be overcome through pre- conditioning of seeds (Saha and Basu, 1984) which allows the seeds to slow hydration and prevent the soaking injury. This technique reported to augment viability (Suneetha et al., 2003) was effective only in stored seeds and the disadvantage has been overcome through solvent infusion (Singh and Amritphale, 1993) and usage of vapours (Mandal et al., 2000). Supplementing with antioxidants, via hydration - dehydration or solvent infusion has an added advantage (Pallavi et al., 2003). In the wake of identification of natural resources rich in anti-oxidants, many medicinal plants have become major candidates for research. By virtue of its higher enzymic and non-enzymic anti-oxidants (Saroja et al., 2000), chicory, a medicinal herb has the potential to play a useful role in seed preservation. This aspect of chicory has not yet been exploited in vigour and viability maintenance of pulse seeds particularly blackgram. In view of the nonresponsiveness of freshly harvested high vigour seeds to fortification, the present study was attempted using one month old seeds. The herbal fortfication treatments using fresh leaf extracts of chicory were effected to blackgram cv. ADT 3 to elucidate their effects on maintenance of vigour, viability and productivity. MATERIALS AND METHODS One month old seeds of blackgram cv. ADT 3 stored under ambient conditions at Agricultural College and Research Institute, Madurai with a moisture content of 10±0.25 % (wet weight basis) were used for the study during Hundred grams of 45 days old fresh chicory leaves were macerated in a mortar and pestle using 30 ml of water, filtered and the volume was made upto 100 ml with water to constitute 100 per cent chicory leaf extract. Different concentrations were prepared using this as stock solution. Seeds retained by 8/64" round perforated metal sieve were *Corresponding author s cvani_seed@yahoo.co.in. Address: National Pulses Research Centre, Vamban, 1 KVK, Vamban.

2 Volume 38 Issue 6 (2015) 805 preconditioned for two hours in chicory leaf extracts at 10, 20 and 25 per cent concentrations (1:0.3 (W/V) seed to herbal extract) along with water and then soaked for two hours in the same concentration (1:1 seed to herbal extract) under aerated conditions at room temperature (26 ± 1 C). Besides leaf extract treatments, water soaking was also given following the same procedure. The treated and water soaked seeds were dried under shade for 24h and then at 30 C for 36 hours to reach the original moisture content. Part of the treated and untreated seeds subjected to accelerated ageing for seven days. The number of days required for reduction in germination to approximately 50 per cent was fixed as 7 days through pilot studies under accelerated ageing (RH 95 ± 2 % and temp.40 ± 1 C). After drying, part of the treated seeds along with control were packed in cloth bags and kept under ambient storage (mean temp. 27 ± 1 C and RH 66 ± 2%) for l2 months. Immediately after treatment and ageing, germination test was carried out in paper medium in quadruplicate using 100 seeds for each treatment with four sub replicates of 25 seeds (ISTA, 1999), following inclined plate method (Punjabi and Basu, 1982) in a germination room maintained at a temperature of 25 ± 1 C and RH 96 ± 2 percent with diffused light (approx. 10 h) during the day. Final count on normal seedlings was recorded on seventh day and percent germination computed. Rate of germination was calculated using the formula of Maguire (1962). X 1 X 2 -X 1 X n -(X n -1) Rate of germination = Y 1 Y 2 Y n where X n = Percentage germination on n th count Y n = Number of days from sowing to n th count The seedling dry weights were determined using normal seedlings after drying at 80 C for 36 h. and mean of dry weight arrived and expressed as mg 10 seedlings -1. The electrical conductivity value was measured by soaking 25 seeds in 50 ml of deionized water for 8 h. The leachate was used for determining amino acids against leucine standard. Catalase activity and lipid peroxide formation were assessed using embryos from seeds preconditioned with moist blotters for 48 h (Sadasivam and Manickam, 1992). All the analysis was made in duplicate. Based on the efficacy of chicory leaf extract as evaluated through vigour and viability parameters under laboratory conditions conducted before and after accelerated ageing, one month old treated seeds alone were forwarded to field trial in both kharif and rabi 2009 season at experimental fields of Agricultural College and Research Institute, Madurai (9 5 North and 78 5 East and altitude of 147 M above MSL). The soil texture of the experimental field is sandy loam, ph and EC is 7.2 and 0.32 dsm -1, respectively. The available N,P and K is 275,16.8 and 255 kg ha -1. Seed rate compensation was done after Manjunathaswamy and Narayanaswamy (1996) to maintain the plant population per unit area. The recommended crop management practices were followed. Dry weight of the plants was recorded at 40 days of sowing after drying in shade and oven at 80 C for 36 h. Leaf area index after 40 days of sowing and net assimilation rate between days of sowing were determined. Seed yield per plant was recorded after drying to uniform moisture content (10 ± 0.25%). The data were subjected to an Analysis of Variance and treatment differences tested (test) for significance (P > 0.05) after Gomez and Gomez (1984). Wherever necessary, the percentage values were transformed to arc sine values. RESULTS AND DISCUSSION The germination of blackgram was unaltered by treatment with chicory leaf extracts at all concentrations when assessed immediately after treatment. However, chicory leaf extract at 20 per cent concentration enhanced the germination after accelerated (by 10 and 13 %) and natural ageing (by 10 and 14%) respectively over water soaking and control. Speed of germination was also hastened by the same concentration immediately (15.84) and after accelerated (8.64) and natural ageing (8.20) compared to control (11.36 and 6.25, respectively). Seedling dry weight also exhibited the same trend (0.244, and g immediately, after accelerated and natural ageing). Treatment of seeds with different concentrations of chicory leaf extract recorded higher values for vigour and viability parameters when compared with water soaking (Table. 1) and the effect was much better than control. Minimal electrical conductivity (0.120, and before and after accelerated and natural ageing) was observed with chicory leaf extract of 20 per cent implying least damage to membrane integrity. Similar trend was observed for leachate amino acids (40.15, and µg). Seeds treated with 20 per cent chicory leaf extract also had the highest catalase activity (2122, 1566 and 1112 units before and after accelerated and natural ageing). While untreated seeds recorded higher lipid peroxidation values, it was the least in treated seeds and especially, the effect was more pronounced in 20 per cent concentration (0.144, and in fresh, aged and naturally aged seeds, respectively) (Table. 2). The efficacy of the chicory leaf extract, when assessed under field conditions, the treatments were very effective during kharif than rabi season. Pooled data indicated highest dry weight (18.52 g) (Table 3) and leaf area index (4.28) (Table 4). In fresh seeds, chicory 20% concentration recorded maximum value for net assimilation

3 806 LEGUME RESEARCH - AN INTERNATIONAL JOURNAL TABLE 1: Effect of chicory leaf extract on germination (%), speed of germination and drymatter production (g 10 seedlings -1 ) in blackgram cv. ADT 3. Treatments Germination (%) Speed of germination Drymatter production (g 10 seedlings -1 ) BA AA NA BA AA NA BA AA NA Control (60.82) (43.86) (41.54) Water (61.85) (44.97) (42.89) Chicory 10% (61.85) (45.72) (44.49) Chicory 20% (62.11) (48.31) (46.36) Chicory 25% (61.85) (46.36) (44.19) SEd (0.08) (0.77) (0.59) CD (P=0.05) NS (1.78) (1.36) Figures in parentheses are arc sine transformations NS Non Significant BA : Before ageing AA : After ageing NA : Natural ageing under ambient conditions for 12 months(mean temp.27±1 0 C, RH 66 ± 2%) TABLE 2: Effect of chicory leaf extract seed fortification on biochemical properties in blackgram cv. ADT 3. Treatments Electrical conductivity Leachate amino acids Catalase (units g -1 ) Lipid peroxidation (dsm -1 ) (mg g -1 ) (OD) BA AA NA BA AA NA BA AA NA BA AA NA Control Water Chicory 10% Chicory 20% Chicory 25% SEd CD (P 0.05) Figures in parentheses are arc sine transformations NS Non Significant BA : Before ageing AA : After ageing NA : Natural ageing under ambient conditions for 12 months(mean temp.27±1 0 C, RH 66 ± 2%) TABLE 3: Effect of chicory leaf extract on dry weight (g plant -1 ) in blackgram cv. ADT.3 Treatments Dry weight (g plant -1 ) Kharif Rabi Pooled Fresh Accelerated Mean Fresh Accelerated Mean Fresh Accelerated Mean seeds aged seeds seeds aged seeds seeds aged seeds Control Water Chicory 10% Chicory 20% Chicory 25% S T SXT S T SXT S T SXT SEd CD(P 0.05) NS S - Season T-Treatment NS - Non Significant ** - Significant at 1% level * - Significant at 5% level

4 Volume 38 Issue 6 (2015) 807 rate (0.41) (Fig 1), yield per plant (6.15 g) (Fig. 2) and yield per plot (410 g) (Fig. 3). Damage to membrane, one of the suggested explanations for loss of viability during ageing is the reflection of seed deterioration, since ageing of dried seeds in storage is thought to be accompanied by alterations in the membranes of aged seeds (Heydecker, 1972). In the present study, the electrical conductivity in general was minimum in treated seeds and the highest in untreated seeds under accelerated and natural ageing. This implies the operation of repair mechanism brought on by treatment with fresh leaf extracts of chicory. Enzymes can be inactivated easily by free radicals and ageing could lead to a reduction in the activity of peroxide scavenging enzymes during hydration (Jeng and Sung, 1994). The loss of enzyme activity in the untreated seeds might partially explain the accumulation of peroxides compared to the treated seeds. Several studies have demonstrated that ageing of seed is associated with loss of antioxidant enzyme activity (Dc Vos et al., 1994 and Bernal - Lugo et al., 2000). Higher catalase activity by hydro-priming has been reported in soybean (Posmyk et al., 2001) indicating the involvement of antioxidant enzymes in seed invigoration. Increased FIG 1: Effect of chicory leaf extract on Net Assimilation Rate (NAR) (mg cm -2 d -1 ) in blackgram cv. ADT.3 FIG 2: Effect of chicory leaf extract on yield per plant in blackgram cv. ADT.3 FIG 3: Effect of chicory leaf extract on yield plot -1 in blackgram cv. ADT.3 catalase activity in chicory leaf extract was associated with presence of high catalase enzyme in chicory leafs (Table 5) and that leads to decreased lipid peroxidation. Conversely, slow germination of aged seeds associated with low catalase activity observed in previous studies on soybean (Sung, 1996) was evident in the present study also, as evidenced by lower values for speed of germination in untreated seeds. Slower germination of aged seeds might be explained in terms of extended time necessary for the cells to reinitiate the antioxidant machinery. In the present investigation, the lipid peroxidation was maximum in untreated control both under accelerated and natural ageing (Table 2) giving credence to the assumption that lipid peroxidation and free radical reactions are basic reactions of deteriorative senescence. It is possible that phosphorous along with other minerals and vitamin C, present in chicory leaf extracts would have helped for substitution of scavenging properties to bring down peroxidative changes ultimately maintaining vigour and viability at a higher level. The beneficial effects of chicory leaf extract in treated seeds may be attributed not only to cellular repair but also to the control of free radical reaction. Under field conditions, the treated seeds of blackgram produced vigorous plants in which, the physiological and yield components were the highest. In order to achieve the highest possible yield, the physiological parameters especially leaf area index must be higher to intercept more solar energy for higher dry matter accumulation (Major and Daynard, 1972). Net assimilation rate as a measure of growth efficiency was highest in treated

5 808 LEGUME RESEARCH - AN INTERNATIONAL JOURNAL Treatments TABLE 4: Effect of chicory leaf extract on Leaf Area Index (LAI) in blackgram cv. ADT.3 Leaf Area Index (LAI) Kharif Rabi Pooled Mean Fresh Accelerated Mean Fresh Accelerated Mean Fresh Accelerated Mean seeds aged seeds seeds aged seeds seeds aged seeds Control Water Chicory 10% Chicory 20% Chicory 25% S T SXT S T SXT S T SXT SEd CD(P 0.05) 0.9 ** 1.42 ** NS 1.01 ** 1.62 ** 0.68 ** 0.65 ** 1.01 ** NS S-Season T-Treatment NS - Non Significant ** - Significant at 1% level * - Significant at 5% level seeds than control (Fig.1) and gain support from the work of Egli and Yu-Zhen Wen (1991). It is the amount of biomass deployed into some storage organs like grain which is the ultimate objective of the best treatment. The reproductive output and drymatter accumulation are positively correlated (Carlos et al., 1995) and in the present study the differences in drymatter accumulation was wider between treated and control (Table 3). Though the effects of vigour on field establishment are many (Halmer and Bewley, 1984), information on the association between vigour and yield is meagre and assumes practical significance. Reduced efficiency of sub cellular organelles such as mitochondria or chloroplast in low vigour seeds would explain the reasons for lesser yield in untreated seeds (Fig 2 and Fig.3) (Powell and Matthews, 1980). If seed vigour affects yield, its influence could be observed through some effect on growth parameters like dry matter production, photosynthetic efficiency and related factors. Higher values for physiological parameters in treated seeds could explain the efficient translocation of assimilates to the sink. The per plant yield in untreated was markedly lower than TABLE 5: Nutrient and antioxidant components in kasini leaf (Cichorium intybus L.) cv. Kalpa after 45 days of sowing. No Details Units Nutrients 1. Nitrogen (mg 100g -1 ) Phosphorus (mg 100g -1 ) Potassium (mg 100g -1 ) Calcium (mg 100g -1 ) Sodium (mg 100g -1 ) Iron (mg 100g -1 ) Manganese (mg 100g -1 ) Copper (mg 100g -1 ) Zinc (mg 100g -1 ) Antioxidants 1. Vitamin C (mg 100g -1 ) Peroxidase (mg g -1 min -1 ) Catalase (Units g -1 ) 2428 that of seeds treated with chicory leaf extracts. Therefore, the reason for the reduced yield in control can be assigned clearly to loss of vigour. Hence, herbal fortification (organic) with 20% chicory leaf extract for four hours is suggested for maintaining vigour, viability and yield in blackgram. REFERENCES Bernal-Lugo, I., Camacho, A. and Carballo A. (2000). Effects of seed ageing on the enzymic antioxidant system of maize cultivars. In: Seed Biology (Black, M. Bradford, K.J. Vazquez-Ramos, J. Eds),. Wallingford, CABI Publishing. pp Carlos, L.B., Scopel, A.L. and Sanchez R.A. (1995). Plant photo morphogenesis in canopies, crop growth and yield. Hort. Sci., 30 : De Vos, C.H.R., Karaak H.L. and Bino R.J. (1994). Ageing of tomato seeds involves glutathione oxidation. Physiol Planta., 92: Egli. D.B. and Yu-Zhen-Wen (1991). Crop growth rate and seeds per unit area in soybean. Crop Sci., 31: Gomez, K.A. and Gomez A.A. (1984). Statistical Procedures for Agricultural Research. John Willey and Sons, New York. Halmer, P. and Bewley J.D. (1984). A physiological perspective on seed vigour testing. Seed Sci &Technol., 12, Heydecker, W. (1972). Vigour. In: Viability of Seeds (ed. Roberts, E.H.)., Chapman and Hall, London. Pp

6 Volume 38 Issue 6 (2015) 809 ISTA.(1999). International Rules for Seed Testing. Seed Sci. & Technol., 13 : Jeng, T.L. and Sung J.M. (1994). Hydration effect on lipid peroxidation and peroxide- scavenging enzymes activity of artificially aged peanut seed. Seed Sci.&Technol., 22: Maguire, I.D. (1962). Speed of germination aid in selection and evaluation for seedling emergence and vigour. Crop Sci., 2 : Major. D.J. and Dayanand S. (1972). Hyperbolic relation between leaf area index and plant population in corn (Zea mays). Can. J. Plant Sci., 52 : Mandal, A.K., Saha, R. B.K. and Basu, R.N. (2000). Seed invigoration treatments for improved storability, field emergence and productivity of soybean (Glycine max L.). Seed Sci. & Technol., 28 : Manjunathaswamy, K.K. and Narayanaswamy R. (1996). Effect of natural ageing and seed rate compensation on crop growth performance and grain yield in hybrid maize (Zea mays L.). Seed Res., 24(2) : Pallavi, M., Kumar, S.S. Dangi K.S. and Reddy A.V. (2003). Effect of seed ageing on physiological, biochemical and yield attributes in sunflower cv. Morden. Seed Res., 31(2) : Posmyk, M.M., Corbineau, F. Vinel, D. Bailly D. and Come D. (2001). Osmoconditioning reduces physiological and biochemical damage induced by chilling in soybean seeds. Physiol. Planta., 111: Powell. A.A. and Matthews S. (1980). The significance of damage during imbibition to the emergence of pea (Pisum Sativum L.) seeds. J. Agric. Sci., 95 : Punjabi, B. and Basu R.N. (1982). Testing germination and seedling growth by an inclined glass plate blotter method. Indian J. Plant Physiol., 25 : Sadasivam, S. and Manickam A. (1992). In: Biochemical methods for Agricultural Sciences, Wiley Eastern Limited, New Delhi: Pp Saha, R. and Basu, R.N. (1984). Invigouration of soybean seed for the alleviation of soaking injury and ageing damage on germinability. Seed Sci. & Technol.,12: Saroja, S., Padma, P.R., Radha P. and Thilagavathy P. (2000). Enzymic and non enzymic antioxidants in Cichorium intybus. Curr. Sci., 2: Singh, B. and Amritphale, D. (1993). Effects of dry permeated gibberrellic acid and benzyladenine on germinability of soybean seeds during storage. Seed Sci. & Technol., 21 : Suneetha, S., Kurdikeri, M.B., Shekhargouda M. and Shashidhar, S.D. (2003). Effect of seed invigoration on storage potential of KBSH hybrid sunflower. Seed Res., 31: Sung, J.M. (1996). Lipid peroxidation and peroxide scavenging in soybean seeds during ageing. Physiol. Planta., 97 :